Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors Reviewed International journal

Loc Dinh Hoang, Eriko Aoyama, Miki Hiasa, Hiroshi Omote, Satoshi Kubota, Takuo Kuboki, Masaharu Takigawa

International Journal of Molecular Sciences 24 ( 24 ) 17294 - 17294 2023.12

Authorship：Last author,　Corresponding author Publishing type：Research paper (scientific journal) Publisher：MDPI AG

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

DOI： 10.3390/ijms242417294

Expression and function of CCN2-derived circRNAs in chondrocytes Reviewed

Soma Kato, Kazumi Kawata, Takashi Nishida, Tomomi Mizukawa, Masaharu Takigawa, Seiji Iida, Satoshi Kubota

Journal of Cell Communication and Signaling 2023.9

Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Abstract

Cellular communication network factor 2 (CCN2) molecules promote endochondral ossification and articular cartilage regeneration, and circular RNAs (circRNAs), which arise from various genes and regulate gene expression by adsorbing miRNAs, are known to be synthesized from CCN2 in human vascular endothelial cells and other types of cells. However, in chondrocytes, not only the function but also the presence of CCN2-derived circRNA remains completely unknown. In the present study, we investigated the expression and function of CCN2-derived circRNAs in chondrocytes. Amplicons smaller than those from known CCN2-derived circRNAs were observed using RT-PCR analysis that could specifically amplify CCN2-derived circRNAs in human chondrocytic HCS-2/8 cells. The nucleotide sequences of the PCR products indicated novel circRNAs in the HCS-2/8 cells that were different from known CCN2-derived circRNAs. Moreover, the expression of several Ccn2-derived circRNAs in murine chondroblastic ATDC5 cells was confirmed and observed to change alongside chondrocytic differentiation. Next, one of these circRNAs was knocked down in HCS-2/8 cells to investigate the function of the human CCN2-derived circRNA. As a result, CCN2-derived circRNA knockdown significantly reduced the expression of aggrecan mRNA and proteoglycan synthesis. Our data suggest that CCN2-derived circRNAs are expressed in chondrocytes and play a role in chondrogenic differentiation.

Graphical abstract

DOI： 10.1007/s12079-023-00782-7

Other Link： https://link.springer.com/article/10.1007/s12079-023-00782-7/fulltext.html

Dual roles of cellular communication network factor 6 (CCN6) in the invasion and metastasis of oral cancer cells to bone via binding to BMP2 and RANKL Reviewed

Hiroaki Hochi, Satoshi Kubota, Masaharu Takigawa, Takashi Nishida

Carcinogenesis 2023.8

Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

Abstract

The acquisition of motility via epithelial–mesenchymal transition (EMT) and osteoclast induction are essential for the invasion and metastasis of oral squamous cell carcinoma (OSCC) to bone. However, the molecule suppressing both EMT and osteoclastogenesis is still unknown. In this study, we found that cellular communication network factor 6 (CCN6) was less produced in a human OSCC cell line, HSC-3 with mesenchymal phenotype, than in HSC-2 cells without it. Notably, CCN6 interacted with bone morphogenetic protein 2 (BMP2) and suppressed the cell migration of HSC-3 cells stimulated by BMP2. Moreover, knockdown of CCN6 in HSC-2 cells led to the promotion of EMT and enhanced the effect of transforming growth factor-β (TGF-β) on the promotion of EMT. Furthermore, CCN6 combined with BMP2 suppressed EMT. These results suggest that CCN6 strongly suppresses EMT in cooperation with BMP2 and TGF-β. Interestingly, CCN6 combined with BMP2 increased the gene expression of receptor activator of nuclear factor-κB ligand (RANKL) in HSC-2 and HSC-3 cells. Additionally, CCN6 interacted with RANKL, and CCN6 combined with RANKL suppressed RANKL-induced osteoclast formation. In metastatic lesions, increasing BMP2 due to the bone destruction led to interference with binding of CCN6 to RANKL, which results in the promotion of bone metastasis of OSCC cells due to continuous osteoclastogenesis. These findings suggest that CCN6 plays dual roles in the suppression of EMT and in the promotion of bone destruction of OSCC in primary and metastatic lesions, respectively, through cooperation with BMP2 and interference with RANKL.

DOI： 10.1093/carcin/bgad057

軟骨細胞におけるCCN2由来circRNAの発現および機能の探索

加藤壮真, 河田かずみ, 西田崇, 水川朋美, 滝川正春, 飯田征二, 久保田聡

日本骨代謝学会学術集会プログラム抄録集 41回 149 - 149 2023.7

Do not overwork: cellular communication network factor 3 for life in cartilage. Reviewed International journal

Satoshi Kubota, Harumi Kawaki, Bernard Perbal, Masaharu Takigawa, Kazumi Kawata, Takako Hattori, Takashi Nishida

Journal of cell communication and signaling 17 ( 2 ) 353 - 359 2023.6

Language：English Publishing type：Research paper (scientific journal)

Cellular communication network factor (CCN) 3, which is one of the founding members of the CCN family, displays diverse functions. However, this protein generally represses the proliferation of a variety of cells. Along with skeletal development, CCN3 is produced in cartilaginous anlagen, growth plate cartilage and epiphysial cartilage. Interestingly, CCN3 is drastically induced in the growth plates of mice lacking CCN2, which promotes endochondral ossification. Notably, chondrocytes in these mutant mice with elevated CCN3 production also suffer from impaired glycolysis and energy metabolism, suggesting a critical role of CCN3 in cartilage metabolism. Recently, CCN3 was found to be strongly induced by impaired glycolysis, and in our study, we located an enhancer that mediated CCN3 regulation via starvation. Subsequent investigations specified regulatory factor binding to the X-box 1 (RFX1) as a transcription factor mediating this CCN3 regulation. Impaired glycolysis is a serious problem, resulting in an energy shortage in cartilage without vasculature. CCN3 produced under such starved conditions restricts energy consumption by repressing cell proliferation, leading chondrocytes to quiescence and survival. This CCN3 regulatory system is indicated to play an important role in articular cartilage maintenance, as well as in skeletal development. Furthermore, CCN3 continues to regulate cartilage metabolism even during the aging process, probably utilizing this regulatory system. Altogether, CCN3 seems to prevent "overwork" by chondrocytes to ensure their sustainable life in cartilage by sensing energy metabolism. Similar roles are suspected to exist in relation to systemic metabolism, since CCN3 is found in the bloodstream.

DOI： 10.1007/s12079-023-00723-4

Angiogenesis Assays for the Analysis of CCN Proteins Invited Reviewed International journal

Tsuyoshi Shimo, Mari Shimatani, Akihiko Tanimura, Masaharu Takigawa

Methods in Molecular Biology 2582 295 - 308 2023

Authorship：Last author Language：English Publishing type：Part of collection (book)

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the actin of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) and CCN2-neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM). In addition, we introduce an autofluorescence imaging of blood vessels in the subcutaneous tumor xenograft mouse model. These assays can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

DOI： 10.1007/978-1-0716-2744-0_20

Protocols for Screening Peptides Binding to CCN Family 2 Proteins and Their Extended Utility Invited Reviewed International journal

Satoshi Kubota, Harumi Kawaki, Masaharu Takigawa

Methods in Molecular Biology 2582 87 - 101 2023

Authorship：Last author Language：English Publishing type：Research paper (scientific journal)

The function of CCN family proteins is determined by their interactions with multiple cofactors that are present in the microenvironment. Therefore, determining these cofactors is critically important in understanding the molecular function of CCN family members. For this objective, a bacteriophage random peptide display library is a suitable tool. In this library, each filamentous bacteriophage is designed to display an oligopeptide of 7-20 random amino acid residues on its surface. Bacteriophage clones that possess peptides that bind to a CCN family protein are selected through several cycles of a process called biopanning or affinity selection. By determining the nucleotide sequence of the DNA that encodes the displayed peptide, the oligopeptides that specifically bind to the CCN family member can be specified. The obtained peptide sequences can be utilized to design peptide aptamers for CCN family proteins, or as a key sequence to determine new CCN family cofactor candidates in silico. Instead of a random peptide cDNA library, an antibody cDNA library from naïve lymphocytes or from B cells immunized by a CCN family protein can be used in order to obtain a highly specific CCN family detection or functional modulation tool.

DOI： 10.1007/978-1-0716-2744-0_8

Evaluation of the Molecular Interaction Between CCN 2 Protein and Its Binding Partners: A Solid-Phase Binding 3 Assay and Surface Plasmon Resonance Invited Reviewed

Eriko Aoyama, Masaharu Takigawa

Methods in Molecular Biology 2582 77 - 86 2023

Imaging of Molecular Interaction Between CCN Protein 2 and Its Binding Partners: An In Situ Proximity Ligation 3 Assay of Interaction Between CCN2 and Rab14 4 in Chondrocytes Reviewed International journal

Mitsuhiro Hoshijima, Eriko Aoyama, Hiroshi Kamioka, Masaharu Takigawa

Methods in Molecular Biology 2582 31 - 37 2023

Authorship：Last author Language：English Publishing type：Research paper (scientific journal)

An in situ proximity ligation assay (PLA) enables visualization of protein interactions in fixed cells. It is a powerful method for investigating protein-protein binding of endogenously expressed proteins. To confirm binding between CCN2 and Rab14 GTPase (Rab14) in chondrocytes, we performed a PLA using chondrocytic HCS-2/8 cells. The protocol in this chapter introduces an optimized technique for visualizing intracellular interactions of CCN2 and Rab14 in fixed cells using a PLA.

DOI： 10.1007/978-1-0716-2744-0_4

Cellular Fluorescence Imaging for the Evaluation 2 of Bioactivity of CCN Family Proteins Reviewed International journal

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 2582 23 - 29 2023

Authorship：Last author Language：English Publishing type：Part of collection (book)

The method of labeling proteins of interest with fluorescent dyes that can specifically stain organelles in living cells provides a tool for investigating various cellular processes under a microscope. Visualization (imaging) of the cells using fluorescence has many advantages, including the ability to stain multiple cell organelles and intracellular proteins simultaneously and discriminately, and is used in many research fields. In this chapter, we describe the observation of cell organelles using fluorescence staining to analyze the functions of CCN family proteins involved in various cellular events.

DOI： 10.1007/978-1-0716-2744-0_3

Novel Cell Biological Assays for Measuring Bone 2 Remodeling Activities of CCN Proteins International journal

Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

Methods in molecular biology (Clifton, N.J.) 2582 255 - 268 2023

Authorship：Last author Language：English Publishing type：Part of collection (book)

Although two-dimensional (2D) cultures from bone lineage cells are often used, it is well-known that this culture system is completely different from the in vivo bone matrix environment. In this paper, we describe a 3D culture method using both the mouse osteocytic cell line, MLO-Y4, and an osteocyte-enriched population of the cells isolated from mice. These cells are embedded in collagen gel with recombinant cellular communication network (CCN) factor proteins; then, osteoblasts or osteoclasts are inoculated and cultured on the collagen gel. Because this method mimics the in vitro bone matrix environment, it is useful for understanding the detailed mechanism of actions of CCN proteins in the bone matrix.

DOI： 10.1007/978-1-0716-2744-0_17

The Evaluation of Meniscus Regenerative Effects 2 of LIPUS-Induced CCN Proteins: Induction by LIPUS of CCN2 3 and Meniscus-Related Genes in Cultured Meniscus Cells 4 and Meniscus Tissues

Yusuke Kamatsuki, Eriko Aoyama, Takayuki Furumatsu, Toshifumi Ozaki, Masaharu Takigawa

Methods in Molecular Biology 2582 223 - 235 2023

Analyses of the Posttranscriptional Regulation of CCN 2 Genes: Approach to Multiple Steps of CCN2 Gene Expression Invited Reviewed International journal

Seiji Kondo, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 2582 127 - 155 2023

Authorship：Last author Language：English Publishing type：Research paper (scientific journal)

Cells generally control the concentration of mRNA via transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation (decay) cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, degradation of the target mRNA, and inhibition of mRNA translation are performed either individually or in combination. From our experience, measurement of the steady-state levels of mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying the ccn2 gene expression. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. The stability of ccn2 mRNAs is then evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Finally, repression of ccn2 mRNA translation can be estimated by comparing the expression of mRNA and protein changes. We herein report the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can, in theory, be used to elucidate the posttranscriptional regulation of other genes belonging to the CCN family.

DOI： 10.1007/978-1-0716-2744-0_10

CCN Proteins (Cellular Communication Network Factors): Expanding Their Repertoire Toward a New Concept Invited Reviewed

Masaharu Takigawa

Methods in Molecular Biology 2582 1 - 10 2023

Mouse Models of Tumor Bone Metastasis and Invasion 2 for Studying CCN Proteins Invited Reviewed International journal

Tsuyoshi Shimo, Tatsuo Okui, Naohiro Horie, Kenji Yokozeki, Masaharu Takigawa, Akira Sasaki

Methods in Molecular Biology 2582 295 - 308 2023

Language：English Publishing type：Part of collection (book)

Bone metastasis and bone destruction are common occurrences in human malignancies, including breast, prostate, and lung cancer, and are associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis and bone destruction are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the left cardiac ventricle. In addition, we introduce mouse model of the bone destruction by injection of SAS oral squamous cell carcinoma cells into the bone marrow space of the right tibial metaphysis. These assays can be applied to studies on roles of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) protein in tumor metastasis and development of treatment strategies targeting CCN proteins.

DOI： 10.1007/978-1-0716-2744-0_24

Elevated Expression of CCN3 in Articular Cartilage Induces Osteoarthritis in Hip Joints Irrespective of Age and Weight Bearing. Reviewed International journal

Kazuki Hirose, Miho Kuwahara, Eiji Nakata, Tomonori Tetsunaga, Kazuki Yamada, Kenta Saiga, Masaharu Takigawa, Toshifumi Ozaki, Satoshi Kubota, Takako Hattori

International journal of molecular sciences 23 ( 23 ) 2022.12

Language：English Publishing type：Research paper (scientific journal)

Osteoarthritis (OA) occurs not only in the knee but also in peripheral joints throughout the whole body. Previously, we have shown that the expression of cellular communication network factor 3 (CCN3), a matricellular protein, increases with age in knee articular cartilage, and the misexpression of CCN3 in cartilage induces senescence-associated secretory phenotype (SASP) factors, indicating that CCN3 promotes cartilage senescence. Here, we investigated the correlation between CCN3 expression and OA degenerative changes, principally in human femoral head cartilage. Human femoral heads obtained from patients who received total hip arthroplasty were categorized into OA and femoral neck fracture (normal) groups without significant age differences. Gene expression analysis of RNA obtained from femoral head cartilage revealed that CCN3 and MMP-13 expression in the non-weight-bearing part was significantly higher in the OA group than in the normal group, whereas the weight-bearing OA parts and normal cartilage showed no significant differences in the expression of these genes. The expression of COL10A1, however, was significantly higher in weight-bearing OA parts compared with normal weight-bearing parts, and was also higher in weight-bearing parts compared with non-weight-bearing parts in the OA group. In contrast, OA primary chondrocytes from weight-bearing parts showed higher expression of CCN3, p16, ADAMTS4, and IL-1β than chondrocytes from the corresponding normal group, and higher ADAMTS4 and IL-1β in the non-weight-bearing part compared with the corresponding normal group. Acan expression was significantly lower in the non-weight-bearing group in OA primary chondrocytes than in the corresponding normal chondrocytes. The expression level of CCN3 did not show significant differences between the weight-bearing part and non-weight-bearing part in both OA and normal primary chondrocytes. Immunohistochemical analysis showed accumulated CCN3 and aggrecan neoepitope staining in both the weight-bearing part and non-weight-bearing part in the OA group compared with the normal group. The CCN3 expression level in cartilage had a positive correlation with the Mankin score. X-ray analysis of cartilage-specific CCN3 overexpression mice (Tg) revealed deformation of the femoral and humeral head in the early stage, and immunohistochemical analysis showed accumulated aggrecan neoepitope staining as well as CCN3 staining and the roughening of the joint surface in Tg femoral and humeral heads. Primary chondrocytes from the Tg femoral head showed enhanced expression of Ccn3, Adamts5, p16, Il-6, and Tnfα, and decreased expression of Col2a1 and -an. These findings indicate a correlation between OA degenerative changes and the expression of CCN3, irrespective of age and mechanical loading. Furthermore, the Mankin score indicates that the expression level of Ccn3 correlates with the progression of OA.

DOI： 10.3390/ijms232315311

Fibroblast Growth Factors and Cellular Communication Network Factors: Intimate Interplay by the Founding Members in Cartilage. Reviewed International journal

Satoshi Kubota, Eriko Aoyama, Masaharu Takigawa, Takashi Nishida

International journal of molecular sciences 23 ( 15 ) 2022.8

Language：English Publishing type：Research paper (scientific journal)

Fibroblast growth factors (FGFs) constitute a large family of signaling molecules that act in an autocrine/paracrine, endocrine, or intracrine manner, whereas the cellular communication network factors (CCN) family is composed of six members that manipulate extracellular signaling networks. FGFs and CCNs are structurally and functionally distinct, except for the common characteristics as matricellular proteins. Both play significant roles in the development of a variety of tissues and organs, including the skeletal system. In vertebrates, most of the skeletal parts are formed and grow through a process designated endochondral ossification, in which chondrocytes play the central role. The growth plate cartilage is the place where endochondral ossification occurs, and articular cartilage is left to support the locomotive function of joints. Several FGFs, including FGF-2, one of the founding members of this family, and all of the CCNs represented by CCN2, which is required for proper skeletal development, can be found therein. Research over a decade has revealed direct binding of CCN2 to FGFs and FGF receptors (FGFRs), which occasionally affect the biological outcome via FGF signaling. Moreover, a recent study uncovered an integrated regulation of FGF and CCN genes by FGF signaling. In this review, after a brief introduction of these two families, molecular and genetic interactions between CCN and FGF family members in cartilage, and their biological effects, are summarized. The molecular interplay represents the mutual involvement of the other in their molecular functions, leading to collaboration between CCN2 and FGFs during skeletal development.

DOI： 10.3390/ijms23158592

Effect of Angiotensin II on Chondrocyte Degeneration and Protection via Differential Usage of Angiotensin II Receptors Invited Reviewed International journal

Takashi Nishida, Sho Akashi, Masaharu Takigawa, Satoshi Kubota

International Journal of Molecular Sciences 22 ( 17 ) 9204 - 9204 2021.8

Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

The renin–angiotensin system (RAS) controls not only systemic functions, such as blood pressure, but also local tissue-specific events. Previous studies have shown that angiotensin II receptor type 1 (AT1R) and type 2 (AT2R), two RAS components, are expressed in chondrocytes. However, the angiotensin II (ANG II) effects exerted through these receptors on chondrocyte metabolism are not fully understood. In this study, we investigated the effects of ANG II and AT1R blockade on chondrocyte proliferation and differentiation. Firstly, we observed that ANG II significantly suppressed cell proliferation and glycosaminoglycan content in rat chondrocytic RCS cells. Additionally, ANG II decreased CCN2, which is an anabolic factor for chondrocytes, via increased MMP9. In Agtr1a-deficient RCS cells generated by the CRISPR-Cas9 system, Ccn2 and Aggrecan (Acan) expression increased. Losartan, an AT1R antagonist, blocked the ANG II-induced decrease in CCN2 production and Acan expression in RCS cells. These findings suggest that AT1R blockade reduces ANG II-induced chondrocyte degeneration. Interestingly, AT1R-positive cells, which were localized on the surface of the articular cartilage of 7-month-old mice expanded throughout the articular cartilage with aging. These findings suggest that ANG II regulates age-related cartilage degeneration through the ANG II–AT1R axis.

DOI： 10.3390/ijms22179204

RFX1‐mediated CCN3 induction that may support chondrocyte survival under starved conditions Reviewed

Tomomi Mizukawa, Takashi Nishida, Sho Akashi, Kazumi Kawata, Sumire Kikuchi, Harumi Kawaki, Masaharu Takigawa, Hiroshi Kamioka, Satoshi Kubota

Journal of Cellular Physiology 236 ( 10 ) 6884 - 6896 2021.3

Bipartite regulation of cellular communication network factor 2 and fibroblast growth factor 1 genes by fibroblast growth factor 1 through histone deacetylase 1 and fork head box protein A1 Reviewed International journal

Abdellatif Elseoudi, Takashi Nishida, Tomomi Mizukawa, Takako Hattori, Kazumi Kawata, Eman A. Taha, Masaharu Takigawa, Satoshi Kubota

Journal of Cell Communication and Signaling 15 ( 1 ) 81 - 91 2021.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities.

DOI： 10.1007/s12079-020-00600-4

Other Link： http://link.springer.com/article/10.1007/s12079-020-00600-4/fulltext.html

Suppression of adipocyte differentiation by low‐intensity pulsed ultrasound via inhibition of insulin signaling and promotion of CCN family protein 2 Reviewed International journal

Takashi Nishida, Yurika Nagao, Satoko Hashitani, Nobuyasu Yamanaka, Masaharu Takigawa, Satoshi Kubota

Journal of Cellular Biochemistry 121 ( 12 ) 4724 - 4740 2020.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

Adipocyte differentiation is regulated by several transcription factors such as the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPARγ). Here, we demonstrate that low-intensity pulsed ultrasound (LIPUS) suppressed differentiation into mature adipocytes via multiple signaling pathways. When C3H10T1/2, a mesenchymal stem cell line, was treated with LIPUS (3.0 MHz, 60 mW/cm2 ) for 20 minutes once a day for 4 days during adipogenesis, and both the number of lipid droplets and the gene expression of PPARγ and C/EBPα were significantly decreased. Furthermore, LIPUS treatment decreased the phosphorylation of the insulin receptor and also that of Akt and ERK1/2, which are located downstream of this receptor. Next, we showed that LIPUS suppressed the gene expression of angiotensinogen (AGT), which is an adipokine produced by mature adipocytes, as well as that of angiotensin-converting enzyme 1 (ACE1) and angiotensin receptor type 1 (AT1 R) during adipogenesis of pre-adipogenic 3T3-L1 cells. Next, the translocation of Yes-associated protein (YAP) into the nucleus of 3T3-L1 cells was promoted by LIPUS, leading to upregulation of CCN family protein 2 (CCN2), a cellular communication network factor. Moreover, forced expression of CCN2 in 3T3-L1 cells decreased PPARγ gene expression, but it did not increase alkaline phosphatase and osterix gene expression. Finally, gene silencing of CCN2 in C3H10T1/2 cells diminished the effect of LIPUS on the gene expression of PPARγ and C/EBPα. These findings suggest that LIPUS suppressed adipogenesis through inhibition of insulin signaling and decreased PPARγ expression via increased CCN2 production, resulting in a possible decrease of mature adipocytes.

DOI： 10.1002/jcb.29680

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jcb.29680

CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage Reviewed International journal

Miho Kuwahara, Koichi Kadoya, Sei Kondo, Shanqi Fu, Yoshiko Miyake, Ayako Ogo, Mitsuaki Ono, Takayuki Furumatsu, Eiji Nakata, Takako Sasaki, Shogo Minagi, Masaharu Takigawa, Satoshi Kubota, Takako Hattori

International Journal of Molecular Sciences 21 ( 20 ) 7556 - 7556 2020.10

Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.

DOI： 10.3390/ijms21207556

Regulation of cellular communication network factor 2 (CCN2) in breast cancer cells via the cell-type dependent interplay between CCN2 and glycolysis Reviewed International journal

Sho Akashi, Takashi Nishida, Tomomi Mizukawa, Kazumi Kawata, Masaharu Takigawa, Seiji Iida, Satoshi Kubota

Journal of Oral Biosciences 62 ( 3 ) 280 - 288 2020.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier BV

OBJECTIVES: Anti-osteoclastic treatments for breast cancer occasionally cause medication-related osteonecrosis of the jaw. Moreover, elevated glycolytic activity, which is known as the Warburg effect, is usually observed in these breast cancer cells. Previously, we found that cellular communication network factor 2 (CCN2) production and glycolysis enhanced each other in chondrocytes. Here, we evaluated the interplay between CCN2 and glycolysis in breast cancer cells, as we suspected a possible involvement of CCN2 in the Warburg effect in highly invasive breast cancer cells. METHODS: Two human breast cancer cell lines with a distinct phenotype were used. Glycolysis was inhibited by using 2 distinct compounds, and gene silencing was performed using siRNA. Glycolysis and the expression of relevant genes were monitored via colorimetric assays and quantitative RT-PCR, respectively. RESULTS: Although CCN2 expression was almost completely silenced when treating invasive breast cancer cells with a siRNA cocktail against CCN2, glycolytic activity was not affected. Notably, the expression of glycolytic enzyme genes, which was repressed by CCN2 deficiency in chondrocytes, tended to increase upon CCN2 silencing in breast cancer cells. Inhibition of glycolysis, which resulted in the repression of CCN2 expression in chondrocytic cells, did not alter or strongly enhanced CCN2 expression in the invasive and non-invasive breast cancer cells, respectively. CONCLUSIONS: High CCN2 expression levels play a critical role in the invasion and metastasis of breast cancer. Thus, a collapse in the intrinsic repressive machinery of CCN2 due to glycolysis may induce the acquisition of an invasive phenotype in breast cancer cells.

DOI： 10.1016/j.job.2020.07.001

Hypoxic induction of CCN2 mRNA through p38 MAP kinase activation in the human chondrosarcoma‐derived cell line, HCS‐2/8 Reviewed

Aya Yoshino, Shiho Hashiguchi, Ryosuke Mano, Seiji Kondo, Satoshi Kubota, Masaharu Takigawa

Oral Science International 2020.7

Publishing type：Research paper (scientific journal) Publisher：Wiley

© 2020 Japanese Stomatological Society CCN2/CTGF (cellular communication network factor 2/connective tissue growth factor) plays critical roles in cartilage development, maintenance, and regeneration. Hypoxia-induced expression of CCN2 mRNA is regulated post-transcriptionally in the human chondrosarcoma-derived cell line, HCS-2/8. In the present study, the hypoxia-induced increase in CCN2 mRNA expression was assessed with quantitative real-time PCR in HCS-2/8 cells in the presence of inhibitors of mitogen-activated protein kinases (MAPKs). Subsequently, the stability of CCN2 mRNA in hypoxia was evaluated with mRNA degradation assays in the presence or absence of the selective p38 MAPK inhibitor, SB203580. We detected phosphorylation of p38 MAPK by immunoblot analysis within 30 minutes in hypoxia, and we observed ~twofold higher CCN2 mRNA levels in hypoxia compared to normoxia. Blockade of p38 MAPK activation with 10 µmol/L SB203580 abolished this CCN2 induction. Furthermore, we found that inhibition of p38 MAPK suppressed the elongation of CCN2 mRNA half-life in hypoxia. Taken together, these findings suggest that the molecular mechanism, by which CCN2 mRNA expression is increased in hypoxia, induces the activation of p38 MAPK leading to the post-transcriptional regulation of the stability of CCN2 mRNA.

DOI： 10.1002/osi2.1076

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/osi2.1076

CTGF/CCN2 facilitates LRP4-mediated formation of the embryonic neuromuscular junction. Reviewed International journal

Bisei Ohkawara, Akinori Kobayakawa, Shunsuke Kanbara, Takako Hattori, Satoshi Kubota, Mikako Ito, Akio Masuda, Masaharu Takigawa, Karen M Lyons, Naoki Ishiguro, Kinji Ohno

EMBO reports 21 ( 8 ) e48462 2020.6

Language：English Publishing type：Research paper (scientific journal)

At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf-/- ) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf-/- embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.

DOI： 10.15252/embr.201948462

Selective Agonists of Nuclear Retinoic Acid Receptor Gamma Inhibit Growth of HCS‐2/8 Chondrosarcoma Cells Reviewed International journal

William P. Shield, Ashley Cellini, Hongying Tian, Kim Wilson, Yang Dan, Joshua M. Abzug, Sonia Garcia, Norifumi Moritani, Ivan Alferiev, Michael Chorny, Masaharu Takigawa, Vincent Y. Ng, Masahiro Iwamoto, Motomi Enomoto‐Iwamoto

Journal of Orthopaedic Research 38 ( 5 ) 1045 - 1051 2020.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

Chondrosarcoma is the second most common primary bone sarcoma. Treatment of chondrosarcoma is limited to surgery due to radiation and chemotherapy resistance of this cancer. An ideal treatment for chondrosarcoma would be a well-tolerated, minimally invasive local or systemic treatment modality to halt or slow tumor growth prior to resection of local, unresectable local, or metastatic disease. Palovarotene, an agonist of nuclear retinoic acid receptor γ (RARγ) has shown therapeutic action for treatment of heterotopic ossification and osteochondroma without serious adverse effects in animal models. We hypothesized that selective agonists of RARγ would have an inhibitory effect on chondrosarcoma. All human chondrosarcoma specimens expressed RARγ as determined by immunohistochemical staining. The ΗCS-2/8 chondrosarcoma cell line, established from low-grade human chondrosarcoma, was used to examine the actions of RARγ agonists. In ΗCS2/8 pellet cultures, RARγ agonist treatment reduced the mass size and significantly decreased total glycosaminoglycan, protein amounts, and gene expression levels of cartilage matrix molecules when compared with control groups. Systemic treatment with RARγ agonists significantly inhibited the growth of ΗCS-2/8 cell transplants in vivo. Furthermore, local injection of RARγ agonist-loaded poly-lactic acid nanoparticles induced regression of the mass size of the transplants. Histologic analysis demonstrated that RARγ agonist treatment inhibited cell proliferation activity and stimulated encapsulation of the tumor. These findings indicate that RARγ agonists, including palovarotene, may have an anti-tumor effect on low-grade chondrosarcomas. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1045-1051, 2020.

DOI： 10.1002/jor.24555

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jor.24555

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes. Reviewed International journal

Mitsuhiro Hoshijima, Takako Hattori, Eriko Aoyama, Takashi Nishida, Satoshi Kubota, Hiroshi Kamioka, Masaharu Takigawa

International journal of molecular sciences 21 ( 8 ) 2020.4

Language：English Publishing type：Research paper (scientific journal)

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.

DOI： 10.3390/ijms21082769

Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor (CCN2/CTGF): CRISPR against Cancer Reviewed

Yuka Okusha, Takanori Eguchi, Manh T. Tran, Chiharu Sogawa, Kaya Yoshida, Mami Itagaki, Eman A. Taha, Kisho Ono, Eriko Aoyama, Hirohiko Okamura, Ken-ichi Kozaki, Stuart K. Calderwood, Masaharu Takigawa, Kuniaki Okamoto

Cancers 12 ( 4 ) 881 - 881 2020.4

Publishing type：Research paper (scientific journal) Publisher：MDPI AG

Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the CCN2/CTGF promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites.

DOI： 10.3390/cancers12040881

Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3/DAT and Reducing STAT3 Reviewed International journal

Chiharu Sogawa, Takanori Eguchi, Manh Tien Tran, Masayuki Ishige, Kilian Trin, Yuka Okusha, Eman Ahmed Taha, Yanyin Lu, Hotaka Kawai, Norio Sogawa, Masaharu Takigawa, Stuart K. Calderwood, Kuniaki Okamoto, Ken-ichi Kozaki

Cancers 12 ( 2 ) 523 - 523 2020.2

Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and β-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

DOI： 10.3390/cancers12020523

Extracellular Oncosomes Rich in Moonlighting Metalloproteinase (MMP3) Are Transmissive, Pro-Tumorigenic, and Induces Cellular Communication Network Factor 2 (CCN2/CTGF): CRISPR against Cancer

Yuka Okusha, Takanori Eguchi, Manh Tien Tran, Chiharu Sogawa, Kaya Yoshida, Mami Itagaki, Eman Ahmed Taha, Kisho Ono, Eriko Aoyama, Hirohiko Okamura, Ken-ichi Kozaki, Stuart K. Calderwood, Masaharu Takigawa, Kuniaki Okamoto

Preprints doi: 10.20944/preprints202002.0281.v1 2020.2

Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

Matrix metalloproteinase 3 (MMP3) plays multiple roles in pro-tumorigenic proteolysis and in intracellular transcription. These include inducing connective tissue growth factor [CTGF, also known as cellular communication network factor 2 (CCN2)] and prompting a new definition of MMP3 as a moonlighting metalloproteinase. Members of the MMP family have been found within tumor-derived extracellular vesicles (EVs) such as oncosomes or exosomes. We here investigated the roles of MMP3-rich oncosomes in tumor progression, molecular transmission, and gene regulation. MMP3 and CCN2/CTGF were significantly co-expressed in tumor samples derived from patients suffering from colorectal adenocarcinoma. We found that oncosomes derived from a rapidly metastatic colon cancer cells (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich oncosomes were highly transmissive into recipient cells and were pro-tumorigenic in an allograft mouse model. Oncosome-derived MMP3 was transmissive into recipient cell nuclei, trans-activated CCN2/CTGF promoter, and induced CCN2/CTGF production at 1 to 6 hours after the addition of oncosomes to culture media. In addition, CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects, including inhibition of migration and invasion of LuM1 cells in vitro, inhibition of tumor growth in vivo, and reduction of CCN2/CTGF and its promoter activity in vitro. These data newly demonstrate that the oncosome-derived moonlighting metalloproteinase promotes metastasis and is pro-tumorigenic at distant sites as well as a transmissive trans-activator for the cellular communication network gene.

DOI： 10.20944/preprints202002.0281.v1

Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer Reviewed International journal

Kisho Ono, Chiharu Sogawa, Hotaka Kawai, Manh Tien Tran, Eman A. Taha, Yanyin Lu, May Wathone Oo, Yuka Okusha, Hirohiko Okamura, Soichiro Ibaragi, Masaharu Takigawa, Ken-Ichi Kozaki, Hitoshi Nagatsuka, Akira Sasaki, Kuniaki Okamoto, Stuart K. Calderwood, Takanori Eguchi

Journal of Extracellular Vesicles 9 ( 1 ) 1769373 - 1769373 2020.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Informa UK Limited

Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones - CDC37, HSP90α and HSP90β play key roles in cancer progression including epithelial-mesenchymal transition (EMT), although their contribution to EVs-mediated cell-cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer-derived EVs (MEV) were enriched with HSP90α HSP90β and cancer-initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/β reversed these MEV-driven malignancy events. In metastatic oral cancer patient-derived tumours, HSP90β was significantly accumulated in infiltrating tumour-associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90-enriched MEV-induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA-mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90β) could potentially be a novel therapeutic strategy to attenuate several EV-driven malignancy events in the tumour microenvironment. Abbreviations: CDC37: cell division control 37; EMT: epithelial-mesenchymal transmission; EV: extracellular vesicles; HNSCC: head and neck squamous cell carcinoma; HSP90: heat shock protein 90; TAM: tumour-associated macrophage.

DOI： 10.1080/20013078.2020.1769373

Jiadifenolide induces expression of cellular communication network factor (CCN) genes, and CCN2 possesses neurotrophic activity in neuronal precursor cells derived from human induced pluripotent stem cells. Reviewed International journal

Shoji M, Ueda M, Nishioka M, Hiroki Minato, M, Kenichi Harada, Kubo M, Fukuyama Y, Suzuki Y, Aoyama E, Takigawa M, Kuzuhara T

Biochem Biophys Res Commun 519 ( 2 ) 309 - 315 2019.11

Language：English Publishing type：Research paper (scientific journal)

Jiadifenolide has been reported to have neurotrophin-like activity in primary rat cortical neurons, and also possesses neurotrophic effects in neuronal precursor cells derived from human induced pluripotent stem cells (hiPSCs), as we have previously reported. However, the molecular mechanisms by which jiadifenolide exerts its neurotrophic effects in rat and human neurons are unknown. Thus, we aimed to investigate the molecular mechanisms and pathways by which jiadifenolide promotes neurotrophic effects. Here, we found that jiadifenolide activated cellular communication network factor (CCN) signaling pathways by up-regulating mRNA level expression of CCN genes in human neuronal cells. We also found that CCN2 (also known as connective tissue growth factor, CTGF) protein promotes neurotrophic effects through activation of the p44/42 mitogen-activated protein kinase signaling pathway. This is the first discovery which links neurotrophic activity with CCN signaling.

DOI： 10.1016/j.bbrc.2019.09.003

Roles of matricellular CCN2 deposited by osteocytes in osteoclastogenesis and osteoblast differentiation. Reviewed

Nishida T, Kubota S, Yokoi H, Mukoyama, M, Takigawa M

Sci Rep 9 ( 1 ) 10913 2019.7

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

In this study, we investigated the effect of CCN2 (cellular communication network factor 2), previously termed connective tissue growth factor, deposited in bone matrix on osteoclastogenesis and osteoblast differentiation. To mimic the bone matrix environment, osteocytic MLO-Y4 cells had been embedded in collagen-gel with recombinant CCN2 (rCCN2), and mouse macrophage-like RAW264.7 cells were inoculated on the gel and treated with receptor activator of NF-kappa B ligand (RANKL). NFATc1 and cathepsin K (CTSK) productions were more increased in the combination of RAW264.7 and MLO-Y4 cells treated with rCCN2 than the combination without rCCN2. Next, we isolated an osteocyte-enriched population of cells and osteoclast progenitor cells from wild type and tamoxifen-inducible Ccn2-deficient (KO) mice and performed similar analysis. NFATc1 and CTSK productions were decreased in the KO osteocyte-enriched population at 6 months after the tamoxifen injection, regardless of the origin of the osteoclast progenitor cells. Interestingly, CTSK production was rather increased in KO osteocytes at 1 year after the injection. Finally, the combination of osteoblastic MC3T3-E1 and MLO-Y4 cells in rCCN2-containing bone matrix revealed the up-regulation of osteoblastic marker genes. These findings suggest that CCN2 supplied by osteocytes regulates both osteoclastogenesis and osteoblast differentiation.

DOI： 10.1038/s41598-019-47285-3

Possible reparative effect of low-intensity pulsed ultrasound (LIPUS) on injured meniscus. Reviewed International journal

Kamatsuki K, Aoyama E, Furumatsu T, Miyazawa S, Maehara A, Yamanaka N, Nishida T, Kubota S, Ozaki T, Takigawa M

J Cell Commun Signal 13 ( 2 ) 193 - 207 2019.6

Language：English Publishing type：Research paper (scientific journal)

Menisci are a pair of crescent-shaped fibrocartilages, particularly of which their inner region of meniscus is an avascular tissue. It has characteristics similar to those of articular cartilage, and hence is inferior in healing. We previously reported that low-intensity pulsed ultrasound (LIPUS) treatment stimulates the production of CCN2/CTGF, a protein involved in repairing articular cartilage, and the gene expression of major cartilage matrices such as type II collagen and aggrecan in cultured chondrocytes. Therefore, in this present study, we investigated whether LIPUS has also favorable effect on meniscus cells and tissues. LIPUS applied with a 60 mW/cm2 intensity for 20 min stimulated the gene expression and protein production of CCN2 via ERK and p38 signaling pathways, as well as gene expression of SOX9, aggrecan, and collagen type II in human inner meniscus cells in culture, and slightly stimulated the gene expression of CCN2 and promoted the migration in human outer meniscus cells in culture. LIPUS also induced the expression of Ccn2, Sox9, Col2a1, and Vegf in rat intact meniscus. Furthermore, histological evaluations showed that LIPUS treatment for 1 to 4 weeks promoted healing of rat injured lateral meniscus, as evidenced by better and earlier angiogenesis and extracellular matrix synthesis. The data presented indicate that LIPUS treatment might prevent meniscus from degenerative change and exert a reparative effect on injured meniscus via up-regulation of repairing factors such as CCN2 and that it might thus be useful for treatment of an injured meniscus as a non-invasive therapy.

DOI： 10.1007/s12079-018-0496-9

CCN2/CTGF binds the small leucine rich proteoglycan protein Tsukushi Reviewed

Ohta K, Aoyama E, Ahmad SAI, Ito N, Anam MB, Kubota S, Takigawa M

J Cell Commun Signal. 13 ( 1 ) 113 - 118 2019.3

The BMP-2 mutant L51P: a BMP receptor IA binding-deficient inhibitor of noggin Reviewed

Hany Mohamed Khattab, Satoshi Kubota, Masaharu Takigawa, Takuo Kuboki, Walter Sebald

JOURNAL OF BONE AND MINERAL METABOLISM 37 ( 2 ) 199 - 205 2019.3

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER JAPAN KK

The antagonist-specific regulation in tissue engineering constitutes important attempts to achieve an improved and rapid bone regeneration by controlling the natural biological response of the natural body growth factors. L51P is molecularly engineered bone morphogentic protein-2 (BMP-2) variant with a substitution of the 51st leucine with a proline residue. L51P is deficient in BMP receptor binding, but maintains its structure and affinity for inhibitory proteins such as noggin, chordin, and gremlin. These modifications convert the BMP-2 variant L51P into a receptor-inactive inhibitor of BMP antagonists. This current approach may prevent the uncontrolled bone overgrowth using high concentration of BMPs and thus regulates the possible growth factor's high-dose side effects. Exploring of L51P biological functions is required to broad our understanding of BMP mutant biological functions and their potential clinical applications. The progress of L51P researches would hopefully lead to the development of multiple applications for using the L51P in bone and fracture healing disorders.

DOI： 10.1007/s00774-018-0925-0

A reporter system evaluates tumorigenesis, metastasis, β-catenin/MMP regulation, and druggability. Reviewed

Sogawa C, Eguchi T, Okusha Y, Ono K, Ohyama K, Iizuka M, Kawasaki R, Hamada Y, Takigawa M, Sogawa N, Okamoto K, Kozaki KI

Tissue Eng Part A 13 ( 2 ) 193 - 207 2019.2

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation. Reviewed

Ishikawa T, Nishida T, Ono M, Takarada T, Nguyen HT, Kurihara S, Furumatsu T, Murase Y, Takigawa M, Oohashi T, Kamioka H, Kubota S

J Cell Physiol 233 ( 6 ) 4825 - 4840 2018.7

Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells. Reviewed International journal

Yuri Namba, Chiharu Sogawa, Yuka Okusha, Hotaka Kawai, Mami Itagaki, Kisho Ono, Jun Murakami, Eriko Aoyama, Kazumi Ohyama, Jun-Ichi Asaumi, Masaharu Takigawa, Kuniaki Okamoto, Stuart K Calderwood, Ken-Ichi Kozaki, Takanori Eguchi

Frontiers in oncology 119 ( 6 ) 4352 - 4360 2018.6

Language：English Publishing type：Research paper (scientific journal)

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10-35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.

DOI： 10.3389/fonc.2018.00376.

Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling. Reviewed International journal

Kenichi Matsumoto, Tsuyoshi Shimo, Naito Kurio, Tatsuo Okui, Soichiro Ibaragi, Yuki Kunisada, Kyoichi Obata, Masanori Masui, Pang Pai, Yuu Horikiri, Nobuyuki Yamanaka, Masaharu Takigawa, Akira Sasaki

Journal of cellular biochemistry 119 ( 6 ) 4352 - 4360 2018.6

Language：English Publishing type：Research paper (scientific journal)

Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling.

DOI： 10.1002/jcb.26418

The BMP-2 mutant L51P: a BMP receptor IA binding-deficient inhibitor of noggin Reviewed

Hany Mohamed Khattab, Satoshi Kubota, Masaharu Takigawa, Takuo Kuboki, Walter Sebald

Journal of Bone and Mineral Metabolism 128 ( 3 ) 1 - 7 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Tokyo

The antagonist-specific regulation in tissue engineering constitutes important attempts to achieve an improved and rapid bone regeneration by controlling the natural biological response of the natural body growth factors. L51P is molecularly engineered bone morphogentic protein-2 (BMP-2) variant with a substitution of the 51st leucine with a proline residue. L51P is deficient in BMP receptor binding, but maintains its structure and affinity for inhibitory proteins such as noggin, chordin, and gremlin. These modifications convert the BMP-2 variant L51P into a receptor-inactive inhibitor of BMP antagonists. This current approach may prevent the uncontrolled bone overgrowth using high concentration of BMPs and thus regulates the possible growth factor’s high-dose side effects. Exploring of L51P biological functions is required to broad our understanding of BMP mutant biological functions and their potential clinical applications. The progress of L51P researches would hopefully lead to the development of multiple applications for using the L51P in bone and fracture healing disorders.

DOI： 10.1007/s00774-018-0925-0

Metabolic regulation of the CCN family genes by glycolysis in chondrocytes Reviewed

Sho Akashi, Takashi Nishida, Abdellatif El-Seoudi, Masaharu Takigawa, Seiji Iida, Satoshi Kubota

Journal of Cell Communication and Signaling 12 ( 1 ) 245 - 252 2018.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Netherlands

The CCN family consists of 6 genes in the mammalian genome and produces multifunctional proteins involved in a variety of biological processes. Recent reports indicate the profound roles of CCN2 in energy metabolism in chondrocytes, and Ccn2 deficiency is known to alter the expression of 2 other family members including Ccn3. However, almost nothing is known concerning the regulation of the CCN family genes by energy metabolism. In order to gain insight into this critical issue, we initially and comprehensively evaluated the effect of inhibition of glycolysis on the expression of all of the CCN family genes in chondrocytic cells. Upon the inhibition of a glycolytic enzyme, repression of CCN2 expression was observed, whereas CCN3 expression was conversely induced. Similar repression of CCN2 was conferred by the inhibition of aerobic ATP production, which, however, did not induce CCN3 expression. In contrast, glucose starvation significantly enhanced the expression of CCN3 in those cells. The results of a reporter gene assay using a molecular construct containing a CCN3 proximal promoter revealed a dose-dependent induction of the CCN3 promoter activity by the glycolytic inhibitor in chondrocytic cells. These results unveiled a critical role of glycolytic activity in the regulation of CCN2 and CCN3, which activity mediated the mutual regulation of these 2 major CCN family members in chondrocytes.

DOI： 10.1007/s12079-017-0420-8

An early history of CCN2/CTGF research: the road to CCN2 via hcs24, ctgf, ecogenin, and regenerin Reviewed

Masaharu Takigawa

Journal of Cell Communication and Signaling 12 ( 1 ) 253 - 264 2018.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Netherlands

The principal aim of this historical review is to present the processes by which the different aspects of CCN2/CTGF/Hcs24 were discovered by different groups and how much CCN2/CTGF, by being integrated into CCN family, has contributed to the establishment of the basic concepts regarding the role and functions of this new class of proteins. This review should be particularly useful to new investigators who have recently entered this exciting field of study and also provides a good opportunity to acknowledge the input of those individuals who participated in the development of this scientific field.

DOI： 10.1007/s12079-017-0414-6

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment Reviewed

Takanori Eguchi, Chiharu Sogawa, Yuka Okusha, Kenta Uchibe, Ryosuke Iinuma, Kisho Ono, Keisuke Nakano, Jun Murakami, Manabu Itoh, Kazuya Arai, Toshifumi Fujiwara, Yuri Namba, Yoshiki Murata, Kazumi Ohyama, Manami Shimomura, Hirohiko Okamura, Masaharu Takigawa, Tetsuya Nakatsura, Ken-ichi Kozaki, Kuniaki Okamoto, Stuart K. Calderwood

PLoS ONE 13 ( 2 ) e0191109 2018.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Public Library of Science

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

DOI： 10.1371/journal.pone.0191109

A Tumor Suppressor Gene Product, Platelet-Derived Growth Factor Receptor-Like Protein Controls Chondrocyte Proliferation and Differentiation Reviewed

Kazumi Kawata, Satoshi Kubota, Takanori Eguchi, Eriko Aoyama, Norifumi H. Moritani, Morihiko Oka, Harumi Kawaki, Masaharu Takigawa

JOURNAL OF CELLULAR BIOCHEMISTRY 118 ( 11 ) 4033 - 4044 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY

The platelet-derived growth factor receptor-like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS-2/8 cells. Interestingly, the proliferation of chondrocytic HCS-2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer-derived MDA-MB-231 cells was inhibited. Of note, in PDGFRL-overexpressing HCS-2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033-4044, 2017. (c) 2017 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.26059

Regulatory mechanism of CCN2 production by serotonin (5-HT) via 5-HT2A and 5-HT2B receptors in chondrocytes Reviewed

Ayaka Hori, Takashi Nishida, Shogo Takashiba, Satoshi Kubota, Masaharu Takigawa

PLOS ONE 12 ( 11 ) 8630 - 8641 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：PUBLIC LIBRARY SCIENCE

Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT(2)Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKC epsilon, PKC zeta, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones.

DOI： 10.1371/journal.pone.0188014

Novel role of CCN3 that maintains the differentiated phenotype of articular cartilage Reviewed

Danilo Janune, Tarek Abd El Kader, Eriko Aoyama, Takashi Nishida, Yasuhiko Tabata, Satoshi Kubota, Masaharu Takigawa

JOURNAL OF BONE AND MINERAL METABOLISM 35 ( 6 ) 582 - 597 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER JAPAN KK

Knowledge of the microenvironment of articular cartilage in health and disease is the key to accomplishing fundamental disease-modifying treatments for osteoarthritis. The proteins comprising the CCN Family are matricellular proteins with a remarkable relevance within the context of cartilage metabolism. CCN2 displays a great capability for regenerating articular cartilage, and CCN3 has been shown to activate the expression of genes related to articular chondrocytes and to repress genes related to endochondral ossification in epiphyseal chondrocytes. Moreover, mice lacking CCN3 protein have been shown to display ostearthritic changes in their knee articular cartilage. In this study, we employed a monoiodoacetic acid (MIA)-induced osteoarthritic model to investigate whether osteoarthritic changes in the cartilage are reciprocally accompanied by CCN3 down-regulation and an inducible overexpression system to evaluate the effects of CCN3 on articular chondrocytes in vitro. Finally, we also investigated the effects of exogenous CCN3 in vivo during the early stages of MIA-induced osteoarthritis. We discovered that CCN3 is expressed by articular chondrocytes in normal rat knees, whereas it is rapidly down-regulated in osteoarthritic knees. In vitro, we also discovered that CCN3 increases the proteoglycan accumulation, the gene expression of type II collagen, tenascin-C and lubricin, as well as the protein production of tenascin-C and lubricin in articular chondrocytes. In vivo, it was discovered that exogenous CCN3 increased tidemark integrity and produced an increased production of lubricin protein. The potential utility of CCN3 as a future therapeutic agent and possible strategies to improve its therapeutic functions are also discussed.

DOI： 10.1007/s00774-016-0793-4

Catabolic effects of FGF-1 on chondrocytes and its possible role in osteoarthritis Reviewed

Abdellatif El-Seoudi, Tarek Abd El Kader, Takashi Nishida, Takanori Eguchi, Eriko Aoyama, Masaharu Takigawa, Satoshi Kubota

JOURNAL OF CELL COMMUNICATION AND SIGNALING 11 ( 3 ) 255 - 263 2017.9

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Fibroblast growth factor 1 (FGF-1) is a classical member of the FGF family and is produced by chondrocytes cultured from osteoarthritic patients. Also, this growth factor was shown to bind to CCN family protein 2 (CCN2), which regenerates damaged articular cartilage and counteracts osteoarthritis (OA) in an animal model. However, the pathophysiological role of FGF-1 in cartilage has not been well investigated. In this study, we evaluated the effects of FGF-1 in vitro and its production in vivo by use of an OA model. Treatment of human chondrocytic cells with FGF-1 resulted in marked repression of genes for cartilaginous extracellular matrix components, whereas it strongly induced matrix metalloproteinase 13 (MMP-13), representing its catabolic effects on cartilage. Interestingly, expression of the CCN2 gene was dramatically repressed by FGF-1, which repression eventually caused the reduced production of CCN2 protein from the chondrocytic cells. The results of a reporter gene assay revealed that this repression could be ascribed, at least in part, to transcriptional regulation. In contrast, the gene expression of FGF-1 was enhanced by exogenous FGF-1, indicating a positive feedback system in these cells. Of note, induction of FGF-1 was observed in the articular cartilage of a rat OA model. These results collectively indicate a pathological role of FGF-1 in OA development, which includes an insufficient cartilage regeneration response caused by CCN2 down regulation.

DOI： 10.1007/s12079-017-0384-8

CATABOLIC EFFECTS OF FGF-1 ON CHONDROCYTES WITH REDUCED CCN2 PRODUCTION THAT PROMOTES CARTILAGE REGENERATION: POSSIBLE ROLE IN OSTEOARTHRITIS

A. Elseoudi, T. Abd El Kader, T. Nishida, E. Aoyama, T. Eguchi, M. Takigawa, S. Kubota

OSTEOPOROSIS INTERNATIONAL 28 S225 - S225 2017.7

UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage-specific manner Reviewed

Tokiro Ishikawa, Takuya Toyama, Yuki Nakamura, Kentaro Tamada, Hitomi Shimizu, Satoshi Ninagawa, Tetsuya Okada, Yasuhiro Kamei, Tomoko Ishikawa-Fujiwara, Takeshi Todo, Eriko Aoyama, Masaharu Takigawa, Akihiro Harada, Kazutoshi Mori

JOURNAL OF CELL BIOLOGY 216 ( 6 ) 1761 - 1774 2017.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ROCKEFELLER UNIV PRESS

The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.

DOI： 10.1083/jcb.201609100

Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation Reviewed

T. Nishida, S. Kubota, E. Aoyama, N. Yamanaka, K. M. Lyons, M. Takigawa

OSTEOARTHRITIS AND CARTILAGE 25 ( 5 ) 759 - 769 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI LTD

Objective: CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism.
Methods: Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm(2) power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca2+ influx was measured by using a Fluo-4 probe.
Results: The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca2+ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BKca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes.
Conclusion: These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes. (C) 2016 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

DOI： 10.1016/j.joca.2016.10.003

Erratum: Proinsulin C-peptide regulates ribosomal RNA expression (The Journal of Biological Chemistry (2010) 285 (3462-3469) DOI: 10.1074/jbc.M109.053587) Reviewed

Emma Lindahl, Ulrika Nyman, Farasat Zaman, Carina Palmberg, Anna Cascante, Jawed Shafqat, Masaharu Takigawa, Lars Sävendahl, Hans Jörnvall, Bertrand Joseph

Journal of Biological Chemistry 285 ( 5 ) 3462 - 3469 2017.3

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Biochemistry and Molecular Biology Inc.

Proinsulin C-peptide is internalized into cells, but a function of its intracellular localization has not been established. We now demonstrate that, upon cellular entry, C-peptide is localized to the nucleoli, where it promotes transcription of genes encoding for ribosomal RNA. We find that C-peptide binds to histones and enhances acetylation of lysine residue 16 of histone H4 at the promoter region of genes for ribosomal RNA. In agreement with synchrony of ribosomal RNA synthesis and cell proliferation, we show that C-peptide stimulates proliferation in chondrocytes and HEK-293 cells. This regulation of ribosomal RNA provides a mechanism by which C-peptide can exert transcriptional effects and implies that the peptide has growth factor activity. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M109.053587

Proinsulin C-peptide regulates ribosomal RNA expression (vol 292, pg 3467, 2017)

Emma Lindahl, Ulrika Nyman, Farasat Zaman, Carina Palmberg, Anna Cascante, Jawed Shafqat, Masaharu Takigawa, Lars Saevendahl, Hans Joernvall, Bertrand Joseph

JOURNAL OF BIOLOGICAL CHEMISTRY 292 ( 10 ) 4382 - 4382 2017.3

Intracellular MMP3 Promotes HSP Gene Expression in Collaboration With Chromobox Proteins Reviewed

Takanori Eguchi, Stuart K. Calderwood, Masaharu Takigawa, Satoshi Kubota, Ken-ichi Kozaki

JOURNAL OF CELLULAR BIOCHEMISTRY 118 ( 1 ) 43 - 51 2017.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Matrix metalloproteinases (MMPs) are crucial factors in tumor progression, inflammatory/immune responses and tissue development/regeneration. Of note, it has been known that MMPs promote genome instability, epithelial-mesenchymal transition, invasion, and metastasis in tumor progression. We previously reported that human MMP3 could translocate into cellular nuclei and control transcription in human chondrosarcoma-derived cells and in articular cartilage (Eguchi et al. [2008] Mol Cell Biol 28(7):2391-2413); however, further transcriptional target genes and cofactors of intranuclear MMP3 have not been uncovered. In this paper, we used transcriptomics analysis in order to examine novel transcriptional target genes regulated by intracellular MMP3. We found that mRNA levels of HSP family members (HSP70B, HSP72, HSP40/DNAJ, and HSP20/CRYAB) are upregulated by the intracellular MMP3 overload. Bioinformatic analysis predicted several transcription factors that possibly interact with MMP3. Among these factors, heat shock factor 1 (HSF1) cooperated with the MMP3 to activate the HSP70B gene promoter in reporter gene assays, while a dominant negative HSF1 blocked the role for MMP3 in the trans-activation. The hemopexin-like repeat (PEX) domain of the human MMP3 was essential for transcriptional induction of the HSP70B gene. In addition, chromobox proteins CBX5/HP1 and CBX3/HP1 cooperated with the PEX domain in induction of HSP70B mRNA. Taken together, this study newly clarified that intracellular MMP3 cooperate with CBXs/HP1s in transcriptional promotion of HSP genes. J. Cell. Biochem. 118: 43-51, 2017. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.25607

Immunohistochemical analysis of CCN proteins in calcified tissues

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 53 - 62 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Immunohistochemistry is a major technique to determine the distribution and localization of differentially produced proteins in the context of an intact tissue. It exploits one of the properties of antibodies, specific binding to an antigen, i.e., to the epitope of its target protein, in combination with a color-developing enzymatic reaction or tagged fluorophore. We have clarified the spatial and temporal expression patterns of CCN family proteins in several different types of animal tissues by using this immunohistochemical technique to support our corresponding data obtained in vitro. In this chapter, we provide our protocol for immunohistochemistry optimized for paraffin-embedded sections after having determined the optimal conditions for the use of antibodies against each member of the CCN family.

DOI： 10.1007/978-1-4939-6430-7_6

Gene expression analysis of CCN proteins: Whole-mount in situ hybridization of Ccn2 in developing calcified tissues

Tomoichiro Yamaai, Masaharu Takigawa

Methods in Molecular Biology 1489 11 - 19 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

A procedure for whole-mount in situ hybridization developed for detecting gene expression of Ccn2 in developing calcified tissues of mouse embryos is presented. In this method, embryos are hybridized with Dig-labeled riboprobes, and the riboprobes are detected by use of the alkaline-phosphatase reaction in the presence of a 4-nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (NBT + BCIP) mixture. Obvious detection of positive signals for Ccn2 in the cartilage of developing phalanges indicates that this method can be applied to gene expression analysis of other Ccn genes in developing calcified tissues.

DOI： 10.1007/978-1-4939-6430-7_2

Western blotting analysis of CCN proteins in calcified tissues

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 43 - 51 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Western blotting is widely used for protein analysis. We routinely perform such analysis for evaluating the production levels of CCN family proteins in a variety of cells under various conditions. In this chapter, we describe our Western blotting protocol to estimate protein production profiles of CCN family members after having assessed the specificity of the antibodies against each CCN member protein to ensure no crossreaction with other CCN member proteins.

DOI： 10.1007/978-1-4939-6430-7_5

The ccn proteins: An overview Invited

Masaharu Takigawa

Methods in Molecular Biology 1489 1 - 8 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

I introduce the general structures and functions of CCN proteins and possible molecular mechanisms regarding the unique biological actions of this new family of signaling regulators, which may be referred to as “signal conductors.” Relevance to pathology is also briefly introduced. The information provided in this overview should be useful for readers of the following chapters.

DOI： 10.1007/978-1-4939-6430-7_1

In vitro transfection with and expression of CCN family of genes

Danilo Janune, Masaharu Takigawa

Methods in Molecular Biology 1489 107 - 113 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

The ability to engineer cells to express a protein of interest in an inducible manner and stably for a long period is a valuable tool in molecular biology and also one that holds promise for regenerative medicine in the future. CCN proteins have been suggested to be involved in tissue regeneration. In this chapter, we describe an in vitro method for stable and inducible expression of CCN protein in a chondroprogenitor cell line and in chondrocytes in primary culture that does not involve the use of any viral vector.

DOI： 10.1007/978-1-4939-6430-7_11

Production of recombinant CCN2 protein by mammalian cells

Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 95 - 105 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Recombinant CCN2 protein (rCCN2) is available from many companies
however, most of them are produced in E. coli. To investigate true functions of rCCN2, glycosylated protein with proper folding needs to be used. Therefore, we use rCCN2 produced by mammalian cells. Conditioned medium (CM) of HeLa cells stably transfected with a CCN2 expression vector are collected, and the recombinant CCN2 protein produced and secreted into the CM is purified by two-step chromatography, first with a heparin affinity column and then with an anti-CCN2 affinity column prepared with a monoclonal antibody against CCN2. The purified rCCN2 shows the bands of 36–38 kDa with sliver staining after gel electrophoresis, which can be confirmed by Western blotting. This chapter describes these methods in detail.

DOI： 10.1007/978-1-4939-6430-7_10

Production of recombinant CCN2 protein in Escherichia coli

Eriko Aoyama, Takako Hattori, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 77 - 84 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Recombinant proteins are important tools for understanding molecular functions in vitro. Recent progress in the generation of recombinant proteins is amazing. However, when we plan to produce them, we should choose the best method according to the nature and the use of the target recombinant protein. Degradation and mis-folding are major problems in producing active recombinant CCN2. The method shown in this chapter describes the appropriate conditions under which we can produce CCN2 and its truncated fragments in Escherichia coli.

DOI： 10.1007/978-1-4939-6430-7_8

Analysis of expression of CCN family genes in skeletal tissue-derived cells

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 33 - 41 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

The quantitative reverse transcription polymerase chain reaction or real-time PCR has become a routine technique for the detection and comparison of amounts of specific mRNA transcripts, done by measuring amplified levels of specific cDNAs. In this chapter, we provide our real-time RT-PCR experimental procedure using SYBR Green I for the quantitative analysis of CCN family gene expression. Especially, we describe the extraction and purification steps for RNA derived from mesenchymal cells, such as chondrocytes and osteoblasts that produce a large amount of extracellular matrix in detail.

DOI： 10.1007/978-1-4939-6430-7_4

In vivo evaluation of cartilage regenerative effects of CCN2 protein

Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 273 - 282 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a unique growth factor that promotes the proliferation and differentiation, but not the hypertrophy of articular chondrocytes in vitro. Based on these fi ndings, we previously evaluated the cartilage-regenerative effects of recombinant CCN2 protein (rCCN2) by using both mono-iodoacetate (MIA) injection into the rat joint cavity and formation of full-thickness defects of rat articular cartilage in vivo, and our results suggested the utility of CCN2 in the regeneration of articular cartilage. This chapter entails helpful tips to apply these two animal models for the evaluation of cartilage-regenerative effects of CCN2 or its derivatives.

DOI： 10.1007/978-1-4939-6430-7_25

骨リモデリングにおける骨細胞の役割 Invited

西田崇, 久保田聡, 滝川正春

Clinical Calcium 27 ( 12 ) 23 - 29 2017

Analysis of pathological activities of CCN proteins in bone metastasis Invited

Tsuyoshi Shimo, Norie Yoshioka, Masaharu Takigawa, Akira Sasaki

Methods in Molecular Biology 1489 505 - 512 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Bone metastasis is a common occurrence in human malignancies, including breast, prostate, and lung cancer, and is associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the heart. This assay can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

DOI： 10.1007/978-1-4939-6430-7_42

Generation and analysis of cartilage-specific CCN2 overexpression in transgenic mice

Takako Hattori, Shinsuke Itoh, Masaharu Takigawa

Methods in Molecular Biology 1489 391 - 403 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Recent progress in gene-editing technology has provided a strong impact for improved our understanding of molecular functions in living organisms. Here we describe our method to generate transgene-overexpressing mouse models, which method involves the use of tissue-specific promoters for analyzing a certain molecule (s) in special tissues. The protocol described in this chapter uses the Col2a1 promoter-enhancer, which is known for driving specific and strong transgene expression in cartilage and is based on several of our studies showing a positive role of the connective tissue growth factor (CCN2) in cartilage-bone development and maintenance of articular cartilage. These mice show strongly accelerated endochondral ossification resulting in enhanced bone elongation, as well as resistance to age-related articular degeneration. This protocol also describes how to analyze the molecular mechanisms of these phenomena by use of chondrocytes isolated from CCN2-overexpressing cartilage.

DOI： 10.1007/978-1-4939-6430-7_32

Analysis of transcytosis of CCN2 by chondrocytes

Kazumi Kawata, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 405 - 413 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Transcytosis is a mechanism for the transcellular transport of biomolecules. Analysis of transcytosis is frequently performed in cells with distinct polarity, such as brain endothelial cells. However, in cells without evident polarity, analysis of transcytosis has not been performed. Here, we describe a method for analyzing transcytosis of a CCN family protein through chondrocytic cells having no apparent polarity.

DOI： 10.1007/978-1-4939-6430-7_33

Cell biological assays for measuring angiogenic activities of CCN proteins

Tsuyoshi Shimo, Masaharu Takigawa

Methods in Molecular Biology 1489 239 - 249 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the action of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of connective tissue growth factor (CTGF/CCN2) and CCN2 neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM).

DOI： 10.1007/978-1-4939-6430-7_22

Preparation of module-specific antibodies against CCN family members

Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 115 - 128 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Specific antibodies against biomolecules are conventional, but robust tools for the structural and functional analysis of target molecules. Since CCN family proteins are composed of four distinct modules that together determine the functionalities as full-length molecules depending upon extracellular microenvironment, specific antibody against independent modules are quite useful in CCN family research. Three distinct strategies are considerable for raising antibodies specific to four modules: IGFBP, VWC, TSP1, and CT modules. In the first strategy, full-length CCN family proteins are used to immunize mice to obtain a number of hybridoma clones producing different monoclonal antibodies, which are to be characterized to locate the epitopes in particular modules. Second methodology is a straightforward one, in which each modular protein fragment or synthetic peptide is prepared and is used for the immunization of animals independently. Finally, DNA immunization technology is recently known to be useful in developing module-specific antibodies against CCN family proteins as well. Preparation of antibodies is a quite classical and established technique, and thus nowadays is managed mostly by professional and commercial facilities. Therefore in this chapter, essentials of each strategy are introduced, rather than experimental details in each process.

DOI： 10.1007/978-1-4939-6430-7_12

Cell biological assays for measuring chondrogenic activities of CCN2 protein

Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 219 - 237 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Growth-plate chondrocytes undergo proliferation, maturation, hypertrophic differentiation, and calcification
and these processes can be reproduced in vitro in a chondrocyte culture system. Using this system, we have shown that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes all stages of proliferation, maturation, hypertrophic differentiation, and calcification, thus suggesting that CCN2 is a multifunctional growth factor for chondrocytes and plays important roles in chondrocyte proliferation and differentiation. In this chapter, we describe how to evaluate CCN2 functions in these processes occurring in cultured chondrocytes. Evaluation strategies for cell proliferation include measuring DNA synthesis by [3 H]-thymidine incorporation, cellular metabolic activity, and cell number with a hemocytometer. Next, evaluation strategies to assess maturation are analysis of the gene expression of markers of mature chondrocytes, and examination of proteoglycan and collagen synthesis by using radioactive compounds. In addition, cytohistochemical detection of glycosaminoglycans (GAGs), such as chondroitin sulfate, by use of alcian blue and toluidine blue staining is useful to evaluate chondrocyte maturation. These methods can be also used for evaluation of physiological functions of CCN2 in permanent chondrocytes such as articular and auricular chondrocytes, which do not calcify under physiological conditions. Next, evaluation of hypertrophic differentiation is performed by detecting type X collagen, which is specific marker of hypertrophic chondrocytes, and by measuring alkaline phosphatase (ALP) activity. Finally, evaluation of calcification is performed by detecting matrix calcification by use of alizarin red staining and by examining the incorporation of 45 Ca into cartilaginous matrix. These methods would be useful for the evaluation not only of CCN2 but also of its derivatives and other CCN proteins.

DOI： 10.1007/978-1-4939-6430-7_21

Protein imaging of CCN2 AND CCN3 in living cells

Takako Hattori, Mitsuhiro Hoshijima, Masaharu Takigawa

Methods in Molecular Biology 1489 211 - 215 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Recent progress in molecular imaging technology has had a strong impact on improving our understanding of molecular translocation, receptor internalization, and interactions in living cells. The protocol in this chapter introduces an optimized technique for intracellular localization of CCN2 and CCN3 in live cells, one using GFP-tagged CCN2 and Halo-tagged CCN3.

DOI： 10.1007/978-1-4939-6430-7_20

Analysis of posttranscriptional regulation of CCN genes

Seiji Kondo, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 187 - 209 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Cells generally control the concentration of mRNA by transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation (“decay”) cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, and degradation of the target mRNA are performed either individually, or in combination. From our experience, measurement of the steady-state levels of the mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying ccn2 gene expression level. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. Finally, the stability of ccn2 mRNAs is evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Here, we describe the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can in theory be applied to elucidating the posttranscriptional regulation of other genes belonging to the CCN family.

DOI： 10.1007/978-1-4939-6430-7_19

Evaluation of molecular interaction between CCN2 protein and its binding partners by surface plasmon resonance (SPR)

Eriko Aoyama, Masaharu Takigawa

Methods in Molecular Biology 1489 169 - 176 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

The surface plasmon resonance (SPR) biosensor is a useful tool to analyze numerically the interaction of certain molecules. The most important advantage of the SPR assay as compared with other protein–protein binding assays is that it can calculate the affinity between protein and its binding partner, for this affinity is necessary to determine the priority of interactions between proteins. Although CCN proteins have been shown to have various binding partners, the affinities of many of them have not yet been determined. Therefore, it is important to determine the unknown affinities of known binding partners and to find new binding partners whose affinities need to be determined. This chapter provides helpful tips to use the instrument for determination of the affinities of binding between CCN proteins and their binding partners.

DOI： 10.1007/978-1-4939-6430-7_17

Promoter analyses of CCN genes

Takanori Eguchi, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 177 - 185 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Promoter analysis is the most basics in the analysis of gene regulation. Luciferase gene is the most commonly used reporter gene in promoter analysis. Luciferase is an enzyme that is used when fi refl y and Renilla reniformis (sea pansy) emit light. The fi rst experimental step in this reporter gene assay is to connect a particular DNA segment to a luciferase gene. The second step is to transfect the reporter construct into the cells. Thereafter, stable luciferase will be produced with the help of transcriptional machinery, mRNA transporters, and translational machinery in the cells. Luciferase assay measures the quantity of light that is emitted by luciferin–luciferase reaction. Consistent with the fact that CCN2 expression has been shown to be altered by a variety of stimuli, the CCN2 promoter region also haa been shown to be bound and regulated by multiple transcription factors such as Smad, MMP3, NF-κB, AP1, TCF/LEF, and Sox9.

DOI： 10.1007/978-1-4939-6430-7_18

Analysis of signaling pathways activated by CCN proteins

Harumi Kawaki, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 139 - 143 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

CCN family proteins activate multiple intracellular phosphorylated kinase cascades to yield the multiple physiological functions of a variety of target cells. In this chapter, we describe our protocol examining the effects of these proteins on signal transduction pathways, especially mitogen-activated protein kinase cascades, activated by CCN member proteins, which examinations have been carried out mainly by using Western blotting methodologies.

DOI： 10.1007/978-1-4939-6430-7_14

Protocols for screening peptide motifs binding to ccn family proteins

Satoshi Kubota, Harumi Kawaki, Masaharu Takigawa

Methods in Molecular Biology 1489 155 - 167 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Function of CCN family proteins is determined by the interactions with multiple cofactors that are present in microenvironment. Therefore, finding out these cofactors is critically important in understanding the molecular function of the CCN family members. For this objective, bacteriophage random peptide display library is a quite feasible tool. In this library, each filamentous bacteriophage is designed to display an oligopeptide of random 12–16 amino acid residues on its surface. Bacteriophage clones that possess the peptides that bind to a CCN family protein are selected through several cycles of a process designated biopanning or affinity selection. By determining the nucleotide sequence of the DNA that encodes the displayed peptide, oligopeptides that specifically bind to the CCN family member can be specified. Obtained peptide sequences can be utilized for designing peptide aptamers for the CCN family protein, or as a key sequence to find out new CCN family cofactor candidates in silico.

DOI： 10.1007/978-1-4939-6430-7_16

Elisa of CCN family proteins in body fluids including serum and plasma

Satoshi Kubota, Harumi Kawaki, Masaharu Takigawa

Methods in Molecular Biology 1489 127 - 138 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Enzyme-linked immunosorbent assay (ELISA) is the most popular methodology for absolute quantification of particular proteins in liquid samples. Especially for CCN family members that are associated with human diseases, utility of ELISA for those proteins in clinics as well as research laboratories is emphasized. However, in order to obtain accurate and stable results in ELISA, particular care should be taken in controlling the quality and quantity of standard CCN family proteins, which bind to various materials and can be unstable in a purified form. Recently, diagnostic value of the CCN family protein fragments in body fluids has been indicated in several diseases. Therefore, module-specific detection system for the CCN family members is desired as a promising tool in clinics. It should be also noted that modular fragments of CCN family members can be more stable than the full-length in purified forms, whose quality may be easier to control than that of the full-length ones.

DOI： 10.1007/978-1-4939-6430-7_13

Protocols for screening for binding partners of CCN proteins: Yeast two-hybrid system

Mitsuhiro Hoshijima, Takako Hattori, Masaharu Takigawa

Methods in Molecular Biology 1489 145 - 154 2017

Language：English Publishing type：Part of collection (book) Publisher：Humana Press Inc.

Yeast two-hybrid screening is a powerful method to identify proteins that interact with a protein of interest. CCN2 consists of four domains, and identification of new proteins that bind to individual domains of CCN2 may reveal a variety of CCN2 functions. To identify CCN2-interactive proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8, with CCN2 cDNA used as a bait. In this chapter, we describe our methods for screening for CCN2 binding partners by this system in detail. This protocol may be applied to other CCN proteins as well.

DOI： 10.1007/978-1-4939-6430-7_15

The role of osteocytes in bone remodeling.

Nishida, T, Kubota, S, Takigawa, M

27 23 - 29 2017

Assessment of CCN2 Independent Modules Regenerative Capacity on Osteoarthritis and Further Selecting the Most Suitable Among them as a Potential Therapeutic Drug Reviewed

Abdelkader Tarek, Aoyama Eriko, Nishida Takashi, Hattori Takako, Janune Danilo, Hara Emilio S, Ono Mitsuaki, Tabata Yasuhiko, Kuboki Takuo, Kubota Satoshi, Takigawa Masaharu

FASEB JOURNAL 30 2016.4

Assessment of CCN2 Independent Modules Regenerative Capacity on Osteoarthritis and Further Selecting the Most Suitable Among them as a Potential Therapeutic Drug Reviewed

Abdelkader Tarek, Aoyama Eriko, Nishida Takashi, Hattori Takako, Janune Danilo, Hara Emilio S, Ono Mitsuaki, Tabata Yasuhiko, Kuboki Takuo, Kubota Satoshi, Takigawa Masaharu

FASEB JOURNAL 30 2016.4

Role of CCN2 in Amino Acid Metabolism of Chondrocytes Reviewed

Yurika Murase, Takako Hattori, Eriko Aoyama, Takashi Nishida, Aya Maeda-Uematsu, Harumi Kawaki, Karen M. Lyons, Akira Sasaki, Masaharu Takigawa, Satoshi Kubota

JOURNAL OF CELLULAR BIOCHEMISTRY 117 ( 4 ) 927 - 937 2016.4

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

CCN2/connective tissue growth factor (CTGF) is a multi-functional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2-null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2-null and wild-type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2-null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2-null chondrocytes. Furthermore, we newly found that expression of two genes encoding two glycolytic enzymes, as well as the previously reported Eno1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2-null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro. Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage. J. Cell. Biochem. 117: 927-937, 2016. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.25377

Matrix remodeling response of human periodontal tissue cells toward fibrosis upon nicotine exposure Reviewed

Hiroko Takeuchi-Igarashi, Satoshi Kubota, Toshiaki Tachibana, Etsuko Murakashi, Masaharu Takigawa, Masataka Okabe, Yukihiro Numabe

ODONTOLOGY 104 ( 1 ) 35 - 43 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

It is widely accepted that fibrosis is frequently observed in the gingiva of smokers. However, the mechanisms by which smoking results in pathological changes in periodontal tissue that lead to fibrosis are not entirely clear. Our former report showed that type I collagen synthesis was promoted by nicotine via CCN family protein 2 in human periodontal tissue cells. Here, we evaluated other aspects of nicotine function from a viewpoint of extracellular matrix (ECM) remodeling. Human gingival fibroblasts (n = 4) and periodontal ligament cells (n = 3) were isolated. The cells were treated with nicotine at a variety of concentrations for 12-48 h. Modulators of matrix remodeling were measured using enzyme-linked immunosorbent assays. Cell migration and morphology were also evaluated. As a result, following treatment with 1 mu g/ml nicotine, tissue inhibitor of metalloproteinase-1 and transforming growth factor-beta 1 production in both cell lysates and supernatants, and matrix metalloproteinases-1 production in cell lysates, were significantly increased (p < 0.05). Compared to controls, cell migration was significantly inhibited (p < 0.005) by nicotine in a time-dependent manner. Electron microscopic analysis revealed the presence of a number of vacuoles in nicotine-treated cells. These results indicate that nicotine not only impairs fibroblast motility, and induces cellular degenerative changes, but also alters ECM-remodeling systems of periodontal cells. Induction of matrix remodeling molecules, combined with type I collagen accumulation, may account for the molecular mechanism of nicotine-induced periodontal fibrosis.

DOI： 10.1007/s10266-014-0177-y

Sorcin Expression in the Epiphyseal Growth Plates of Mice Reviewed

Mariko Kawai, Ning Liu, Takako Hattori, Yo-Hei Kataoka, Masaharu Takigawa, Satoshi Kubota, Toshio Yamamoto, Kiyoshi Ohura

JOURNAL OF HARD TISSUE BIOLOGY 25 ( 1 ) 57 - 61 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL HARD TISSUE BIOLOGY

Sorcin is a small calcium-binding protein that is widely expressed in many tissues. Sorcin regulates cardiac myocyte apoptosis by modulating mitochondrial Ca2+ cycling and regulates fibroblast cell cycling and apoptosis via Ca2+ signaling through the endoplasmic reticulum (ER). During endochondral ossification, some chondrocytes undergo apoptosis after their terminal differentiation; however, Sorcin's expression in these cells has not been characterized. Here we examined Sorcin's gene expression in the chondrocytes derived from mouse growth plate by reverse transcription PCR (RT-PCR), and its protein localization in the chondrocytes of femoral growth plate using immunohistochemistry. Sorcin protein was detected in the chondrocytes, and was particularly abundant in the cytoplasm and nuclei of proliferative zone chondrocytes and in the nuclei of hypertrophic chondrocytes. Apoptotic analysis of the growth plate revealed that many of the hypertrophic chondrocytes undergo the DNA fragmentation. We report for the first time the localization of Sorcin in the epiphyseal growth plate in which one of the apoptotic phenomenon was detected.

DOI： 10.2485/jhtb.25.57

CCN4/WISP-1 positively regulates chondrogenesis by controlling TGF-beta 3 function Reviewed

Yoshioka, Yuya, Ono, Mitsuaki, Maeda, Azusa, Kilts, Tina M., Hara, Emilio Satoshi, Khattab, Hany, Ueda, Junji, Aoyama, Eriko, Oohashi, Toshitaka, Takigawa, Masaharu, Young, Marian F., Kuboki, Takuo

Bone 83 162 - 170 2016

Involvement of multiple CCN family members in platelets that support regeneration of joint tissues Reviewed

Chikako Hara, Satoshi Kubota, Takashi Nishida, Miki Hiasa, Takako Hattori, Eriko Aoyama, Yoshinori Moriyama, Hiroshi Kamioka, Masaharu Takigawa

MODERN RHEUMATOLOGY 26 ( 6 ) 940 - 949 2016

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Objectives: Platelet-rich plasma (PRP) has been widely used to enhance the regeneration of damaged joint tissues, such as osteoarthritic and rheumatoid arthritic cartilage. The aim of this study is to clarify the involvement of all of the CCN family proteins that are crucially associated with joint tissue regeneration.
Methods: Cyr61-CTGF-NOV (CCN) family proteins in human platelets and megakaryocytic cells were comprehensively analyzed by Western blotting analysis. Production of CCN family proteins in megakaryocytes in vivo was confirmed by immunofluorescence analysis of mouse bone marrow cells. Effects of CCN family proteins found in platelets on chondrocytes were evaluated by using human chondrocytic HCS-2/8 cells.
Results: Inclusion of CCN2, a mesenchymal tissue regenerator, was confirmed. Of note, CCN3, which counteracts CCN2, was newly found to be encapsulated in platelets. Interestingly, these two family members were not detectable in megakaryocytic cells, but their external origins were suggested. Furthermore, we found for the first time CCN5 and CCN1 that inhibits ADAMTS4 in both platelets and megakaryocytes. Finally, application of a CCN family cocktail mimicking platelets onto HCS-2/8 cells enhanced their chondrocytic phenotype.
Conclusions: Multiple inclusion of CCN1, 2 and 3 in platelets was clarified, which supports the harmonized regenerative potential of PRP in joint therapeutics.

DOI： 10.3109/14397595.2016.1155255

Physical interaction of CCN2 with diverse growth factors involved in chondrocyte differentiation during endochondral ossification Reviewed

Hany Mohamed Khattab, Eriko Aoyama, Satoshi Kubota, Masaharu Takigawa

JOURNAL OF CELL COMMUNICATION AND SIGNALING 9 ( 3 ) 247 - 254 2015.9

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

CCN family member 2 (CCN2) has been shown to promote the proliferation and differentiation of chondrocytes, osteoblasts, osteoclasts, and vascular endothelial cells. In addition, a number of growth factors and cytokines are known to work in harmony to promote the process of chondrogenesis and chondrocyte differentiation toward endochondral ossification. Earlier we showed that CCN2 physically interacts with some of them, suggesting that multiple effects of CCN2 on various differentiation stages of chondrocytes may be attributed to its interaction with these growth factors and cytokines. However, little is known about the functional interaction occurring between CCN2 and other growth factors and cytokines in promoting chondrocyte proliferation and differentiation. In this study we sought to shed light on the binding affinities between CCN2 and other essential growth factors and cytokines known to be regulators of chondrocyte differentiation. Using the surface plasmon resonance assay, we analyzed the dissociation constant between CCN2 and each of the following: TGF-beta 1, TGF-beta 3, IGF-I, IGF-II, PDGF-BB, GDF5, PTHrP, and VEGF. We found a strong association between CCN2 and VEGF, as well as a relatively high association with TGF-beta 1, TGF-beta 3, PDGF-BB, and GDF-5. However, the sensorgrams obtained for possible interaction between CCN2 and IGF-I, IGF-II or PTHrP showed no response. This study underlines the correlation between CCN2 and certain other growth factors and cytokines and suggests the possible participation of such interaction in the process of chondrogenesis and chondrocyte differentiation toward endochondral ossification.

DOI： 10.1007/s12079-015-0290-x

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes. Reviewed International journal

Murata H, Hattori T, Maeda H, Takashiba S, Takigawa M, Kido J, Nagata T

Journal of periodontal research 50 ( 4 ) 452 - 460 2015.8

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND AND OBJECTIVE: Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. MATERIAL AND METHODS: To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. RESULTS: Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. CONCLUSION: We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression.

DOI： 10.1111/jre.12227

CCN2 enhances RANKL-induced osteoclast differentiation via direct binding to RANK and OPG Reviewed

Eriko Aoyama, Satoshi Kubota, Hany Mohamed Khattab, Takashi Nishida, Masaharu Takigawa

BONE 73 242 - 248 2015.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a multi-potent factor for mesenchymal cells such as chondrocytes, osteoblasts, osteoclasts, and endothelial cells. CCN2 is also known as a modulator of other cytokines and receptors via direct molecular interactions with them. We screened additional factors binding to CCN2 and found receptor activator of NF-kappa B (RANK) as one of them. RANK is also known as TNF-related activation-induced cytokine (TRANCE) receptor, and its signaling plays a critical role in osteoclastogenesis. Notable affinity between CCN2 and RANK was confirmed by using surface plasmon resonance (SPR) analysis. In fact, CCN2 enhanced the RANK-mediated signaling, such as occurs in NF-kappa B, p38 and JNK pathways, in pre-osteoclastic RAW264.7 cells; whereas CCN2 had no influence on RANK RANK ligand (RANKL) binding. Moreover, CCN2 also significantly bound to osteoprotegerin (OPG), which is a decoy receptor of RANKL. Of note, OPG markedly inhibited the binding between CCN2 and RANK; and CCN2 canceled the inhibitory effect of OPG on osteoclast differentiation. These findings suggest CCN2 as a candidate of the fourth factor in the RANK/RANKL/OPG system for osteodastogenesis, which regulates OPG and RANK via direct interaction. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2014.12.058

CCN family protein 2 (CCN2) promotes the early differentiation, but inhibits the terminal differentiation of skeletal myoblasts Reviewed

Takashi Nishida, Satoshi Kubota, Eriko Aoyama, Danilo Janune, Karen M. Lyons, Masaharu Takigawa

JOURNAL OF BIOCHEMISTRY 157 ( 2 ) 91 - 100 2015.2

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Many studies have reported that CCN family protein 2 (also known as connective tissue growth factor) induces fibrotic response in skeletal muscle, thus emphasizing the pathological role of CCN2 in muscle tissues. However, the physiological role of CCN2 in myogenesis is still unknown. This study clarified the CCN2 functions during myogenesis. Recombinant CCN2 (rCCN2) promoted proliferation and MyoD production in C2C12 cells and primary myoblasts, but inhibited myogenin production. In accordance with these findings, the gene expression levels of myosin heavy chain, which is a marker of terminally differentiated myoblasts and desmin, which is the main intermediate filament protein of muscle cells, were decreased by rCCN2 treatment. In vivo analyses with Ccn2-deficient skeletal muscle revealed decreased proliferating cell nuclear antigen (PCNA)/MyoD double positive cells and muscle hypoplasia. Consistent with this finding, myogenic marker genes and myotube formation were repressed in Ccn2-deficient myoblasts. The protein production of CCN2 was increased in C2C12 myoblasts treated with tumor necrosis factor-alpha, which is a pro-inflammatory cytokine, suggesting its role in muscle regeneration after inflammation. These findings indicate that CCN2 promotes proliferation and early differentiation but inhibits the terminal differentiation of myoblasts, thus suggesting that CCN2 plays a physiological role in myogenesis.

DOI： 10.1093/jb/mvu056

Cellular and molecular actions of CCN2/CTGF and its role under physiological and pathological conditions

Satoshi Kubota, Masaharu Takigawa

CLINICAL SCIENCE 128 ( 3 ) 181 - 196 2015.2

Language：English Publisher：PORTLAND PRESS LTD

CCN family protein 2 (CCN2), also widely known as connective tissue growth factor (CTGF), is one of the founding members of the CCN family of matricellular proteins. Extensive investigation on CCN2 over decades has revealed the novel molecular action and functional properties of this unique signalling modulator. By its interaction with multiple molecular counterparts, CCN2 yields highly diverse and context-dependent biological outcomes in a variety of microenvironments. Nowadays, CCN2 is recognized to conduct the harmonized development of relevant tissues, such as cartilage and bone, in the skeletal system, by manipulating extracellular signalling molecules involved therein by acting as a hub through a web. However, on the other hand, CCN2 occasionally plays profound roles in major human biological disorders, including fibrosis and malignancies in major organs and tissues, by modulating the actions of key molecules involved in these clinical entities. In this review, the physiological and pathological roles of this unique protein are comprehensively summarized from a molecular network-based viewpoint of CCN2 functionalities.

DOI： 10.1042/CS20140264

New functional aspects of CCN2 revealed by trans-omic approaches Reviewed

Kubota, S, Maeda-Uematsu, A, Nishida, T, Takigawa, M

Journal of Oral Biosciences 57 37 - 43 2015

Role of CCN2 in Amino Acid Metabolism of Chondrocytes.

Murase Y, Hattori T, Aoyama E, Nishida T, Maeda-Uematsu A, Kawaki H, Lyons KM, Sasaki A, Takigawa M, Kubota S

J Cell Biochem 151 - 155 2015

Fluocinolone Acetonide Is a Potent Synergistic Factor of TGF-beta 3-Associated Chondrogenesis of Bone Marrow-Derived Mesenchymal Stem Cells for Articular Surface Regeneration Reviewed

Hara, Emilio Satoshi, Ono, Mitsuaki, Hai Thanh Pham, Sonoyama, Wataru, Kubota, Satoshi, Takigawa, Masaharu, Matsumoto, Takuya, Young, Marian F., Olsen, Bjorn R., Kuboki, Takuo

Journal of Bone and Mineral Research 30 ( 9 ) 1585 - 1596 2015

Exosomes mediate intercellular transfer of pro-fibrogenic connective tissue growth factor (CCN2) between hepatic stellate cells, the principal fibrotic cells in the liver Reviewed

Alyssa Charrier, Ruju Chen, Li Chen, Sherri Kemper, Takako Hattori, Masaharu Takigawa, David R. Brigstock

SURGERY 156 ( 3 ) 548 - 555 2014.9

Language：English Publishing type：Research paper (scientific journal) Publisher：MOSBY-ELSEVIER

Background. Fibrogenic pathways in the liver are principally regulated by hepatic stellate cells (HSC), which produce and respond to fibrotic mediators such as connective tissue growth factor (CCN2). The aim of this study was to determine whether CCN2 is shuttled between HSC in membranous nanovesicles, or "exosomes."
Methods. Exosomes were incubated with HSC after isolation from conditioned medium of control or CCN2-green fluorescent protein (GFP)-transfected primary mouse HSC or human LX-2 HSC. Some exosomes were stained fluorescently with PKH26. HSC co-culture experiments were performed in the presence of GW4869 exosome inhibitor. CCN2 or CCN2-GFP were evaluated by quantitative real-time polymerase. chain reaction or Western blot.
Results. HSC-derived exosomes contained CCN2 or CCN2 mRNA, each of which increased in concentration during HSC activation or after transfection of HSC with CCN2-GFP. Exosomes, stained with either PKH26 or purified from CCN2-GFP transfected cells, were taken up by activated or quiescent HSC resulting in CCN2-GFP delivery, as shown by their direct addition to recipient cells or by the GW4869-dependency of donor HSC.
Conclusion. CCN2 is packaged into secreted, nano-sized exosomes that mediate its intercellular transfer between HSC. Exosomal CCN2 may amplify or fine tune fibrogenic signaling and, in conjunction with other exosome constituents, may have utility as a noninvasive biomarker to assess hepatic fibrosis.

DOI： 10.1016/j.surg.2014.04.014

Direct interaction between CCN family protein 2 and fibroblast growth factor 1

Tarek Abd El Kader, Satoshi Kubota, Ken Anno, Saho Tanaka, Takashi Nishida, Takayuki Furumatsu, Eriko Aoyama, Takuo Kuboki, Masaharu Takigawa

JOURNAL OF CELL COMMUNICATION AND SIGNALING 8 ( 2 ) 157 - 163 2014.6

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

In an attempt to find out a new molecular counterpart of CCN family protein 2 (CCN2), a matricellular protein with multiple functions, we performed an interactome analysis and found fibroblast growth factor (FGF) -1 as one of the candidates. Solid-phase binding assay indicated specific binding between CCN2 and FGF-1. This binding was also confirmed by surface plasmon resonance (SPR) analysis that revealed a dissociation constant (Kd) of 3.98 nM indicating strong molecular interaction between the two. RNA analysis suggested that both FGF-1 and CCN2 could be produced by chondrocytes and thus their interaction in the cartilage is possible. These findings for the first time indicate the direct interaction of CCN2 and FGF-1 and suggest the co-presence of these molecules in the cartilage microenvironment. CCN2 is a well-known promoter of cartilage development and regeneration, whereas the physiological and pathological role of FGF-1 in cartilage mostly remains unclear. Biological role of FGF-1 itself in cartilage is also suspected.

DOI： 10.1007/s12079-014-0232-z

Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during chronic pancreatitis and are exported in PSC-derived exosomes Reviewed

Alyssa Charrier, Ruju Chen, Li Chen, Sherri Kemper, Takako Hattori, Masaharu Takigawa, David R. Brigstock

JOURNAL OF CELL COMMUNICATION AND SIGNALING 8 ( 2 ) 147 - 156 2014.6

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Pancreatitis is an inflammatory condition of the pancreas which, in its chronic form, involves tissue destruction, exocrine and endocrine insufficiency, increased risk of pancreatic cancer, and an extensive fibrotic pathology which is due to unrelenting collagen deposition by pancreatic stellate cells (PSC). In response to noxious agents such as alcohol-excessive consumption of which is a major cause of pancreatitis in the West-normally quiescent PSC undergo a phenotypic and functional transition to activated myofibroblasts which produce and deposit collagen at high levels. This process is regulated by connective tissue growth factor (CCN2), expression of which is highly up-regulated in activated PSC. We show that CCN2 production by activated PSC is associated with enhanced expression of microRNA-21 (miR-21) which was detected at high levels in activated PSC in a murine model of alcoholic chronic pancreatitis. A positive feedback loop between CCN2 and miR-21 was identified that resulted in enhancement of their respective expression as well as that of collagen alpha 1(I). Both miR-21 and CCN2 mRNA were present in PSC-derived exosomes, which were characterized as 50-150 nm CD9-positive nanovesicles. Exosomes from CCN2-GFP- or miR-21-GFP-transfected PSC were taken up by other PSC cultures, as shown by direct fluorescence or qRT-PCR for GFP. Collectively these studies establish miR-21 and CCN2 as participants in a positive feedback loop during PSC activation and as components of the molecular payload in PSC-derived exosomes that can be delivered to other PSC. Thus interactions between cellular or exosomal miR-21 and CCN2 represent novel aspects of fibrogenic regulation in PSC. Summary Chronic injury in the pancreas is associated with fibrotic pathology which is driven in large part by CCN2-dependent collagen production in pancreatic stellate cells. This study shows that CCN2 up-regulation in PSC is associated with increased expression of miR-21 which, in turn, is able to stimulate CCN2 expression further via a positive feedback loop. Additionally miR-21 and CCN2 were identified in PSC-derived exosomes which effected their delivery to other PSC. The cellular and exosomal miR-21-CCN2 axis is a novel component in PSC fibrogenic signaling.

DOI： 10.1007/s12079-014-0220-3

CCN2 as a novel molecule supporting energy metabolism of chondrocytes. Reviewed

Maeda-Uematsu A, Kubota S, Kawaki H, Kawata K, Miyake Y, Hattori T, Nishida T, Moritani N, Lyons KM, Iida S, Takigawa M

Journal of cellular biochemistry 115 ( 5 ) 854 - 865 2014.5

Differential Expression of Vascular Endothelial Growth Factor in High- and Low-Metastasis Cell Lines of Salivary Gland Adenoid Cystic Carcinoma Reviewed

Seiji Kondo, Yoshiki Mukudai, Daisuke Soga, Takashi Nishida, Masaharu Takigawa, Tatsuo Shirota

ANTICANCER RESEARCH 34 ( 2 ) 671 - 677 2014.2

Language：English Publishing type：Research paper (scientific journal) Publisher：INT INST ANTICANCER RESEARCH

We used high- (ACCM) and low- (ACC2) metastasis cell lines of human adenoid cystic carcinoma (ACC) as an experimental model to study metastatic mechanisms and compare their expression levels for angiogenic-related factor vascular endothelial growth factor (VEGF). By using a series of extensive analyses, hypoxia-inducible factor-1 (HIF-1) alpha-dependent VEGF expression levels were observed to be higher in ACCM cell lines, increasing the possible development of tumor metastasis, compared to ACC2 cell lines. Our findings provide the novel insight that HIF-1 alpha-dependent VEGF overexpression under hypoxic conditions shows to some extent associations with the metastatic tendency of ACC cells and may function as a potential target for ACC therapy.

Dexamethasone differentially regulates Bcl-2 family proteins in human proliferative chondrocytes: Role of pro-apoptotic Bid Reviewed

Farasat Zaman, Dionisios Chrysis, Kirsten Huntjens, Andrei Chagin, Masaharu Takigawa, Bengt Fadeel, Lars Savendahl

TOXICOLOGY LETTERS 224 ( 2 ) 196 - 200 2014.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

Glucocorticoids (GCs) are widely used to treat inflammatory diseases and cancers. A multitude of undesired side effects have been reported in GC-treated patients including decreased linear bone growth. We have previously reported that GCs activate the caspase cascade and trigger Bax-mediated mitochondrial apoptosis in growth plate chondrocytes causing growth retardation in young mice. To further explore the role of mitochondrial apoptosis in GC-induced bone growth retardation, a number of pro- and anti-apoptotic proteins were studied in ex vivo cultures of human growth plate cartilage and human HCS-2/8 proliferative chondrocytes exposed to dexamethasone. Dexamethasone was found to increase the pro-apoptotic proteins Bcl-xS, Bad, and Bak as well as the proteolysis of Bid. Anti-Bid small interfering RNA partially rescued the chondrocytes from dexamethasone-induced apoptosis. Taken together, our data suggest that GC treatment differentially regulates Bcl-2 family member proteins to facilitate mitochondrial apoptosis in proliferative chondrocytes thereby contributing to GC-induced bone growth impairment. Prevention of this imbalance between pro- and anti-apoptotic Bcl-2 family proteins may provide a new strategy to protect from adverse effects of GCs on bone growth. (C) 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.toxlet.2013.10.020

The regenerative effects of CCN2 independent modules on chondrocytes in vitro and osteoarthritis models in vivo Reviewed

El Kader, Tarek Abd, Kubota Satoshi, Nishida Takashi, Hattori Takako, Aoyama Eriko, Janune Danilo, Hara Emilio S, Ono Mitsuaki, Tabata Yasuhiko, Kuboki Takuo, Takigawa Masaharu

Bone 59 180 - 188 2014

E6-AP/UBE3A Protein Acts as a Ubiquitin Ligase toward SOX9 Protein Reviewed International journal

Hattori Takako, Kishino Tetsuya, Stephen Shelley, Eberspaecher Heidi, Maki Sayumi, Takigawa Masaharu, de Crombrugghe Benoit, Yasuda Hideyo

JOURNAL OF BIOLOGICAL CHEMISTRY 288 ( 49 ) 35138 - 35148 2013.12

Language：English Publishing type：Research paper (scientific journal)

SOX9 is a transcription factor that acts as a key regulator at various stages of cartilage differentiation. There is ample evidence that intracellular SOX9 protein levels are tightly regulated both by sumoylation and by degradation through the ubiquitin-proteasome pathway. Using a proteomics approach, here we report the identification of a SOX9-binding protein, E6-AP/UBE3A, that may act as a ubiquitin ligase toward Sox9. E6-AP bound SOX9 through the region consisting mostly of its high mobility group domain in vitro. In nuclear lysates, FLAG-tagged E6-AP coprecipitated with Sox9 and its high mobility group domain. This finding was estimated using nuclear lysates from a chondrocytic cell line that endogenously expresses E6-AP and SOX9. Accordingly, ectopically expressed E6-AP and SOX9 colocalized in the nucleus. We show that E6-AP ubiquitinates SOX9 in vitro and in vivo and that SOX9 levels are enhanced after addition of the proteasome inhibitor bortezomib. Similar, siRNA knockdown of E6-AP and the E2 ligase Ubc9 increased cellular SOX9 amounts, supporting the notion that SOX9 may be ubiquitinated in hypertrophic chondrocytes by E6-AP and degraded by proteasomes. This is in accordance with the distribution of SOX9 levels, which are high in proliferating and prehypertrophic chondrocytes but low in hypertrophic chondrocytes, whereas E6-AP levels are high in hypertrophic chondrocytes and low in prehypertrophic chondrocytes. Furthermore, E6-AP-deficient mice showed SOX9 accumulation in chondrocytes and the brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.

DOI： 10.1074/jbc.M113.486795

The CCN family acting throughout the body: Recent research developments Reviewed

Satoshi Kubota, Masaharu Takigawa

Biomolecular Concepts 4 ( 5 ) 477 - 494 2013.10

Language：English Publishing type：Research paper (scientific journal)

The animal body is composed of a variety of cells and extracellular matrices that are organized and orchestrated in a harmonized manner to support life. Therefore, the critical importance of a comprehensive understanding of the molecular network surrounding and integrating the cells is now emphasized. The CCN family is a novel group of matricellular proteins that interact with and orchestrate a number of extracellular signaling and matrix molecules to construct and maintain living tissues. This family comprises six distinct members in mammals, which are characterized by a unique and conserved modular structure. These proteins are not targeted to limited and specific receptors to execute specific missions, but manipulate a vast number of biomolecules in the network by serving as a molecular hub at the center. The unified nomenclature, CCN, originates from a simple acronym of the three classical members, which helps us to avoid having any preconception about their pleiotropic and anonymous functional nature. In this review, after a brief summary of the general molecular concepts regarding the CCN family, new aspects of each member uncovered by recent research are introduced, which represent, nevertheless, only the tip of the iceberg of the profound functionality of these molecules. © 2013 Walter de Gruyter GmbH.

DOI： 10.1515/bmc-2013-0018

Novel role of miR-181a in cartilage metabolism. Reviewed

Sumiyoshi K, Kubota S, Ohgawara T, Kawata K, Abd El Kader T, Nishida T, Ikeda N, Shimo T, Yamashiro T, Takigawa M

Journal of cellular biochemistry 114 ( 9 ) 2094 - 2100 2013.9

Publishing type：Research paper (scientific journal)

Micro RNA (miRNA) is a small non-coding post-transcriptional RNA regulator that is involved in a variety of biological events. In order to specify the role of miRNAs in cartilage metabolism, we comparatively analyzed the expression profile of known miRNAs in chicken sternum chondrocytes representing early and late differentiation stages. Interestingly, none of the miRNAs displaying strong expression levels showed remarkable changes along with differentiation, suggesting their roles in maintaining the homeostasis rather than cytodifferentiation of chondrocytes. Among these miRNAs, miR-181a, which is known to play critical roles in a number of tissues, was selected and was further characterized. Human microarray analysis revealed remarkably stronger expression of miR-181a in human HCS-2/8 cells, which strongly maintained a chondrocytic phenotype, than in HeLa cells, indicating its significant role in chondrocytes. Indeed, subsequent investigation indicated that miR-181a repressed the expression of two genes involved in cartilage development. One was CCN family member 1 (CCN1), which promotes chondrogenesis; and the other, the gene encoding the core protein of aggrecan, a major cartilaginous proteoglycan, aggrecan. Based on these findings, negative feedback system via miR-181a to conserve the integrity of the cartilaginous phenotype may be proposed. © 2013 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.24556

Other Link： http://orcid.org/0000-0002-4419-9643

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes Reviewed

Itoh Shinsuke, Hattori Takako, Tomita Nao, Aoyama Eriko, Yutani Yasutaka, Yamashiro Takashi, Takigawa Masaharu

PLOS ONE 8 ( 8 ) e71156 2013.8

Publishing type：Research paper (scientific journal)

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage. © 2013 Itoh et al.

DOI： 10.1371/journal.pone.0071156

CCN2: a master regulator of the genesis of bone and cartilage Reviewed

Masaharu Takigawa

JOURNAL OF CELL COMMUNICATION AND SIGNALING 7 ( 3 ) 191 - 201 2013.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

CCN family member 2 (CCN2), also known as connective tissue growth factor (CTGF), has been suggested to be an endochondral ossification genetic factor that has been termed "ecogenin", because in vitro studies revealed that CCN2 promotes the proliferation and differentiation of growth-plate chondrocytes, osteoblasts, and vascular endothelial cells, all of which play important roles in endochondral ossification. In addition to its action toward these three types of cells, CCN2 was recently found to promote the formation of osteoclasts in vitro, which cells play an important role in the replacement of cartilage by bone during endochondral ossification, thus strengthening the "ecogenin" hypothesis. For confirmation of this hypothesis, transgenic mice over-expressing CCN2 in cartilage were generated. The results proved the hypothesis; i.e., the over-expression of CCN2 in cartilage stimulated the proliferation and differentiation of growth-plate chondrocytes, resulting in the promotion of endochondral ossification. In addition to its "ecogenin" action, CCN2 had earlier been shown to promote the differentiation of various cartilage cells including articular cartilage cells. In accordance with these findings, cartilage-specific overexpression of CCN2 in the transgenic mice was shown to protect against the development of osteoarthritic changes in aging articular cartilage. Thus, CCN2 may also play a role as an anti-aging (chondroprotective) factor, stabilizing articular cartilage. CCN2 also had been shown to promote intramembranous ossification, regenerate cartilage and bone, and induce angiogenesis in vivo. For understanding of the molecular mechanism underlying such multifunctional actions, yeast two-hybrid analysis, protein array analysis, solid-phase binding assay, and surface plasmon resonance (SPR) analysis have been used to search for binding partners of CCN2. ECMs such as fibronectin and aggrecan, growth factors including BMPs and FGF2 and their receptors such as FGFR1 and 2 and RANK, as well as CCN family members themselves, were shown to bind to CCN2. Regarding the interaction of CCN2 with some of them, various binding modules in the CCN2 molecule have been identified. Therefore, the numerous biological actions of CCN2 would depend on what kinds of binding partners and what levels of them are present in the microenvironment of different types of cells, as well as on the state of differentiation of these cells. Through this mechanism, CCN2 would orchestrate various signaling pathways, acting as a signal conductor to promote harmonized skeletal growth and regeneration.

DOI： 10.1007/s12079-013-0204-8

Cartilage-Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth Reviewed

Tomita Nao, Hattori Takako, Itoh Shinsuke, Aoyama Eriko, Yao Mayumi, Yamashiro Takashi, Takigawa Masaharu

PLOS ONE 8 ( 3 ) e59226 2013.3

Publishing type：Research paper (scientific journal)

Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage-related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones. Our results also indicate the possible involvement of locally enhanced IGF-I or IGF-II in this extended bone growth. © 2013 Tomita et al.

DOI： 10.1371/journal.pone.0059226

Anti-fibrotic effect of CCN3 accompanied by altered gene expression profile of the CCN family Reviewed International journal

El Kader, Tarek Abd, Kubota Satoshi, Janune Danilo, Nishida Takashi, Hattori Takako, Aoyama Eriko, Perbal Bernard, Kuboki Takuo, Takigawa Masaharu

JOURNAL OF CELL COMMUNICATION AND SIGNALING 7 ( 1 ) 11 - 18 2013.3

Language：English Publishing type：Research paper (scientific journal)

CCN family proteins 2 and 3 (CCN2 and CCN3) belong to the CCN family of proteins, all having a high level of structural similarity. It is widely known that CCN2 is a profibrotic molecule that mediates the development of fibrotic disorders in many different tissues and organs. In contrast, CCN3 has been recently suggested to act as an anti-fibrotic factor in several tissues. This CCN3 action was shown earlier to be exerted by the repression of the CCN2 gene expression in kidney tissue, whereas different findings were obtained for liver cells. Thus, the molecular action of CCN3 yielding its anti-fibrotic effect is still controversial. Here, using a general model of fibrosis, we evaluated the effect of CCN3 overexpression on the gene expression of all of the CCN family members, as well as on that of fibrotic marker genes. As a result, repression of CCN2 gene expression was modest, while type I collagen and α-smooth muscle actin gene expression was prominently repressed. Interestingly, not only CCN2, but also CCN4 gene expression showed a decrease upon CCN3 overexpression. These findings indicate that fibrotic gene induction is under the control of a complex molecular network conducted by CCN family members functioning together.

DOI： 10.1007/s12079-012-0180-4

The CCN2-inducer harmine promotes chondrogenesis and protects against TNFα-induced ablation of chondrocytic phenotype Reviewed

Hara, E.S, M. Ono, S. Kubota, W. Sonoyama, Y. Oida, T. Hattori, T. Nishida, T. Furumatsu, T. Ozaki, M. Takigawa, T. Kuboki

Biochimie 95 374 - 381 2013

miRNA-720 Controls Stem Cell Phenotype, Proliferation and Differentiation of Human Dental Pulp Cells Reviewed

Hara, Emilio Satoshi, Ono, Mitsuaki, Eguchi, Takanori, Kubota, Satoshi, Hai Thanh Pham, Sonoyama, Wataru, Tajima, Shoji, Takigawa, Masaharu, Calderwood, Stuart K., Kuboki, Takuo

Plos One 8 ( 12 ) e83545 2013

Novel chondrogenic and chondroprotective effects of the natural compound harmine Reviewed

Hara, Emilio Satoshi, Ono, Mitsuaki, Kubota, Satoshi, Sonoyama, Wataru, Oida, Yasutaka, Hattori, Takako, Nishida, Takashi, Furumatsu, Takayuki, Ozaki, Toshifumi, Takigawa, Masaharu, Kuboki, Takuo

Biochimie 95 ( 2 ) 374 - 381 2013

CCN2/CTGF binds to fibroblast growth factor receptor 2 and modulates its signaling Reviewed

Eriko Aoyama, Satoshi Kubota, Masaharu Takigawa

FEBS LETTERS 586 ( 24 ) 4270 - 4275 2012.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

CCN2 plays a critical role in the development of mesenchymal tissues such as cartilage and bone, and the binding of CCN2 to various cytokines and receptors regulates their signaling. By screening a protein array, we found that CCN2 could bind to fibroblast growth factor receptors (FGFRs) 2 and 3, with a higher affinity toward FGFR2. We ascertained that FGFR2 bound to CCN2 and that the binding of FGFR2 to FGF2 and FGF4 was enhanced by CCN2. CCN2 and FGF2 had a collaborative effect on the phosphorylation of ERK and the differentiation of osteoblastic cells. The present results indicate the biological significance of the binding of CCN2 to FGFR2 in bone metabolism.
Structured summary of protein interactions:
FGFR2 binds to CCN2 by protein array (View interaction)
FGFR1OP binds to CCN2 by protein array (View interaction)
FGFR3 binds to CCN2 by protein array (View interaction) (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2012.10.038

Mechanical stretch increases Smad3-dependent CCN2 expression in inner meniscus cells Reviewed

Takayuki Furumatsu, Tomoko Kanazawa, Yoshiaki Miyake, Satoshi Kubota, Masaharu Takigawa, Toshifumi Ozaki

JOURNAL OF ORTHOPAEDIC RESEARCH 30 ( 11 ) 1738 - 1745 2012.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

The intrinsic zone-specific properties of the menisci are determined by biomechanical environments. In this study, we examined mechanical stretch-dependent expression of multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, and investigated the role of CCN2 in meniscus cells. Uni-axial cyclic tensile strain (CTS) was applied using a STB-140 system. CTS-induced expression of CCN2 and a1(I) collagen (COL1A1) was assessed by quantitative real-time PCR analysis. The distribution of CCN2 and Smad2/3 in stretched cells was investigated by immunohistochemical analysis. Smad2/3-dependent CCN2 transactivation was measured by luciferase reporter assay. The relationship between Smad2/3 and CTS-induced CCN2 transcription was investigated by chromatin immunoprecipitation. CTS stimulated gene expression of CCN2 and COL1A1 in inner meniscus cells, but not in outer meniscus cells. Recombinant CCN2 increased COL1A1 expression only in inner meniscus cells. CCN2 synthesis and nuclear translocalization of phosphorylated Smad2/3 in inner meniscus cells were stimulated by CTS. The CCN2 promoter activity was synergistically enhanced by overexpressed Smad3 in stretched inner meniscus cells, but was not by Smad2. Chromatin immunoprecipitation revealed that CTS increased the association between Smad3 and the Smad-binding element on the CCN2 proximal promoter in inner meniscus cells. Our results suggest that stretch-induced CCN2 may have a crucial role in regulating COL1A1 expression in the inner meniscus. (c) 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:17381745, 2012

DOI： 10.1002/jor.22142

Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes Reviewed

Mitsuhiro Hoshijima, Takako Hattori, Eriko Aoyama, Takashi Nishida, Takashi Yamashiro, Masaharu Takigawa

FEBS JOURNAL 279 ( 19 ) 3584 - 3597 2012.10

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2CCN2 and CCN2CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 x 10-9 m for CCN2 and CCN2, and 1.95 x 10-9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent proteinCCN2 and HaloCCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3.

DOI： 10.1111/j.1742-4658.2012.08717.x

Role of LRP1 in transport of CCN2 protein in chondrocytes. Reviewed International journal

Kawata K, Kubota S, Eguchi T, Aoyama E, Moritani NH, Kondo S, Nishida T, Takigawa M

Journal of cell science 125 ( Pt 12 ) 2965 - 2972 2012.6

Language：English Publishing type：Research paper (scientific journal)

Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-β-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

DOI： 10.1242/jcs.101956

A selective estrogen receptor modulator inhibits tumor necrosis factor-alpha-induced apoptosis through the ERK1/2 signaling pathway in human chondrocytes Reviewed

Yosuke Hattori, Toshihisa Kojima, Daizo Kato, Hiroyuki Matsubara, Masaharu Takigawa, Naoki Ishiguro

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 421 ( 3 ) 418 - 424 2012.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine mediating inflammatory as well as cell death activities, and is thought to induce chondrocytic chondrolysis in inflammatory and degenerative joint diseases. Selective estrogen receptor modulators (SERMs), such as raloxifene, which are commonly used in clinical settings act as estrogen agonists or antagonists. It is assumed that estrogens have a potential role in cartilage protection; however, the precise molecular mechanism for the protective effects of estrogens is unclear. This study was designed to examine whether raloxifene inhibits TNF-alpha-induced apoptosis in human chondrocytes and to clarify the mechanisms involved. We also investigated the signaling pathways responsible for the anti-apoptotic effect of raloxifene. Apoptosis in chondrocytes was determined by DNA fragmentation assay and caspase-3 activation. Raloxifene significantly inhibited TNF-alpha-induced caspase-3 activation and cell DNA fragmentation levels in chondrocytes. The inhibitory effect of raloxifene was abolished by the estrogen receptor antagonist ICI 182,780. Extracellular signal-regulated kinase 1/2 (ERK1/2) regulates apoptosis, acting as an apoptotic or anti-apoptotic signal. TNF-alpha-induced apoptosis was significantly enhanced by the ERK1/2 pathway inhibitor PD98059. Raloxifene stimulated a further increase in ERK1/2 phosphorylation in TNF-alpha-treated chondrocytes. Furthermore, the anti-apoptotic effects of raloxifene were inhibited by PD98059. In addition, the anti-apoptotic effects of raloxifene were completely abolished in ERK1/2 siRNA-treated chondrocytes. These results suggest that raloxifene prevents caspase-3-dependent apoptosis induced by TNF-alpha in human chondrocytes by activating estrogen receptors and the ERK1/2 signaling pathway. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.03.111

Role of low-density lipoprotein receptor related protein 1 (LRP1) in CCN2/connective tissue growth factor (CTGF) protein transport in chondrocytes

Kawata., K, Kubota, S, Eguchi, T, Aoyama, E, Moritani, N, Kondo, S, Nishida, T, Takigawa, M

J Cell Sci 15 2965 - 2972 2012

Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress Reviewed

Honjo T, Kubota S, Kamioka H, Sugawara Y, Ishihara Y, Yamashiro T, Takigawa M, Takano-Yamamoto T

J Cell Commun Signal 6 ( 4 ) 225 - 232 2012

Induction of CCN2/CTGF by laminar fluid flow stress, which is mediated by the actin cytoskeleton in osteoblastic cells Reviewed

Honjo, T, S. Kubota, H. Kamioka, Y. Sugawara, Y. Ishihara, T. Yamashiro, M. Takigawa, T. Takano-Yamamoto

J Cell Commun Signal. 6 225 - 232 2012

Association of the metastatic phenotype with CCN family members among breast and oral cancer cells Reviewed

Toshihiro Ohgawara, Satoshi Kubota, Harumi Kawaki, Naito Kurio, Tarek Abd El Kader, Mitsuhiro Hoshijima, Danilo Janune, Tsuyoshi Shimo, Bernard Perbal, Akira Sasaki, Masaharu Takigawa

JOURNAL OF CELL COMMUNICATION AND SIGNALING 5 ( 4 ) 291 - 299 2011.12

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

The CCN family of proteins consists of six members with conserved structural features. These proteins play several roles in the physiology and pathology of cells. Among the pathological roles of the CCN family, one of the most important and controversial ones is their role in the expansion and metastasis of cancer. Up to now a number of reports have described the possible role of each CCN family member independently. In this study, we comprehensively analyzed the roles of all six CCN family members in cell growth, migration and invasion of breast cancer cells in vitro and in vivo. As a result, we found the CCN2/CCN3 ratio to be a parameter that is associated with the metastatic phenotype of breast cancer cells that are highly metastatic to the bone. The same analysis with cell lines from oral squamous carcinomas that are not metastatic to the bone further supported our notion. These results suggest the functional significance of the interplay between CCN family members in regulating the phenotype of cancer cells.

DOI： 10.1007/s12079-011-0133-3

Novel pathogenic role of fibrin as revealed by a case study on ligneous gingivitis Reviewed

Tsuyoshi Shimo, Akiyoshi Nishiyama, Satoshi Kubota, Naito Kurio, Tatsuo Okui, Naoki Katase, Nur Mohammad Monsur Hassan, Tatsuki Honami, Koji Kishimoto, Hiroshi Mese, Masaharu Takigawa, Akira Sasaki

Oral Science International 8 ( 2 ) 44 - 49 2011.11

Language：English Publishing type：Research paper (scientific journal)

Purpose of the research: Ligneous gingivitis is a rare disease characterized by nodular gingival enlargement secondary to fibrin deposits induced by micro-injury in the gingiva, which disorder results from plasminogen (PLG) deficiency. Although none have investigated the association of wound healing factors with ligneous gingivitis. In this study, in addition to a histopathologic examination of ligneous gingivitis in a case of type I PLG deficiency, we further present data showing the effect of wound healing factors in association with fibrin in vitro to clarify the pathobiology of ligneous gingivitis in PLG-deficient patients. Principle results: Immunohistochemical analysis revealed that transforming growth factor (TGF)-β1, connective tissue growth factor/CCN2 (CCN2), and endothelin-1 (ET-1) had accumulated in the extracellular matrix around the epithelial and fibroblastic cells near the fibrin deposition. Consistent with these results, fibrin and TGF-β1 synergistically up-regulated CCN2 and ET-1 gene expression in human dermal fibroblasts. Major conclusions: Fibrin plays a vicious role in ligneous gingivitis pathobiology by up-regulating CCN2 and ET-1 expression through the TGF-β signaling pathway. © 2011 Japanese Stomatological Society.

DOI： 10.1016/S1348-8643(11)00025-5

Effect of CCN2 on FGF2-Induced Proliferation and MMP9 and MMP13 Productions by Chondrocytes Reviewed

Takashi Nishida, Satoshi Kubota, Eriko Aoyama, Danilo Janune, Azusa Maeda, Masaharu Takigawa

ENDOCRINOLOGY 152 ( 11 ) 4232 - 4241 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

CCN2 (also known as connective tissue growth factor) interacts with several growth factors involved in endochondral ossification via its characteristic four modules and modifies the effect of such growth factors. Presently we investigated whether CCN2 interacts with fibroblast growth factor 2 (FGF2). Solid-phase binding assay, immunoprecipitation-Western blot analysis, and surface plasmon resonance (SPR) spectroscopy revealed that the C-terminal module of CCN2 (CT) directly bound to FGF2 with a dissociation constant of 5.5 nM. Next, we examined the combinational effects of CCN2 and FGF2 on the proliferation of and matrix metalloproteinase (MMP)-9 and -13 productions by cultured chondrocytes. FGF2 promoted not only the proliferation but also the production of MMP9 and -13, however, combined of FGF2 with CT module nullified the enhancement of both MMP productions and proliferation. To clarify the mechanism, we investigated the binding of CCN2 or its CT module to FGF receptor 1. As a result, we found that CCN2 bound to FGF receptor 1 with a dissociation constant of 362 nM, whereas the CT module did not. In addition, when we tested FGF signaling in chondrocytic HCS-2/8 cells stimulated by the combination of FGF2 with CT module, the level of ERK1/2, p38 MAPK, and c-Jun N-terminal kinase phosphorylation was decreased compared with that found with FGF2 alone. These findings suggest that CCN2 may regulate the proliferation and matrix degradation of chondrocytes by forming a complex with FGF2 as a novel modulator of FGF2 functions. (Endocrinology 152: 4232-4241, 2011)

DOI： 10.1210/en.2011-0234

Differential roles of CCN family proteins during osteoblast differentiation: Involvement of Smad and MAPK signaling pathways Reviewed

Harumi Kawaki, Satoshi Kubota, Akiko Suzuki, Makoto Suzuki, Kumiko Kohsaka, Kenji Hoshi, Toshiya Fujii, Noureddine Lazar, Toshihiro Ohgawara, Takeyasu Maeda, Bernard Perbal, Teruko Takano-Yamamoto, Masaharu Takigawa

BONE 49 ( 5 ) 975 - 989 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

CCN family proteins play diverse roles in many aspects of cellular processes such as proliferation, differentiation, adhesion, migration, angiogenesis and survival. In the bone tissue of vertebrate species, the expression of most CCN family members has been observed in osteoblasts. However, their spatial and temporal distributions, as well as their functions, are still only partially understood. In this study, we evaluated the localization of CCN family members in skeletal tissue in vivo and comparatively analyzed the gene expression patterns and functions of the members in murine osteoblasts in primary culture. Immunofluorescent analyses revealed that the CCN family members were differentially produced in osteoblasts and osteocytes. The presence of all Ccn transcripts was confirmed in those osteoblasts. Among the members, CCN1, CCN2, CCN4 and CCN5 were found in osteocytes. CCN4 and CCN5 were distributed in osteocytes located inside of bone matrix as well. Next, we investigated the expression pattern of Ccn family members during osteoblast differentiation. Along with differentiation, most of the members followed proper gene expression patterns: whereas, Ccn4 and Ccn5 showed quite similar patterns. Furthermore, we evaluated the effects of CCN family members on the osteoblastic activities by using recombinant CCN proteins and RNA interference method. Five members of this family displayed positive effects on osteoblast proliferation or differentiation. Of note, CCN3 drastically inhibited the osteoblast activities. Each Ccn specific siRNA could modulate osteoblast activities in a manner expected by the observed effect of respective recombinant CCN protein. In addition, we found that extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinase pathways were critically involved in the CCN family member-mediated modification of osteoblast activities.
Collectively, all Ccn family members were found to be differentially expressed along with differentiation and therefore could participate in progression of the osteoblast lineage. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2011.06.033

Novel effects of CCN3 that may direct the differentiation of chondrocytes Reviewed

Danilo Janune, Satoshi Kubota, Takashi Nishida, Harumi Kawaki, Bernard Perbal, Seiji Iida, Masaharu Takigawa

FEBS LETTERS 585 ( 19 ) 3033 - 3040 2011.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Identification and characterization of local molecules directing the differentiation of chondrocytes to either transient or permanent cartilage are major issues in cartilage biology. Here, we found CCN family protein 3 (CCN3) was abundantly produced in rat developing epiphyseal cartilage. Evaluations in vitro showed that CCN3 repressed epiphyseal chondrocyte proliferation, while promoting matrix production in multiple assays performed. Furthermore, CCN3 enhanced the articular chondrocytic phenotype; whereas it repressed the one representing endochondral ossification. Additionally, the phenotype of growth plate chondrocytes and chondrogenic progenitors also appeared to be affected by CCN3 in a similar manner. These findings suggest a significant role of CCN3 in inducing chondrocytes to articular ones during joint formation. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2011.08.024

The role of CCN2 in cartilage and bone development

Satoshi Kubota, Masaharu Takigawa

JOURNAL OF CELL COMMUNICATION AND SIGNALING 5 ( 3 ) 209 - 217 2011.8

Language：English Publisher：SPRINGER

CCN2, a classical member of the CCN family of matricellular proteins, is a key molecule that conducts cartilage development in a harmonized manner through novel molecular actions. During vertebrate development, all cartilage is primarily formed by a process of mesenchymal condensation, while CCN2 is induced to promote this process. Afterwards, cartilage develops into several sub-types with different fates and missions, in which CCN2 plays its proper roles according to the corresponding microenvironments. The history of CCN2 in cartilage and bone began with its re-discovery in the growth cartilage in long bones, which determines the skeletal size through the process of endochondral ossification. CCN2 promotes physiological developmental processes not only in the growth cartilage but also in the other types of cartilages, i.e., Meckel's cartilage representing temporary cartilage without autocalcification, articular cartilage representing hyaline cartilage with physical stiffness, and auricular cartilage representing elastic cartilage. Together with its significant role in intramembranous ossification, CCN2 is regarded as a conductor of skeletogenesis. During cartilage development, the CCN2 gene is dynamically regulated to yield stage-specific production of CCN2 proteins at both transcriptional and post-transcriptional levels. New functional aspects of known biomolecules have been uncovered during the course of investigating these regulatory systems in chondrocytes. Since CCN2 promotes integrated regeneration as well as generation (=development) of these tissues, its utility in regenerative therapy targeting chondrocytes and osteoblasts is indicated, as has already been supported by experimental evidence obtained in vivo.

DOI： 10.1007/s12079-011-0123-5

CCN3-mediated promotion of sulfated proteoglycan synthesis in rat chondrocytes from developing joint heads Reviewed

Danilo Janune, Satoshi Kubota, Noureddine Lazar, Bernard Perbal, Seiji Iida, Masaharu Takigawa

JOURNAL OF CELL COMMUNICATION AND SIGNALING 5 ( 3 ) 167 - 171 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Chondrocytes forming articular cartilage are embedded in a vast amount of extracellular matrix having physical stiffness and elasticity, properties that support the mechanical load from bones and enable the flexible movement of synovial joints. Unlike chondrocytes that conduct the growth of long bones by forming the growth plate, articular chondrocytes show suppressed cell proliferation, unless these cells are exposed to pathological conditions such as mechanical overload. In the present study, we found that one of the members of the CCN family, CCN3, was significantly expressed in chondrocytes isolated from the epiphyseal head in developing rat synovial joints. Evaluation of the effect of recombinant CCN3 on those chondrocytes revealed that CCN3 promoted proteoglycan synthesis, whereas this factor repressed the proliferation of the same cells. These results suggest a critical role for CCN3 in the regulation of the biological properties of articular chondrocytes.

DOI： 10.1007/s12079-011-0135-1

Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice Reviewed

Takahiro Kodama, Tetsuo Takehara, Hayato Hikita, Satoshi Shimizu, Minoru Shigekawa, Hinako Tsunematsu, Wei Li, Takuya Miyagi, Atsushi Hosui, Tomohide Tatsumi, Hisashi Ishida, Tatsuya Kanto, Naoki Hiramatsu, Satoshi Kubota, Masaharu Takigawa, Yoshito Tomimaru, Akira Tomokuni, Hiroaki Nagano, Yuichiro Doki, Masaki Mori, Norio Hayashi

JOURNAL OF CLINICAL INVESTIGATION 121 ( 8 ) 3343 - 3356 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC CLINICAL INVESTIGATION INC

The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mchm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

DOI： 10.1172/JCI44957

Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells Reviewed

Yoshiaki Miyake, Takayuki Furumatsu, Satoshi Kubota, Kazumi Kawata, Toshifumi Ozaki, Masaharu Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 409 ( 2 ) 247 - 252 2011.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates alpha 1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.04.138

CCN Family 2/Connective Tissue Growth Factor (CCN2/CTGF) Promotes Osteoclastogenesis via Induction of and Interaction with Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Reviewed

Takashi Nishida, Kenji Emura, Satoshi Kubota, Karen M. Lyons, Masaharu Takigawa

JOURNAL OF BONE AND MINERAL RESEARCH 26 ( 2 ) 351 - 363 2011.2

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF-kappa B ligand (GST-RANKL), and a combination of recombinant CCN2 (rCCN2) and GST-RANKL significantly enhanced tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cell formation compared with GST-RANKL alone. Therefore, we suspected the involvement of CCN2 in cell-cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real-time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid-phase binding assay of CCN2 and dendritic cell-specific transmembrane protein (DC-STAMP), which is involved in cell-cell fusion. The results showed that CCN2 induced and interacted with DC-STAMP. Furthermore, GST-RANKL-induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC-STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC-STAMP. (C) 2011 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.222

Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA Reviewed

Seiji Kondo, Satoshi Kubota, Yoshiki Mukudai, Takashi Nishida, Yasuto Yoshihama, Tatsuo Shirota, Satoru Shintani, Masaharu Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 405 ( 3 ) 382 - 387 2011.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

CCN2/connective tissue growth factor (CTGF) can be induced by hypoxia and promotes tumor angiogenesis. Our previous studies revealed that hypoxia-induced gene expression of human ccn2 mRNA is regulated post-transcriptionally in human chondrosarcoma-derived cell line, HCS-2/8, in which a minimal cis-element, entitled CAESAR, in the 3'-untranslated region (UTR) of ccn2 mRNA and a 35-kDa protein counterpart play an important role by determining the stability of ccn2 mRNA. In the present study, we identified this corresponding protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by utilizing RNA affinity chromatography combined with mass spectrometry. The results of an RNA binding assay revealed the specific binding of GAPDH to this cis-element. To further characterize the interaction between GAPDH and ccn2 mRNA, we examined the roles of redox conditions and glycolytic coenzyme in the binding of GAPDH to the ccn2 mRNA. An oxidizing agent, diamide, abolished the GAPDH-RNA interaction in a concentration-dependent manner; whereas this effect could be reversed by subsequent treatment with 2-mercaptoethanol (2-ME). In addition, nicotinamide-adenine dinucleotide (NAD), a coenzyme of GAPDH, inhibited the GAPDH-RNA binding. Taken together, these findings suggest that the glycolytic enzyme GAPDH regulates the gene expression of ccn2 mRNA in trans by acting as a sensor of oxidative stress and redox signals, leading to CCN2 overexpression under the condition of hypoxia and promotion of angiogenesis. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.01.034

A Coding RNA Segment That Enhances the Ribosomal Recruitment of Chicken ccn1 mRNA Reviewed

Yoshiki Mukudai, Satoshi Kubota, Takanori Eguchi, Kumi Sumiyoshi, Danilo Janune, Seiji Kondo, Satoru Shintani, Masaharu Takigawa

JOURNAL OF CELLULAR BIOCHEMISTRY 111 ( 6 ) 1607 - 1618 2010.12

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

CCN1, a member of the CCN family of proteins, plays important physiological or pathological roles in a variety of tissues. In the present study, we initially found a highly guanine-cytosine (GC)-rich region of approximately 200 bp near the 5'-end of the open reading frame, which was always truncated by amplification of the corresponding cDNA region through the conventional polymerase chain reaction. An RNA in vitro folding assay and selective ribonuclease digestion of the corresponding segment of the ccn1 mRNA confirmed the involvement of a stable secondary structure. Subsequent RNA electromobility-shift assays demonstrated the specific binding of some cytoplasmic factor(s) in chicken embryo fibroblasts to the RNA segment. Moreover, the corresponding cDNA fragment strongly enhanced the expression of the reporter gene in cis at the 5'-end, but did not do so at the 3'-end. According to the results of a ribosomal assembly test, the effect of the mRNA segment can predominantly be ascribed to the enhancement of transport and/or entry of the mRNA into the ribosome. Finally, the minimal GC-rich mRNA segment that was predicted and demonstrated to form a secondary structure was confirmed to be a functional regulatory element. Thus, we here uncover a novel dual-functionality of the mRNA segment in the eat ! open reading frame, which segment acts as a cis-element that mediates posttranscriptional gene regulation, while retaining the information for the amino acid sequence of the resultant protein. J. Cell. Biochem. 111: 1607-1618, 2010. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jcb.22894

Design and utility of CCN2 anchor peptide aptamers

Harumi Kawaki, Satoshi Kubota, Eriko Aoyama, Naoya Fujita, Hiroshi Hanagata, Akira Miyauchi, Kenta Nakai, Masaharu Takigawa

BIOCHIMIE 92 ( 8 ) 1010 - 1015 2010.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) consists of 4 conserved modules that are highly interactive with a number of biomolecules. With such interaction, CCN2 exerts multiple functions by forming an extracellular information network. In the present study, we screened for dodecapeptide sequences that bound to each module of human CCN2 by using a bacteriophage display library. Thereafter, consensus amino acid sequences for the binding to individual modules were extracted in silico and utilized to design anchor peptide aptamers that would facilitate the interaction between CCN2 and other molecules. Direct binding of a few peptides to CCN2 was confirmed by surface plasmon resonance analysis. Subsequent biological assay indicated that one such peptide was capable of promoting the proliferation of CCN2-producing chondrocytic cells. This cell biological activity was found to be sequence specific and CCN2 dependent. Since CCN2/CTGF was shown to be effective in articular cartilage/bone regeneration in vivo, utility of such peptide aptamers in CCN2-associated regenerative therapeutics is suggested herein. (C) 2010 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2010.04.021

Thrombopoietic-mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2

Kumi Sumiyoshi, Satoshi Kubota, Rika A. Furuta, Kazuta Yasui, Eriko Aoyama, Harumi Kawaki, Kazumi Kawata, Toshihiro Ohgawara, Takashi Yamashiro, Masaharu Takigawa

JOURNAL OF CELL COMMUNICATION AND SIGNALING 4 ( 1 ) 5 - 14 2010.3

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.

DOI： 10.1007/s12079-009-0067-1

Proinsulin C-peptide Regulates Ribosomal RNA Expression Reviewed

Emma Lindahl, Ulrika Nyman, Farasat Zaman, Carina Palmberg, Anna Cascante, Jawed Shafqat, Masaharu Takigawa, Lars Savendahl, Hans Jorvall, Bertrand Joseph

JOURNAL OF BIOLOGICAL CHEMISTRY 285 ( 5 ) 3462 - 3469 2010.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Proinsulin C-peptide is internalized into cells, but a function of its intracellular localization has not been established. We now demonstrate that, upon cellular entry, C-peptide is localized to the nucleoli, where it promotes transcription of genes encoding for ribosomal RNA. We find that C-peptide binds to histones and enhances acetylation of lysine residue 16 of histone H4 at the promoter region of genes for ribosomal RNA. In agreement with synchrony of ribosomal RNA synthesis and cell proliferation, we show that C-peptide stimulates proliferation in chondrocytes and HEK-293 cells. This regulation of ribosomal RNA provides a mechanism by which C-peptide can exert transcriptional effects and implies that the peptide has growth factor activity.

DOI： 10.1074/jbc.M109.053587

Role of the Low-Density Lipoprotein Receptor-Related Protein-1 in Regulation of Chondrocyte Differentiation Reviewed

Kazumi Kawata, Satoshi Kubota, Takanori Eguchi, Norifumi H. Moritani, Tsuyoshi Shimo, Seiji Kondo, Takashi Nishida, Shogo Minagi, Masaharu Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 222 ( 1 ) 138 - 148 2010.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

The low-density lipoprotein receptor-related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown Irpl in chondrocytic cells and obtained findings indicating a critical role therein. As a result of IrpI knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axing, which is known to be induced by activation of the WNT/beta-catenin (beta cat) signaling pathway. Thereby, we found that Axing promoter activity was enhanced in the Irpl knockdown cells. Furthermore, when the WNT/beta-catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3 beta, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) zeta was also decreased in the Irp1 knockdown cells. When the phosphorylation of PKC zeta was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1. J. Cell. Physiol. 222: 138-148, 2010. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jcp.21930

Nicotine-induced CCN2: from Smoking to Periodontal Fibrosis Reviewed

H. Takeuchi, S. Kubota, E. Murakashi, Y. Zhou, K. Endo, P. S. Ng, M. Takigawa, Y. Numabe

JOURNAL OF DENTAL RESEARCH 89 ( 1 ) 34 - 39 2010.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SAGE PUBLICATIONS INC

Since fibrosis is observed in smokers' gingiva, it was hypothesized that fibrosis was caused by nicotine in the periodontium. Therefore, in this study, we investigated the effects of nicotine on the induction of a profibrotic molecule, connective tissue growth factor (CCN2/CTGF), in human gingival fibroblasts (HGFs) and periodontal ligament (PDL) cells. With 1 mu g/mL nicotine, vacuolization and attenuated proliferation were observed. Interestingly, 1 mu g/mL nicotine increased the production of CCN2/CTGF protein in both cells without increasing mRNA expression. Furthermore, type I collagen mRNA and protein were also increased and were significantly blocked by a CCN2/CTGF neutralizing antibody. This is the first report to describe a relationship between nicotine and CCN2/CTGF in periodontal tissue cells. Analysis of our data also indicated that nicotine was cytotoxic, while it increased CCN2/CTGF and, eventually, type I collagen production. These findings suggest that periodontal fibrosis can be promoted by nicotine from smoking via effects on CCN2/CTGF.

DOI： 10.1177/0022034509353403

Nucleophosmin/B23: A Multifunctional Regulator that Determines the Fate of CCN2 mRNA Reviewed

Satoshi Kubota, Yoshiki Mukudai, Harumi Kawaki, Seiji Kondo, Takanori Eguchi, Kumi Sumiyoshi, Toshihiro Ohgawara, Tsuyoshi Shimo, Masaharu Takigawa

CCN PROTEINS IN HEALTH AND DISEASE: AN OVERVIEW OF THE FIFTH INTERNATIONAL WORKSHOP ON THE CCN FAMILY OF GENES 41 - + 2010

Language：English Publishing type：Research paper (international conference proceedings) Publisher：SPRINGER-VERLAG BERLIN

CCN2/CTGF is a multifunctional molecule that has been shown to play a central role in chondrocyte differentiation. During this process, the expression of ccn2 is tightly regulated to confer a maximal level at prehypertrophic - hypertrophic stages, in which the 3'-untranslated region (UTR) of the mRNA is critically involved in mediating its post-transcriptional regulation. In our previous studies, we found that a 40-kDa protein binding specifically to an RNA cis-element, 3'-100/50, in the 3'-UTR of the chicken ccn2 mRNA regulated the intracellular stability of the mRNA. The interaction of this 40-kDa protein with 3'-100/50 was enhanced in proliferating chondrocytes, in which ccn2 mRNA is rapidly degraded; whereas a prolonged half life of ccn2 mRNA is observed in hypertrophic chondrocytes, where the interaction of the 40 kDa-protein and 3'-100/50 is diminished. Collectively, the data suggested that this 40-kDa protein acts as a ccn2-specific mRNA destabilizer during chondrocyte differentiation.
In this present study we finally identified this 40-kDa protein as nucleophosmin (NPM)/B23. NPM is a nuclear-cytoplasmic shuttling protein that is characterized by its multiple functionality. This protein is known to be a histone chaperone, a regulator of ribosomal RNA transcription, as well as an RNA-binding post-transcriptional regulator of gene expression. In our hands, direct binding of NPM to 3'-100/50 was confirmed not only by RNA EMSA and UV crosslinking assays, but also by RNA immunoprecipitation analysis. By using recombinant chicken NPM, we could successfully reconstitute the post-transcriptional regulation of ccn2 by NPM in vitro and found that this regulation was more robust in chondrocytes than in fibroblasts. Furthermore, siRNA-mediated gene silencing of NPM in vivo clearly showed enhanced ccn2 gene expression and a prolonged half life of the ccn2 mRNA, confirming the functional property of NPM as a specific destabilizer of the ccn2 mRNA in living cells.
The 5'-100/50 element, a target of NPM, is evolutionally conserved among vertebrate species. Therefore, we consider NPM to be a critical post-transcriptional regulator of ccn2 acting via 3'-UTR during endochondral ossification and possibly, in other physiological and pathological states as well.

DOI： 10.1007/978-90-481-3779-4_4

Other Link： http://orcid.org/0000-0002-9600-4430

Cooperative Regulation of Cell Proliferation and Differentiation by CCN2 and CCN3 Reviewed

Masaharu Takigawa, Harumi Kawaki, Satoshi Kubota, Karen M. Lyons, Bernard Perbal

CCN PROTEINS IN HEALTH AND DISEASE: AN OVERVIEW OF THE FIFTH INTERNATIONAL WORKSHOP ON THE CCN FAMILY OF GENES 105 - + 2010

Language：English Publishing type：Research paper (international conference proceedings) Publisher：SPRINGER-VERLAG BERLIN

In this chapter, we introduce a new trend in the field of CCN proteins research, that is, the yin/yang effects of CCN2 and CCN3 and the mutual regulation of ccn2 and ccn3 gene expression by these two proteins. These findings point out the need for a more thorough investigation of functional interactions between CCN proteins in normal and pathological conditions

DOI： 10.1007/978-90-481-3779-4_8

結合組織成長因子(CTGF/CCN2)と軟骨形成 Invited

久保田聡, 滝川正春

骨粗鬆症治療 9 ( 4 ) 287 - 290 2010

Identification of miR-1 as a micro RNA that supports late-stage differentiation of growth cartilage cells Reviewed

Sumiyoshi K, Kubota S, Ohgawara T, Kawata K, Nishida T, Shimo T, Yamashiro T, Takigawa M

Biochemical and Biophysical Research Communications 402 ( 2 ) 286 - 290 2010

Publishing type：Research paper (scientific journal)

The process of endochondral ossification is strictly regulated by a variety of extracellular and intracellular factors. Recently, it has become recognized that specific miRNAs are involved in this process by regulating the expression of the relevant genes at the post-transcriptional level. In this present study we obtained the first evidence of the involvement of a specific micro RNA (miRNA) in the regulation of the chondrocyte phenotype during late stages of differentiation. By use of the microarray technique, miR-1 was identified as this miRNA, the expression of which was most repressed upon hypertrophic differentiation. Transfection of human chondrocytic HCS-2/8 cells and chicken normal chondrocytes with miR-1 led to repressed expression of aggrecan, the major cartilaginous proteoglycan gene. Therefore, miR-1 was found to be involved in the regulation of the chondrocytic phenotype and thus to play an important role in chondrocytes during the late stage of the differentiation process, maintaining the integrity of the cartilage tissue. © 2010 Elsevier Inc.

DOI： 10.1016/j.bbrc.2010.10.016

Other Link： http://orcid.org/0000-0002-4419-9643

Novel Transcriptional Regulation of CCN2/CTGF by Nuclear Translocation of MMP3 Reviewed

Takanori Eguchi, Satoshi Kubota, Kazumi Kawata, Yoshiki Mukudai, Junji Uehara, Toshihiro Ohgawara, Soichiro Ibaragi, Akira Sasaki, Takuo Kuboki, Masaharu Takigawa

CCN PROTEINS IN HEALTH AND DISEASE: AN OVERVIEW OF THE FIFTH INTERNATIONAL WORKSHOP ON THE CCN FAMILY OF GENES 255 - + 2010

Language：English Publishing type：Research paper (international conference proceedings) Publisher：SPRINGER-VERLAG BERLIN

CCN2/CTGF, previously known as Connective Tissue Growth Factor, is a crucial regulator of extra-cellular matrix (ECM), which promotes ECM synthesis and stabilization. As their family name clearly implies, matrix metalloproteases (MMPs) are also localized in the ECM, where they function as proteases, modulating cell signaling by cleaving proteins such as matrix proteins, growth factors and growth factor receptors. Strong expression of CCN2/CTGF in chondrocytic cells occurs through transcription enhancer dominant in chondrocytes (TRENDIC). Matrix metalloprotease-3 (MMP3) is a novel TRENDIC-binding transcription factor for CCN2/CTGF expression. First, MMP3 cDNA was cloned as a TRENDIC-binding factor by Southwestern screening. The interaction between MMP3 and TRENDIC was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by transfected MMP3, whereas a TRENDIC mutant for the promoter lost the response. In addition, the knockdown of MMP3 suppressed CCN2/CTGF expression. Cytochemical and histochemical analyses demonstrated that MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 was observed in 30 min after the addition, and six putative nuclear localization signals were found in MMP3. These results indicated a novel trans-activation mechanism of CCN2/CTGF by the nuclear translocation of MMP3 through binding with TRENDIC in chondrocytes. Although MMPs historically had been recognized as a protease for extra-cellular proteins, this study indicated that it also stimulates ECM synthesis through CCN2/CTGF trans-activation. This novel regulatory role of the ECM may contribute to understanding the mechanism of not only the development, but also the pathogenesis of arthritis fibrosis and periodontitis.

DOI： 10.1007/978-90-481-3779-4_19

Other Link： http://orcid.org/0000-0002-9600-4430

CCN proteins in health and disease: An overview of the fifth international workshop on the CCN family of genes

Annick Perbal, Masaharu Takigawa, Bernard Perbal

CCN Proteins in Health and Disease: An Overview of the Fifth International Workshop on the CCN Family of Genes 1 - 338 2010

Language：English Publisher：Springer Netherlands

The CCN family of genes currently comprises six secreted proteins (designated CCN16 i.e., Cyr61/CCN1
ctgf/CCN2
Nov/CCN3
WISP1/CCN4
WISP2/CCN5, and WISP3/CCN6) showing a strikingly conserved primary structure, with four modules sharing partial identity with IGF binding proteins, Von Willebrand protein, thrombospondin and several matricellular proteins and growth factors. The current view is that CCN proteins modulate signaling pathways that involve regulatory components of the extracellular matrix. As such, they likely act as a central hub in the regulation of mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. The 5th international workshop on the CCN family of genes, that was held in Toronto in 2008 brought together scientists from around the world who have an interest in the biological roles of this emerging family of proteins. On an educational point of view, the workshop was a unique place for an efficient diffusion of scientific information. The present book comprises a series of selected manuscripts that are based on the original communications that were presented at the meeting by worldwide leaders in the field of CCN biology. All major aspects of CCN proteins biology in both normal and pathological conditions are covered in this volume, from structure-functions analysis up to the involvement of CCN proteins in complex physiological functions. In addition to reports that support the Yin-Yang concept of CCN proteins driving opposite effects on the same biological process, this book also comprises several contributions that point to CCN proteins as amenable targets for therapeutic manipulation of disease processes. Together with the special issue of Journal of Cell Communication and Signaling in which authors have extended on the original data presented at the meeting, the present Proceedings provide an instant picture and unique update of the state of the art in the CCN field. © Springer Science+Business Media B.V. 2010.

DOI： 10.1007/978-90-481-3779-4

低密度リポタンパク受容体関連タンパク-1(LRP1)の軟骨細胞分化における多面的作用機構

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 森谷徳文, 近藤誠二, 西田崇, 皆木省吾, 滝川正春

日本生化学会大会プログラム・講演要旨集 82回 4T4p - 9 2009.9

All-trans retinoic acid-induced ADAM28 degrades proteoglycans in human chondrocytes Reviewed

Yuichi Hikichi, Koji Yoshimura, Masaharu Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 386 ( 2 ) 294 - 299 2009.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

In order to elucidate the mechanism of cartilage degradation in osteoarthritis (OA), we established a cell assay system. Under the stimulation of all-trans retinoic acid (ATRA), the human chondrosarcoma, cell line HCS-2/8 increased proteoglycan release from inactivated bovine nasal cartilage (BNC) and the results suggested the involvement of membrane-bound metalloproteinase(s). Therefore, we focused on the induction of a disintegrin and metalloproteinase (ADAM) superfamily upon ATRA stimulation. Of all ADAMs tested, only ADAM28 was induced by ATRA in HCS-2/8 cells and also in human primary chondrocytes. We found that transfection of ADAM28 or its alternatively spliced soluble form augmented proteoglycan release in the cell assay; however, a mutant soluble form in which a portion of the disintegrin domain was deleted did not have proteoglycan-releasing activity, implying the importance of the domain for enzyme localization and substrate recognition for cartilage degradation in OA. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.06.052

N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan Reviewed

Eriko Aoyama, Takako Hattori, Mitsuhiro Hoshijima, Daisuke Araki, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa

BIOCHEMICAL JOURNAL 420 413 - 420 2009.6

Language：English Publishing type：Research paper (scientific journal) Publisher：PORTLAND PRESS LTD

CCN2/CTGF (CCN family 2/connective tissue growth factor) is a multi-cellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production, and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF-interactive proteins capable of modulating its action in cartilage, we carried Out a yeast two-hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. In the present paper, we report the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, its a CCN2/CTGF-binding protein. Among the four domains of CCN2/CTGF, the IGFBP [IGF (insulin-like growth factor)-binding protein-like] and/or VWC (von Willebrand factor type C) domains had a direct interaction with aggrecan in a yeast two-hybrid assay. The results of a solid-phase-binding assay using aggrecan-coated plates also showed binding to recombinant CCN2/CTGF in a dose-dependent manner. rIGFBP (recombinant IGFBP) and rVWC (recombinant VWC) module peptides had stronger binding to aggrecan compared with rTSP1 (recombinant thrombospondin type I repeat) and rCT (recombinant C-terminal cystine knot) module peptides. SPR (surface plasmon resonance) analysis showed the direct interaction between the CCN2/CTGF and aggrecan. and ectopically overexpressed CCN2/CTGF and AgG3 (G3 domain of aggrecan) confirmed their binding in vivo. Indirect immunofluorescence analysis indicated that CCN2/CTGF was extracellularly co-localized with aggrecan on HCS-2/8 cells. The rIGFBP-rVWC peptide effectively enhanced tire production and release of aggrecan compared with the rTSP-rCT peptide in chondrocytes. These results indicate that CCN2/CTGF binds to aggrecan through its N-terminal IGFBP and VWC Modules. and this binding may be related to the CCN2/CTGF-enhanced production and secretion of aggrecan by chondrocytes.

DOI： 10.1042/BJ20081991

CTGF and Apoptosis in Mouse Osteocytes Induced by Tooth Movement

Y. Sakai, T. A. Balam, S. Kuroda, N. Tamamura, T. Fukunaga, M. Takigawa, T. Takano-Yamamoto

JOURNAL OF DENTAL RESEARCH 88 ( 4 ) 345 - 350 2009.4

Language：English Publishing type：Research paper (scientific journal) Publisher：SAGE PUBLICATIONS INC

It is known that experimental tooth movement stimulates the gene expression of connective tissue growth factor (CTGF) and induces apoptosis in osteocytes in rats. We hypothesized that there is a relationship between CTGF expression and the induction of apoptosis in osteocytes, to play a significant role in triggering bone remodeling during experimental tooth movement. In this study, CTGF mRNA expression was detected at 2 hours in osteocytes on the pressure side, followed by apoptosis at 6 hours after tooth movement in mice. The number of empty lacunae significantly increased on day 1 after mechanical stimulation. Thereafter, the number of osteoclasts significantly increased on the pressure side of the alveolar bone on day 3. Tooth movement increased rapidly on day 10. These findings suggest that CTGF expression, followed by apoptosis in osteocytes in response to mechanical stimulation, might play a significant role in triggering bone remodeling during tooth movement.

DOI： 10.1177/0022034509334649

Effect of transforming growth factor-beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cells Reviewed

H. Takeuchi, S. Kubota, E. Murakashi, T. Fukada, S. Hashimoto, M. Takigawa, Y. Numabe

JOURNAL OF PERIODONTAL RESEARCH 44 ( 2 ) 161 - 169 2009.4

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL PUBLISHING, INC

Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro.
Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription-polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy.
In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization.
The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue.

DOI： 10.1111/j.1600-0765.2008.01093.x

Regulation of chondrocytic phenotype by micro RNA 18a: Involvement of Ccn2/Ctgf as a major target gene

Toshihiro Ohgawara, Satoshi Kubota, Harumi Kawaki, Seiji Kondo, Takanori Eguchi, Naito Kurio, Eriko Aoyama, Akira Sasaki, Masaharu Takigawa

FEBS LETTERS 583 ( 6 ) 1006 - 1010 2009.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

We searched for miRNAs that were down-regulated in chondrocytic cells and predicted to target CCN2/connective tissue growth factor (CCN2/CTGF) that promotes endochondral ossification. Among them, expression of miR-18a was most strongly repressed in chondrocytic cells. Reporter gene analysis confirmed the functionality of an miR-18a target in the 3'-untranslated region of Ccn2 mRNA, which was predicted in silico. Indeed, introduction of miR-18a efficiently repressed the CCN2 production from chondrocytic cells. Finally, transfected miR-18a significantly repressed the mature chondrocytic phenotype. Our present study revealed a regulatory role for miR-18a in chondrocytic differentiation through CCN2. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2009.02.025

CCN Family 2/Connective Tissue Growth Factor Modulates BMP Signalling as a Signal Conductor, Which Action Regulates the Proliferation and Differentiation of Chondrocytes

Azusa Maeda, Takashi Nishida, Eriko Aoyama, Satoshi Kubota, Karen M. Lyons, Takuo Kuboki, Masaharu Takigawa

JOURNAL OF BIOCHEMISTRY 145 ( 2 ) 207 - 216 2009.2

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Both CCN family 2/connective tissue growth factor (CCN2/CTGF) and bone morphogenetic protein (BMP)-2 play an important role in cartilage metabolism. We evaluated whether or not CCN2 would interact with BMP-2, and examined the combination effect of CCN2 with BMP-2 (CCN2-BMP-2) on the proliferation and differentiation of chondrocytes. Immunoprecipitation-western blotting analysis, solid-phase binding assay and surface plasmon resonance (SPR) spectroscopy showed that CCN2 directly interacted with BMP-2 with a dissociation constant of 0.77 nM as evaluated by SPR. An in vivo study revealed that CCN2 was co-localized with BMP-2 at the pre-hypertrophic region in the E18.5 mouse growth plate. Interestingly, CCN2-BMP-2 did not affect the BMP-2/CCN2-induced phosphorylation of p38 MAPK but caused less phosphorylation of ERK1/2 in cultured chondrocytes. Consistent with these results, cell proliferation assay showed that CCN2-BMP-2 stimulated cell growth to a lesser degree than by either CCN2 or BMP-2 alone, whereas the expression of chondrocyte marker genes and proteoglycan synthesis, representing the mature chondrocytic phenotype, was increased collaboratively by CCN2-BMP-2 treatment in cultured chondrocytes. These findings suggest that CCN2 may regulate the proliferating and differentiation of chondrocytes by forming a complex with BMP-2 as a novel modulator of BMP signalling.

DOI： 10.1093/jb/mvn159

CCN family 2/connective tissue growth factor (CCN2/CFGF) regulates the expression of Vegf through Hif-1 alpha expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition

Takashi Nishida, Seiji Kondo, Azusa Maeda, Satoshi Kubota, Karen M. Lyons, Masaharu Takigawa

BONE 44 ( 1 ) 24 - 31 2009.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Vascular endothelial growth factor (VEGF) is essential for establishing vascularization and regulating chondrocyte development and survival. We have demonstrated that VEGF regulates the expression of CCN2/connective tissue growth factor (CCN2/CTGF) an essential mediator of cartilage development and angiogenesis, suggesting that CCN2 functions in down-stream of VEGF, and that VEGF function is mediated in part by CCN2. On the other hand, the phenotype of Ccn2 mutant growth plates, which exhibit decreased expression of VEGF in the hypertrophic zone, indicates that Vegf expression is dependent on Ccn2 expression as well. Therefore, we investigated the molecular mechanisms underlying the induction of VEGF by CCN2 using a human chondrocytic cell line, HCS-2/8. Hypoxic stimulation (5% O(2)) of HCS-2/8 cells increased VEGF mRNA levels by similar to 8 fold within 6 h as compared with the cells cultured under normoxia. In addition, VEGF expression was further up-regulated under hypoxia in HCS-2/8 cells transfected with a Ccn2 expression plasmid. Hypoxia-inducible factor (HIF)-1 alpha mRNA and protein levels were increased by stimulation with recombinant CCN2 (rCCN2). Furthermore, the activity of a VEGF promoter that contained a HIF-1 binding site was increased in HCS-2/8, when the cells were stimulated by rCCN2. These results suggest that CCN2 regulates the expression of VEGF at a transcriptional level by promoting HIF-1 alpha activity. In fact, HIF-1 alpha was detected in the nuclei of proliferative and pre-hypertrophic chondrocytes of wild-type mice, whereas it was not detected in Ccn2 Mutant chondrocytes in vivo. This activation cascade from CCN2 to VEGF may therefore play a critical role in chondrocyte development and survival. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2008.08.125

Cooperative Regulation of Chondrocyte Differentiation by CCN2 and CCN3 Shown by a Comprehensive Analysis of the CCN Family Proteins in Cartilage

Harumi Kawaki, Satoshi Kubota, Akiko Suzuki, Noureddine Lazar, Tomohiro Yamada, Tatsushi Matsumura, Toshihiro Ohgawara, Takeyasu Maeda, Bernard Perbal, Karen M. Lyons, Masaharu Takigawa

JOURNAL OF BONE AND MINERAL RESEARCH 23 ( 11 ) 1751 - 1764 2008.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members. and its null mice display skeletal dysmorphisms. However, little is known concerning roles of the other CCN members in chondrocytes. Using both in vivo and in vitro approaches, We conducted a comparative analysis of CCN2-null and wildtype mice to study the roles of CCN2 and the other CCN proteins in cartilage development. Immunohistochemistry was used to evaluate the localization of CCN proteins and other chondrocyte-associated molecules in the two types of mice. Moreover, gene expression levels and the effects of exogenous CCN proteins oil chondrocyte proliferation, differentiation, and the expression of chondrocyte-associated genes in their primary chondrocytes were evaluated. Ccn3 was dramatically upregulated in CCN2-null cartilage and chondrocytes. This upregulation was associated with diminished cell proliferation and delayed differentiation. Consistent with the in vivo findings, CCN2 deletion entirely retarded chondrocyte terminal differentiation and decreased the expression of several chondrocyte-associated genes ill vitro. whereas CcO expression drastically increased. In contrast, the addition Of exogenous CCN2 promoted differentiation strongly and induced the expression of the associated genes. whereas decreasing, the CcO expression. These findings collectively indicate that CCN2 induces chondrocyte differentiation by regulating the expression of chondrocyte-associated genes but that these effects are counteracted by CCN3. The lack of CCN2 caused upregulation of CCN3 in CCN2-null mice, which resulted in the observed phenotypes, Such as the resultant delay of terminal differentiation. The involvement of the PTHrP-Ihh loop in the regulation of CCN3 expression is also suggested.

DOI： 10.1359/JBMR.080615

低密度リポタンパク受容体関連タンパク-1(LRP1)の軟骨細胞分化における機能とその作用機構

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 皆木省吾, 滝川正春

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 81回・31回 4T13 - 2 2008.11

Posttranscriptional regulation of chicken ccn2 gene expression by nucleophosmin/B23 during chondrocyte differentiation

Yoshiki Mukudai, Satoshi Kubota, Harumi Kawaki, Seiji Kondo, Takanori Eguchi, Kumi Sumiyoshi, Toshihiro Ohgawara, Tsuyoshi Shimo, Masaharu Takigawa

MOLECULAR AND CELLULAR BIOLOGY 28 ( 19 ) 6134 - 6147 2008.10

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

CCN2/CTGF is a multifunctional factor that plays a crucial role in the growth and differentiation of chondrocytes. The chicken ccn2 gene is regulated not only at the transcriptional level but also by the interaction between a posttranscriptional element in the 3' untranslated region (3'-UTR) and a cofactor. In the present study, we identified a nucleophosmin (NPM) (also called B23) as this cofactor. Binding of NPM to the element was confirmed, and subsequent analysis revealed a significant correlation between the decrease in cytosolic NPM and the increased stability of the ccn2 mRNA during chondrocyte differentiation in vivo. Furthermore, recombinant chicken NPM enhanced the degradation of chimeric RNAs containing the posttranscriptional cis elements in a chicken embryonic fibroblast extract in vitro. It is noteworthy that the RNA destabilization effect by NPM was far more prominent in the cytosolic extract of chondrocytes than in that of fibroblasts, representing a chondrocyte-specific action of NPM. Stimulation by growth factors to promote differentiation changed the subcellular distribution of NPM in chondrocytes, which followed the expected patterns from the resultant change in the ccn2 mRNA stability. Therefore, the present study reveals a novel aspect of NPM as a key player in the posttranscriptional regulation of ccn2 mRNA during the differentiation of chondrocytes.

DOI： 10.1128/MCB.00495-08

軟骨細胞分化における低密度リポタンパク受容体関連タンパク-1(LRP1)の関与

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 皆木省吾, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 26回 234 - 234 2008.10

Distribution, gene expression, and functional role of EphA4 during ossification

Chisa Kuroda, Satoshi Kubota, Kazumi Kawata, Eriko Aoyama, Kumi Sumiyoshi, Morihiko Oka, Miho Inoue, Shogo Minagi, Masaharu Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 374 ( 1 ) 22 - 27 2008.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

EphA4 receptor tyrosine kinase has been shown to be critically involved in neural tissue development. Here, we found EphA4 was also distributed among hypertrophic chondrocytes and osteoblasts in the growth plate of developing mouse long bones. In vitro evaluation revealed that ephA4 expression was elevated Upon hypertrophic differentiation of chondrocytes and that markedly stronger expression was observed in osteoblastic SaOS-2 than chondrocytic HCS-2/8 cells. Of note, RNAi-mediated silencing of ephA4 in SaOS-2 cells resulted in the repression of osteocalcin gene expression and alkaline phosphatase activity. Interestingly, confocal laser-scanning Microscopic analysis revealed the presence of EphA4 molecules in the nucleus as well as on the surface of SaOS-2 cells. These findings are the first indication of a critical role of EphA4 in ossification, especially at the final stage in which osteoblasts and hypertrophic chondrocytes play major roles. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.06.089

Increased expression of matrilin-3 not only in osteoarthritic articular cartilage but also in cartilage-forming tumors, and down-regulation of SOX9 via epidermal growth factor domain 1-dependent signaling

Jean-Baptiste Vincourt, Jean-Michel Vignaud, Frederic Lionneton, Francois Sirveaux, Harumi Kawaki, Sophie Marchal, Sandra Lomazzi, Francois Plenat, Francois Guillemin, Patrick Netter, Masaharu Takigawa, Didier Mainard, Jacques Magdalou

ARTHRITIS AND RHEUMATISM 58 ( 9 ) 2798 - 2808 2008.9

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Objective. To identify regulators of the cartilaginous phenotype, on the basis of their differential expression in human conventional chondrogenic tumors compared with articular cartilage.
Methods. Differential proteomics analysis revealed matrilin-3 (MATN3) as a candidate regulator of the cartilaginous phenotype. Its capacity to modulate gene expression was investigated in human HCS-2/8 chondrosarcoma cells and transfected chondrocytes, using cell culture fractionation, reverse transcription-polymerase chain reaction, and Western blot analyses.
Results. Increased expression of the cartilage-specific matrix protein MATN3 was specifically observed in enchondromas and conventional chondrosarcomas. A substantial fraction of MATN3 was found in cytoplasmic structures of tumor cells, as demonstrated by immunohistochemistry. Analyses of intracellular MATN3 revealed that it corresponded to an imperfectly maturated MATN3 polypeptide, both in HCS-2/8 human chondrosarcoma cells and in transfected human chondrocytes. Moderately increased expression of MATN3 resulted in its intracellular retention. Antibody-mediated blockade of soluble, extracellular MATN3 in HCS-2/8 cell cultures resulted in increased expression of MATN3 and the chondrogenic transcription factor SOX9. Conversely, increased ectopic expression of MATN3 resulted in decreased expression of MATN3 and SOX9 in primary chondrocytes, while a mutant MATN3 lacking its first epidermal growth factor (EGF)-like domain failed to down-regulate SOX9.
Conclusion. Aberrant expression and processing of MATN3 are hallmarks of conventional cartilaginous neoplasms. A particular step in the maturation of MATN3 limits its processing through the secretion machinery, resulting in its intracellular accumulation upon increased expression. Soluble, secreted MATN3, however, down-regulates SOX9 at the messenger RNA and protein levels. The first EGF-like domain of MATN3 is a critical determinant of its regulatory activity toward SOX9. These activities of MATN3 suggest that its increased expression in osteoarthritis might contribute to the degeneration of articular cartilage.

DOI： 10.1002/art.23761

Induction of hepatocyte growth factor expression by maleic acid in human fibroblasts through MAPK activation

Takahiro Motoki, Yoshihiro Sugiura, Yohsuke Matsumoto, Tomoe Tsuji, Satoshi Kubota, Masaharu Takigawa, Eiichi Gohda

JOURNAL OF CELLULAR BIOCHEMISTRY 104 ( 4 ) 1465 - 1476 2008.7

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Carboxylic acids have various biological activities and play critical roles in cellular metabolic pathways such as the tricarboxylic acid (TCA) cycle. It has been shown that some carboxylic acids induce cell proliferation and production of cytokines or growth factors. However,there have been no reports on effects of carboxylic acids on hepatocyte growth factor (HGF) expression. In this study, we found that only maleic acid among various carboxylic acids examined markedly induced HGF production from human dermal fibroblasts. Maleic acid also induced HGF production from human lung fibroblasts and neuroblastoma cells. The stimulatory effect was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) but not in phosphorylation of p38 was observed from 6 h and up to 24 h after maleic acid addition. The ERK kinase inhibitor PD98059 and the JNK inhibitor SP6001 25 potently inhibited maleic acid-induced HGF production, while the p38 inhibitor SB203580 did not significantly inhibit the production. The protein synthesis inhibitor cycloheximicle completely inhibited upregulation of HGF mRNA induced by maleic acid but superinduced HGF mRNA expression upregulated by I 2-0-tetradecanoylphorbol I 3-acetate (TPA). These resu Its suggest that maleic acid indirectly induced HGF expression from human dermal fibroblasts through activation of ERK and JNK and thatcle novo protein synthesis is required for maleic acid-induced upregulation of HGF mRNA.

DOI： 10.1002/jeb.21724

Clinical significance and pathogenic function of connective tissue growth factor (CTGF/CCN2) in osteolytic mandibular squamous cell carcinoma

Tsuyoshi Shimo, Satoshi Kubota, Takeshi Goda, Yasuto Yoshihama, Naito Kurio, Takashi Nishida, Poh-Sing Ng, Koki Endo, Masaharu Takigawa, Akira Sasaki

ANTICANCER RESEARCH 28 ( 4C ) 2343 - 2348 2008.7

Language：English Publishing type：Research paper (scientific journal) Publisher：INT INST ANTICANCER RESEARCH

Background: Mandibular bone destruction is a frequent occurrence in oral squamous cell carcinoma. However, the relationship between the bone destruction and associated factors is unclear. Here, the role and diagnostic utility of connective tissue growth factor (CCN2) in bone destruction of the mandible was investigated. Patients and Methods: The production of CCN2 was explored by using immunohistochemistry on paraffin-embedded tissues from 20 cases of mandibular squamous cell carcinoma. The effect of CCN2 on osteoclastogenesis was examined in vitro by using total bone marrow cell populations from male mice. Results: Immunohistochemical analysis showed that CCN2-positive signals were closely associated with destructive invasion of the mandible by oral squamous cell carcinomas. Consistent with these results, recombinant human CCN2 (rCCN2) stimulated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cell formation in vitro. Conclusion: CCN2 can be considered a diagnostic marker and target for treatment in oral osteolytic mandibular squamous cell carcinoma.

CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes

T. Fujisawa, T. Hattori, M. Ono, J. Uehara, S. Kubota, T. Kuboki, M. Takigawa

OSTEOARTHRITIS AND CARTILAGE 16 ( 7 ) 787 - 795 2008.7

Language：English Publishing type：Research paper (scientific journal) Publisher：W B SAUNDERS CO LTD

Objectives: CCN family 2/connective tissue growth factor (CCN2/CTGF) is an atypical growth factor for growth plate chondrocytes. It plays an important role in their proliferation and differentiation in vitro, but does not stimulate hypertrophy or calcification of articular chondrocytes. We herein report for the first time that CCN2/CTGF promotes growth and differentiation of auricular chondrocytes and maintains their molecular phenotype in vitro and in vivo.
Methods: Auricular chondrocytes were isolated from rabbit auricular cartilage by trypsin-collagenase treatment, and treated with human recombinant CCN2/CTGF or infected with adenovirus harboring the ccn2/ctgf gene. Cell proliferation was measured by [3 H] thymidine incorporation and MTS assay, and changes in gene expression of auricular chondrocyte markers were monitored by real-time polymerase chain reaction, Northern hybridization, and histological analysis. For in vivo studies, auricular chondrocytes were cultured as pellets and implanted subcutaneously after treatment of recombinant human CCN2/CTGF. Ectopically formed cartilage was subjected to histological analysis. Cell death was monitored by in situ TUNEL analysis.
Results: CCN2/CTGF stimulated proliferation, differentiation and synthesis of elastin and proteoglycans of rabbit primary auricular chondrocytes in a dose-dependent manner. CCN2/CTGF caused a 2.5-fold increase in the expression of elastin in comparison to the control, resulting in enhanced deposition of elastin fibers in a monolayer culture of auricular chondrocytes. Mineralization was not induced; in contrast, CCN2/CTGF stimulated expression of matrix gla protein which is known to impair mineralization. Furthermore, pretreatment of pellets of auricular chondrocytes with CCN2/CTGF and subcutaneous implantation significantly enhanced the growth of ectopic auricular cartilage pieces expressing phenotypic markers of auricular chondrocytes including type 11 and X collagen. Notably, chondrocyte apoptosis was impaired by CCN2/CTGF.
Conclusions: These findings show that CCN2/CTGF may be a suitable agent for promoting differentiation and growth of auricular chondrocytes, while preventing mineralization and apoptosis, and suggests that CCN2/CTGF may be useful for the repair or reconstruction Of Elastic cartilage. (C) 2007 Ostecarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2007.11.001

Promotion of bone regeneration by CCN2 incorporated into gelatin hydrogel

Takeshi Kikuchi, Satoshi Kubota, Koji Asaumi, Harumi Kawaki, Takashi Nishida, Kazumi Kawata, Shigeru Mitani, Yasuhiko Tabata, Toshifumi Ozaki, Masaharu Takigawa

TISSUE ENGINEERING PART A 14 ( 6 ) 1089 - 1098 2008.6

Language：English Publishing type：Research paper (scientific journal) Publisher：MARY ANN LIEBERT, INC

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a unique molecule that promotes the entire endochondral ossification process and regeneration of damaged articular cartilage. Also, CCN2 has been shown to enhance the adhesion and migration of bone marrow stromal cells as well as the growth and differentiation of osteoblasts; hence, its utility in bone regeneration has been suggested. Here, we evaluated the effect of CCN2 on the regeneration of an intractable bone defect in a rat model. First, we prepared two recombinant CCN2s of different origins, and the one showing the stronger effect on osteoblasts in vitro was selected for further evaluation, based on the result of an in vitro bioassay. Next, to obtain a sustained effect, the recombinant CCN2 was incorporated into gelatin hydrogel that enabled the gradual release of the factor. Evaluation in vivo indicated that CCN2 continued to be released at least for up to 14 days after its incorporation. Application of the gelatin hydrogel-CCN2 complex, together with a collagen scaffold to the bone defect prepared in a rat femur resulted in remarkable induction of osteoblastic mineralization markers within 2 weeks. Finally, distinct enhancement of bone regeneration was observed 3 weeks after the application of the complex. These results confirm the utility of CCN2 in the regeneration of intractable bone defects in vivo when the factor is incorporated into gelatin hydrogel.

DOI： 10.1089/ten.tea.2007.0167

CCN2遺伝子関連ノンコーディングRNAの軟骨細胞様HCS-2/8細胞における発現

大河原敏博, 久保田聡, 川木晴美, 近藤誠二, 佐々木朗, 滝川正春

岡山歯学会雑誌 27 ( 1 ) 63 - 63 2008.6

CCNファミリー2/結合組織成長因子(CCN2/CTGF)が骨芽細胞において骨形成因子(BMP)-2刺激による骨芽細胞分化を修飾する

前田あずさ, 西田崇, 川木晴美, 久保田聡, 窪木拓男, 滝川正春

岡山歯学会雑誌 27 ( 1 ) 63 - 64 2008.6

Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5

Takako Hattori, Francoise Coustry, Shelley Stephens, Heidi Eberspaecher, Masaharu Takigawa, Hideyo Yasuda, Benoit de Crombrugghe

NUCLEIC ACIDS RESEARCH 36 ( 9 ) 3011 - 3024 2008.5

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Sox9 is a transcription factor of the SRY family required for several steps of chondrogenesis. It activates the expression of various chondrocyte-specific genes, but the mechanisms and role of cofactors involved in Sox9-regulated gene transcription are not fully understood. Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes.

DOI： 10.1093/nar/gkn150

Interleukin-4 downregulates the cyclic tensile stress-induced matrix metalloproteinases-13 and cathepsin B expression by rat normal chondrocytes

Hideyuki Doi, Keiichiro Nishida, Masanori Yorimitsu, Takamitsu Komiyama, Yasutaka Kadota, Tomonori Tetsunaga, Aki Yoshida, Satoshi Kubota, Masaharu Takigawa, Toshifumi Ozaki

ACTA MEDICA OKAYAMA 62 ( 2 ) 119 - 126 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：OKAYAMA UNIV MED SCHOOL

Mechanical stress plays a key role in the pathogenesis of cartilage destruction seen in osteoarthritis (OA). We investigated the effect of cyclic tensile stress (CTS) on the anabolic and catabolic gene expression of rat cultured normal chondrocytes using the Flexercell strain unit. The effects of interleukin (IL)-4, a chondroprotective cytokine, on the changes in gene expression induced by CTS were also investigated. CTS (7% elongation at 0.5 Hz) for 24 h did not affect the expression of aggrecan and type 11 collagen, whereas CTS significantly upregulated matrix metalloproteinase (MMP)-13 and cathepsin B mRNA expression by chondrocytes. IL-1 beta expression was also significantly upregulated by CTS up to 12 h. The upregulation of MMP-13 was observed at 3 h, which was earlier than that of IL-1 beta. Furthermore, pre-treatment with IL-4 (10 ng/ml) suppressed both MMP-13 and cathepsin B induction by mechanical stress, as well as CTS-induced IL-1 beta expression. Our results suggest that IL-4 might have a therapeutic value in the treatment of OA by downregulation of mechanical stress-induced MMP-13 and cathepsin B expression by chondrocytes.

Connective tissue growth factor is overexpressed in muscles of human muscular dystrophy

Guilian Sun, Kazuhiro Haginoya, Yanling Wu, Yoko Chiba, Tohru Nakanishi, Akira Onuma, Yuko Sato, Masaharu Takigawa, Kazuie Iinuma, Shigeru Tsuchiya

JOURNAL OF THE NEUROLOGICAL SCIENCES 267 ( 1-2 ) 48 - 56 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The detailed process of how dystrophic muscles are replaced by fibrotic tissues is unknown. In the present study, the immunolocalization and mRNA expression of connective tissue growth factor (CTGF) in muscles from normal and dystrophic human muscles were examined with the goal of elucidating the pathophysiological function of CTGF in muscular dystrophy. Biopsies of frozen muscle from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, congenital muscular dystrophy, spinal muscular atrophy, congenital myopathy were analyzed using anti-CTGF polyclonal antibody. Reverse transcription-polymerase chain reaction (RT-PCR) was also performed to evaluate the expression of CTGF mRNA in dystrophic muscles. In normal muscle, neuromuscular junctions and vessels were CTGF-immunopositive, which suggests a physiological role for CTGF in these sites. In dystrophic muscle, CTGF immunoreactivity was localized to muscle fiber basal lamina, regenerating fibers, and the interstitium. Triple immunolabeling revealed that activated fibroblasts were immunopositive for CTGF and transforming growth factor-betal (TGF-beta1). RT-PCR analysis revealed increased levels of CTGF mRNA in the muscles of DMD) patients. Co-localization of TGF-beta1 and CTGF in activated fibroblasts suggests that CTGF expression is regulated by TGF-beta1 through a paracrine/autocrine mechanism. In conclusion, TGF-beta1-CTGF pathway may play a role in the fibrosis that is commonly observed in muscular dystrophy. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jns.2007.09.043

Effects of alendronate and pamidronate on cultured rat metatarsal bones: Failure to prevent dexamethasone-induced growth retardation

Terhi J. Heino, Andrei S. Chagin, Masaharu Takigawa, Lars Savendahl

BONE 42 ( 4 ) 702 - 709 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Bisphosphonates are widely used anti-resorptive drugs in the adult population. In children, their use has mainly been limited to patients with osteogenesis imperfecta. However, the powerful effects of bisphosphonates on bone turnover have raised concern about their long-term effects on the growing skeleton.
We aimed to study the effects of two commonly used bisphosphonates, alendronate (Aln) and pamidronate (Pam) on normal bone growth as well as their potential to prevent glucocorticoid-induced growth retardation.
Effects on bone growth were studied in fetal rat metatarsal bones (day E20) that were cultured for 5-47 days and measured every 2-7 days. Cellular mechanisms were investigated in metatarsal bones and also in the human chondrocytic cell line HCS-2/8. Chondrocyte viability (WST-1), proliferation (BrdU incorporation), differentiation (collagen type X immunohistochemistry) and apoptosis (TUNEL and Cell Death ELISA) were determined.
At a clinically relevant concentration of bisphosphonates (1 mu M), metatarsal bone growth was stimulated by both Aln (p<0.001 for length and p < 0.05 for width) and Pam (p < 0.05 for both length and width) from day 19 of culture. The growth-stimulatory effect was associated with increased chondrocyte proliferation (+21% with Aln and +24% with Pam), while cell differentiation and apoptosis were not affected. Despite the finding that both Aln and Pam (1 mu M) rescued HCS-2/8 cells from undergoing dexamethasone-induced apoptosis, neither of them was able to prevent dexamethasone-induced growth retardation of fetal rat metatarsal bones.
Aln and Pam have the capacity to stimulate the growth of cultured fetal rat metatarsal bones; an effect associated with increased proliferation of growth plate chondrocytes. Our experimental data suggest that bisphosphonates are ineffective in preventing glucocorticoid-induced growth retardation. Nevertheless, based on our in vitro data, both Aln and Pam appear safe to use in growing children, at least with regard to their effects on linear bone growth. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2008.01.001

Novel transcription factor-like function of human matrix metalloproteinase 3 regulating the CTGF/CCN2 gene

Takanori Eguchi, Satoshi Kubota, Kazumi Kawata, Yoshiki Mukudai, Junji Uehara, Toshihiro Ohgawara, Soichiro Ibaragi, Akira Sasaki, Takuo Kuboki, Masaharu Takigawa

MOLECULAR AND CELLULAR BIOLOGY 28 ( 7 ) 2391 - 2413 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

Matrix metalloproteinase 3 (MMP3) is well known as a secretory endopeptidase that degrades extracellular matrices. Recent reports indicated the presence of MMPs in the nucleus (A. J. Kwon et al., FASEB J. 18:690-692, 2004); however, its function has not been well investigated. Here, we report a novel function of human nuclear MMP3 as a trans regulator of connective tissue growth factor (CCN2/CTGF). Initially, we cloned MMP3 cDNA as a DNA-binding factor for the CCN2/CTGF gene. An interaction between MMP3 and transcription enhancer dominant in chondrocytes (TRENDIC) in the CCN2/CTGF promoter was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by overexpressed MMP3, whereas a TRENDIC mutant promoter lost the response. Also, the knocking down of MMP3 suppressed CCN2/CTGF expression. By cytochemical and histochemical analyses, MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 and six putative nuclear localization signals in MMP3 also were shown. Furthermore, we determined that heterochromatin protein gamma coordinately regulates CCN2/CTGF by interacting with MMP3. The involvement of this novel role of MMP3 in the development, tissue remodeling, and pathology of arthritic diseases through CCN2/CTGF regulation thus is suggested.

DOI： 10.1128/MCB.01288-07

Plasma connective tissue growth factor is a novel potential biomarker of cardiac dysfunction in patients with chronic heart failure

Norimichi Koitabashi, Masashi Arai, Kazuo Niwano, Atai Watanabe, Michiko Endoh, Masahiko Suguta, Tomoyuki Yokoyama, Hiroshi Tada, Takuji Toyama, Hitoshi Adachi, Shigeto Naito, Shigeru Oshima, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa, Masahiko Kurabayashi

EUROPEAN JOURNAL OF HEART FAILURE 10 ( 4 ) 373 - 379 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Background: Connective tissue growth factor (CTGF) has been recently reported as a mediator of myocardial fibrosis; however, the significance of plasma CTGF concentration has not been evaluated in patients with heart failure. The aim of this study was to investigate the clinical utility of plasma CTGF concentration for the diagnosis of heart failure.
Methods and results: We evaluated fifty-two patients with chronic heart failure. The plasma concentration of CTGF and other markers of fibrosis were assessed and compared with clinical and echocardiographic data. Plasma CTGF was significantly elevated in symptomatic patients in proportion to their NYHA classes and was significantly correlated with plasma brain natriuretic peptide (BNP) concentration (r=0.395, P<0.01). Plasma CTGF was also correlated with plasma transforming growth factor beta (TGF-beta) (r=0.512, P<0.01), matrix metalloproteinase (MMP)-2 (r=0.391, P<0.05) and tissue inhibitor of MMP (TIMP)-2 (r=0.354, P<0.05) concentrations. Interestingly, plasma CTGF was correlated with E/E' value evaluated by tissue Doppler echocardiography (r=0.593, P=0.012), but not with systolic function and left ventricular mass.
Conclusion: Our study suggests that plasma CTGF concentration is a novel diagnostic marker for cardiac dysfunction and may provide additional specific information about myocardial fibrosis in chronic heart failure patients. (C) 2008 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejheart.2008.02.011

Functional requirement of CCN2 for intramembranous bone formation in embryonic mice

Harumi Kawaki, Satoshi Kubota, Akiko Suzuki, Tomohiro Yamada, Tatsushi Matsumura, Toshiko Mandal, Mayumi Yao, Takeyasu Maeda, Karen M. Lyons, Masaharu Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 366 ( 2 ) 450 - 456 2008.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and Ccn2 null mutant mice display skeletal dysmorphisms. However, little is known concerning the roles of CCN2 during bone formation. We herein present a comparative analysis of wild-type and Ccn2 null mice to investigate the roles of CCN2 in bone development. Multiple histochemical methods were employed to analyze the effects of CCN2 deletion in vivo, and effects of CCN2 on the osteogenic response were evaluated with the isolated and cultured osteoblasts. As a result, we found a drastic reduction of the osteoblastic phenotype in Ccn2 null mutants. Importantly, addition of exogenous CCN2 promoted every step of osteoblast differentiation and rescued the attenuated activities of the Ccn2 null osteoblasts. These results suggest that CCN2 is required not only for the regulation of cartilage and subsequent events, but also for the normal intramembranous bone development. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.11.155

Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

Yohsuke Matsumoto, Takahiro Motoki, Satoshi Kubota, Masaharu Takigawa, Hirohito Tsubouchi, Eiichi Gohda

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 366 ( 1 ) 110 - 116 2008.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E-2 without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.11.089

Effects of tensile and compressive strains on response of a chondrocytic cell line embedded in type I collagen gel

Yuji Hirano, Naoki Ishiguro, Masahiro Sokabe, Masaharu Takigawa, Keiji Naruse

JOURNAL OF BIOTECHNOLOGY 133 ( 2 ) 245 - 252 2008.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Tensile and compressive strains are commonly used in mechanobiological models. Here we report on the development of a novel three-dimensional cell-culture method, which allows both tensile and compressive loads to be applied. Preliminary results were obtained using HCS2/8 chondrocytic cells embedded in type I collagen gel. This construct was subjected to either 16% tension or 14% compression. Confocal laser scanning microscopy showed that both tension and compression caused significant cell deformation. The collagen gel-embedded HCS2/8 cells were subjected to static tension, dynamic tension, static compression or dynamic compression for 24 h. Dynamic compression led to significantly decreased 5-bromo-2'-deoxyuridine incorporation compared with the control group. PCR analysis revealed upregulation of type II collagen caused by dynamic tension, upregulation of aggrecan caused by static compression, and downregulation of type II collagen and aggrecan caused by dynamic compression. Nitric oxide production was significantly increased by static tension and static compression compared with the control group. Our experimental system effectively applied several types of strain to HCS2/8 cells embedded in collagen gel. Our results suggest that the mode of mechanical strain affects the response of HCS2/8 cells. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotee.2007.07.955

Role of mechanical-stress inducible protein Hcs24/CTGF/CCN2 in cartilage growth and regeneration: Mechanical stress induces expression of Hcs24/CTGF/CCN2 in a human chondrocytic cell line HCS-2/8, rabbit costal chondrocytes and meniscus tissue cells

Takashi Nishida, Azusa Maeda, Satoshi Kubota, Masaharu Takigawa

BIORHEOLOGY 45 ( 3-4 ) 289 - 299 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：IOS PRESS

Mechanical stress plays an important role in the cartilage metabolism. The aim of this study is to determine the influence of mechanical load magnitude and frequency on cartilage metabolism in terms of the expression of hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), as an essential mediator of extracellular matrix (ECM) production. When a human chondrocytic cell line, HCS-2/8 was exposed to uni-axial cyclic mechanical force (6% elongation, 10 times/min) only for 30 min, the expression level of Hcs24/CTGF/CCN2 (CCN2) increased, and c-Jun N-terminal protein kinase (JNK) was activated. These findings suggest that stretch-induced CCN2 may be mediated by the JNK pathway. When HCS-2/8 cells were subjected to cyclic tension force at 15 kPa, 30 cycles/min, which has been reported to be a degradation force for HCS-2/8 cells, the expressions of CCN2 and aggrecan were inhibited, and such expressions remained unchanged in rabbit hyaline costal cartilage cells. However, these expressions increased in rabbit meniscus tissue cells. These findings suggest that the sensitivity of mechanical stretch may be different depending on the type of cells. Furthermore, CCN2 was co-localized with aggrecan in this meniscus tissue region exposed to mechanical stress in vivo. These findings suggest that CCN2 induced by mechanical stress may therefore play some role in meniscus growth and regeneration.

DOI： 10.3233/BIR-2008-0478

Promotion of hydroxyapatite-associated, stem cell-based bone regeneration by CCN2

Mitsuaki Ono, Satoshi Kubota, Takuo Fujisawa, Wataru Sonoyama, Harumi Kawakij, Kentaro Akiyama, Kengo Shimono, Masarnitsu Oshima, Takashi Nishida, Yasuhiro Yoshida, Kazuomi Suzuki, Masaharu Takigawa, Takuo Kuboki

CELL TRANSPLANTATION 17 ( 1-2 ) 231 - 240 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：COGNIZANT COMMUNICATION CORP

Multiple roles have been already recognized for CCN2 in cartilage development and regeneration. However, the effects of CCN2 on bone regeneration remain to be elucidated. In this study, the utility of CCN2 on bone regeneration was examined in vitro and in vivo in combination with hydroxyapatite (HAp) as a scaffold. Human bone marrow stromal cells (hBMSCs) were isolated from human iliac bone marrow aspirates of healthy donors and expanded, and the effects of CCN2 on their proliferation and migration were examined in vitro. The proliferation of hBMSCs on a plastic or HAp plate was significantly enhanced by CCN2. Moreover, the migration of hBMSCs also dramatically increased by CCN2. Interestingly, a C-terminal signal modular fragment of CCN2 (CT-module) also enhanced the cell proliferation and migration as efficiently as the full-length CCN2. Next, in order to estimate the effect of CCN2 on the migration and survival of hBMSCs and bone formation inside the HAp scaffold in vivo, two experiments were performed. First, the porous HAp carrier was cultured with hBMSCs for a week, and the cell-scaffold hybrid was transplanted with or without CCN2 subcutaneously into immunocompromised mice. CCN2 accelerated the hBMSC-like cell migration and survival inside the porous HAp within 4 weeks after transplantation. Second, the porous HAp carrier with or without CCN2 was directly implanted into bone defects within a rabbit mandible, and bone regeneration inside was evaluated. As a result, CCN2 efficiently induced the cell invasion and bone formation inside the porous HAp scaffold. These findings suggest that CCN2 and its CT-module fragment could be useful for regeneration and reconstruction of large-scale bone defects.

DOI： 10.3727/000000008783907143

Gene expression and distribution of connective tissue growth factor (CCN2/CTGF) during secondary ossification center formation

Morihiko Oka, Satoshi Kubota, Seiji Kondo, Takanori Eguchi, Chisa Kuroda, Kazumi Kawata, Shogo Minagi, Masaharu Takigawa

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 55 ( 12 ) 1245 - 1255 2007.12

Language：English Publishing type：Research paper (scientific journal) Publisher：HISTOCHEMICAL SOC INC

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metal loproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

DOI： 10.1369/jhc.7A7263.2007

Proteasome inhibition up-regulates p53 and apoptosis-inducing factor in chondrocytes causing severe growth retardation in mice

Farasat Zaman, Victoria Menendez-Benito, Emma Eriksson, Andrei S. Chagin, Masaharu Takigawa, Bengt Fadeel, Nico P. Dantuma, Dionisios Chrysis, Lars Saevendahl

CANCER RESEARCH 67 ( 20 ) 10078 - 10086 2007.10

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Proteasome inhibitors (PI), a novel class of anticancer drugs, are relatively well tolerated and have recently been introduced into the clinic for the treatment of multiple myeloma. The tumor selectivity and low toxicity of PIs are surprising, given the crucial role of the ubiquitin/proteasome system in a multitude of cellular processes. Here, we show that systemic administration of PIs specifically impairs the ubiquitin/ proteasome system in growth plate chondrocytes. Importantly, young mice displayed severe growth retardation during treatment as well as 45 days after the cessation of treatment with clinically relevant amounts of MG262 (0.2 [mu mol/kg body weight/injection) or bortezomib (1.0 mg/kg body weight/ injection). Dysfunction of the ubiquitin/proteasome system was accompanied by the induction of apoptosis of stem-like and proliferative chondrocytes in the growth plate. These results were recapitulated in cultured fetal rat metatarsal bones and chondrocytic cell lines (rat, human). Apoptosis was associated with up-regulation of the proapoptotic molecules, p53 and apoptosis-inducing factor (AIF), both in vitro and in vivo. In addition to the observation that AIF is expressed in the growth plate, we also provide evidence that AIF serves as a direct target protein for ubiquitin, thus explaining its prominent up-regulation upon proteasome inhibition. Suppression of p53 or AIF expression with small interfering RNAs partly rescued chondrocytes from proteasome inhibition-induced apoptosis (35% and 41%, respectively). Our observations show that proteasome inhibition may selectively target essential cell populations in the growth plate causing significant growth failure. These findings could have important implications for the use of proteasome inhibitors in the treatment of childhood cancer. [Cancer Res 2007;67(20):10078-86]

DOI： 10.1158/0008-5472.CAN-06-3982

CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes

Takashi Nishida, Harumi Kawaki, Ruth M. Baxter, R. Andrea DeYoung, Masaharu Takigawa, Karen M. Lyons

JOURNAL OF CELL COMMUNICATION AND SIGNALING 1 ( 1 ) 45 - 58 2007.6

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

The matricellular protein CCN2 (Connective Tissue Growth Factor; CTGF) is an essential mediator of ECM composition, as revealed through analysis of Ccn2 deficient mice. These die at birth due to complications arising from impaired endochondral ossification. However, the mechanism(s) by which CCN2 mediates its effects in cartilage are unclear. We investigated these mechanisms using Ccn2(-/-) chondrocytes. Expression of type II collagen and aggrecan were decreased in Ccn2(-/-) chondrocytes, confirming a defect in ECM production. Ccn2(-/-) chondrocytes also exhibited impaired DNA synthesis and reduced adhesion to fibronectin. This latter defect is associated with decreased expression of alpha 5 integrin. Moreover, CCN2 can bind to integrin alpha 5 beta 1 in chondrocytes and can stimulate increased expression of integrin alpha 5. Consistent with an essential role for CCN2 as a ligand for integrins, immuno-fluorescence and Western blot analysis revealed that levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 phosphorylation were reduced in Ccn2(-/-) chondrocytes. These findings argue that CCN2 exerts major effects in chondrocytes through its ability to (1) regulate ECM production and integrin alpha 5 expression, (2) engage integrins and (3) activate integrin-mediated signaling pathways.

DOI： 10.1007/s12079-007-0005-z

Report on the fourth international workshop on the CCN family of genes

S. Kubota, H. Yeger, B. Perbal, M. Takigawa

JOURNAL OF CELL COMMUNICATION AND SIGNALING 1 ( 1 ) 59 - 65 2007.6

Tamoxifen induces permanent growth arrest through selective induction of apoptosis in growth plate chondrocytes in cultured rat metatarsal bones

Andrei S. Chagin, Elham Karimian, Farasat Zaman, Masaharu Takigawa, Dionisios Chrysis, Lars Savendahl

BONE 40 ( 5 ) 1415 - 1424 2007.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Estrogen affects skeletal growth and promotes growth plate fusion in humans. High doses of estrogen have been used to limit growth in girls with predicted extreme tall stature; a treatment which has been associated with severe side effects. Selective estrogen receptor modulators (SERMs) could potentially be used as an alternative treatment. We chose to study the effects of Tamoxifen (Tam), a first generation SERM that has been used in the treatment of pubertal gynecomastia or McCune-Albright syndrome.
Cultured fetal rat metatarsal bones were used to study the effects of Tam on longitudinal bone growth. In sectioned bones, chondrocyte apoptosis and proliferation were analyzed by TUNEL assay and BrdU incorporation, respectively. We also used a human chondrocytic cell line, HSC-2/8, to study the effects of Tam on apoptosis (FACS analysis and Cell Death detection ELISA) and caspase activation (caspase substrate cleavage and Western immunoblotting).
Tam caused a dose-dependent growth retardation of cultured metatarsal bones. No catch-up growth was observed after Tam was removed from the culture medium. Detailed analysis of sectioned growth plate cartilage revealed increased apoptosis of chondrocytes within the resting and hypertrophic zones. HCS-2/8 cells also underwent apoptosis upon Tam treatment. Tam-induced apoptosis was caspase-dependent and completely abrogated by either caspase-8 or -9 inhibitors. A substrate assay revealed that caspase-8 is first activated followed by caspase-9 and -3. Finally, FasL secretion was stimulated by Tam and blocking of either FasL or Fas decreased Tam-induced apoptosis in chondrocytes.
We here describe a novel mechanism of tamoxifen-induced apoptosis in chondrocytes, involving the activation of caspases and the FasL/Fas pathway, which diminishes the potential for bone growth. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2006.12.066

Promotion of attachment of human bone marrow stromal cells by CCN2

Mitsuaki Ono, Satoshi Kubota, Takuo Fujisawa, Wataru Sonoyama, Harumi Kawaki, Kentaro Akiyama, Masamitsu Oshima, Takashi Nishida, Yasuhlro Yoshida, Kazuomi Suzuki, Masaharu Takigawa, Takuo Kuboki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 357 ( 1 ) 20 - 25 2007.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Cell attachment is a crucial step in tissue regeneration. In this study, human bone marrow stromal cells (hBMSCs) were isolated, and the effects of CCN2 on their attachment were examined. CCN2 significantly enhanced the hBMSC attachment, and this enhanced cell attachment was mainly regulated by the C-terminal module of CCN2. This enhancement was negated by the anti-integrin alpha(v)beta(3) antibody and p38 MAPK inhibitor, and phosphorylation of p38 MAPK was detected upon the enhanced cell attachment mediated by CCN2. We thus conclude that CCN2 enhances hBMSC attachment via integrin-p38 MAPK signal pathway. Enhanced hBMSC attachment on hydroxyapatite plates by CCN2 further indicated the utility of CCN2 in bone regeneration. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.03.052

Increased connective tissue growth factor relative to brain natriuretic peptide as a determinant of myocardial fibrosis

Norimichi Koitabashi, Masashi Arai, Shinya Kogure, Kazuo Niwano, Atai Watanabe, Yasuhiro Aoki, Toshitaka Maeno, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa, Masahiko Kurabayashi

HYPERTENSION 49 ( 5 ) 1120 - 1127 2007.5

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF, and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression. Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G protein - coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas aldosterone and transforming growth factor-beta preferentially induced expression of the CTGF gene. Finally, exogenous BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure.

DOI： 10.1161/HYPERTENSIONAHA.106.077537

Expression and physiological role of CCN4/Wnt-induced secreted protein 1 mRNA splicing variants in chondrocytes

Takeshi Yanagita, Satoshi Kubota, Harumi Kawaki, Kazumi Kawata, Seiji Kondo, Teruko Takano-Yamamoto, Shinji Tanaka, Masaharu Takigawa

FEBS JOURNAL 274 ( 7 ) 1655 - 1665 2007.4

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

CCN4/Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF/Cyr61/Nov) family proteins. CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lacking certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4/WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. The expression of CCN4/WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and primary rabbit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2/8, as well as full-length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4/WISP1 mRNA along chondrocyte differentiation, RGC cells were cultured and sampled until they were mineralized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal differentiation in HCS-2/8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2/8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas.

DOI： 10.1111/j.1742-4658.2007.05709.x

A functional polymorphism in the 5 ' UTR of GDF5 is associated with susceptibility to osteoarthritis

Yoshinari Miyamoto, Akihiko Mabuchi, Dongquan Shi, Toshikazu Kubo, Yoshio Takatori, Susumu Saito, Mikihiro Fujioka, Akihiro Sudo, Atsumasa Uchida, Seizo Yamamoto, Koichi Ozaki, Masaharu Takigawa, Toshihiro Tanaka, Yusuke Nakamura, Qing Jiang, Shiro Ikegawa

NATURE GENETICS 39 ( 4 ) 529 - 533 2007.4

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Osteoarthritis (MIM 165720), characterized by degeneration of articular cartilage, is the most common form of human arthritis and a major concern for aging societies worldwide(1-3). Epidemiological and genetic studies have shown that osteoarthritis is a polygenic disease(1,4,5). Here, we report that the gene encoding growth differentiation factor 5 (GDF5) is associated with osteoarthritis in Asian populations. A SNP in the 5' UTR of GDF5 (+104T/ C; rs143383) showed significant association (P = 1.8 x 10(-13)) with hip osteoarthritis in two independent Japanese populations. This association was replicated for knee osteoarthritis in Japanese (P = 0.0021) and Han Chinese (P = 0.00028) populations. This SNP, located in the GDF5 core promoter, exerts allelic differences on transcriptional activity in chondrogenic cells, with the susceptibility allele showing reduced activity. Our findings implicate GDF5 as a susceptibility gene for osteoarthritis and suggest that decreased GDF5 expression is involved in the pathogenesis of osteoarthritis.

DOI： 10.1038/ng2005

Different transcriptional strategies for ccn2/ctgf gene induction between human chondrocytic and breast cancer cell lines

Takanori Eguchi, Satoshi Kubota, Kazumi Kawata, Yoshiki Mukudai, Toshihiro Ohgawara, Kohei Miyazono, Kyouji Nakao, Seiji Kondo, Masaharu Takigawa

BIOCHIMIE 89 ( 3 ) 278 - 288 2007.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

Connective tissue growth factor (CTGF/CCN2) plays a critical role in endochondral bone formation; however, CCN2 also promotes angiogenesis and bone metastasis in breast cancer. Chondrocytic HCS-2/8 cells and breast cancer MDA231 cells produce over 6 times more CCN2 than any other cell type. In this study, we demonstrate that these cell lines employ different transcriptional strategies for ccn2 gene induction. Four tandem copies of the dominant transcriptional enhancer in chondrocytes (4 x TRENDIC) were chimerically connected to an SV40 pro-moter-luciferase construct and subsequently analyzed. The enhancement of the promoter activity by 4 x TRENDIC was greater in the HCS-2/8 cells (7-fold) than in the other 4 cell lines (3-4 fold). The TRENDIC-binding protein complex was detected at a higher signal in the HCS-2/8 cells than in the other cell lines. In addition, the HCS-2/8 nuclear factors strongly targeted not only TRENDIC, but also the previously reported basal control element and a novel enhancer element in the ccn2 promoter. In contrast, high-level ccn2 gene induction in MDA231 cells was largely dependent on Smad signaling through the Smad-binding element in the ccn2 promoter. Based on these results, we propose a model of differential transcription of the ccn2 gene between the chondrocytic cell line and the breast cancer cell line, and therefore imply that these cells utilize distinct transcriptional strategies to obtain the enhanced CCN2 production that is not observed in other types of cells. (c) 2007 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2006.12.006

CCN family proteins and angiogenesis: From embryo to adulthood

Satoshi Kubota, Masaharu Takigawa

Angiogenesis 10 ( 1 ) 1 - 11 2007.3

Language：English

The CCN family is a novel class of extracellular signal modulators that has been recently established. Typical members are composed of four conserved modules connected tandem, each of which is rich in cysteines and highly interactive with other molecules. The mammalian CCN family consists of six members, most of which have been described as multifunctional factors for the developmental process of mesenchymal tissue including blood vessel formation/induction. Particularly, the angiogenic properties of the three classical members, CCN1, 2 and 3 have so far been characterized, and their physiological and pathological significance has thus been indicated. Recent research has uncovered a unique mechanism regarding these proteins in promoting and/or modulating developmental, physiological and pathological angiogenic events. Namely, CCN proteins exert their ability to drive angiogenesis, not by stimulating a particular behavior of a particular type of cells, but by manipulating the cell communication networks that integrate most of the associated molecules/cells toward angiogenesis. In this article, the role of the CCN proteins in a variety of angiogenic events as an organizer of microenvironmental cell society is comprehensively described, together with a brief summary of the recent findings on each CCN family member relevant to angiogenesis including cardiovascular development and diseases. © 2006 Springer Science + Business Media B.V.

DOI： 10.1007/s10456-006-9058-5

Prostaglandin E-2 downregulates TNF-alpha-induced production of matrix metalloproteinase-1 in HCS-2/8 chondrocytes by inhibiting Raf-1/MEK/ERK cascade through EP4 prostanoid receptor activation

Kazunari Fushimi, Shigeru Nakashima, Fukka You, Masaharu Takigawa, Katsuji Shimizu

JOURNAL OF CELLULAR BIOCHEMISTRY 100 ( 3 ) 783 - 793 2007.2

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Matrix metalloproteinase-1 (MMP-1, collagenase-1) plays a pivotal role in the process of joint destruction in degenerative joint diseases. We have examined the regulation of MMP-1 production in human chondrocytic HCS-2/8 cells stimulated by tumor necrosis factor-alpha (TNF-alpha). In response to TNF-alpha, MMP-1 is induced and actively released from HCS-2/8 cells. The induction of MMP-1 expression correlates with activation of ERK1/2, MEK, and Raf-1, and is potently prevented by U0126, a selective inhibitor of MEK1/2 activation. In contrast, SB203580, a selective p38 mitogen-activated protein kinases (MAPK) inhibitor, had no effects on TNF-alpha-induced MMP-1 release. A serine/threonine kinase, Akt was not activated in TNF-alpha-stimulated HCS-2/8 cells. TNF-alpha stimulated the production of PGE(2) in addition to MMP-1 in HCS-2/8 cells. Exogenously added PGE(2) potently inhibited TNF-alpha-induced both MMP-1 production and activation of ERK1/2. The effects of PGE(2) were mimicked by ONO-AE1-329, a selective EP4 receptor agonist but not by butaprost, a selective EP2 agonist. In contrast, blockade of endogenously produced PGE(2) signaling by ONO-AE3-208, a selective EP4 receptor antagonist, enhanced TNF-alpha-induced MMP-1 production. Furthermore, the suppression of MMP-l production by exogenously added PGE(2) was reversed by ONO-AE3-208. Activation of EP4 receptor resulted in cAMP-mediated phosphorylation of Raf-1 on Ser259, a negative regulatory site, and blocked activation of Raf-1/MEK/ERK cascade. Taken together, these findings indicate that Raf-1/MEK/ERK signaling pathway plays a crucial role in the production of MMP-1 in HCS-2/8 cells in response to TNF-alpha, and that the produced PGE(2) downregulates the expression of MMP-1 by blockage of TNF-alpha-induced Raf-1 activation through EP4-PGE(2) receptor activation.

DOI： 10.1002/jcb.21099

Dopamine Receptor Presence in the Rat Area Postrema Identified by RT-PCR, Immunohistochemistry, and In Situ Hybridization

Izawa Shunji, Yamaai Tomoichiro, Mukudai Yoshiki, Yamaji Kozo, Nishitani Yoshihiro, Itota Toshiyuki, Matsuo Ryuji, Takigawa Masaharu, Yoshiyama Masahiro

Japanese Journal of Oral Biology 49 ( 4 ) 259 - 268 2007

Language：English Publisher：Japanese Association for Oral Biology

Dopamine (DA) is a major central nervous system (CNS) neurotransmitter with many important physiological activities. Investigations into the neuroanatomy and neurologic functions of the dopaminergic neural systems have generated much debate. Regarding neuroanatomy, physiological and pharmacological criteria have divided DA receptors into D1 and D2 subtypes. The genes encoding these subtypes have been cloned and classified into a D1 subfamily encompassing D1 and D5 receptors and a D2 subfamily with D2, D3, and D4. Based on the sequences of the cloned receptors, we prepared antibodies and riboprobes to elucidate the expression of the corresponding proteins and mRNAs in the rat area postrema (AP) by immunohistochemistry and in situ hybridization (ISH). The AP was obtained from adult male Sprague-Dawley rats undergoing brain surgery, and tissue samples were used for RT-PCR, immunohistochemistry, and ISH. The results showed that D2 and D5 receptors and their mRNAs exist in the rat AP. On the other hand, D1, D3, and D4 receptors and their mRNAs were not detected.

DOI： 10.2330/joralbiosci.49.259

結合組織成長因子CTGF/CCN2 Invited

服部高子, 久保田聡, 滝川正春

The Lung perspectives 15 ( 3 ) 331 - 335 2007

Language：Japanese Publisher：(株)メディカルレビュー社

結合組織成長因子(connective tissue growth factor;CTGF/CCN2)は前肥大軟骨細胞層が特異的に発現する遺伝子として、また、血管内皮細胞の培養上清中に存在する線維芽細胞の増殖・遊走促進因子として単離されたが、その発現部位は軟骨組織、血管内皮細胞だけでなく、脳をはじめとする神経系にも広がり、また、血小板に大量に蓄積されて体内を循環している。生理的には、正常な内軟骨性骨形成、血管新生、胚発育に必須であることが明らかになっており、細胞外マトリックス成分の合成を強く促進し、細胞外マトリックスに富む軟骨組織で高発現していることと密接に関連している。また、肺をはじめ、心臓、肝臓、腎臓、筋、膵臓などの組織における線維芽細胞では、トランスフォーミング増殖因子(TGF)-βの誘導に伴って遺伝子発現が誘導され、各組織の線維化、線維症の形成を促進することから、そのメディエーターとしての働きが注目されている。また、乳癌細胞で高度に発現していることが観察されているが、低酸素状態によってCTGF/CCN2の発現が誘導され、これにより血管新生が惹起されることが明らかになっている。(著者抄録)

Role of CCN2/CTGF/Hcs24 in bone growth Reviewed

Satoshi Kubota, Masaharu Takigawa

INTERNATIONAL REVIEW OF CYTOLOGY - A SURVEY OF CELL BIOLOGY, VOL 257 257 1 - 41 2007

Language：English Publishing type：Part of collection (book) Publisher：ELSEVIER ACADEMIC PRESS INC

Our bones mostly develop through a process called endochondral ossification. This process is initiated in the cartilage prototype of each bone and continues through embryonic and postnatal development until the end of skeletal growth. Therefore, the central regulator of endochondral ossification is the director of body construction, which is, in other words, the determinant of skeletal size and shape. We suggest that CCN2/CTGF/Hcs24 (CCN2) is a molecule that conducts all of the procedures of endochondral ossification. CCN2, a member of the CCN family of novel modulator proteins, displays multiple functions by manipulating the local information network, using its conserved modules as an interface with a variety of other biomolecules. Under a precisely designed four-dimensional genetic program, 002 is produced from a limited population of chondrocytes and acts on all of the mesenchymal cells inside the bone callus to promote the integrated growth of the bone. Furthermore, the utility of CCN2 as regenerative therapeutics against connective tissue disorders, such as bone and cartilage defects and osteoarthritis, has been suggested. Over the years, the pathological action of CCN2 has been suggested. Nevertheless, it can also be regarded as another aspect of the physiological and regenerative function of CCN2, which is discussed as well.

DOI： 10.1016/S0074-7696(07)57001-4

Multiple activation of mitogen-activated protein kinases by purified independent CCN2 modules in vascular endothelial cells and chondrocytes in culture

S. Kubota, H. Kawaki, S. Kondo, G. Yosimichi, M. Minato, T. Nishida, H. Hanagata, A. Miyauchi, M. Takigawa

BIOCHIMIE 88 ( 12 ) 1973 - 1981 2006.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

CCN2 consists of 4 distinct modules that are conserved among various CCN family protein members. From the N-terminus, insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C repeat (VWC), thrombospondin type 1 repeat (TSP1) and C-terminal cysteine-knot (CT) modules are all aligned tandem therein. The multiple functionality of CCN2 is thought to be enabled by the differential use of these modules when interacting with other molecules. In this study, we independently prepared all 4 purified module proteins of human CCN2, utilizing a secretory production system with Brevibacillus choshinensis and thus evaluated the cell biological effects of such single modules. In human umbilical vascular endothelial cells (HUVECs), VWC, TSP and CT modules, as well as a full-length CCN2, were capable of efficiently activating the ERK signal transduction cascade, whereas IGFBP was not. In contrast, the IGFBP module was found to prominently activate JNK in human chondrocytic HCS-2/8 cells, while the others showed similar effects at lower levels. In addition, ERK1/2 was modestly, but significantly activated by IGFBP and VWC in those cells. No single module, but a mixture of the 4 modules provoked a significant activation of p38 MAPK in HCS-2/8 cells, which was activated by the full-length CCN2. Therefore, the signals emitted by CCN2 can be highly differential, depending upon the cell types, which are thus enabled by the tetramodular structure. Furthermore, the cell biological effects of each module on these cells were also evaluated to clarify the relationship among the modules, the signaling pathways and biological outcomes. Our present results not only demonstrate that single CCN2 modules were potent activators of the intracellular signaling cascade to yield a biological response per se, while also providing new insight into the module-wise structural and functional relationship of a prototypic CCN family member, CCN2. (c) 2006 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2006.07.007

Expression of neurotrophins and their receptors tropomyosin-related kinases (Trk) under tension-stress during distraction osteogenesis

Ayako Aiga, Koji Asaumi, You-Jin Lee, Hiroaki Kadota, Shigeru Mitani, Toshifumi Ozaki, Masaharu Takigawa

ACTA MEDICA OKAYAMA 60 ( 5 ) 267 - 277 2006.10

Language：English Publishing type：Research paper (scientific journal) Publisher：OKAYAMA UNIV MED SCHOOL

The localization and expression of neurotrophins and their receptors during distraction osteogenesis was investigated in 72 male rat femurs (11 weeks old) to further clarify the concurrence of cellular and molecular events of new bone formation. After osteotomy, a 7-day lag phase was followed by distraction at the rate of 0.25 mm/12 h for 21 days (distraction phase), and a 7-day consolidation phase. The localization of neurotrophins (NGF, BDNF and NT-3) and their receptors tropomyosin-related kinases (TRKA, TRKB and TRKC) by immunostaining showed positive staining in bone forming cells in each stage, although the presence and staining intensity varied by cell type and phase. The expressions of NGF, BDNF and NT-3 by real-time polymerase chain reaction (real-time PCR) showed that the peak of the mRNA expression of NGF occurred 10 days after distraction. NT-3 increased during bone extension, but decreased when distraction stopped. In contrast, BDNF continued to increase gradually throughout the distraction and consolidation phases. These findings suggest that neurotrophins and their receptors may play different roles in endochondral and intramembranous ossification in distraction osteogenesis. The tension stress caused by distraction may stimulate the expression of neurotrophins and their receptors, and promote osteogenesis.

[Autoantigen and molecular chaperone in rheumatoid arthritis--their roles in metabolism of chondrocytes]. Reviewed

Hattori T, Takigawa M

Clinical calcium 16 ( 9 ) 1553 - 1556 2006.9

Language：Japanese Publishing type：Research paper (scientific journal)

The endoplasmic reticulum chaperone; heat shock protein/rheumatoid arthritis-related antigen (HSP47/RA-A47), in addition to its important intercellular functions for collagen maturation and secretion, has chondrocytes-destructive roles such as induction of endoplasmic reticulum (ER)-stress and metabolic gene expressions result in apoptosis when HSP47/RA-A47 is downregulated. Extracellular HSP47/RA-A47 may act as an autoantigen, but also regulate autoimmune inflammation. It is, therefore, a potential new biologic therapy for rheumatoid arthritis.

硬組織モジュレーターCCN/CTGFのIGFBPモジュレーターを介した軟骨細胞増殖・分化促進効果

川木晴美, 久保田聡, 湊雅直, 近藤誠二, 滝川正春

Journal of Oral Biosciences 48 ( Suppl. ) 113 - 113 2006.9

Pathogenic role of connective tissue growth factor (CTGF/CCN2) in osteolytic metastasis of breast cancer

T Shimo, S Kubota, N Yoshioka, S Ibaragi, S Isowa, T Eguchi, A Sasaki, M Takigawa

JOURNAL OF BONE AND MINERAL RESEARCH 21 ( 7 ) 1045 - 1059 2006.7

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BONE & MINERAL RES

Introduction: Connective tissue growth factor (CTGF/CCN2) is a mediator of local angiogenesis induced by breast cancer, but its role in osteolytic metastasis has not been evaluated. PTH-related peptide (PTHrP) is another critical factor in the development of the osteolytic metastasis. Using both in vivo and in vitro approaches, we studied whether/how neutralization of CCN2 prevented bone metastasis and how PTHrP signaling is related.
Materials and Methods: A mouse model of bone metastasis by human breast cancer cell line MDA231 was treated with a CCN2-neutralizing antibody, and osteolytic bone metastases were assessed on radiographs and immunohistochemistry. Ccn2 gene expression and transcription were examined by Northern blot and luciferase analysis. Immunoblot analysis and kinase inhibitors were used to identify the signaling pathways implicated. Anti-angiogenic/osteoclastogenic effects of ccn2 downregulation were also evaluated.
Results: Treatment of mice with a CCN2-neutralizing antibody greatly decreased osteolytic bone metastasis, microvasculature, and osteoclasts involved. The antibody also suppressed the growth of subcutaneous tumor in vivo and proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. Downregulation of ccn2 also repressed osteoclastogenesis. CCN2 expression was specifically observed in cancer cells producing PTHrP and type I PTH/PTHrP receptor (PTH1R) invaded the bone marrow, and PTHrP strongly upregulated ccn2 in MDA231 cells in vitro. Activation of protein kinase C (PKC) and protein kinase A (PKA) was necessary and sufficient for the stimulation of ccn2 by PTHrP. Indeed, inhibition of the extracellular signal-regulated kinase (ERK1/2), PKC, or PKA by specific inhibitors counteracted the stimulation of ccn2 expression. Incubation of MDA231 cells with PTHrP induced the activation of ERK1/2. Consistent with these findings, inhibition of PKC prevented PTHrP-induced ERK1/2 activation, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA), a stimulator of PKC, upregulated it.
Conclusions: CCN2 was critically involved in osteolytic metastasis and was induced by PKA- and PKC-dependent activation of ERK1/2 signaling by PTHrP. Thus, CCN2 may be a new molecular target for anti-osteolytic therapy to shut off the PTHrP-CCN2 signaling pathway.

DOI： 10.1359/JBMR.060416

結合組織成長因子CCN2/CTGFによる骨髄由来間質細胞の細胞接着、遊走の亢進とシグナル伝達経路の活性化

大野充昭, 藤澤拓生, 久保田聡, 園山亘, 秋山謙太郎, 西田崇, 滝川正春, 窪木拓男

日本骨代謝学会学術集会プログラム抄録集 24回 248 - 248 2006.7

Possible role of LRP1, a CCN2 receptor, in chondrocytes

K Kawata, T Eguchi, S Kubota, H Kawaki, M Oka, S Minagi, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 345 ( 2 ) 552 - 559 2006.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Low density lipoprotein receptor (LDLR)-related protein 1 (LRP1/CD91) is one of the receptors of CCN2 that conducts endochondral ossification and cartilage repair. LRP1 is a well-known endocytic receptor, but its distribution among chondrocytes remains to be elucidated. We herein demonstrate for the first time that the distribution of LRP1 in chondrocytes except for hypertrophic chondrocytes in vivo and in vitro. Interestingly, the LRP1 levels were higher in mature chondrocytic HCS-2/8 and osteoblastic SaOS-2 than in other cells, whereas the other LDLR family members involved in ossification were detected at lower levels in HCS-2/8. It was interesting to note that in HCS-2/8, LRP1 was observed not only on the cell surface and in the cytoplasm, but also in the nucleus. Exogenously added CCN2 was incorporated into HCS-2/8, which was partially co-localized with LRP1, and targeted to the recycling endosomes and nucleus as well as the lysosomes. These findings suggest specific roles of LRP1 in cartilage biology. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.04.109

Roles of PKC, PI3K and JNK in multiple transduction of CCN2/CTGF signals in chondrocytes

G Yosimichi, S Kubota, T Nishida, S Kondo, T Yanagita, K Nakao, T Takano-Yamamoto, M Takigawa

BONE 38 ( 6 ) 853 - 863 2006.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

CCN2/connective tissue growth factor (CCN2/CTGF) is known to promote both the proliferation and differentiation of chondrocytes, which actions are mediated by ERK and p38 MAPK, respectively. In this study, we first re-evaluated the involvement of multiple MAPKs therein and found that JNK also mediated such CCN2 signals. Thereafter, we further analyzed the roles of upstream kinases. The involvement of PKC, PI3K and PKA in the CCN2 signaling to promote the maturation, proliferation and terminal differentiation of a human chondrocytic cell line, HCS-2/8 and rabbit primary growth cartilage cells was investigated. As a result, the PKC inhibitor calphostin C repressed all of the effects of CCN2, which were represented by increased synthesis of DNA and proteoglycans and the display of alkaline phosphatase activity. In addition, evaluation of the effect of the PI3K inhibitor wortmannin disclosed the contribution of PI3K in transducing CCN2 signals to promote chondrocyte hypertrophy. This signal was known to be mediated by PKB, which was translocated into the nucleus upon CCN2 stimulation. Of note, calphostin C showed inhibitory effects on the activation of p38 MAPK, ERK and also PKB, whereas it exerted no effect on JNK activation. These results suggest that PKC is a driver of multiple signal transducing kinases that promote the proliferation and differentiation of chondrocytes. The requirement of PI3K in transmitting the signal for terminal differentiation and PKC-independent signaling pathways for the promotion of chondrocytic growth and differentiation, which was mediated by JNK, were also uncovered. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2005.11.016

Dexamethasone induces connective tissue growth factor expression in renal tubular epithelial cells in a mouse strain-specific manner

H Okada, T Kikuta, T Inoue, Y Kanno, S Ban, T Sugaya, M Takigawa, H Suzuki

AMERICAN JOURNAL OF PATHOLOGY 168 ( 3 ) 737 - 747 2006.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC

Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor-beta 1, mediates mesangial cell/fibroblast proliferation and extracellular matrix production by renal cells. Here, we show that renal tubular epithelial cells from patients with minimal change nephritic syndrome produced CTGF after glucocorticoid treatment. in addition, the glucocorticoid dexamethasone (DEX) increased CTGF mRNA levels in the kidneys of C57B6 but not SJL mice and produced intermediate CTGF mRNA levels in the kidneys of F1 (C57B6 X SJL) mice, midway between the levels found for parental strains. DEX also increased CTGF mRNA levels in cultured tubular epithelial cells derived from C57B6 (mProx24) but not SJL (MCT) mice via transcriptional up-regulation of CTGF mRNA. Transient transfection experiments using luciferase reporter constructs bearing CTGF promoter fragments revealed that the -897-to-628-bp fragment contained DEX-responsive positive regulatory elements, which were active in mProx24 but not MCT cells. Long-term DEX treatment resulted in fibronectin deposition in the kidneys of C57B6 but not SJL mice, and this effect was inhibited by co-administration of CTGF anti-sense oligodeoxynucleotides. Thus, glucocorticoid-induced renal fibrogenesis seems to be influenced by genetic background, with the critical DEX-responsive elements in the -897-to-628-bp region of the CTGF promoter.

DOI： 10.2353/ajpath.2006.050656

Transcriptional regulation of the cartilage intermediate layer protein (CILP) gene Reviewed

M Mori, M Nakajima, Y Mikami, S Seki, M Takigawa, T Kubo, S Ikegawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 341 ( 1 ) 121 - 127 2006.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Cartilage intermediate layer protein (CILP) is an extracellular matrix protein abundant in cartilaginous tissues. CILP is implicated in common musculoskeletal disorders, including osteoarthritis and lumbar disc disease. Regulation of the CILP gene is largely unknown, however. We have found that CILP mRNA expression is induced by TGF-beta 1 and dependent upon signaling via TGF-beta receptors. TGF-beta 1 induction of CILP is mediated by Smad3, which acts directly through cis-elements in the CILP promoter region. Pathways other than Smad3 also arc involved in TGF-beta 1 induction of CILP. These observations, together with the finding that CILP protein binds and inhibits TGF-beta 1, suggest that CILP and TGF-beta 1 may form a functional feedback loop that controls chondrocyte metabolism. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.12.159

Locally produced estrogen promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation and decreased apoptosis

AS Chagin, D Chrysis, M Takigawa, EM Ritzen, L Savendahl

JOURNAL OF ENDOCRINOLOGY 188 ( 2 ) 193 - 203 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC ENDOCRINOLOGY

The importance of estrogens for the regulation of longitudinal bone growth is unequivocal. However, any local effect of estrogens in growth plate cartilage has been debated. Recently, several enzymes essential for estrogen synthesis were shown to be expressed in rat growth plate chondrocytes. Local production of 17 beta-estradiol (E2) has also been demonstrated in rat costal chondrocytes. We aimed to determine the functional role of locally produced estrogen in growth plate cartilage. The human chondrocyte-like cell line HCS-2/8 was used to study estrogen effects on cell proliferation (3 H-labeled thymidine uptake) and apoptosis (cell death detection ELISA kit). Chondrocyte production of E2 was measured by RIA and organ cultures of fetal rat metatarsal bones were used to study the effects of estrogen on longitudinal growth rate. We found that significant amounts of E2 were produced by HCS-2/8 chondrocytes (64(.)1 +/- 5(.)3 fmol/3 days/10(6) cells). The aromatase inhibitor letrozole (1 mu M) and the pure estrogen receptor antagonist ICI 182,780 (10 mu M) inhibited proliferation of HCS-2/8 chondrocytes by 20% (P < 0(.)01) and almost 50% (P < 0(.)001), respectively. Treatment with ICI 182,780 (10 mu M) increased apoptosis by 228% (P < 0(.)05). Co-treatment with either caspase-3 or pan-caspase inhibitors completely blocked ICI 182,780-induced apoptosis (P < 0(.)001 vs ICI 182,780 only). Moreover, both ICI 182,780 (10 mu M) and letrozole (1 mu M) decreased longitudinal growth of fetal rat metatarsal bones after 7 days of culture (P < 0-01). In conclusion, our data clearly show that chondrocytes endogenously produce E2 and that locally produced estrogen stimulates chondrocyte proliferation and protects from spontaneous apoptosis. In addition, longitudinal growth is promoted by estrogens locally produced within the epiphyseal growth plate.

DOI： 10.1677/joe.1.06364

Hypoxic regulation of stability of connective tissue growth factor/CCN2 mRNA by 3 '-untranslated region interacting with a cellular protein in human chondrosarcoma cells

S Kondo, S Kubota, Y Mukudai, N Moritani, T Nishida, H Matsushita, S Matsumoto, T Sugahara, M Takigawa

ONCOGENE 25 ( 7 ) 1099 - 1110 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Connective tissue growth factor (CTGF/CCN2) can be induced by various forms of stress such as exposure to high glucose, mechanical load, or hypoxia. Here, we investigated the molecular mechanism involved in the induction of ctgf/ccn2 by hypoxia in a human chondrosarcoma cell line, HCS-2/8. Hypoxia increased the ctgf/ccn2 mRNA level by altering the 3'-untranslated region (UTR)-mediated mRNA stability without requiring de novo protein synthesis. After a series of extensive analyses, we eventually found that the cis-repressive element of 84 bases within the 3'-UTR specifically bound to a cytoplasmic/nuclear protein. By conducting a UV crosslinking assay, we found the cytoplasmic/nuclear protein to be a 35 kDa molecule that bound to the cis-element in a hypoxia-inducible manner. These results suggest that a cis-element in the 3'-UTR of ctgf/ccn2 mRNA and trans-factor counterpart(s) play an important role in the post-transcriptional regulation by determining the stability of ctgf/ccn2 mRNA.

DOI： 10.1038/sj.onc.1209129

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha 5 beta 1

M Hoshijima, T Hattori, M Inoue, D Araki, H Hanagata, A Miyauchi, M Takigawa

FEBS LETTERS 580 ( 5 ) 1376 - 1382 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha 5 beta 1 integrin (alpha 5 beta 1), but not by one against anti-alpha v beta 3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha 5 beta 1 is involved in this adhesion. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2006.01.061

Cartducin, a paralog of Acrp30/adiponectin, is induced during chondrogenic differentiation and promotes proliferation of chondrogenic precursors and chondrocytes

T Maeda, A Jikko, M Abe, T Yokohama-Tamaki, H Akiyama, S Furukawa, M Takigawa, S Wakisaka

JOURNAL OF CELLULAR PHYSIOLOGY 206 ( 2 ) 537 - 544 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

We previously reported that CORS26 gene, isolated from C3H10T1/2 cells treated with transforming growth factor-beta 1, was predominantly expressed in cartilage. Because the gene product is a kind of secretory protein produced by cartilage tissue, we named it "cartducin". Cartducin shares a similar modular organization to adipocyte-derived hormone, adiponectin. In this Study, we investigated cartducin function during chondrogenesis and cartilage development. In situ hybridization analysis showed that cartducin transcripts were restricted to the proliferating chondrocytes in the growth plate cartilage. Whole-mount in situ hybridization revealed that the first significant induction of cartducin expression occurred in the sclerotome, which contains a chondrogenic cell lineage between days 9.5 and 10.5 postcoitus (p.c.) during mouse embryogenesis. Chondrogenic differentiation by combined treatment with bone morphogenetic protein-2 and insulin induced cartducin expression along with type II and IX collagen expression in chondrogenic progenitor N1511 cells. To elucidate the direct action of cartducin on the cells, recombinant cartducin protein was expressed in and purified from Escherichia coli. The recombinant cartducin potentially forms homo-oligomers and promoted the proliferation of chondrogenic progenitor N1511 cells, and chondrocytic HCS-2/8 cells in a dose-dependent manner. On the other hand, cartducin did not affect the production of sulfated glycosarninoglycan (sGAG) in these cells. These findings indicate that cartducin is a novel growth factor and plays important roles in regulating both chondrogenesis and cartilage development by its direct stimulatory action on the proliferation of chondrogenic precursors and chondrocytes.

DOI： 10.1002/jcp.20493

Novel angiogenic inhibitor DN-9693 that inhibits post-transcriptional induction of connective tissue growth factor (CTGF/CCN2) by vascular endothelial growth factor in human endothelial cells

S Kondo, N Tanaka, S Kubota, Y Mukudai, G Yosimichi, T Sugahara, M Takigawa

MOLECULAR CANCER THERAPEUTICS 5 ( 1 ) 129 - 137 2006.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Connective tissue growth factor (CTGF/CCN2) is a potent angiogenic factor. In this report, we describe for the first time that vascular endothelial growth factor (VEGF)mediated induction of the ctgf/ccn2 gene was a posttranscriptional event that was inhibited by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells. Steady-state mRNA levels of ctgf/ccn2 were remarkably increased by VEGF in a concentration-dependent manner, whereas the activity of the ctgf/ccn2 promoter was not responsive to VEGF as confirmed by a reporter gene assay and quantitative real-time PCR analysis. By employing a RNA degradation assay, we eventually found that the observed increase in the ctgf/ccn2 mRNA level was due to an increased stability of the mRNA induced by VEGF. DN-9693 at a dose of 0.1 to 2 ng/mL did not affect basal levels of ctgf/ccn2 mRNA; however, enhancement of ctgf/ccn2 mRNA expression by VEGF was specifically inhibited by DN-9693. Of importance, the inhibitory effects could be also ascribed to post-transcriptional regulation, because the VEGF-mediated increase in stability of ctgf/ccn2 mRNA was suppressed by DN-9693. Furthermore, we investigated the effects of DN-9693 on VEGF-induced activation of three subgroups of mitogen-activated protein kinase pathways and found that DN-9693 blocked the activation of these pathways by VEGF. These results suggest that VEGF increases ctgf/ccn2 mRNA stability through mitogen-activated protein kinase-mediated intracellular signaling cascade(s), which can be inhibited posttranscriptionally by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells.

DOI： 10.1158/1535-7163.MCT-05-0097

軟骨組織の発生分化とCCNファミリー遺伝子

久保田聡, 滝川正春

Clinical Calcium 16,486-492 2006

関節リウマチにおける自己抗原と分子シャペロン?軟骨細胞におけるその役割

服部高子, 滝川正春

Clinical Calcium 16,1553-1556 ( 9 ) 1553 - 1556 2006

Language：Japanese Publisher：(株)医薬ジャーナル社

小胞体シャペロンheat shock protein/rheumatoid arthritis-related antigen(HSP47/RA-A47)は細胞内のコラーゲン成熟、分泌の機能を持ち、細胞をストレスから防御し、未成熟あるいは正常な合成に失敗したコラーゲンの細胞外への分泌を妨げる役割を担っているが、リウマチ軟骨ではその発現量が減少しており、細胞に小胞体ストレスを誘発している。また、軟骨細胞破壊につながる細胞障害性因子の放出を促し、その結果軟骨細胞はアポトーシスにより死ぬ。また、この時細胞外にHSP47/RA-A47が放出される。細胞外HSP47/RA-A47は自己抗原として認識される可能性があるが、抗原抗体反応およびそれにかかわる炎症反応を制御している可能性もある。HSP47/RA-A47の細胞内外の量のコントロールがリウマチ治療の鍵となろう。(著者抄録)

Expression and regulation of an antisense RNA transcript of the human connective tissue growth factor gene in human tumour cells

Seiji Kondo, Satoshi Kubota, Harumi Kawaki, Norifumi Moritani, Toshimasa Kagawa, Takaaki Ueno, Toshio Sugahara, Masaharu Takigawa

Asian Journal of Oral and Maxillofacial Surgery 18 ( 3 ) 172 - 179 2006

Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Communications International Ltd

Objective: To characterise a natural antisense transcript of connective tissue growth factor. Materials and Methods: RNA from several cell lines was analysed by RNase protection assay to detect antisense transcripts. To characterise the regulatory aspect of the transcribed area, chimeras were constructed in which the firefly luciferase gene was fused with the corresponding segment of ctgf/ccn2, and the gene expression was monitored. Results: A natural antisense transcript complementary to the 3′-untranslated region of ctgf/ccn2 mRNA in cultured human tumour cells was detected. The luciferase gene fused with the full-length antisense 3′-untranslated region showed strikingly low levels of luciferase expression compared with the control. Based on a series of deletion analyses, the major repressive element was located in the middle portion within the 3′-untranslated region, which corresponded to the antisense transcribed area. Conclusion: These results suggest that controlled expression of antisense RNAs against the 3′-untranslated region of ctgf/ccn2 mRNA may be involved in the determination of phenotypes in certain oral malignancies. © 2006 Asian Association of Oral and Maxillofacial Surgeons.

DOI： 10.1016/S0915-6992(06)80014-2

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells

Masahiro Asano, Satoshi Kubota, Tohru Nakanishi, Takashi Nishida, Tomoichiro Yamaai, Gen Yosimichi, Kazumi Ohyama, Tomosada Sugimoto, Yoji Murayama, Masaharu Takigawa

Cell Communication and Signaling 3 11(Epub) 2005.10

Language：English Publishing type：Research paper (scientific journal)

Background: CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro. Results: In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium
whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis. Conclusion: These results taken together suggest important roles of CCN2/ CTGF in the development and regeneration of periodontal tissue including the periodontal ligament. © 2005 Asano et al
licensee BioMed Central Ltd.

DOI： 10.1186/1478-811X-3-11

The human chondrosarcoma HCS-2/8 cell line is responsive to BMP-7, but not to IL-1beta

J Saas, M Gebauer, C Jacobi, J Haag, M Takigawa, T Aigner

FRONTIERS IN BIOSCIENCE 10 2027 - 2035 2005.9

Language：English Publishing type：Research paper (scientific journal) Publisher：FRONTIERS IN BIOSCIENCE INC

Cultures of primary chondrocytes as in vitro model systems for studying the cellular behavior of chondrocytes are notoriously difficult to cultivate and propagate. One way to circumvent these problems appears to be the use of immortalized/immortal chondrocytic cell lines. In the present study, we were interested whether the chondrosarcoma derived HCS-2/8 cells are suitable for studying major cellular reaction pattern in response to key anabolic (BMP-7) and catabolic (IL-1beta) factors. Therefore, we used cDNA array and real-time PCR technology in order to evaluate gene expression triggerd by stimulation with IL-1beta (0,1-100 ng/ml) and BMP-7 in confluent monolayer cultures. HCS-2/8 cells hardly responded to IL-1beta, but showed good responsiveness to BMP-7. We found 12 genes up- and 17 significantly down-regulated by BMP-7 ( out of 340 investigated genes). Besides the expected activation of anabolic genes chondrocytic cells after BMP-stimulationtry to neutralize activation of the BMP-signalling cascade by expressing intra- and extracellular BMP-antagonists. Chondrosarcoma derived cell lines are a potential substitute for primary articular chondrocytes promising consistent expression of a differentiated chondrocyte phenotype with sufficient proliferative capacity. However, as shown by this study one needs to carefully select the cell line depending on the effects which one intends to study. In this respect, HCS-2/8 cells are a validated tool for studying BMP-effects on chondrocytes, but not e. g. effects of interleukin-1.

軟骨細胞におけるCCN familyメンバーのdexahamethasoneによる遺伝子発現調節

川木晴美, 久保田聡, 近藤誠二, 滝川正春

Journal of Oral Biosciences 47 ( Suppl. ) 86 - 86 2005.9

新規血管新生阻害剤DN-9693の作用機序 VEGFによる血管新生因子CTGF/CCN2の発現レベルの上昇に対する阻害効果

近藤誠二, 田中紀子, 久保田聡, 菅原利夫, 滝川正春

日本癌学会総会記事 64回 134 - 134 2005.9

ヒト軟骨肉腫由来軟骨細胞株 (HCS-2/8)におけるWnt誘導分泌タンパク質1 (Wisp1/CCN4)mRNAスプライス変異体(Writ-induced secreted protein 1(Wispl/CCN4) mRNA splicing variants in a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8))

Yanagita Takeshi, Kubota Satoshi, Hattori Takako, Hoshijima Mitsuhiro, Kawata Kazumi, Takano-Yamamoto Teruko, Takigawa Masaharu

生化学 77 ( 8 ) 1014 - 1014 2005.8

Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development

T Yamaai, T Nakanishi, M Asano, K Nawachi, G Yoshimichi, K Ohyama, T Komori, T Sugimoto, M Takigawa

JOURNAL OF BONE AND MINERAL METABOLISM 23 ( 4 ) 280 - 288 2005.7

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER TOKYO

Connective tissue growth factor (CTGF/CCN2), one of the most recently described growth factors, is produced by chondrocytes, vascular endothelial cells, and transforming growth factor (TGF)-beta-stimulated fibroblasts. CTGF was isolated from a chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and found to be normally expressed in cartilage tissues, especially in hypertrophic chondrocytes, and also to stimulate both the proliferation and the differentiation of chondrocytes in vitro. Therefore, CTGF is thought to be one of the most important regulators of endochondral ossification in vivo. Herein we describe the expression pattern of the ctgf gene in the calcifying tissues of normal developing mouse embryos in comparison with that in core binding factor a1 (Cbfa1)-targeted mutant (cbfa1-null) mouse embryos, in which impaired development and growth were characteristically observed in the skeletal system. After 15 days of development (E15), the expression of ctgf was detected in the zone of hypertrophy and provisional calcification, in which ossification proceeds toward the epiphysis during the skeletal development of the mouse embryo. Furthermore, ctgf was expressed in developing molar and incisal tooth germs around the perinatal stage. However, no expression of the gene was found in the cbfa1-null mouse embryos. These results indicate that CTGF may have certain important roles in the development of the calcifying tissues in the mouse embryo.

DOI： 10.1007/s00774-004-0600-5

Translational repression by the cis-acting element of structure-anchored repression (CAESAR) of human ctgf/ccn2 mRNA

S Kubota, Y Mukudai, NH Moritani, K Nakao, K Kawata, M Takigawa

FEBS LETTERS 579 ( 17 ) 3751 - 3758 2005.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The cis-acting element of structure-anchored repression (CAESAR) is a post-transcriptional regulatory element of gene expression, which is located in the 3'-untranslated region (UTR) of the human ccn2 gene (ctgflccn2). In this report, the repression mechanism of CAESAR, as well as the structural requirement, was investigated. Removal of minor stem-loops from CAESAR resulted in proportional attenuation of the repressive function, whereas removal of the single bulge or modification of primary nucleotide sequence did not affect its functionality. In light of functional mechanism, CAESAR exerted no significant effects on stability or nuclear export of the cis-linked mRNA. However, this element significantly interfered with the association of such mRNA on ribosome and slowed down the translation process thereafter in vitro. A translation repression mechanism by RNA secondary structure to determine the basal ctgflccn2 expression level was uncovered herein. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.05.068

CONNECTIVE TISSUE GROWTH FACTOR MEDIATES PROFIBROTIC EFFECTS OF TRANSFORMING GROWTH FACTOR-B PRODUCED BY TUBULAR EPITHELIAL CELLS IN RESPONSE TO HIGH GLUCOSE

Nobutaka Kato, Hirokazu Okada, Tatsuya Kobayashi, Tsutomu Inoue, Tomohiro Kikuta, Yoshihiko Kanno, Yusuke Watanabe, Soichi Sugahara, Hitoshi Hoshi, Keita Sueyoshi, Hiromichi Suzuki

NEPHROLOGY 10 A24 - A24 2005.6

二次骨化中心形成過程に発現する結合組織成長因子CTGF/CCN2のパールカン陽性軟骨細胞への特異的集積

岡森彦, 久保田聡, 近藤誠二, 江口傑徳, 河田かずみ, 黒田知沙, 皆木省吾, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 23回 229 - 229 2005.6

低酸素組織,軟骨における肥大軟骨特異的蛋白24/結合組織成長因子/CCNファミリー2mRNAの安定化機構核および細胞質タンパク結合を介した3'-非翻訳領域(UTR)の関与

近藤誠二, 久保田聡, 椋代義樹, 森谷徳文, 西田崇, 菅原利夫, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 23回 159 - 159 2005.6

結合組織成長因子CCN2/CTGF/Hcs24はヒト骨髄由来間質細胞の細胞接着を促進させる

大野充昭, 園山亘, 藤澤拓生, 秋山謙太郎, 前川賢治, 完山学, 西田崇, 久保田聡, 滝川正春, 窪木拓男

日本骨代謝学会学術集会プログラム抄録集 23回 225 - 225 2005.6

軟骨由来多機能成長因子CCN2/CTGF/Hcs24は耳介軟骨細胞の形質発現を増強する

藤澤拓生, 中尾匡志, 服部高子, 久保田聡, 窪木拓男, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 23回 262 - 262 2005.6

軟骨細胞における低密度リポタンパク受容体関連タンパク1(LRP1)の遺伝子発現とタンパク質局在

河田かずみ, 江口傑徳, 久保田聡, 川木晴美, 岡森彦, 皆木省吾, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 23回 260 - 260 2005.6

各種軟骨細胞におけるM-CSFの産生とその生理的役割

中尾匡志, 久保田聡, 藤澤拓生, 岡森彦, 江口傑徳, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 23回 177 - 177 2005.6

Connective tissue growth factor causes persistent pro alpha 2(l) collagen gene expression induced by transforming growth factor-beta in a mouse fibrosis model

S Chujo, F Shirasaki, S Kawara, Y Inagaki, T Kinbara, M Inaoki, M Takigawa, K Takehara

JOURNAL OF CELLULAR PHYSIOLOGY 203 ( 2 ) 447 - 456 2005.5

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteine-rich mitogenic peptide that is implicated in various fibrotic disorders and induced in fibroblasts after activation with transforming growth factor-beta (TGF-beta). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF-beta transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF-beta caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF-beta and CTGF in vivo, we investigated in this study, the effects of growth factors on the promoter activity of the pro alpha 2 (1) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial P-galactosidase gene. Serial injections of CTGF after TGF-beta resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF-beta alone on day 8. We also demonstrated that the number of fibroblasts with activated COL1A2 transcription was increased by serial injections of CTGF after TGF-beta in comparison with the injection of TGF-beta alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF-beta-induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated fibroblasts. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis. (c) 2004 Wiley-Liss, Inc.

DOI： 10.1002/jcp.20251

Collaborative action of M-CSF and CTGF/CCN2 in articular chondrocytes: Possible regenerative roles in articular cartilage metabolism

K Nakao, S Kubota, H Doi, T Eguchi, M Oka, T Fujisawa, T Nishida, M Takigawa

BONE 36 ( 5 ) 884 - 892 2005.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

It is known that expression of the macrophage colony-stimulating factor (M-CSF) gene is induced in articular chondrocytes upon inflammation. However, the functional role of M-CSF in cartilage has been unclear. In this study, we describe possible roles of M-CSF in the protection and maintenance of the articular cartilage based on the results of experiments using human chondrocytic cells and rat primary chondrocytes. Connective tissue growth factor (CTGF/CCN2) is known to be a potent molecule to regenerate damaged cartilage by promoting the growth and differentiation of articular chondrocytes. Here, we uncovered the fact that M-CSF induced the mRNA expression of the ctgf/ccn2 gene in those cells. Enhanced production of CTGF/CCN2 protein by M-CSF was also confirmed. Furthermore, M-CSF could autoactivate the m-csf gene, forming a positive feed-back network to amplify and prolong the observed effects. Finally, promotion of proteoglycan synthesis was observed by the addition of M-CSF. These findings taken together indicate novel roles of M-CSF in articular cartilage metabolism in collaboration with CTGF/CCN2, particularly during an inflammatory response. Such roles of M-CSF were further supported by the distribution of M-CSF producing chondrocytes in experimentally induced rat osteoarthritis cartilage in vivo. &COPY; 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2004.10.015

Comparable response of ccn1 with ccn2 genes upon arthritis: An in vitro evaluation with a human chondrocytic cell line stimulated by a set of cytokines

Norifumi H. Moritani, Satoshi Kubota, Toshio Sugahara, Masaharu Takigawa

Cell Communication and Signaling 3 6 - e-Pub 2005.4

Language：English Publishing type：Research paper (scientific journal)

Background: The chondrosarcoma-derived HCS-2/8 has been known to be an excellent model of human articular chondrocytes. By mimicking the arthritic conditions through the treatment of HCS-2/8 cells with cytokines, we estimated the gene expression response of ccn1 and ccn2 during the course of joint inflammation in vitro. Results: In order to mimic the initiation of inflammation, HCS-2/8 cells were treated with tumor necrosis factor (TNF)-α. To induce pro-inflammatory or reparative responses, TGF-β was employed. Effects of an anti-inflammatory glucocorticoid were also evaluated. After stimulation, expression levels of ccn1 and ccn2 were quantitatively analyzed. Surprisingly, not only ccn2, but also ccn1 expression was repressed upon TNF-α stimulation, whereas both mRNAs were uniformly induced by transforming growth factor (TGF)-β and a glucocorticoid. Conclusion: These results describing the same response during the course of inflammation suggest similar and co-operative roles of these 2 ccn family members in the course of arthritis. © 2005 Moritani et al
licensee BioMed Central Ltd.

DOI： 10.1186/1478-811X-3-6

Methylation status of EXT1 and EXT2 promoters and two mutations of EXT2 in chondrosarcoma

T Tsuchiya, T Osanai, A Ogose, G Tamura, T Chano, Y Kaneko, A Ishikawa, H Orui, T Wada, T Ikeda, M Namba, M Takigawa, H Kawashima, T Hotta, A Tsuchiya, T Ogino, T Motoyama

CANCER GENETICS AND CYTOGENETICS 158 ( 2 ) 148 - 155 2005.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Germline mutation and functional loss of EXT1 or EXT2 are commonly found in multiple osteochondrornas and predispose to the development of chondrosarcoma. Mutations of EXT1 and EXT2 have rarely been detected in sporadic secondary chondrosarcomas from osteochondrorna; these frequently display loss of heterozygosity at the EXT1 and EXT2 loci, but primary chondrosarcomas typically do not. To evaluate promoter methylation (which is an epigenetic gene silencing mechanism) of EXT1 and EXT2, we performed methylation-specific polymerase chain reaction (PCR) for 20 chondrosarcoma cases (12 primary, 3 secondary to osteochondroma, 2 secondary to enchondromatosis, 2 extraskeletal ordinary. and I clear cell) and in five cell lines. In addition, mutation analysis of the EXT1 and EXT2 coding regions was performed using PCR-single-strand conformation polymorphism and sequencing analysis for 12 of the 20 chondrosarcoma cases (8 primary, I secondary to enchondromatosis, I secondary to osteochondroma, and 2 extraskeletal ordinary) and five cell lines. Promoter methylation of EXT1 and EXT2 was not detected in any of the cases, and both EXT1 and EXT2 were expressed in all cell lines. Two missense mutations in EXT2 (D227E and R299H) were detected among the chondrosarcoma cases. When considering tumor development in primary chondrosarcoma, we should include mutations in EXT2, along with the status of other members of the EXT gene family. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cancergentyto.2004.08.031

Dexamethasone induces apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt-phosphatidylinositol 3 '-kinase signaling pathway

D Chrysis, F Zaman, AS Chagin, M Takigawa, L Savendahl

ENDOCRINOLOGY 146 ( 3 ) 1391 - 1397 2005.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

Although glucocorticoids are known to induce apoptosis in chondrocytes, the mechanisms for this effect and the potential antiapoptotic role of IGF-I are unknown. To address this, we studied the effects of dexamethasone ( Dexa) on apoptosis in the HCS-2/8 chondrocytic cell line. Dexa (25 muM) increased apoptosis (cell death ELISA) by 39% and 45% after 48 and 72 h, respectively (P < 0.01 and P < 0.05, respectively). IGF-I (100 ng/ml) decreased Dexa-induced apoptosis to levels similar to control cells. Apoptosis was associated with cleavage of poly-ADP-ribose polymerase (PARP) and alpha-fodrin and activation of caspases-8, -9, and -3 ( Western), an effect that was counteracted when chondrocytes were cocultured with Dexa + IGF-I. Inhibitors for caspases-8, -9, and -3 (50 muM each) equally suppressed Dexa-induced apoptosis (P < 0.01). Time-response experiments showed that caspase-8 was activated earlier ( at 12 h) than caspase-9 ( at 36 h). We studied the phosphatidylinositol 3'-kinase (PI3K) pathway to further investigate the mechanisms of Dexa-induced apoptosis. Dexa decreased Akt phosphorylation by 93% (P<0.001) without affecting total Akt and increased the p85alpha subunit 4-fold. The Akt inhibitor SH-6 (10 muM) increased apoptosis by 54% (P < 0.001). When combining Dexa with SH-6, apoptosis was not further increased, showing that Dexa-induced apoptosis is mediated through inhibition of the PI3K pathway. Addition of IGF-I to SH-6- or Dexa + SH-6-treated cells decreased apoptosis by 21.2% (P < 0.001) and 20.6% ( P < 0.001), respectively. We conclude that Dexa-induced apoptosis is caspase dependent with an early activation of caspase-8. IGF-I can rescue chondrocytes from Dexa-induced apoptosis partially through the activation of other pathways than the PI3K signaling pathway. Based on our in vitro data, we speculate that in vivo treatment with glucocorticoids may diminish longitudinal growth by increasing apoptosis of proliferative growth plate chondrocytes.

DOI： 10.1210/en.2004-1152

Regulation of chicken ccn2 gene by interaction between RNA cis-element and putative trans-factor during differentiation of chondrocytes

Y Mukudai, S Kubota, T Eguchi, S Kondo, K Nakao, M Takigawa

JOURNAL OF BIOLOGICAL CHEMISTRY 280 ( 5 ) 3166 - 3177 2005.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3'-untranslated region (3'-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3'-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.

DOI： 10.1074/jbc.M411632200

Downregulation of rheumatoid arthritis-related antigen RA-A47 (HSP47/colligin-2) in chondrocytic cell lines induces apoptosis and cell-surface expression of RA-A47 in association with CD9

T Hattori, K von der Mark, H Kawaki, Y Yutani, S Kubota, T Nakanishi, H Eberspaecher, B de Crombrugghe, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 202 ( 1 ) 191 - 204 2005.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Previously, we showed that gene expression of the rheurnatoid arthritis-related antigen RA-A47, which is identical to human heat shock protein (HSP)47, was downregulated in chondrocytes by inflammatory cytokines such as TNFalpha. Associated with this phenomenon, RA-A47 appeared on the cell surface concomitant with upregulation of metabolic factors related to cartilage destruction. The upregulation of the metabolic factors could be achieved by clownregulation of RA-A47 expression with ra-a47-specific anti-senseoligonucleotide. Here, we show that the enhanced surface expression of RA-A47 on a chondrocytic cell line, HCS-2/ 8 was also a direct result of RA-A47 downregulation by ra-a47 anti-sense oligonucleoticle, independent of the cytokine effects. Moreover, cell-surface expression of CD9, a l integrin-associated transmembrane protein that is involved in cell adhesion and cell motility events, was enhanced in the ra-a47 anti-sense oligonucleoticle-treated cells. The CD9 was colocalized with RA-A47 on the cell surface, where it may have affected integrin signaling. Furthermore, Annexin-V binding to the cell surface and the level of a number of apoptosis-related genes including caspase-9 were increased after ra-a47 anti-sense oligonucleoticle treatment, suggesting that enhanced surface expression of RA-A47 and CD9 may be initiating apoptosis. Differential screening using a cDNA gene array showed induction of metal lothionein-III and chemokine receptor CXCR4 and of factors of the Notch signaling pathway by the anti-sense treatment, but not by TNFalpha.. Thus, here we show for the first time an alternative mechanism of inducing apoptosis by downregulating molecular chaperones, independent of the action of TNFalpha. The surface-exposed RA-A47 may induce autoantibodies and inflammatory reactions in autoimmune disease situations such as rheurnatoid arthritis. J. Cell. Physiol. 202: 191 -204, 2005. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/jcp.20112

Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis

H Okada, T Kikuta, T Kobayashi, T Inoue, Y Kanno, M Takigawa, T Sugaya, JB Kopp, H Suzuki

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 16 ( 1 ) 133 - 143 2005.1

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Connective tissue growth factor (CTGF) is one of the candidate factors that are thought to mediate the downstream profibrotic action of TGF-beta. However, its precise role in renal interstitial fibrogenesis has not yet been clarified. It was demonstrated previously that CTGF was expressed in tubular epithelial cells that had been engulfed by interstitial fibrosis in the remnant kidney of the subtotal nephrectomy (SNx) model. In the present study, co-cultures of tubular epithelial cells (mProx24) and tubulointerstitial fibroblasts (TFB) that mimic the subepithelial mesenchyme in the kidney were used to study the profibrotic effects of TGF-beta1-induced CTGF. In these co-cultures, TGF-beta1 treatment resulted in significantly increased mRNA levels of type I collagen and fibronectin in the TFB. These effects were both direct and indirect, with the latter being mediated by CTGF derived from the co-cultured mProx24. Then TGF-beta1 transgenic mice were subtotally nephrectomized and treated with CTGF antisense oligodeoxynucleotide, and their kidneys were analyzed for fibrosis. Intravenous administration of CTGF antisense oligodeoxynucleotide significantly blocked CTGF expression in the proximal tubular epithelial cells in the remnant kidney of these animals despite the sustained level of TGF-beta1 mRNA. This reduction in CTGF mRNA level paralleled a reduction in mRNA levels of matrix molecules as well as proteinase inhibitors plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase-1, suppressing renal interstitial fibrogenesis. In conclusion, tubular CTGF acts as a downstream mediator of the profibrotic effects of TGF-beta1 in the remnant kidney, which is a promising target for antifibrotic drugs designed to treat TGF-beta1- dependent interstitial fibrosis.

DOI： 10.1681/ASN.2004040339

Introduction for CCN proteins.

Perbal, B, Takigawa, M

In CCN Proteins: A New Family of Cell Growth and Differentiation Regulators (Perbal B. & Takigawa M. eds.) 1 - 18 2005

Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage

M Gebauer, J Saas, J Haag, U Dietz, M Takigawa, E Bartnik, T Aigner

ARTHRITIS RESEARCH & THERAPY 7 ( 2 ) R274 - R284 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：BIOMED CENTRAL LTD

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal ( n = 9) and osteoarthritic ( n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1 beta and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage ( P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression.

DOI： 10.1186/ar1479

Increased levels of CTGF mRNA expression in a murine model of allergic airway inflammation

Hong Mei Piao, Kohei Yamauchi, Li-Hua Pan, Toshihide Nakadate, Harumasa Ito, Takashi Mouri, Hitoshi Kobayashi, Takashi Sawai, Tohru Nakanishi, Masaharu Takigawa, Hiroshi Inoue

Allergology International 54 ( 1 ) 107 - 115 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing

Background: Connective tissue growth factor (CTGF) is known to play a direct role in fibrosis in various organs as a downstream mediator of TGF-β. Objective: To evaluate a role in subepithelial fibrosis in the asthmatic airway, we investigated CTGF mRNA expression and CTGF producing cells in the airways of a murine asthma model with allergic inflammation. Methods: After repetitive inhalation challenges with ovalbumin (OVA), cell numbers and TGF-β1 concentrations in bronchoalveolar lavage fluid from immunized mice were measured. Collagen deposition in lung tissue was estimated by measuring hydroxyproline content. CTGF mRNA and GAPDH mRNA levels were determined by quantitative RT-PCR method. Immunohistochemistry for CTGF with anti-CTGF antibody was performed. Results: Numbers of eosinophils and TGF-β1 concentration increased markedly in BALF on the 7th day and 14 th day after inhalation challenge with OVA. Hydroxyproline content in lung tissue increased significantly on the 14th day after inhalation challenge of OVA compared to control. The ratio of CTGF mRNA /GAPDH mRNA in lung tissue in mice exposed to OVA increased 10-fold compared to those exposed to saline. Immunohistochemistry revealed that the number of CTGF-positive cells increased in bronchial submucosa after inhalation challenge of OVA. Conclusions: Our results suggested that CTGF might be one of the potential molecules involved in subepithelial fibrosis in murine airways with allergic inflammation. ©2005 Japanese Society of Allergology.

DOI： 10.2332/allergolint.54.107

CCNファミリー：その構造と機能. Invited

滝川正春

細胞 37,462-465 2005

Roles of CCN2/CTGF in the control of growth and regeneration.

Takigawa, M, Nishida, T, Kubota, S

In CCN Proteins: A New Family of Cell Growth and Differentiation Regulators (Perbal B. & Takigawa M. eds.) 19 - 59 2005

Cartducin, a Paralog of acrp30/adiponectin, is induced during chondrogenic differentiation and promotes proliferation of chondrogenic precursors and chondrocytes.

Maeda, T, Jikko, A, Abe, M, Yokohama-Tamaki, T, Akiyama, H, Furukawa, S, Takigawa, M, Wakisaka, S

J. Cell. Physiol. 206(2), 537 - 544, 2005

Molecular phenotyping of HCS-2/8 cells as an in vitro model of human chondrocytes

J Saas, K Lindauer, B Bau, M Takigawa, T Aigner

OSTEOARTHRITIS AND CARTILAGE 12 ( 11 ) 924 - 934 2004.11

Language：English Publishing type：Research paper (scientific journal) Publisher：W B SAUNDERS CO LTD

Objective: Cultures of primary articular chondrocytes for studying chondrocyte biology are notoriously difficult to handle. One alternative is the use of chondrocytic cell lines. Because the HCS-2/8 cells are the most widely used cell line in cartilage research, we investigated the molecular phenotype of these cells by mRNA-expression profiling.
Design: Monolayers of HCS-2/8 cells were cultured to sub-confluence, confluence and over-confluence; primary human chondrocytes were grown in monolayer culture and alginate-bead cultures and several other chondrocytic cell lines were cultured as monolayers. RNA was isolated and analyzed by cDNA array profiling using Affymetrix GeneChips (U95A/U95Av2) and quantitative PCR.
Results: Important similarities, but also remarkable differences between the HCS-2/8 cells and adult human articular chondrocytes were detected: Aggrecan and several cartilage typical collagens as well as SOX9 transcripts were strongly expressed in HCS-2/8 cells, whereas HCS-2/8 cells expressed hardly any chondrocyte-typical cartilage matrix degrading enzymes. Of all culturing conditions, clustering analysis showed that HCS-2/8 cultured at confluence are most closely related to primary chondrocytes.
Conclusion: Our study confirms how careful one needs to be in choosing in vitro model systems for investigating effects of interest. The major issue of chondrocyte cell lines appears to be that they mainly proliferate and show less expression of genes of matrix synthesis and turnover. A successful approach will have to select suitable chondrocyte cell lines and to validate findings obtained using primary chondrocytes. This allows to establish a reproducible in vitro model showing the property of interest and subsequently to relate back the obtained results to the physiologic situation. (C) 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2004.08.002

Implication of prostaglandin E-2 in TNF-alpha-induced release of m-calpain from HCS-2/8 chondrocytes. Inhibition of m-calpain release by NSAIDs

K Fushimi, S Nakashima, Y Banno, A Akaike, M Takigawa, K Shimizu

OSTEOARTHRITIS AND CARTILAGE 12 ( 11 ) 895 - 903 2004.11

Language：English Publishing type：Research paper (scientific journal) Publisher：W B SAUNDERS CO LTD

Objective: Calpains are known as Ca2+-dependent intracellular neutral cysteine proteases. However, m-calpain is detected in synovial fluid of arthritic joints and is shown to possess the proteoglycanase activity in vitro. The mechanism of m-calpain release into the extracellular spaces during arthritis has not yet been well characterized. In the present study, we have analyzed m-calpain release from cultured chondrocytes stimulated by a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on m-calpain release were also examined.
Methods: Human chondrocytic HCS-2/8 cells were stimulated by TNF-alpha in the presence or absence of an NSAID. m-Calpain in the cells and culture medium was quantified by Western blot analysis using an anti-m-calpain antibody. Western blots were subjected to densitometric analysis and band intensities were determined.
Results: TNF-alpha (10 ng/ml) stimulated m-calpain release with transient increase in cellular m-calpain in HCS-2/8 cells. NSAIDs examined (aspirin, loxoprofen-SRS, diclofenac sodium, indomethacin and NS398) inhibited m-calpain release and production of prostaglandin E-2 (PGE(2)) induced by 10 ng/ml TNF-alpha. Exogenously added PGE(2) accelerated the release of m-calpain in response to a lower concentration of TNF-alpha (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-alpha-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-alpha.
Conclusions: These results suggest that TNF-alpha stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE(2) receptor by produced PGE(2) is deeply involved in this process. (C) 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2004.08.001

Expression and localization of connective tissue growth factor (CTGF/Hcs24/CCN2) in osteoarthritic cartilage

S Omoto, K Nishida, Y Yamaai, M Shibahara, T Nishida, T Doi, H Asahara, T Nakanishi, H Inoue, M Takigawa

OSTEOARTHRITIS AND CARTILAGE 12 ( 10 ) 771 - 778 2004.10

Language：English Publishing type：Research paper (scientific journal) Publisher：W B SAUNDERS CO LTD

Objective: The investigation of the expression and localization of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24/CCN family member 2 (CTGF/Hcs24/CCN2) in normal and osteoarthritic (OA) cartilage, and quantification of CTGF/Hcs24-positive cells.
Methods: Cartilage samples of patients (n = 20) with late stage CA were obtained at total joint replacement surgery. Morphologically normal cartilage was harvested for comparison purposes from the femoral heads of 6 other patients with femoral neck fracture. Paraffin -embedded sections were stained by Safranin O. The severity of the OA lesions was divided into four stages (normal, early, moderate, and severe). The localization of protein and mRNA for CTGF/Hcs24 was investigated by immunohistochemistry and in situ hybridization, respectively. The population of CTGF/Hcs24-positive chondrocytes in CA cartilage and chondro-osteophyte was quantified by counting the number of the cells under light microscopy.
Results: Signals for CTGF/Hcs24 were detected in a small percentage of chondrocytes throughout the layers of normal cartilage. In early stage CA cartilage, the CTGF/Hcs24-positive chondrocytes were localized mainly in the superficial layer. In moderate to severe OA cartilage, intense staining for CTGF/Hcs24 was observed in proliferating chondrocytes forming cell clusters next to the cartilage surface. In chondro-osteophyte, strong signals were found in the chondrocytes of the proliferative and hypertrophic zones.
Conclusion: CTGF/Hcs24 expression was detected in both normal and OA chondrocytes of human samples. The results of the current study suggested that expression of CTGF/Hcs24 was concomitant with development of CA lesions and chondrocyte differentiation in chondro-osteophyte. (C) 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2004.06.009

Regeneration of defects in the articular cartilage in rat knee joints by connective tissue growth factor hypertrophic chondrocyte-specific gene product 24 CCN family member 2 (CTGF/Hcs24/CCN2).

T Nishida, S Kubota, T Kuboki, K Nakao, T Kushibiki, Y Tabata, M Takigawa

JOURNAL OF BONE AND MINERAL RESEARCH 19 S216 - S216 2004.10

Abundant retention and release of connective tissue growth factor (CTGF/CCN2) by platelets

S Kubota, K Kawata, T Yanagita, H Doi, T Kitoh, M Takigawa

JOURNAL OF BIOCHEMISTRY 136 ( 3 ) 279 - 282 2004.9

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE BIOCHEMICAL SOC

Wound healing and tissue regeneration are usually initiated by coagulation followed by fibrous tissue formation. In the present study, we discovered an abundance of connective tissue growth factor (CTGF/CCN2) in human platelets, which was released along with the coagulation process. The CTGF/CCN2 content in platelets was 10-fold higher than that in arterial tissue. Furthermore, the CTGF/CCN2 content in a single platelet was computed to be more than 20-fold higher than that of any other growth factor reported. Considering that CTGF/CCN2 promotes angiogenesis, cartilage regeneration, fibrosis and platelet adhesion, it may be now regarded as one of the major functional components of platelets.

DOI： 10.1093/jb/mvh126

Connective tissue growth factor causes persistent proa2(I) collagen gene expression induced by transforming growth factor-b in a mouse fibrosis model

S Chujo, F Shirasaki, T Kinbara, K Takehara, S Kawara, Y Inagaki

ARTHRITIS AND RHEUMATISM 50 ( 9 ) S624 - S625 2004.9

Differential regulation of biglycan and decorin synthesis by connective tissue growth factor in cultured vascular endothelial cells

T Kaji, C Yamamoto, M Oh-I, T Nishida, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 322 ( 1 ) 22 - 28 2004.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

It is possible that connective tissue growth factor (CTGF) serves as either an independent regulator or a downstream effector of transforming growth factor-beta (TGF-beta) on the proteoglycan synthesis in vascular endothelial cells. Since TGF-beta regulates endothelial proteoglycan synthesis in a cell density-dependent manner, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [S-35]sulfate or S-35-labeled amino acids in the presence of CTGF, and the labeled proteoglycans were characterized by biochemical techniques. The results indicate that CTGF suppresses the synthesis of biglycan but newly induced that of decorin in the cells when the cell density is low; in addition, no change was observed in the hydrodynamic size and the glycosaminoglycan chain length of these two small chondroitin/dermatan sulfate proteoglycans. The regulation of endothelial proteoglycan synthesis by CTGF is completely different from that by TGF-beta, suggesting that CTGF is not a downstream effector of TGF-beta but an independent regulator in vascular endothelial cells with respect to the proteoglycan synthesis. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.07.078

低酸素におけるCTGF mRNAの安定化機構核内タンパク質結合を介した3'-非翻訳領域(UTR)の関与

近藤誠二, 久保田聡, 森谷徳文, 滝川正春

日本癌学会総会記事 63回 400 - 400 2004.9

Regeneration of defects in articular cartilage in rat knee joints by CCN2 (connective tissue growth factor) Reviewed

T Nishida, S Kubota, S Kojima, T Kuboki, K Nakao, T Kushibiki, Y Tabata, M Takigawa

JOURNAL OF BONE AND MINERAL RESEARCH 19 ( 8 ) 1308 - 1319 2004.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BONE & MINERAL RES

CTGF/CCN2, a hypertrophic chondrocyte-specific gene product, possessed the ability to repair damaged articular cartilage in two animal models, which were experimental osteoarthritis and full-thickness defects of articular cartilage. These findings suggest that CTGF/CCN2 may be useful in regeneration of articular cartilage.
Introduction: Connective tissue growth factor (CTGF)/CCN2 is a unique growth factor that stimulates the proliferation and differentiation, but not hypertrophy, of articular chondrocytes in vitro. The objective of this study was to investigate the therapeutic use of CTGF/CCN2.
Materials and Methods: The effects of recombinant CTGF/CCN2 (rCTGF/CCN2) on repair of damaged cartilage were evaluated by using both the monoiodoacetic acid (MIA)-induced experimental rat osteoarthritis (OA) model and full-thickness defects of rat articular cartilage in vivo.
Results: In the MIA-induced OA model, quantitative real-time RT-PCR assays showed a significant increase in the level of CTGF/CCN2 mRNA, and immunohistochemical analysis and in situ hybridization revealed that the clustered chondrocytes, in which Clustering indicates an attempt to repair the damaged cartilage, produced CTGF/CCN2. Therefore, CTGF/CCN2 was suspected to play critical roles in cartilage repair. In fact, a single injection of rCTGF/CCN2 incorporated in gelatin hydrogel (rCTGF/CCN2-hydrogel) into the joint cavity of MIA-induced OA model rats repaired their articular cartilage to the extent that it became histologically similar to normal articular cartilage. Next, to examine the effect of rCTGF/CCN2 on the repair of articular cartilage, we created defects (2 mm in diameter) on the surface of articular cartilage in situ and implanted rCTGF/CCN2-hydrogel or PBS-hydrogel therein with collagen sponge. In the group implanted with rCTGF/CCN2-hydrogel collagen, new cartilage filled the defect 4 weeks postoperatively. In contrast, only soft tissue repair occurred when the PBS-hydrogel collagen was implanted. Consistent with these in vivo effects, rCTGF/CCN2 enhanced type II collagen and aggrecan mRNA expression in mouse bone marrow-derived stromal cells and induced chondrogenesis in vitro.
Conclusion: These findings suggest the utility of CTGF/CCN2 in the regeneration of articular cartilage.

DOI： 10.1359/JBMR.040322

二次骨化中心形成過程における結合組織成長因子CTGF/CCN2の発現血管新生因子としての関与

岡森彦, 久保田聡, 江口傑徳, 河田かずみ, 黒田知沙, 皆木省吾, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 22回 199 - 199 2004.8

ニワトリ軟骨細胞の分化過程におけるCCN2/CTGF遺伝子の転写後発現調節機構の解析

椋代義樹, 久保田聡, 江口傑徳, 近藤誠二, 中尾匡志, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 22回 159 - 159 2004.8

Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) during distraction osteogenesis

H Kadota, T Nakanishi, K Asaumi, T Yamaai, E Nakata, S Mitani, K Aoki, A Aiga, H Inoue, M Takigawa

JOURNAL OF BONE AND MINERAL METABOLISM 22 ( 4 ) 293 - 302 2004.7

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER JAPAN KK

To investigate the localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24/CCN family member 2 (CTGF/Hcs24/CCN2) during distraction osteogenesis in the rat femur, we studied a total of 54 male rats (11 weeks old). We performed osteotomy in the midshaft of the right femur. After 7 days (lag phase), distraction was started, at the rate of 0.25 mm/12 h for 21 days (distraction phase) by using a small external fixator, and this was followed by a 7-day consolidation phase. Localization and expression of CTGF/Hcs24 during distraction osteogenesis in the femur were examined by immunostaining, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). Immunostaining showed the localization of CTGF/Hcs24 in various cells located in the bone-forming area around the osteotomy site. During the distraction phase, in situ hybridization showed that CTGF/Hcs24 mRNA was expressed not only in hypertrophic chondrocytes and osteoblasts but also in fibroblast-like cells and mesenchymal cells at sites of end-ochondral ossification, and not only in osteoblasts but also in pre-osteoblasts and fibroblast-like cells at sites of intramembranous ossification. RT-PCR showed higher level expression of CTGF/Hcs24 mRNA in the distracted group than in the nondistracted group. These results revealed an elevated pattern of CTGF/Hcs24 mRNA expression during distraction osteogenesis, and suggest that CTGF/Hcs24 may play some roles in the endochondral and intramembranous ossification processes that occur during distraction osteogenesis.

DOI： 10.1007/s00774-004-0486-2

[Cartilage and mechanical stress from the point of the view of development, growth, pathology and therapeutic aspects].

Takuo Fujisawa, Masaharu Takigawa, Takuo Kuboki

Clinical calcium 14 ( 7 ) 29 - 35 2004.7

Language：Japanese Publishing type：Research paper (scientific journal)

Articular cartilage is always subjected to various types, magnitudes and cycles of mechanical stress. While it has been recognized that these stresses regulate the chondrocyte growth and differentiation, the mechanism is still unclear. Here, we summarize the effect of mechanical stress on cartilage metabolism from the point of view of development, growth, pathology and therapeutic aspect.

Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis

H Yokoi, M Mukoyama, T Nagae, K Mori, T Suganami, K Sawai, T Yoshioka, M Koshikawa, T Nishida, M Takigawa, A Sugawara, K Nakao

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 15 ( 6 ) 1430 - 1440 2004.6

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Connective tissue growth factor (CTGF/CCN2) is one of the candidate factors mediating fibrogenic activity of TGF-beta. It was shown previously that the blockade of CTGF by antisense oligonucleotide (ODN) inhibits TGF-beta-induced production of fibronectin and type I collagen in cultured renal fibroblasts. The in vivo contribution of CTGF in renal interstitial fibrosis, however, remains to be clarified. With the use of a hydrodynamics-based gene transfer technique, the effects of CTGF antisense ODN are investigated in rat kidneys with unilateral ureteral obstruction (UUO). FITC-labeled ODN injection via the renal vein showed that the ODN was specifically introduced into the interstitium. At day 7 after UUO, the gene expression of CTGF, fibronectin, fibronectin ED-A, and alphal(l) collagen in untreated or control ODN-treated obstructed kidneys was prominently upregulated. CTGF antisense ODN treatment, by contrast, markedly attenuated the induction of CTGF, fibronectin, fibronectin ED-A, and alphal(l) collagen genes, whereas TGF-beta gene upregulation was not affected. The antisense treatment also reduced interstitial deposition of CTGF, fibronectin ED-A, and type I collagen and the interstitial fibrotic areas. The number of myofibroblasts determined by the expression of alpha-smooth muscle actin was significantly decreased as well. Proliferation of tubular and interstitial cells was not altered with the treatment. These findings indicate that CTGF expression in the interstitium plays a crucial role in the progression of interstitial fibrosis but not in the proliferation of tubular and interstitial cells during UUO. CTGF may become a potential therapeutic target against tubulointerstitial fibrosis.

DOI： 10.1097/01.ASN.0000130565.69170.85

Gene expression profile of human chondrocyte HCS-2/8 cell line by EST sequencing analysis

YK Jung, JH Jeong, HM Ryoo, HN Kim, YJ Kim, EK Park, HJ Si, SY Kim, M Takigawa, BH Lee, RW Park, IS Kim, JY Choi

GENE 330 85 - 92 2004.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Large-scale single-pass sequencing of randomly selected cDNA clones from cell type specific libraries has proven to be a powerful approach for the discovery of novel gene functions, identification of novel gene family members, and definition of gene expression profiles. HCS-2/8 chondrocyte has been used as a cell culture model to study chondrocyte differentiation. Here we performed 3350 single-pass sequencing reactions obtained from the 5' ends of cDNAs from HCS-2/8 cells. To define the expression profiles of HCS-2/ 8 chondrocytes, we analyzed the identity of these representative cDNA sequences using database searches (BLAST). The sequences represent 1927 unique genes with known function (i.e., unigene clusters), 38 transcripts that are similar to genes with known function, 739 expressed genes with unknown function (i.e., expressed sequence tags), and 18 cDNAs which have not previously been sequenced. Interestingly, many transcripts,were expressed from chromosome 12 compared with total genes, while the fewer numbers of cDNAs were derived front genes on chromosomes 14, IS and Y. The chondrocytic phenotype of HCS-2/8 cells is reflected by abundant expression of,genes related to cell structure and motility and the 20 most frequently expressed unigenes reflect a chondrocyte-related gene expression signature. Thus. our data establish a representative set of more than 2000 genes expressed in a chondrocytic cell line. This finding provides a framework for understanding cell growth and differentiation of chondrocytes and their metabolic function in the formation and remodeling of cartilage. C (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2004.01.007

Module-specific antibodies against human connective tissue growth factor: Utility for structural and functional analysis of the factor as related to chondrocytes

M Minato, S Kubota, H Kawaki, T Nishida, A Miyauchi, H Hanagata, T Nakanishi, T Takano-Yamamoto, M Takigawa

JOURNAL OF BIOCHEMISTRY 135 ( 3 ) 347 - 354 2004.3

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE BIOCHEMICAL SOC

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1. antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.

DOI： 10.1093/jb/mvh042

Connective tissue growth factor(CTGF)の軟骨細胞特異的な転写調節機構の探索

江口傑徳, 久保田聡, 椋代義樹, 森谷徳文, 中尾匡志, 滝川正春

生化学 76 ( 3 ) 303 - 303 2004.3

軟骨とメカニカルストレス－発生、成長、病態、治療などとの観点から－。 Invited

藤澤拓生, 窪木拓男, 滝川正春

Clinical Calcium 14, 1049-1055 ( 7 ) 1049 - 1055 2004

Language：Japanese Publisher：(株)医薬ジャーナル社

関節はさまざまな種類,大きさ,頻度のメカニカルストレスにさらされている.メカニカルストレスは軟骨細胞に対してアナボリックにもカタボリックにも作用し,軟骨細胞の増殖,分化を調節していることが明らかになっているが,その詳細は不明である.そこで,軟骨の発生,成長,病態,治療などとの観点からメカニカルストレスの作用について考察し述べた

Downregulation of rheumatoid arthritis-related antigen RA-A47 (=HSP47/Colligin-2) in chondrocytic cell lines induces apoptosis and cell-surface expression of RA-A47 in association with CD9.

Hattori, T, von der, Mark, K, Kawaki, H, Yutani, Y, Kubota, S, Nakanishi, T, Eberspeacher, H, de Crombrugghe, B, Takigawa, M

J. Cell. Physiol. 202, 191 - 204, 2004

CCNファミリー． Invited

滝川正春

Molecular Medicine 41, 756-758 ( 6 ) 756 - 758 2004

Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis.

Okada H, Kikuta T, Kobayashi T, Inoue T, Kanno Y, Takigawa M, Sugaya T, Kopp JB, Suzuki H

J. Am. Soc. Nephrol. 16(1), 133 - 143, 2004

Regulation of chicken ccn2 gene by interaction between RNA cis-element and putative trans-factor during differentiation of chondrocytes.

Mukudai, Y, Kubota, S, Takanori, E, Kondo, S, Nakao, K, Takigawa, M

J. Biol. Chem. 280, 3166 - 3177, 2004

Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression

T Fukunaga, T Yamashiro, S Oya, N Takeshita, M Takigawa, T Takano-Yamamoto

BONE 33 ( 6 ) 911 - 918 2003.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2003.07.010

Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction

M Kanyama, T Kuboki, K Akiyama, K Nawachi, FM Miyauchi, H Yatani, S Kubota, T Nakanishi, M Takigawa

ARCHIVES OF ORAL BIOLOGY 48 ( 10 ) 723 - 730 2003.10

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Objective: To understand bone regeneration process after tooth extraction could be a clue to develop a new strategy for alveolar bone reconstruction. Recently, accumulated evidences support that connective tissue growth factor (CTGF) is implicated in tissue repair of many tissues. In this study, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets. Design: Five weeks old wild type mate rats (weighing 120 g) were used for this experiment. Expression of CTGF was determined by immunohistochemistry and in situ hybridization in the rat upper molar tooth extraction sockets at 2, 4, 7, 10 and 14 days after tooth extraction. Results: CTGF was expressed strongly in the endothelial. cells migrating into the granulation tissue at the bottom of the sockets during 4 days after tooth extraction. During the reparative process, no apparent chondrocyte-like cell appeared in the sockets, while osteoblast-like cells proliferated in the sockets with low CTGF expression at 7, 10, 14 days after extraction. As expected, no staining was observed with the preimmune rabbit IgG and CTGF sense probe. CTGF may play an important rote in angiogenesis and granulation tissue formation specifically at early heating stage after tooth extraction to initiate alveolar bone repair. Conclusion: CTGF was expressed at early heating stage of the rat tooth extraction wound. (C) 2003 Elsevier Ltd. All. rights reserved.

DOI： 10.1016/S0003-9969(03)00153-5

Novel enzyme-linked immunosorbent assay systems for the quantitative analysis of connective tissue growth factor (CTGF/Hcs24/CCN2): Detection of HTLV-I tax-induced CTGF from a human carcinoma cell line

H Kawaki, S Kubota, M Minato, NH Moritani, T Hattori, H Hanagata, M Kubota, A Miyauchi, T Nakanishi, M Takigawa

DNA AND CELL BIOLOGY 22 ( 10 ) 641 - 648 2003.10

Language：English Publishing type：Research paper (scientific journal) Publisher：MARY ANN LIEBERT INC PUBL

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.

Transcriptional induction of connective tissue growth factor/hypertrophic chondrocyte-specific 24 gene by dexamethasone in human chondrocytic cells Reviewed

S Kubota, NH Moritani, H Kawaki, H Mimura, M Minato, M Takigawa

BONE 33 ( 4 ) 694 - 702 2003.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Connective tissue growth factor (CTGF/Hcs24) is a critical growth factor for chondrocytic growth and differentiation. In this report, we describe for the first time glucocorticoid-mediated induction of the CTGF/Hcs24 gene in a chondrocytic cell line, HCS-2/8. Steady-state mRNA levels of CTGF/Hcs24 were remarkably increased after treatment with 50 nM dexamethasone, as confirmed by Northern blotting and quantitative real-time polymerase chain reaction (PCR) analysis. Corresponding to the increase in mRNA, production of CTGF/Hcs24 protein was remarkably enhanced, following a time course of up to 6 h. The observed increase in mRNA can be ascribed to transcriptional enhancement, since the stability of CTGF/Hcs24 mRNA was not affected by the same concentration of dexamethasone, which was indicated by the results of an mRNA degradation assay. However, unexpectedly, the prototypic ctgf/hcs24 promoter was not responsible for the dexamethasone stimulation, suggesting the glucocorticoid receptor binding site(s) to be elsewhere in the CTGF/Hcs24 gene. Enhancement of the prototypic promoter activity by dexamethasone was observed in murine fibroblastic cells, demonstrating the complexity of the regulatory mechanism of ctgf/hcs24 gene expression. Of importance, dexamethasone at the same concentration significantly stimulated proteoglycan synthesis in HCS-2/8 cells up to the same levels as exogenously added CTGF/Hcs24. These findings represent a novel effect of glucocorticoid on the production of CTGF/Hcs24 by chondrocytic cells, and indicate that CTGF/Hcs24 may mediate the stimulative effect of dexamethasone on chondrocytic phenotypes. Also, our results shed light on the complex mechanism of CTGF/Hcs24 induction by glucocorticoids. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S8756-3282(03)00227-8

Downregulation of a rheumatoid arthritis-related antigen (RA-A47) by ra-a47 antisense oligonucleotides induces inflammatory factors in chondrocytes

T Hattori, H Kawaki, S Kubota, Y Yutani, B De Crombrugghe, K Von Der Mark, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 197 ( 1 ) 94 - 102 2003.10

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Previously we have shown that the expression of RA-A47 (rheumatoid arthritis-related antigen) which is identical to HSP47, a collagen-binding chaperon, is downregulated in chondrocytes by tumor necrosis factor alpha (TNFalpha). RA-A47 was also found on the surface of chondrocytes where it is recognized as an antigen in the serum of rheumatoid arthritis (RA) patients. Its translocation to the cell surface from endoplasmic reticulum membrane where it is normally located was also enhanced by TNFalpha. To understand the significance of RA-A47 downregulation in chondrocytes independent from other effects of TNFalpha, we used an antisense oligonucleotide approach and investigated the effect of this treatment on the expression of molecules related to matrix degradation and production of growth factors for chondrocytic, endothelial, and synovial cells. Here we show that treatment of rabbit chondrocyes and human chondrosarcoma cells HCS-2/8 by ra-a47 antisense S-oligonucleotides significantly reduced the expression of ra-a47 both at mRNA and protein level. Interestingly, this TNFalpha-independent RA-A47 clownregulation was associated with a strong induction of matrix metalloproteinase (MMP)-9 mRNA and inducible NO synthase (iNOS) mRNA. The induction of active-type MMP-9 was further detected by gelatin zymography. Under the same conditions, the release of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) from HCS-2/8 cells into the conditioned medium (CM) was strongly enhanced. These effects were not a result of TNFalpha upregulation, since the ra-a47 antisense oligonucleotide treatment did not enhance TNFalpha synthesis. These observations indicate that clownregulation of RA-A47 induces TNFalpha-independent cartilage-degrading pathways involving iNOS and MMP-9. Furthermore, the stimulation of bFGF and CTGF release from chondrocytes may stimulate the proliferation of adjacent endothelial and/or synovial cells. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/jcp.10341

CTGFは軟骨においてI型コラーゲンと共発現する

福永智広, 山城隆, 大矢伸治, 竹下信郎, 滝川正春, 山本照子

日本矯正歯科学会大会プログラム・抄録集 62回 201 - 201 2003.10

A repetitive, steady mouth opening induced an osteoarthritis-like lesion in the rabbit temporomandibular joint

T Fujisawa, T Kuboki, T Kasai, W Sonoyama, S Kojima, J Uehara, C Komori, H Yatani, Hattori, I, M Takigawa

JOURNAL OF DENTAL RESEARCH 82 ( 9 ) 731 - 735 2003.9

Language：English Publishing type：Research paper (scientific journal) Publisher：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R

Although excessive mechanical stress is assumed to be one of the factors contributing to pathogenesis of temporomandibular joint (TMJ) osteoarthritis (OA), no pure mechanical-stress-induced OA model has been developed without surgical manipulation or puncture of the joint cavity. The purpose of this study was to establish a genuine mechanical-stress-induced OA model of the rabbit TMJ. In the experimental rabbits, repetitive, forced jaw-opening, 3 hrs/day for 5 days, was applied with the use of a general anesthesia protocol. By histological assessment of the TMJ articular tissues, partial eburnation of the articular cartilage, reactive marginal proliferation of the articular cartilage chondrocytes, and nested proliferation of chondrocytes in the subchondral bone area were observed at 7 days after the repetitive, forced-jaw-opening period. These results suggest that the repetitive, forced-jaw-opening protocol without surgical intervention can induce evident OA-like lesions in the rabbit TMJ, and this OA model may greatly contribute to the elucidation of the cartilage degradation mechanism in TMJ OA.

CTGF/Hcs24/CCN2, hypertrophic chondrocyte-specific gene product, interacts with perlecan in regulating the proliferation and differentiation of chondrocytes.

T Nishida, S Kubota, T Fukunaga, S Kondo, G Yosimichi, T Nakanishi, T Takano-Yamamoto, M Takigawa

JOURNAL OF BONE AND MINERAL RESEARCH 18 S108 - S108 2003.9

CTGF/Hcs24, hypertrophic chondrocyte-specific gene product, interacts with perlecan in regulating the proliferation and differentiation of chondrocytes Reviewed

T Nishida, S Kubota, T Fukunaga, S Kondo, G Yosimichi, T Nakanishi, T Takano-Yamamoto, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 196 ( 2 ) 265 - 275 2003.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) plays important roles in the control of the proliferation and differentiation of chondrocytes in vitro. To clarify the mechanisms of regulation by CTGF/Hcs24 with respect to cartilage metabolism, we investigated the interaction between CTGF/Hcs24 and heparan sulfate proteoglycan perlecan. An immunofluorescence study showed that CTGF/Hcs24 was colocalized with heparan sulfate and perlecan in human chondrosarcoma-derived chondrocytic cell line HCS-2/8 in vitro. Northern blot analysis showed that perlecan, syndecan-1, -2, and -4 transcripts were detected in HCS-2/8 cells. Particularly, expression of the perlecan gene increased markedly in HCS-2/8 cells by recombinant CTGF/Hcs24 (rCTGF/Hcs24) treatment. We also found that CTGF/Hcs24 interacted with perlecan from HCS-2/8 cells in vitro. Furthermore, CTGF/Hcs24-stimulated gene expression of the aggrecan gene, as well as DNA/proteoglycan synthesis, was diminished when HCS-2/8 cells were pretreated with heparinase, indicating that the effects of CTGF/Hcs24 on chondrocytes occurred through the interaction between CTGF/Hcs24 and heparan sulfate on the cells. An in vivo study using mouse growth plate revealed that CTGF/Hcs24 produced by hypertrophic chondrocytes was localized from the proliferative to the hypertrophic zone, whereas perlecan was predominantly localized in the prehyphertrophic zone. Consistent with such findings in vivo, the binding of I-125-rCTGF/Hcs24 to maturing chondrocytes was at higher levels than that to chondrocytes in hypertrophic stages. These findings suggest that CTGF/Hcs24 produced in the hypertrophic region may act on chondrocytes in the proliferative and maturative zone via some heparan sulfate proteoglycan, such as perlecan. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/jpc.10277

ヒト軟骨肉腫培養細胞株における結合組織成長因子(CTGF)の低酸素による誘導はp38 MAPK経路を介している

近藤誠二, 久保田聡, 森谷徳文, 滝川正春

日本癌学会総会記事 62回 86 - 86 2003.8

Cbfa1/Runx2 gene expression in articular chondrocytes of the mice temporomandibular and knee joints in vivo

T Kuboki, M Kanyama, T Nakanishi, K Akiyama, K Nawachi, H Yatani, K Yamashita, T Takano-Yamamoto, M Takigawa

ARCHIVES OF ORAL BIOLOGY 48 ( 7 ) 519 - 525 2003.7

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Healthy articular cartilage is thought to be maintained by the modulation of Cbfa1 expression, although little is currently known about Cbfa1 expression in such tissues. Therefore, we examined in vivo Cbfa1 transcript levels in the temporomandibular (TM) and knee joints of 3- and 10-week-old mate ICR mice (weighing 50-70 g). A digoxigenin-11-UTP-labeled single-stranded RNA probe (0.6 kbp PstI-HindIII fragment of the 3' of untranslated region in exon 8 of mouse Cbfa1 cDNA) was prepared and in situ hybridization was performed on paraffin-embedded TM and knee joint sections. The antisense probe detected Cbfa1 transcripts in prehypertrophic chondrocytes, but not in the articular surface layer chondrocytes, of 3- and 10-week-old mice TMJs. Despite the intense Cbfa1 expression in prehypertrophic chondrocytes, articular surface layer chondrocytes of the knee joints expressed tow and undetectable level of Cbfa1 in the 3- and 10-week-old mice, respectively. These results indicate that Cbfa1 are highly expressed in the prehypertrophic chondrocytes presumably for articular tissue remodeling during the entire lifespan of the mouse, whereas Cbfa1 expression is suppressed in the articular surface chondrocytes in the adult mouse TM and knee joints to obtain the permanent cartilage phenotype. (C) 2003 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0003-9969(03)00088-8

Transcriptional induction of connective tissue growth factor hypertrophic chondrocyte-specific 24 gene by dexamethasone in human chondrocytic cells

S Kubota, NH Moritami, H Kawaki, H Mimura, M Minato, M Takigawa

BONE 32 ( 5 ) S98 - S98 2003.5

Proposal for a unified CCN nomenclature

DR Brigstock, R Goldschmeding, K Katsube, SCT Lam, LF Lau, K Lyons, C Naus, B Perbal, B Riser, M Takigawa, H Yeger

JOURNAL OF CLINICAL PATHOLOGY-MOLECULAR PATHOLOGY 56 ( 2 ) 127 - 128 2003.4

Language：English Publishing type：Research paper (scientific journal) Publisher：BRITISH MED JOURNAL PUBL GROUP

A proposal is put forth to unify the nomenclature of the CCN family of secreted, cysteine rich regulatory proteins. In the order of their description in the literature, CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) constitute a family of matricellular proteins that regulate cell adhesion, migration, proliferation, survival, and differentiation, at least in part through integrin mediated mechanisms. This proposal is endorsed by the International CCN Society and will serve to eliminate confusion from the multiple names that have been given to these molecules.

顎関節ならびに膝関節の関節軟骨におけるCbfα1/Runx2遺伝子の発現

窪木拓男, 完山学, 中西徹, 秋山謙太郎, 縄稚久美子, 矢谷博文, 山下和夫, 山本照子, 滝川正春

日本顎関節学会雑誌 15 ( 1 ) 112 - 112 2003.4

Suppressive effect of overexpressed connective tissue growth factor on tumor cell growth in a human oral squamous cell carcinoma-derived cell line

NH Moritani, S Kubota, T Nishida, H Kawaki, S Kondo, T Sugahara, M Takigawa

CANCER LETTERS 192 ( 2 ) 205 - 214 2003.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI IRELAND LTD

Connective tissue growth factor (CTGF) is known to be a multifunctional growth factor that is overexpressed. in several types of malignancies. In this study, effects of CTGF gene overexpression on the phenotypes of oral squamous cell carcinoma cells were investigated by using a cell line with undetectable endogenous CTGF expression. Surprisingly, our results indicated that CTGF-overexpressed clones were characterized by attenuated cell growth and less potent tumorigenicity, with coincidental downregulation of prothymosin a gene. Although CTGF is known to promote cell proliferation in mesenchymal cells, our present results suggest that CTGF acts as a negative regulator of the cell growth in oral squamous cell carcinoma possibly through its interaction with growth modifiers inside the cell. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3835(02)00718-8

Role of CTGF/HCS24/ecogenin in skeletal growth control

M Takigawa, T Nakanishi, S Kubota, T Nishida

JOURNAL OF CELLULAR PHYSIOLOGY 194 ( 3 ) 256 - 266 2003.3

Language：English Publisher：WILEY-LISS

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for chondrocytes, osteoblasts, and vascular endothelial cells. CTGF/Hcs24 promotes the proliferation and maturation of growth cartilage cells and articular cartilage cells in culture and hypertrophy of growth cartilage cells in culture. The factor also stimulates the proliferation and differentiation of cultured osteoblastic cells. Moreover, CTGF/Hcs24 promotes the adhesion, proliferation, and migration of vascular endothelial cells, as well as induces tube formation by the cells and strong angiogenesis in vivo. Because angiogenesis is critical for the replacement of cartilage with bone at the final stage of endochondral ossification and because gene expression of CTGF/Hcs24 predominates in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor should be the promotion of the entire process of endochondral ossification, with the factor acting on the above three types of cells as a paracrine factor. Thus, CTGF/Hcs24 should be called "ecogenin: endochondral ossification genetic factor." In addition to hypertrophic chondrocytes, osteoblasts activated by various stimuli including wounding also express a significantly high level of CTGF/Hcs24. These findings in conjunction with in vitro findings about osteoblasts mentioned above suggest the involvement of CTGF/Hcs24 in intramembranous ossification and bone modeling/remodeling. Because angiogenesis is also critical for intramembranous ossification and bone remodeling, CTGF/Hcs24 expressed in endothelial cells activated by various stimuli including wounding may also play important roles in direct bone formation. In conclusion, although the most important physiological role of CTGF/Hcs24 is ecogenin action, the factors also play important roles in skeletal growth and modeling/remodeling via its direct action on osteoblasts under both physiological and pathological conditions. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/jcp.10206

CTGF/Hcs24 as a multifunctional growth factor for fibroblasts, chondrocytes and vascular endothelial cells

M Takigawa

DRUG NEWS & PERSPECTIVES 16 ( 1 ) 11 - 21 2003.1

Language：English Publisher：PROUS SCIENCE, SA-THOMSON REUTERS

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24), a member of the CCN family, is a multifunctional growth factor for fibroblasts, chondrocytes and vascular endothelial cells. Depending on the type of cell with which it interacts, it promotes chemotaxis, migration, adhesion, proliferation, differentiation and/or extracellular matrix formation. Because gene expression of CTGF/Hcs24 is maximal in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor should be the promotion of endochondral ossification. On the other hand, its expression is up-regulated during wound healing, indicating the involvement of this factor in this process as well. When overexpressed, CTGF/Hcs24 may cause pathological states such as fibrotic disorders and angiogenic diseases. This review focuses on the physiological and pathological significances of his novel type of growth factor. (C) 2003 Prous Science. All rights reserved.

Conserved repressive regulation of connective tissue growth factor/hypertrophic chondrocyte-specific gene 24 (ctgf/hcs24) enabled by different elements and factors among vertebrate species

Y Mukudai, S Kubota, M Takigawa

BIOLOGICAL CHEMISTRY 384 ( 1 ) 1 - 9 2003.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WALTER DE GRUYTER & CO

CTGF/Hcs24 is a multifunctional growth factor that potentiates the growth and differentiation of various cells. Our previous study revealed that the 3'-UTR of mammalian CTGF/Hcs24 mRNA contains a small segment that represses the gene expression in cis fashion. In this study, we isolated and characterized a chicken CTGF/Hcs24 cDNA clone. Chicken ctgf/hcs24 mRNA showed highly conserved homology in the ORF to that of mammalian species, whereas the homology in the 3'-UTR was relatively low. Northern blotting analysis revealed that chicken ctgf/hcs24 mRNA was expressed most strongly in cartilage, and also in brain, lung, heart, but faintly in liver. Thereafter we analyzed the functional potential of the 3'-UTR of ctgf/hcs24 cDNA to regulate its gene expression by reporter gene assay, and found that it repressed gene expression in cis fashion, specifically in avian cells, but not in mammalian cells. Conversely, the mammalian 3'-UTR showed less repressive activity in avian cells than in mammalian cells. Deletion analysis showed that a segment near the polyadenyl tail of the 3'-UTR of chicken ctgf/hcs24 played an important functional role, unlike in the mammalian species. Thus, we uncovered a novel mode of functional conservation of the ctgf/hcs24 3'-UTR among vertebrate species mediated by different factors.

成長因子と軟骨細胞 Invited

久保田聡, 滝川正春

腎と骨代謝 16, 103-110 2003

Interaction of AP-1 and the ctgf gene: a possible driver of chondrocyte hypertrophy in growth cartilage

NH Moritani, S Kubota, T Eguchi, T Fukunaga, T Yamashiro, T Takano-Yamamoto, H Tahara, K Ohyama, T Sugahara, M Takigawa

JOURNAL OF BONE AND MINERAL METABOLISM 21 ( 4 ) 205 - 210 2003

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER-VERLAG TOKYO

The expression of the connective tissue growth factor (ctgf) gene increases along with the differentiation of growth cartilage cells, and the highest expression is observed in the hypertrophic stage. Similarly, recent reports demonstrated c-fos expression in chondrocytes in the early hypertrophic zone of growth cartilage, and suggested that the c-fos gene may play a crucial role in the regulation of hypertrophic differentiation. A chondrocytic human cell line, HCS-2/8, is known to retain a variety of chondrocytic phenotypes. When such cells were kept overconfluent, they expressed increasing levels of c-fos transcripts along a time course phenotypically similar to that of hypertrophic differentiation. Moreover, by using a competitive electromobility-shift assay, we found that AP-1, a Fos/Jun heterodimer, in HCS-2/8 was capable of binding not only to a typical AP-1-binding DNA fragment but also to the enhancer fragment of the ctgf gene. Based on the findings above, we hypothesize that, prior to hypertrophic differentiation, AP-1-related oncogenes are activated and that their gene products subsequently activate ctgf gene expression, which might eventually induce hypertrophy.

DOI： 10.1007/s00774-003-0410-1

Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and hydrostatic pressure. A cDNA array study

HM Karjalainen, RK Sironen, MA Elo, K Kaarniranta, M Takigawa, HJ Helminen, MJ Lammi

BIORHEOLOGY 40 ( 1-3 ) 93 - 100 2003

Language：English Publishing type：Research paper (scientific journal) Publisher：IOS PRESS

Mechanical forces have a profound effect on cartilage tissue and chondrocyte metabolism. Strenuous loading inhibits the cellular metabolism, while optimal level of loading at correct frequency raises an anabolic response in chondrocytes. In this study, we used Atlas Human Cancer cDNA array to investigate mRNA expression profiles in human chondrosarcoma cells stretched 8% for 6 hours at a frequency of 0.5 Hz. In addition, cultures were exposed to continuous and cyclic (0.5 Hz) 5 MPa hydrostatic pressure. Cyclic stretch had a more profound effect on the gene expression profiles than 5 MPa hydrostatic pressure. Several genes involved with the regulation of cell cycle were increased in stretched cells, as well as mRNAs for PDGF-B, glucose-1-phosphate uridylyltransferase, Tiam1, cdc37 homolog, Gem, integrin alpha(6), and matrix metalloproteinase-3. Among down-regulated genes were plakoglobin, TGF-alpha, retinoic acid receptor-alpha and Wnt8b. A smaller number of changes was detected after pressure treatments. Plakoglobin was increased under cyclic and continuous 5 MPa hydrostatic pressure, while mitogen-activated protein kinase-9, proliferating cell nuclear antigen, Rad6, CD9 antigen, integrins alpha(E) and beta(8), and vimentin were decreased. Cyclic and continuous pressurization induces a number of specific changes. In conclusion, a different set of genes were affected by three different types of mechanical stimuli applied on chondrosarcoma cells.

Hepatocyte growth factor counteracts transforming growth factor-beta1, through attenuation of connective tissue growth factor induction, and prevents renal fibrogenesis in 5/6 nephrectomized mice.

Inoue T, Okada H, Kobayashi T, Watanabe Y, Kanno Y, Kopp J B, Nishida T, Takigawa M, Ueno M, Nakamura T, Suzuki H

FASEB J 17, 268 - 270, 2003

CTGF/Hcs24 interacts with the cytoskeletal protein actin in chondrocytes

G Yosimichi, S Kubota, T Hattori, T Nishida, K Nawachi, T Nakanishi, M Kamada, T Takano-Yamamoto, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 299 ( 5 ) 755 - 761 2002.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) displays multiple functions in several types of mesenchymal cells, including the promotion of proliferation and differentiation of chondrocytes. Recently, the internalization and intracellular function of CTGF/Hcs24 were indicated as well. In this study, a binding protein for this factor was purified from the cytosolic fraction of human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by CTGF/Hcs24-affinity chromatography. The apparent molecular weight of the protein was 42 kDa and determination of the internal amino acid sequence revealed this protein to be beta- or gamma-actin. An in vitro competitive binding assay of I-125-labeled recombinant CTGF/Hcs24 with cold-rCTGF/Hcs24 showed that the binding between actin and I-125-CTGF/Hcs24 was specific. Immunoprecipitation analysis also showed that CTGF/Hcs24 bound to actin in HCS-2/8 cells. However, rCTGF/Hcs24 had no effects on the expression level of gamma-actin mRNA or total actin protein. These findings suggest that a significant portion of intracellular CTGF/Hcs24 may regulate certain cell biological events in chondrocytes through the interaction with this particular cytoskeletal protein. (C) 2002 Elsevier Science (USA). All rights reserved.

CD44 stimulation by fragmented hyaluronic acid induces upregulation of urokinase-type plasminogen activator and its receptor and subsequently facilitates invasion of human chondrosarcoma cells

H Kobayashi, M Suzuki, N Kanayama, T Nishida, M Takigawa, T Terao

INTERNATIONAL JOURNAL OF CANCER 102 ( 4 ) 379 - 389 2002.12

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

It has been established that fragmented hyaluronic acid (HA), but not native high molecular weight HA, can induce angiogenesis, cell proliferation and migration. We have studied the outside-in signal transduction pathways responsible for fragmented HA-mediated cancer cell invasion. In our study, we have studied the effects of CD44 stimulation by ligation with HA upon the expression of matrix metalloproteinases (MMPs)-2 and -9 as well as urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) and the subsequent induction of invasion of human chondrosarcoma cell line HCS-2/8. Our study indicates that (i) CD44 stimulation by fragmented HA upregulates expression of uPA and uPAR mRNA and protein but does not affect MMPs secretion or PAW mRNA expression; (ii) the effects of HA fragments are critically HA size dependent: high molecular weight HA is inactive, but lower molecular weight fragmented HA (Mr 33 kDa) is active; (iii) cells can bind avidly Mr 3.5 kDa fragmented HA through a CD44 molecule, whereas cells do not effectively bind higher Mr HA; (iv) a fragmented HA induces phosphorylation of MAP kinase proteins (MEK1/2, ERK1/2 and c-Jun) within 30 min; (v) CD44 is critical for the response (activation of MAP kinase and upregulation of uPA and uPAR expression); and (vi) cell invasion induced by CD44 stimulation with a fragmented HA is inhibited by anti-CD44 mAb, MAP kinase inhibitors, neutralizing anti-uPAR pAb, anti-catalytic anti-uPA mAb or amiloride. Therefore, our study represents the first report that CD44 stimulation induced by a fragmented HA results in activation of MAP kinase and, subsequently, enhances uPA and uPAR expression and facilitates invasion of human chondrosarcoma cells. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/ijc.10710

軟骨細胞におけるチロシンキナーゼ型レセプターErbB4遺伝子の発現

縄稚久美子, 久保田聡, 井上美穂, 西田崇, 吉道玄, 中西徹, 完山学, 窪木拓男, 矢谷博文, 山合友一郎, 滝川正春

日本骨形態計測学会雑誌 12 ( 3 ) 63 - 63 2002.12

軟骨細胞におけるチロシンキナーゼ型レセプターErbB4遺伝子の発現

縄稚久美子, 久保田聡, 井上美穂, 西田崇, 吉道玄, 中西徹, 完山学, 窪木拓男, 矢谷博文, 山合友一郎, 滝川正春

生化学 74 ( 11 ) 1413 - 1413 2002.11

Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing

E Nakata, T Nakanishi, A Kawai, K Asaumi, T Yamaai, M Asano, T Nishida, S Mitani, H Inoue, M Takigawa

BONE 31 ( 4 ) 441 - 447 2002.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing in mouse ribs were investigated. In situ hybridization demonstrated that CTGF/Hcs24 mRNA was remarkably expressed, especially in hypertrophic chondrocytes and proliferating chondrocytes, in the regions of regenerating cartilage on days 8 and 14 after fracture. CTGF/Hcs24 mRNA was also expressed in proliferating periosteal cells in the vicinity of the fracture sites on days 2 and 8, and in cells in fibrous tissue around the callus on day 8. Northern blot analysis showed that expression of CTGF/Hcs24 mRNA was 3.9 times higher on day 2 of fracture healing than that on day 0. On day 8, it reached a peak of 8.6 times higher than that on day 0. It then declined to a lower level. Immunostaining showed that CTGF/Hcs24 was localized in hypertrophic chondrocytes and proliferating chondrocytes in the regions of regenerating cartilage, and in active osteoblasts in the regions of intramembranous ossification. Although CTGF/Hcs24 was abundant in the proliferating and differentiating cells (on days 8 and 14), immunostaining decreased as the cells differentiated to form bone (on day 20). CTGF/Hcs24 was also detected in cells in fibrous tissue, vascular endothelial cells in the callus, and periosteal cells around the fracture sites. These results suggest that CTGF/Hcs24 plays some role in fracture healing.

ラット下顎枝骨折治癒過程の膜性骨化と内軟骨性骨化における結合組織成長因子(CTGF)の経時的発現

福永智広, 山城隆, 小橋紀之, 滝川正春, 山本照子

日本矯正歯科学会大会プログラム・抄録集 61回 165 - 165 2002.10

Tyrosine kinase-type receptor ErbB4 in chondrocytes: interaction with connective tissue growth factor and distribution in cartilage

K Nawachi, M Inoue, S Kubota, T Nishida, G Yosimichi, T Nakanishi, M Kanyama, T Kuboki, H Yatani, T Yamaai, M Takigawa

FEBS LETTERS 528 ( 1-3 ) 109 - 113 2002.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

In order to identify receptor molecules that participate in the growth and differentiation of cliondrocytes, we cloned a number of cDNA fragments from HCS-2/8 chondrocytic cells, by using tyrosine kinase-specific primers for amplification. The mRNA expression of one such receptor, ErbB4, was increased by connective tissue growth factorthypertrophic chondrocyte-specific gene product (CTGF/Hes24), which promotes all stages of the endochondral ossification in vitro. ErbB4 expression was observed through all stages of chondrocytic differentiation in vitro, corresponding to the wide distribution of CTGF/Hcs24 target cells. Furthermore, positive signals for erbB4 mRNA were detectable throughout most populations of chondrocytes, in growth and articular cartilage in vivo. These results demonstrate for the first time that ErbB4 is expressed in chondrocytes and may play some roles in chondrocytic growth and differentiation along with CTGF/Hcs24. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

TGF-beta 1 and HGF coordinately facilitate collagen turnover in subepithelial mesenchyme

T Inoue, H Okada, T Kobayashi, Y Watanabe, T Kikuta, Y Kanno, M Takigawa, H Suzuki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 297 ( 2 ) 255 - 260 2002.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

We have employed co-culture of proximal tubular epithelial cells (PTEC) and renal tubulo-interstitial fibroblasts (TFB) to study the role of transforming growth factor-beta1 (TGF-beta1) and hepatocyte growth factor (HGF) in epithelial-mesenchymal interactions. In co-culture, TGF-beta1 stimulated TFB to produce type I collagen (COLI). This effect was both direct and indirect, via connective tissue growth factor (CTGF) produced by the co-cultured PTEC. Co-administration of TGF-beta1 and HGF significantly increased overall COLI production by TFB by 24 h. However, in detail, this co-administration enhanced CTGF induction in PTEC during the first 8 h, and then decreased its expression, resulting in a rapid decrease in expression of the alpha1 (1) procollagen gene in TFB by 24 h. Additionally, tissue inhibitor of metalloproteinase-1 was induced in PTEC by TGF-beta1 with or without co-administration of HGF, which contributed to the COLI accumulation. In contrast, HGF alone or co-administered with TGF-beta1 significantly increased collagenolytic activity derived from PTEC. Therefore, TGF-beta1 and HGF seem to coordinately modulate epithelial-mesenchymal interactions to facilitate COLI turnover in subepithelial mesenchyme. (C) 2002 Elsevier Science (USA). All rights reserved.

Possible roles of CTGF/Hcs24 in the initiation and development of ossification of the posterior longitudinal ligament

Y Yamamoto, KI Furukawa, K Ueyama, T Nakanishi, M Takigawa, S Harata

SPINE 27 ( 17 ) 1852 - 1857 2002.9

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Study Design. A biochemical and histochemical study investigating the role of CTGF/Hcs24 in the ossification of the posterior longitudinal ligament (OPLL) was conducted.
Objective. To clarify the involvement of CTGF/Hcs24 in ectopic bone formation in OPLL through endochondral ossification using human tissue.
Summary of Background Data. Previous studies have shown that various cytokines are involved in the occurrence or development of ectopic bone formation in OPLL. Recently, the authors cloned an mRNA predominantly expressed in chondrocytes by differential display PCR and found,that it's gene, hcs24, is identical to that of connective tissue growth factor. It has been shown that CTGF/Hcs24 plays a major role in endochondral ossification.
Methods. Ossified ligament tissues were taken from seven male OPLL patients during surgery. Immunohistochemical staining was performed using an antibody specific for CTGF/Hcs24. Spinal ligament cells were isolated from five OPLL patients as well as five non-OPLL patients. The cells were incubated with recombinant human CTGF/Hcs24 or TGFbeta. The expression of ALP was analyzed by RT-PCR. For the effects of TGFbeta, the expression of CTGF/Hcs24 mRNA was analyzed.
Results. Immunohistochemical staining showed that chondrocytes in the transitional region from nonossified to ossified ligament were stained with an antibody against,,CTGF/Hcs24. It was found that CTGF/Hcs24 enhanced the expression ALP mRNA in OPLL cells, whereas the expression remained unchanged in non-OPLL cells. The-expression of CTGF/Hcs24 mRNA in OPLL and non-OPLL cell lines was increased by TGFbeta, and there was no significant difference between the two groups. However, TGFbeta and CTGF/Hcs24 enhanced the expression of ALP mRNA only in OPLL cells.
Conclusions. According to the study results, CTGF/Hcs24 may not only be an important factor in the development of endochondral ossification in OPLL, but may also be responsible for initiating osteogenesis in spinal ligament cells.

DOI： 10.1097/01.BRS.0000025725.06173.C6

ヒト口腔扁平上皮癌細胞株における結合組織成長因子(CTGF)の腫瘍細胞増殖抑制効果

森谷徳文, 久保田聡, 近藤誠二, 西田崇, 川木晴美, 菅原利夫, 滝川正春

歯科基礎医学会雑誌 44 ( 5 ) 395 - 395 2002.9

Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade

H Kobayashi, P Suzuki, N Kanayama, T Nishida, M Takigawa, T Terao

EUROPEAN JOURNAL OF BIOCHEMISTRY 269 ( 16 ) 3945 - 3957 2002.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-1 and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.

DOI： 10.1046/j.1432-1033.2002.03068.x

CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

M Suzuki, H Kobayashi, N Kanayama, T Nishida, M Takigawa, T Terao

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1591 ( 1-3 ) 37 - 44 2002.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the mug/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met. (C) 2002 Elsevier Science B.V. All rights reserved.

cDNA array reveals mechanosensitive genes in chondrocytic cells under hydrostatic pressure

RK Sironen, HM Karjalainen, MA Elo, K Kaarniranta, K Torronen, M Takigawa, HJ Helminen, MJ Lammi

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1591 ( 1-3 ) 45 - 54 2002.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Hydrostatic pressure (HP) has a profound effect on cartilage metabolism in normal and pathological conditions, especially in weight-bearing areas of the skeletal system. As an important component of overall load, HP has been shown to affect the synthetic capacity and wellbeing of chondrocytes, depending on the mode, duration and magnitude of pressure. In this study we examined the effect of continuous HP on the gene expression profile of a chondrocytic cell line (HCS-2/8) using a cDNA array containing 588 well-characterized human genes under tight transcriptional control. A total of 51 affected genes were identified, many of them not previously associated with mechanical stimuli. Among the significantly up-regulated genes were immediate-early genes, and genes involved in heat-shock response (hsp70, hsp40, hsp27), and in growth arrest (GADD45, GADD153, p21(Cip1/Waf1), tob). Markedly down-regulated genes included members of the Id family genes (dominant negative regulators of basic helix-loop-helix transcription factors), and cytoplasmic dynein light chain and apoptosis-related gene NIP3. These alterations in the expression profile induce a transient heat-shock gene response and activation of genes involved in growth arrest and cellular adaptation and/or differentiation. (C) 2002 Elsevier Science B.V. All rights reserved.

軟骨由来成長因子CTGF/Hcs24の細胞種特異的遺伝子発現抑制機構の解析

森谷徳文, 久保田聡, 江口傑徳, 近藤誠二, 菅原利夫, 滝川正春

生化学 74 ( 8 ) 913 - 913 2002.8

結合組織成長因子CTGF/Hcs24は細胞内で細胞骨格蛋白質と結合する

吉道玄, 久保田聡, 服部高子, 西田崇, 縄稚久美子, 中西徹, 山本照子, 滝川正春

生化学 74 ( 8 ) 797 - 797 2002.8

CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, stimulates proliferation and differentiation, but not hypertrophy of cultured articular chondrocytes Reviewed

T Nishida, S Kubota, T Nakanishi, T Kuboki, G Yosimichi, S Kondo, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 192 ( 1 ) 55 - 63 2002.7

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTCF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTCF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/jcp.10113

Autoimmunity against YKL-39, a human cartilage derived protein, in patients with osteoarthritis

JI Tsuruha, K Masuko-Hongo, T Kato, M Sakata, H Nakamura, T Sekine, M Takigawa, K Nishioka

JOURNAL OF RHEUMATOLOGY 29 ( 7 ) 1459 - 1466 2002.7

Language：English Publishing type：Research paper (scientific journal) Publisher：J RHEUMATOL PUBL CO

Objective. Our previous study revealed that some patients with rheumatoid arthritis (RA) possessed autoantibodies to YKL-39, a cartilage related protein. We investigated whether patients with osteoarthritis (OA) also displayed autoimmunity to YKL-39.
Methods. Autoantibodies to recombinant YKL-39 as well as human cartilage glycoprotein-39 were detected by ELISA and Western blotting. The tested serum samples were derived from 117 patients with OA, 94 patients with RA, and 2 groups of 50 arthropathy-free healthy donors who matched the OA and RA groups for age and sex. We determined autoepitopes on YKL-39 using 3 overlapping fragments of YKL-39 (designated F1, F2, F3). T cell proliferation response to YKL-39 was analyzed using the H-3-thymidine incorporation assay.
Results. Autoantibodies to YKL-39 were detected in 13 (11.1%) patients with OA and 11 (11.8%) with RA. In the epitope mapping, all the 3 fragments of YKL-39 were found to carry autoepitopes, but F1 was recognized most frequently. Proliferative responses of peripheral blood mononuclear cells against YKL-39 were detected in 6 (46%) of the 13 OA patients who were positive for the anti-YKL-39 autoantibodies and in 2 (17%) of the 11 antibody positive RA patients.
Conclusion. These results show that autoimmunity to YKL-39 in patients with OA was present at equal or somewhat higher frequency than in patients with RA. The cellular and humoral immune responses to YKL-39 may be involved in the pathological process of OA as well as RA.

A novel cis-element that enhances connective tissue growth factor gene expression in chondrocytic cells

T Eguchi, S Kubota, S Kondo, T Kuboki, H Yatani, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 295 ( 2 ) 445 - 451 2002.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

To clarify the chondrocyte-specific regulatory mechanism of connective tissue growth factor (ctgf) gene expression, we analyzed the functionality and DNA-protein interaction of the CTGF promoter. Comparative luciferase assay of the CTGF promoter deletion mutants among HCS-2/8 chondrocytic cells and fibroblastic cells revealed that a 110-bp region in the promoter was crucial for the HCS-2/8-specific transcriptional enhancement. Subsequent competitive gel shift assay revealed that transcription factors in HCS-2/8 nuclei bound to a 60-bp portion in the corresponding region. Relative luciferase activity from a CTGF promoter with mutant TGF-beta response element (TbRE) was 16.9% lower than that from an intact promoter. On the other hand, relative luciferase activity from a CTGF promoter with 4 bp point mutations at 30 bp upstream of the TbRE was 47.7% lower than that from the intact one. The binding activity of HCS-2/8 nuclear factor(s) to the sequence over the 4-bp was remarkably higher than that of any nuclear extract from other types of cells. Therefore, we entitled the sequence 'TRENDIC', a transcription enhancer dominant in chondrocytes, which stands for a novel enhancer for chondrocyte-specific CTGF gene expression. (C) 2002 Elsevier Science (USA). All rights reserved.

結合組織成長因子(CTGF)の構造・機能解析のためのELISAシステムの開発

川木晴美, 久保田聡, 湊雅直, 森谷徳文, 服部高子, 大山和美, 中西徹, 滝川正春

岡山歯学会雑誌 21 ( 1 ) 182 - 183 2002.6

YKL-39, a human cartilage-related protein, induces arthritis in mice

M Sakata, K Masuko-Hongo, J Tsuruha, T Sekine, H Nakamura, M Takigawa, K Nishioka, T Kato

CLINICAL AND EXPERIMENTAL RHEUMATOLOGY 20 ( 3 ) 343 - 350 2002.5

Language：English Publishing type：Research paper (scientific journal) Publisher：CLINICAL & EXPER RHEUMATOLOGY

Objective
To determine whether YKL-39, a recently cloned secretory protein of articular chondrocytes, is arthritogenic in mice. Methods Recombinant YKL-39 (rYKL-39) was expressed and purified from E. coli. To induce arthritis in mice, rYKL-39 (1, 10 or 50 mug in Freund's incomplete adjuvant) was injected into the right footpad of mice from four different strains (BALB/c, DBA/1J, C57BL/6 and ICR). The mice received a second immunization with rYKL-39 by intradermal injection into the root of the tail 10 days after the first immunization. Severin, of arthritis was assessed by scoring each paw on a scale from 0 to 4. Sixty days after the first immunization, the mice were sacrificed and the joints were examined by immunohistochemistry and radiography. The anti-YKL-39 and anti type II-collagen (CII) antibody titres were also assayed using ELISA.
Results
Immunization with YKL-39 induced arthritis in all strains of mice tested, among which BALB/c was most susceptible. Histological examination showed synovial proliferation and irregularity of the cartilage surface in YKL-39-injected BALB/c mice. Moreover radiographic analysis revealed pathological changes in these mice. The YKL-39-immunised mice produced not only anti-YKL-39 antibody but also antibody against AN H collagen, suggesting a spreading of autoimmunity after YKL-39.
Conclusions
YKL-39, a cartilage-related protein, is found to induce arthritis accompanied by pathologic changes in bone and cartilage. A better understanding of the immune response against cartilage-related components including YKL-39 may help to elucidate the pathological processes of arthritic disorders.

Connective tissue growth factor increased by hypoxia may initiate angiogenesis in collaboration with matrix metalloproteinases

S Kondo, S Kubota, T Shimo, T Nishida, G Yosimichi, T Eguchi, T Sugahara, M Takigawa

CARCINOGENESIS 23 ( 5 ) 769 - 776 2002.5

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here we investigated how CTGF and matrix metalloproteinases (MMPs) are involved in the early stage of hypoxia-induced angiogenesis using human breast cancer cell line, MDA231, and vascular endothelial cells. Hypoxic stimulation (5% O-2) of MDA231 cells increased their steady-state level of ctgf mRNA by similar to2-fold within 1.5 h, and the levels remained at a plateau up to 6 h, and then decreased by 12 h as compared with the cells cultured under the normoxic condition. Membrane-type 1 MMP (MT1-MMP) mRNA levels was also increased within a few hours of the exposure to hypoxia. Indeed, ELISA revealed that the CTGF protein/cell in medium conditioned by MDA231 cells exposed to hypoxia was maximally greater at 24 h than in the medium from normoxic cultures and that the secretion rate (supernatant CTGF/cell layer CTGF) increased in a time-dependent manner from 24 to 72 h of hypoxic exposure. Hypoxic induction of CTGF was also confirmed by immunohistochemical analyses. Furthermore, zymogram analysis revealed that the production of active MMP-9 was also induced in MDA231 cells incubated under hypoxic conditions. Finally, we found that recombinant CTGF also increased the expression of a number of metalloproteinases that play a role in the vascular invasive processes and decreased the expression of tissue inhibitors of metalloproteinases by vascular endothelial cells. These findings suggest that hypoxia stimulates MDA231 cells to release CTGF as an angiogenic modulator, which initiates the invasive angiogenesis cascade by modulating the balance of extracellular matrix synthesis and degradation via MMPs secreted by endothelial cells in response to CTGF. This cascade may play critical roles in the hypoxia-induced neovascularization that accompanies tumor invasion in vivo.

軟骨由来軟骨成長因子(CTGF/Hcs24)を用いた関節軟骨再生療法の検討

小島俊司, 西田崇, 小森千尋, 藤沢拓生, 窪木拓男, 矢谷博文, 中西徹, 滝川正春

日本顎関節学会雑誌 14 ( 1 ) 81 - 81 2002.4

ウサギ変形性顎関節症モデルにおける軟骨細胞のアポトーシスの関与

藤沢拓生, 笠井照夫, 窪木拓男, 矢谷博文, 服部高子, 滝川正春

日本顎関節学会雑誌 14 ( 1 ) 110 - 110 2002.4

Kunitz-type protease inhibitor bikunin disrupts phorbol ester-induced oligomerization of CD44 variant Isoforms containing epitope v9 and subsequently suppresses expression of urokinase-type plasminogen activator in human chondrosarcoma cells

M Suzuki, H Kobayashi, M Fujie, T Nishida, M Takigawa, N Kanayama, T Terao

JOURNAL OF BIOLOGICAL CHEMISTRY 277 ( 10 ) 8022 - 8032 2002.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses phorbol ester (PMA)-stimulated expression of urokinase-type plasminogen activator (uPA). In the present study, we tried to answer this mechanism using human chondrosarcoma HCS-2/8 cells. Our results showed the following novel findings: (a) the standard form of CD44 (CD44s; 85 kDa) is expressed in both unstimulated and PMA-stimulated cells, while CD44v isoforms containing epitope v9 (110 kDa) are strongly up-regulated in response to treatment with PMA, (b) CD44v isoforms containing epitope v9 present on the same cell exclusively form aggregates in stimulated cells; (c) induction of uPA mRNA expression could be achieved by using a second cross-linker antibody to cross-link Fab monomers of anti-CD44; (d) co-treatment of stimulated cells with anti-CD44 mAb alone or anti-CD44v9 mAb alone suppresses PMA-induced clustering of CD44, which results in inhibition of uPA overexpression; (e) bikunin efficiently disrupts PMA-induced clustering of CD44, but does not prevent PMA-induced up-regulation of CD44v isoforms containing epitope v9; and (f) after exposure to bik, similar to150-kDa band is mainly detected with immunoprecipitation and this band is shown to be a heterodimer composed of the 110-kDa v9-containing CD44v isoforms and a 45-kDa bik receptor (bik-R). In conclusion, we provide, for the first time, evidence that the bik-R can physically interact with the CD44v isoforms containing epitope v9 and function as a repressor to down-regulate PMA-stimulated uPA expression, at least in part, by preventing clustering of CD44v isoforms containing epitope v9.

DOI： 10.1074/jbc.M108545200

骨格成長と骨折治癒過程におけるCTGF/Hcs24の遺伝子発現

滝川正春, 中西徹, 西田崇, 縄稚久美子, 中田英二

厚生労働省特定疾患対策研究事業研究報告書脊柱靭帯骨化症に関する調査研究班平成13年度 69 - 76 2002.3

Language：Japanese Publisher：厚生労働省特定疾患対策研究班

肥大軟骨細胞由来の成長因子CTGF/Hcs24の生理的,病理的役割を解明するため,胎生10,15,17日齢,生後1日齢のマウスを用いて骨格成長過程と骨折治癒過程におけるCTGF/Hcs24の遺伝子発現について検討した.マウス胎生期における発現は,胎生7日の初期に高く,胎生10日で一旦低下した後,胎生15日〜17日で再び増強した.マウスでは多くの内軟骨性骨化が胎生13日で始まることから,後期の遺伝子発現は内軟骨性骨化と関連すると考えられるが,初期の遺伝子発現の亢進は幼若細胞の分化と関連すると考えられた.また,CTGF/Hcs24は骨折治癒過程においても肥大軟骨細胞から産生され肥大軟骨細胞層を中心として軟骨細胞層全体に存在することが明らかとなり,CTGF/Hcs24は軟骨細胞にとってオートクリン・パラクリン因子であることが明らかとなった.更に,骨折後初期において,増殖しつつある骨膜細胞や血管内皮細胞にも発現していることから,組織再生の際には肥大軟骨細胞だけでなく種々の細胞が発現・産生する因子であることが示唆された

Development of Dentin Regeneration Therapy : Expression of Type I Collagen and Alkaline Phosphatase Induced by CTGF in Human Cultured Dental Pulp

Hirotoshi SHIMIZU, Yoshihiro NISHITANI, Tomiko YAMADA, Takashi NISHIDA, Masaharu TAKIGAWA, Masahiro YOSHIYAMA, Department of Operative Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Operative Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Operative Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Biochemistry and Molecular Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Biochemistry and Molecular Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Operative Dentistry Okayama University Graduate School of Medicine and Dentistry

Journal of hard tissue biology = Journal of hard tissue biology 11, ( 2 ) 62 - 67 2002

High pressure effects on cellular expression profile and mRNA stability. A cDNA array analysis

RK Sironen, HM Karjalainen, K Torronen, MA Elo, K Kaarniranta, M Takigawa, HJ Helminen, MJ Lammi

BIORHEOLOGY 39 ( 1-2 ) 111 - 117 2002

Language：English Publishing type：Research paper (scientific journal) Publisher：IOS PRESS

Hydrostatic pressure has a profound effect on cartilage tissue and chondrocyte metabolism. Depending on the type and magnitude of pressure various responses can occur in the cells. The mechanisms of mechanotransduction at cellular level and the events leading to specific changes in gene expression are still poorly understood. We have previously shown that induction of stress response in immortalized chondrocytes exposed to high static hydrostatic pressure increases the stability of heat shock protein 70 mRNA. In this study, our aim was to examine the effect of high pressure on gene expression profile and to study whether stabilization of mRNA molecules is a general phenomenon under this condition. For this purpose a cDNA array analysis was used to compare mRNA expression profile in pressurized vs. non-pressurized human chondrosarcoma cells (HCS 2/8). mRNA stability was analyzed using actinomycin-treated and nontreated samples collected after pressure treatment. A number of immediate-early genes, and genes regulating cell cycle and growth were up-regulated due to high pressure. Decrease in osteonectin, fibronectin, and collagen types VI and XVI mRNAs was observed. Also bikunin, cdc37 homologue and Tiam1, genes linked with hyaluronan metabolism, were down-regulated. In general, stability of down-regulated mRNA species appeared to increase. However, no increase in mRNA above control level due to stabilization was noticed in the genes available in the array. On the other hand, mRNAs of certain immediate-early genes, like c-jun, jun-B and c-myc, became destabilized under pressure treatment. Increased accumulation of mRNA on account of stabilization under high pressure conditions seems to be a tightly regulated, specific phenomenon.

CTGF/Hcs24, a hypertrophic chondrocyte specific gene product, stimulates the proliferation and expression of the cartilage phenotype but not hypertrophy or calcification or articular cartilage in culture.

Nishida T, Kubota S, Nakanishi T, Kuboki T, Yosimichi G, Kondo S, Takigawa M

J Cell Physiol 192, 55 - 63 2002

岡山大学歯学部におけるチュートリアル教育(2) －5年次生に導入された「Evidence-based medicine(EBM)に基づく歯科医療」の解析と評価－

宮本学, 窪木拓男, 高務朋将, 西谷佳浩, 鷲尾憲文, 水口一, 若狭享, 多田徹, 原哲也, 高木慎, 福永城司, 真野隆充, 山田庸介, 尾形小霧, 松尾龍二, 永井教之, 矢谷博文, 滝川正春, 山本照子

岡山歯学会雑誌 21 ( 2 ) 247 - 253 2002

Connective tissue growth factor as a major angiogenic agent that is induced by hypoxia in a human breast cancer cell line

T Shimo, S Kubota, S Kondo, T Nakanishi, A Sasaki, H Mese, T Matsumura, M Takigawa

CANCER LETTERS 174 ( 1 ) 57 - 64 2001.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here, we present the evidence that the hypoxic induction of angiogenesis by human breast cancer cells (MDA-231) can be ascribed at least in part to CTGF, Our results indicate that (i) CTGF is abundantly present in MDA-231 cells in vitro and in vivo, (ii) its secretion is up-regulated by hypoxia, and (iii) its gene expression is enhanced in MDA-231 cells cultured under hypoxic conditions. These data suggest CTGF may stimulate angiogenesis by paracrine mechanisms, thereby contributing to the invasion of breast cancer cells. This is the first evidence that human cancer cells differentially express CTGF protein and mRNA under the control of hypoxic conditions. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK) Reviewed

G Yosimichi, T Nakanishi, T Nishida, T Hattori, T Takano-Yamamoto, M Takigawa

EUROPEAN JOURNAL OF BIOCHEMISTRY 268 ( 23 ) 6058 - 6065 2001.12

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE LTD

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24) promotes proliferation and differentiation of chondrocytes in culture. We investigated the roles of two major types of mitogen activated protein kinase (MAPK) in the promotion of proliferation and differentiation by CTGF/Hcs24. Here we report the effects of the MAPKK/MEK 1/2 inhibitor, PD098059, and p38 MAPK inhibitor, SB203580, in a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) and rabbit growth cartilage (RGC) cells treated with CTGF/Hcs24. In the proliferation phase, CTGF/Hcs24 induced a approximate to fivefold increase in the phosphorylation of p44/42 MAPK/ERK and a approximate to twofold increase in that of p38 MAPK in an in vivo kinase assay. These inhibitors of MAPKK and MAPK suppressed phosphorylation of ets-like gene-1 (Elk-1) and nuclear activating transcription factor-2 (Atf-2) induced by CTGF/Hcs24 in a dose-dependent manner, respectively. Western blot analysis showed that phosphorylation of ERK was induced from 30 to 60 min and phosphorylation of p38 MAPK from 10 to 15 min after the addition of CTGF/Hcs24 in confluence HCS-2/8 cells. PD098059 suppressed the DNA synthesis of HCS-2/8 cells and RGC cells, while SB203580 did not. On the other hand, the p38 MAPK inhibitor, SB203580, completely inhibited the CTGF/Hcs24-induced synthesis of proteoglycans in HCS-2/8 cells and RGC cells but the MEK1/2 inhibitor, PD098059, did not. These results suggest that ERK mediates the CTGF/Hcs24-induced proliferation of chondrocytes, and that p38 MAPK mediates the CTGF/Hcs24-induced differentiation of chondrocytes.

遺伝子発現の抑圧的調節を仲介するマウスctgf3'-UTR segmentの性質

近藤誠二, 久保田聡, 江口傑徳, 服部高子, 中西徹, 菅原利夫, 滝川正春

日本口腔科学会雑誌 50 ( 6 ) 568 - 568 2001.11

Cell-type-specific trans-activation of herpes simplex virus thymidine kinase promoter by the human T-cell leukemia virus type I Tax protein

S Kubota, Y Mukudai, T Hattori, T Eguchi, S Kondo, M Takigawa

DNA AND CELL BIOLOGY 20 ( 9 ) 563 - 568 2001.9

Language：English Publishing type：Research paper (scientific journal) Publisher：MARY ANN LIEBERT INC PUBL

The human T-cell leukemia virus type I Tax protein (HTLV-I Tax) is known as a trans-activating factor for a variety of genes, including those of cytokines. Here, we show that Tax is capable of activating the herpes simplex virus thymidine kinase (HSV-TK) promoter in certain mammalian cell lines. In murine NIH 3T3 fibroblasts and human HeLa cells, trans-activation by Tax was remarkably strong, whereas in human chondrocytic HCS-2/8 and monkey kidney Cos-7 cells, the responsiveness of the TK promoter to Tax was poor. Deletion analysis revealed that one of the two previously described Sp1 sites is required for the Tax responsiveness, whereas the CTF binding site is not. The results suggest possible interactions between the oncogenic Tax protein and the viral TK in coinfected cells in vivo. Care should be taken in the context of HTLV-I research, as the HSV-TK promoter has been widely used in molecular biology and gene therapeutics.

Novel mode of processing and secretion of connective tissue growth factor/ecogenin (CTGF/Hcs24) in chondrocytic HCS-2/8 cells

S Kubota, T Eguchi, T Shimo, T Nishida, T Hattori, S Kondo, T Nakanishi, M Takigawa

BONE 29 ( 2 ) 155 - 161 2001.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

The synthesis, processing, and secretion of human connective tissue growth factor (CTGF/Hcs24) in a human chondrocytic cell line, HCS-2/8, were analyzed immunochemically. By metabolic pulse-labeling, chasing, and subsequent immunoprecipitation analyses, active synthesis of CTGF was observed not only in growing HCS-2/8 cells, but also in confluent cells. However, secretion and processing of CTGF were found to be regulated differentially, depending upon the growth status. During phases of growth, HCS-2/8 cells released CTGF molecules immediately without sequestering them within the cell layer. In contrast, after the cells reached confluence, the secretion slowed, resulting in an accumulation of CTGF in the cells or extracellular matrices (ECMs). Also, in confluent cell layers, a 10 kDa protein that was reactive to an anti-CTGF serum was observed. This CTGF-related small protein was not detected immediately after labeling, but gradually appeared within 6 h after chase, which suggests its entity as a processed subfragment of CTGF. Surprisingly, the 10 kDa protein was stable even 48 h after synthesis, and was not released by ECM digestion, suggesting an intracellular maintenance and function. Taken together, the behavior of CTGF in HCS-2/8 cells is remarkably different from that reported in fibroblasts, which may represent unique roles for CTGF in the growth and differentiation of chondrocytes. (C) 2001 by Elsevier Science Inc. All rights reserved.

多機能成長因子CTGF/Hcs24遺伝子の転写後制御エレメント,CAESARの作用機序

久保田聡, 近藤誠二, 椋代義樹, 江口傑徳, 服部高子, 中西徹, 滝川正春

生化学 73 ( 8 ) 710 - 710 2001.8

Tumor necrosis factor alpha induces expression of genes for matrix degradation in human chondrocyte-like HCS-2/8 cells through activation of NF-kappa B: Abrogation of the tumor necrosis factor alpha effect by proteasome inhibitors

T Sakai, F Kambe, H Mitsuyama, N Ishiguro, K Kurokouchi, M Takigawa, H Iwata, H Seo

JOURNAL OF BONE AND MINERAL RESEARCH 16 ( 7 ) 1272 - 1280 2001.7

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BONE & MINERAL RES

Tumor necrosis factor alpha (TNF-alpha) has been suggested to induce chondrocytic chondrolysis in both inflammatory and degenerative joint diseases. However, its intracellular signaling pathway leading to the chondrolysis has not been studied in detail. Thus, we investigated whether TNF-alpha activates a transcription factor nuclear factor KB (NF-KB) in human chondrocyte-like cells (HCS-2/8) and induces the expression of genes involved in the degradation of cartilage matrix. Treatment of the cells with TNF-alpha markedly increased the levels of matrix metalloproteinase 1 (MMP-1), MMP3, intercellular adhesion molecule 1 (ICAM-1), and cyclo-oxygenase 2 (COX-2) messenger RNAs (mRNAs). The increase in the mRNAs was associated with the activation of p65/p50 heterodimer NF-kappaB. I kappaB-alpha and I kappaB-beta, cytoplasmic molecules preventing the nuclear translocation of NF-kappaB, were degraded rapidly by TNF-alpha followed by their synthesis to the basal level. Treatment with proteasome inhibitors inhibited the degradation of both IKB-alpha and IKB-beta and prevented the TNF-alpha -dependent nuclear translocation of p65. Furthermore, the inhibitors completely prevented the TNF-alpha -dependent induction of MMP-1, MMP3, ICAM-1, and COX-2 mRNAs. Thus, it is shown that the activation of p65/p50 NF-KB by TNF-a! plays a cardinal role in inducing the expression of MMP-1, MMP-3, ICAM-1, and COX-2 genes, which are involved in matrix degradation and inflammatory reaction in chondrocytes, leading to chondrocytic chondrolysis.

ヒト軟骨肉腫由来軟骨様細胞株HCS-2/8における結合組織成長因子CTGF-Hcs24の遺伝子発現制御機構(Regulatory Mechanism of Human Connective Tissue Growth Factor (CTGF/Hcs24) Gene Expression in a Human Chondrocytic Cell Line, HCS-2/8)

江口傑徳, 久保田聡, 近藤誠二, 志茂剛, 服部高子, 中西徹, 窪木拓男, 矢谷博文, 滝川正春

The Journal of Biochemistry 130 ( 1 ) 79 - 87 2001.7

Language：English Publisher：(公社)日本生化学会

多機能成長因子CTGF/Hcs24の軟骨における遺伝子発現制御機構を明らかにするために,軟骨細胞様HCS-2/8細胞を用い分子生物学的検討を行った.HCS-2/8細胞はCTGF-Hcs24を高発現しており,これはプロモーターのTGF-β応答領域を介した,TGF-βによる転写活性の上昇が関与していることが示された.しかし一方で,別の転写因子の関与も同時に推察された

In vitro及びin vivoにおける肥大軟骨細胞由来の成長因子CTGF/Hcs24の関節軟骨細胞に対する作用

西田崇, 中西徹, 久保田聡, 吉道玄, 近藤誠二, 滝川正春

日本骨代謝学会雑誌 19 ( 2 ) 30 - 30 2001.7

CTGF/Hcs24軟骨強制発現トランスジェニックマウスの解析

縄稚久美子, 中西徹, 吉道玄, 中田英二, 服部高子, 小守壽文, 滝川正春

日本骨代謝学会雑誌 19 ( 2 ) 30 - 30 2001.7

Type II alveolar epithelial cells and interstitial fibroblasts express connective tissue growth factor in IPF

LH Pan, K Yamauchi, M Uzuki, T Nakanishi, M Takigawa, H Inoue, T Sawai

EUROPEAN RESPIRATORY JOURNAL 17 ( 6 ) 1220 - 1227 2001.6

Language：English Publishing type：Research paper (scientific journal) Publisher：EUROPEAN RESPIRATORY SOC JOURNALS LTD

Connective tissue growth factor (CTGF) is a growth and chemotactic factor for fibroblasts encoded by an immediate early gene that is transcriptionally activated by transforming growth factor-beta, Previous studies have shown that both CTGF messenger ribonuclear acid (mRNA) and protein are expressed in renal fibrosis and bleomycin-induced pulmonary fibrosis in mice. The aim of the present study,ras to investigate the localization of CTGF protein and its mRNA expression in the fibrotic lung tissue of patients with idiopathic pulmonary fibrosis (IPF),
Using human fibrotic lung tissue obtained from eight autopsy cases and four biopsy cases with IPF, immunohistochemical staining, ib situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) were performed.
The cellular immunoreactivity for CTGF was markedly increased in the lung tissue of patients with IPF, compared to normal lungs. The immunolocalization of CTGF was confined predominantly to proliferating type II alveolar epithelial cells and activated fibroblasts, In the normal lung, type II alveolar epithelial cells stained for CTGF were sparsely distributed. CTGF mRNA was localized in proliferating type II alveolar epithelial cells and activated fibroblasts in the interstitium of fibrotic lung tissues. RT-PCR analysis showed that CTGF mRNA was expressed at a higher level in fibrotic lungs than in normal lungs.
In both an autocrine and a paracrine manner, type II alveolar epithelial cells and activated fibroblasts may play a critical role in pulmonary fibrosis by producing connective tissue growth factor which modulates fibroblast proliferation and extracellular matrix production.

Characterization of binding properties of urinary trypsin inhibitor to cell-associated binding sites on human chondrosarcoma cell line HCS-2/8

Y Hirashima, H Kobayashi, M Suzuki, Y Tanaka, N Kanayama, M Fujie, T Nishida, M Takigawa, T Terao

JOURNAL OF BIOLOGICAL CHEMISTRY 276 ( 17 ) 13650 - 13656 2001.4

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness, It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP(40)), which:is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP(40)). Here we characterize binding properties of UTI-BPs UTI complexes in the cells, In vitro ligand blot, cell binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP(40) and UTP-BP(45) bind (125)I-UTL A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP(40) Additional experiments, using various reagents to block binding of (125)I-UTI and NG-UTI to the UTI-BP(40) and UTI-BP(45) confirm that the chondroitin sulfate Side chain of UTI is required for its binding to UTI-BP(45) analysis of binding of (125)I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP(40) (which can bind NG-UTI), and the high affinity sites are the UTI-BP(45), In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.

Overexpression of connective tissue growth factor hypertrophic chondrocyte-specific gene product 24 decreases bone density in adult mice and induces dwarfism

T Nakanishi, T Yamaai, M Asano, K Nawachi, M Suzuki, T Sugimoto, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 281 ( 3 ) 678 - 681 2001.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for fibroblasts, chondrocytes, and vascular endothelial cells. In the present study, we established transgenic (Tg) mice that overproduce CTGF/Hcs24 under the control of mouse type XI collagen promoter. Tg mice could develop and their embryonic and neonatal growth occurred normally. But they showed dwarfism within a few months of birth. X-ray analysis revealed that their bone density was decreased compared with normal mice. The femurs in the hindlimbs in particular showed an apparent low density. These results indicated that overexpression of CTGF/Hcs24 affects certain steps of endochondral ossification. In addition, the testes were much smaller than normal and fertility was affected in Tg mice, indicating that CTGF/Hcs24 may also regulate the embryonic development of the testis. (C) 2001 Academic Press.

Mitogenic effects of neurotrophins on a periodontal ligament cell line Reviewed

Y Tsuboi, T Nakanishi, T Takano-Yamamoto, M Miyamoto, T Yamashiro, M Takigawa

JOURNAL OF DENTAL RESEARCH 80 ( 3 ) 881 - 886 2001.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC DENTAL RESEARCH

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are three representative neurotrophins responsible for the differentiation and survival of neurons, and their high-affinity receptors are tropomyosin-receptor-kinase (TRK)A, TRKB, and TRKC, respectively. In this study, we investigated the expression of neurotrophins in a mouse periodontal ligament cell line (MPL), by reverse transcription-polymerase chain-reaction (RT-PCR) and enzyme-linked immunoabsorbent assay (ELISA). We also studied the expression of TRK receptors on MPL by immunostaining and the effects of neurotrophins on the proliferation of MPL, with a hypothesis of autocrine mechanism of neurotrophins. Each neurotrophin and TRK receptor was expressed, and neurotrophins enhanced the proliferation of MPL. These findings suggest that the MPL has functional neurotrophin receptors involved in an autocrine function of neurotrophins. The expression level of neurotrophins and TRKs showed the reverse pattern, and we propose an auto-regulatory mechanism of ligands and receptors in accordance with the level of synthesized neurotrophins.

DOI： 10.1177/00220345010800030701

Other Link： http://orcid.org/0000-0002-4419-9643

Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes

T Hattori, S Kubota, Y Yutani, T Fujisawa, T Nakanishi, K Takahashi, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 186 ( 2 ) 268 - 281 2001.2

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

We previously isolated a rheumatoid arthritis-related antigen (RA-A47) protein that had reactivity with RA sera from a human chondrosarcoma-derived chondrocytic cell Line, HCS-2/8. Sequencing analysis of ra-a47 cDNA revealed RA-A47 as a product of the colligin-2 gene, which is also known as the human heat shock protein (HSP) 47 gene. Expression of hsp47 has been shown to be cooperatively altered with that of collagen genes upon stimulation. in this study, it was confirmed that the mRNA expression of ra-a47 and COL2A1, a type II collagen gene, was upregulated on stimulation with transforming growth factor (TGF) beta in chondrocytes. However, in contrast, inflammatory cytokines such as tumor necrosis factor (TNF) alpha, interferon (IFN) beta, and interleukin (IL)-6 downregulated the expression of ra-a47 mRNA, whereas the expression of COL2A1 mRNA was not repressed, or even upregulated, in HCS-2/8 cells. Of note, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP)-9 mRNAs were strongly stimulated by TNF alpha. We also found that cell-surface type II collagen disappeared upon such a stimulation, suggesting that decrement of RA-A47 may inhibit the secretion of type II collagen and lead to its accumulation inside the cells. RA-A47 was detected in the cultured medium of TNF alpha -treated HCS-2/8 cells and of IL-1-treated rabbit chondrocytes by Western blot analysis. Under the same conditions, RA-A47 was detected on the cell surface by immunofluorescence staining. These findings demonstrate that the RA-A47 chaperone protein is specifically downregulated, causing the intracellular accumulation of unsecretable type II collagen, while the extracellular matrix (ECM) is degraded by MM Ps and iNOS th rough the stimulation of chondrocytes by TNF alpha. The altered localization of RA-A47 to the surface or outside of cells may represent the mechanism for the recognition of RA-A47 as an autoantigen during rheumatoid arthritis. J. Cell. Physiol. 186:268-281, 2001. (C) 2001 Wiley-Liss, Inc.

Expression of transduced HSP70 gene protects chondrocytes from stress

T Kubo, Y Arai, K Takahashi, T Ikeda, S Ohashi, Kitajima, I, O Mazda, M Takigawa, J Imanishi, Y Hirasawa

JOURNAL OF RHEUMATOLOGY 28 ( 2 ) 330 - 335 2001.2

Language：English Publishing type：Research paper (scientific journal) Publisher：J RHEUMATOL PUBL CO

Objective. To investigate thr efficacy of adenovirus vector mediated transduction of heat shock protein 70 (HSP70) gene to human chondrocyte-like cell (HCS-2/8) against heat stress.
Methods. Two adenovirus vectors that contain wild-type (AxSHEwt) or mutant-type (AxSHEmt) HSP70 gene, and that are regulated by SR alpha promoter, were constructed. The mutant-type lacks the area that expresses stress durability. One of the 2 adenovirus vectors was added to the cultures of human chondrocyte-like cells (HCS-2/8). Heat stress (48 degreesC) was applied to the transduced cells for 2 h. and the efficacy of adenovirus vector mediated transduction of HSP70 gene against heat stress in the chondrocytes was investigated using alamar blue assay and MTT assay.
Results. Absorbance levels at 48 degreesC were 300.3 +/- 51.9 and 1.173 +/- 0.011 in the controls, 278.5 +/- 33.8 and 1.217 +/- 0.018 in the AxSHEmt transduced cells, and 349 +/- 14.7 and 1.371 +/- 0.033 in the AxSHEwt transduced cells. The level in the AxSHEwt transduced cells was significantly higher than in the other 2 groups (p < 0.05). With 37<degrees>C treatment, no significant difference was observed.
Conclusion. Chondrocytes to which HSP70 gene was transduced had a significantly higher metabolic activity and viability under heat stress.

Mechanical stimulation induces CTGF expression in rat osteocytes

T Yamashiro, T Fukunaga, N Kobashi, H Kamioka, T Nakanishi, M Takigawa, T Takano-Yamamoto

JOURNAL OF DENTAL RESEARCH 80 ( 2 ) 461 - 465 2001.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC DENTAL RESEARCH

Connective tissue growth factor (CTGF), which is encoded by an immediate early gene and a member of the CCN family, has been shown to be expressed in osteoblasts, fibroblasts, and chondrocytes. Although CTGF is expressed in bone and cartilage tissues, we tested the hypothesis that CTGF is regulated in mechanotransduction. In the alveolar bone during experimental tooth movement, CTGF mRNA was expressed in osteoblasts and in osteocytes localized around the periodontal ligament under control conditions. Interestingly, 12 hrs after the start of experimental tooth movement, the expression of CTGF mRNA in osteocytes and osteoblasts became more intense around the periodontal ligament, and the intense expression of CTGF extended to osteocytes situated deep in alveolar bone matrix apart from periodontal ligament in both tension and compression sides. Our present findings indicate that CTGF could play a role in regulation of osteocyte function during the mechanical stimulation of bone.

Recognition of YKL-39, a human cartilage related protein, as a target antigen in patients with rheumatoid arthritis

T Sekine, K Masuko-Hongo, T Matsui, H Asahara, M Takigawa, K Nishioka, T Kato

ANNALS OF THE RHEUMATIC DISEASES 60 ( 1 ) 49 - 54 2001.1

Language：English Publishing type：Research paper (scientific journal) Publisher：BRITISH MED JOURNAL PUBL GROUP

Objective-To investigate whether autoimmunity to YKL-39, a recently cloned cartilage protein, occurs in patients with rheumatoid arthritis (RA).
Methods-Autoantibody to YKL-39 was assayed by enzyme Linked immunosorbent assay (ELISA) and western blotting in serum samples from patients with RA, systemic lupus erythematosus (SLE), and healthy donors, using recombinant YKL-39 protein. This reactivity was compared with that against a YKL-39 homologue, YKL-40 (human cartilage gp-39/ chondrex), which has been reported to be an autoantigen in RA.
Results-Autoantibody to YKL-39 was detected in seven of 87 patients with RA (8%), but not in serum samples from patients with SLE or healthy donors. YKL-40 reactivity was found in only one of 87 RA serum samples (1%), with no cross reactivity to YKL-39.
Conclusion-The existence of anti-YKL-39 antibody in a subset of patients with RA is reported here for the first time. Further, it was shown that the immune response to YKL-39 was independent of that to YKL-40. Clarification of the antibody and T cell responses to autoantigens derived from chondrocyte, cartilage, or other joint components may lead to a better understanding of the pathophysiology of joint destruction in patients with RA.

Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells

Suzuyo Ohashi, Toshikazu Kubo, Takumi Ikeda, Yuji Arai, Kenji Takahashi, Yasusuke Hirasawa, Masaharu Takigawa, Etsuko Satoh, Jiro Imanishi, Osam Mazda

Journal of Orthopaedic Science 6 ( 1 ) 75 - 81 2001

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Japan

The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent
Qiagen), linear polyethylenimine (PEI: ExGen 500: Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively. A plasmid vector containing the Escherichia coli LacZ (pSES.β) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by an O-nitrophenyl β-D-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal: Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2μg of pSES.β was combined with either 21 μg of dendrimer, 1.7 μg of linear PEI, or 2.0 μg of branched PEI. At the same DNA/polymer ratios, the proportions of X-galstained cells were also the highest (31.3 ± 7.5%, 30.3 ± 9.0%, and 8.3 ± 3.1%, respectively). LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes.

DOI： 10.1007/s007760170028

軟骨細胞とMMPs-生理的役割を中心に Invited

久保田聡, 滝川正春

The Bone 5, 39-43 2001

Involvement of CTGF, a hypertrophic chondrocyte-specific gene product, in tumor angiogenesis

T Shimo, T Nakanishi, T Nishida, M Asano, A Sasaki, M Kanyama, T Kuboki, T Matsumura, M Takigawa

ONCOLOGY 61 ( 4 ) 315 - 322 2001

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Connective tissue growth factor (CTGF) is a potent secreted signaling factor which functions in multiple stages of angiogenesis. In the present study, we examined the role of CTGF in tumor angiogenesis and made the following observations: (1) Histological analysis of human breast cancer (MDA231) cell and human fibrosarcoma (HT1080) cell xenografts in BALB/c nude mice showed a high level of neovascularization. Human squamous cell carcinoma (A431) xenografts induced only a low level of neovascularization. (2) CTGF mRNA was strongly expressed in MDA231 and in HT1080 cells in vivo and in vitro, but not in A431 cells. (3) CTGF protein was markedly produced in MDA231 cells and HT1080 cells and secreted into culture medium, and its production was greater during phases of growth rather than confluency. (4) Production of CTGF in bovine aorta endothelial cells was induced by CTGF, VEGF, bFGF and TGF-beta. (5) Neovascularization induced by HT1080 cells or MDA231 cells on chicken chorioallantoic membrane was suppressed in the presence of neutralizing CTGF-specific polyclonal antibody. These results suggest that CTGF regulates progression in tumor angiogenesis and the release or secretion of CTGF from tumor cells is essential for the angiogenesis. Copyright (C) 2001 S. Karger AG, Basel.

CTGF/Hcs24 induces chondrocyte differentiation through p44/42 MAPK/exracellular-signal regulated kinase (ERK).

Yosimichi, G, Nakanishi, T, Nishida, T, Hattori, T, Takano-Yamamoto, T, Takigawa, M

Eur. J. Biochem. 268, 1 - 9, 2001

Cell density-dependent proliferative effects of transforming growth factor (TGF)-beta 1, beta 2, and beta 3 in human chondrosarcoma cells HCS-2/8 are associated with changes in the expression of TGF-beta receptor type I

K Boumediene, M Takigawa, JP Pujol

CANCER INVESTIGATION 19 ( 5 ) 475 - 486 2001

Language：English Publishing type：Research paper (scientific journal) Publisher：MARCEL DEKKER INC

In this study, the growth properties of the human chondrosarcoma cell line HCS-2/8, its response to transforming growth factor (TGF)-beta isoforms 1, 2, and 3, and its expression of TGF-beta receptors I and II were examined. We demonstrated that these tumor cells are not contact-inhibited and that they can proliferate in the absence of additional serum growth factors. In sparse cultures, all TGF-beta forms inhibited the growth of HCS-2/8 cells, whereas they induced a 2-fold increase of DNA synthesis in serum-fed confluent cultures. In serum-free confluent conditions only TGF-beta1 stimulated the proliferation rate, whereas TGF-beta2 was without effect and TGF-beta3 was rather inhibitory. The bimodal effect of TGF-beta forms was associated with a greater level of TGF-beta receptor I mRNA in confluent HCS-2/8 than in sparse cultures suggesting that the growth response to TGF-beta forms is dependent on the receptor profile expressed.

Mitogenetic effect of neurotrophins on periodonatal ligament cell line.

Tsuboi, Y, Nakanishi, T, Takano-Yamamoto, T, Miyamoto, M, Yamashiro, T, Takigawa, M

J. Dent. Res. 80(3), 881 - 886, 2001

Characterization of a mouse ctgf 3 '-UTR segment that mediates repressive regulation of gene expression

S Kondo, S Kubota, T Eguchi, T Hattori, T Nakanishi, T Sugahara, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 278 ( 1 ) 119 - 124 2000.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC

We isolated a small segment of the 3'-untranslated region (3'-UTR) in the mouse connective tissue growth factor (ctgf/fisp12) gene and evaluated its functionality. Comparison of the nucleotide sequences of human and mouse ctgf 3'-UTRs revealed a conserved small segment of 91 bases, The corresponding segments of the 3'-UTRs shared as much as 82.4% homology, whereas the overall homology between the 3'-UTRS was 71.8%. To study the functionality of the conserved segment, the corresponding region of mouse ctgf cDNA was amplified from NIH3T3 cells. When it was fused downstream of a marker gene, it showed remarkable repressive effects on gene expression. The repressive effect of the sense form was more prominent than that of the antisense form. Computer analyses of these sequence predicted stable secondary structures, suggesting that they act at the RNA level. The predicted structures of the sense and antisense forms appeared to be slightly different, which is consistent with the difference in repressive function. These findings defined the conserved small element in the mouse ctgf gene as a potent negative regulator of gene expression, which may act at a posttranscriptional level. (C) 2000 Academic Press.

Expression of connective tissue growth factor in cartilaginous tumors

T Shakunaga, T Ozaki, N Ohara, K Asaumi, T Doi, K Nishida, A Kawai, T Nakanishi, M Takigawa, H Inoue

CANCER 89 ( 7 ) 1466 - 1473 2000.10

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

BACKGROUND. Connective tissue growth factor (CTGF) predominantly is expressed in hypertrophic chondrocytes and its specific receptors are demonstrated on chondrocytic cells. Therefore, CTGF may be involved in the proliferation and/or differentiation of cartilage cells. In the current study, CTGF expression was examined both in chondrosarcoma and enchondroma to clarify the relation between the expression of CTGF and the grade of malignancy.
METHODS. The expression of CTGF and proliferating cell nuclear antigen (PCNA] were analyzed immunohistochemically in 34 cartilaginous tumor specimens. Eighteen tumors were determined to be chondrosarcoma including 8 Grade 1 tumors, 6 Grade 2 tumors, and 4 Grade 3 tumors. The percentage of CTGF positive and PCNA positive cells was quantified using at least 500 cells.
RESULTS, CTGF was expressed in 70.1% of enchondroma cells, 84.0% of Grade 1 chondrosarcoma cells, 53.7% of Grade 2 tumor cells, and 26.8% of Grade 3 tumor cells (rho = -0.501; P = 0.0053). In chondrosarcoma cases, CTGF expression was correlated closely with tumor grade (rho = -0.920; P = 0.0001). There was a strong correlation between PCNA expression and tumor grade (rho = 0.907; P < 0.0001) and a strong negative correlation between CTGF and PCNA expression (rho = -0.493; P = 0.0061]. In chondrosarcoma cases, patients with high expression of CTGF (greater than or equal to 30%) showed higher overall survival compared with those with low expression (< 30%) (P = 0.004).
CONCLUSIONS. The current study revealed a correlation between the histologic grade of chondrosarcoma and prognosis, and the concomitant association between CTGF immunostaining and tumor grade and prognosis. Therefore, immunohistochemical staining with CTGF is a useful procedure for assessing the tumor grade and clinical course in patients with chondrosarcoma. Cancer 2000;89: 1466-73. (C) 2000 American Cancer Society.

Regulation of human COL2A1 gene expression in chondrocytes - Identification of C-Krox-responsive elements and modulation by phenotype alteration

C Ghayor, JF Herrouin, C Chadjichristos, L Ala-Kokko, M Takigawa, JP Pujol, P Galera

JOURNAL OF BIOLOGICAL CHEMISTRY 275 ( 35 ) 27421 - 27438 2000.9

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

To identify control motifs involved in human type II collagen gene transcription in both differentiated and dedifferentiated rabbit articular chondrocytes, transient transfection experiments were performed, A 715-base pair (bp) region of the first intron (+2127/+2842), including a 153-bp sequence so far uncharacterized (+2689/+2842), was found to mediate enhancer activity. In dedifferentiated chondrocytes, this enhancer activity was shown to be less effective than in primary cultures but still present. We then demonstrated that a zinc finger protein, C-Krox, activates COL2A1 gene transcription in differentiated chondrocytes through the enhancer region, whereas in subcultured cells, it inhibited the gene activity via a 266-bp promoter. Multicopies of the C-Krox binding site were found to mediate transactivation in both primary cultures and passaged cells, whereas C-Krox overexpression inhibited transcription in dedifferentiated chondrocytes. Additionally, we showed that C-Krox binds to several cis sequences that mediate its transcriptional effects. During chondrocyte dedifferentiation, the protein levels and binding activity of C-Krox were reduced, whereas those of NF-KB were increased. This was not associated with variations of mRNA levels, suggesting that post-transcriptional regulatory mechanisms could be involved in C-Krox expression. These results suggest that C-Krox plays a major role in type II collagen expression and the chondrocyte phenotype modulation.

Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) gene: Similarities to retroviral RNA-protein interactions

S Kubota, S Kondo, T Eguchi, T Hattori, T Nakanishi, RJ Pomerantz, M Takigawa

ONCOGENE 19 ( 41 ) 4773 - 4786 2000.9

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

The repressive effect of the 3'-untranslated region (3'-UTR) in human connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) mRNA on gene expression had been demonstrated in our previous study. Here, we identified a minimal RNA element in the 3'-UTR, which acts as a cis-acting element of structure-anchored repression (CAESAR). Deletion analyses of the 3'-UTR led us to minimize the element of 84 bases at the junction of the coding region and the 3'-UTR. The minimized RNA segment is predicted, and actually capable of forming a stable secondary structure in vitro. Mutational analyses disclosed a significant relationship between the predicted structure and repressive effect. The utility of CAESAR as a post-transcriptional regulatory element was represented by the fact that steady-state mRNA levels were not affected by CAESAR linked in cis, while protein levels from such a chimeric gene were markedly reduced. Of note, the CAESAR sequence exerted no effect, when it was placed upstream of the promoter. Finally, RNA gel electromobility-shift analyses demonstrated a nuclear factor that interacts with the folded CAESAR. Taken together, it was uncovered that CAESAR of ctgf is a novel post-transcriptional structured RNA regulatory element, probably acting through direct interactions with a nuclear factor as observed in retroviral RNA elements with certain proteins.

Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro

T Nishida, T Nakanishi, M Asano, T Shimo, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 184 ( 2 ) 197 - 206 2000.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product Hcs24 (CTGF/Hcs24) promotes the proliferation and differentiation of chondrocytes and endothelial cells which are involved in endochondral ossification (Shimo et al., 1998, J Biochem 124:130-140; Shimo el al., 1999, J Biochem 126:137-145; Nakanishi et at., 2000, Endocrinology 141:264-273). To further clarify the role of CTGF/Hcs24 in endochondral ossification, here we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of osteoblastic cell lines in vitro. A binding study using I-125-labeled recombinant CTGF/Hcs24 (rCTGF/Hcs24) disclosed two classes of specific binding sites on a human osteosarcoma cell line, Saos-2. The apparent dissociation constant (Kd) value of each binding sire was 17.2 and 391 nM, respectively. A cross-linking study revealed the formation of I-125-rCTGF/Hcs24-receptor complex with an apparent molecular weight of 280 kDa. The intensity of I-125-rCTGF/Hcs24-receptor complex decreased on the addition of increasing concentrations of unlabeled rCTGF/Hcs24, but not platelet-derived growth factor-BB homodimer or basic fibroblast growth factor. These findings suggest that osteoblastic cells have specific receptor molecules for CTGF/Hcs24. rCTGF/Hcs24 promoted the proliferation of Saos-2 cells and a mouse osteoblast cell line MC3T3-E1 in a dose- and time-dependent manner. rCTGF/Hcs24 also increased mRNA expression of type I collagen, alkaline phosphatase, osteopontin, and osteocalcin in both Saos-2 cells and MC3T3-E1 cells. Moreover, rCTGF/Hcs24 increased alkaline phosphatase activity in both cells, it also stimulated collagen synthesis in MC3T3-E1 cells. Furthermore, rCTGF/Hcs24 stimulated the matrix mineralization on MC3T3-E1 cells and its stimulatory effect was comparable to that of bone morphogenetic protein-2. These findings indicate that CTGF/Hcs24 is a novel, potent stimulator for the proliferation and differentiation of osteoblasts in addition to chondrocytes and endothelial cells. Because of these functions, we are re-defining CTGF/Hcs24 as a major factor to promote endochondral ossification to be called "ecogenin: endochondral ossification genetic factor." J. Cell. Physiol. 184:197-206, 2000. (C) 2000 Wiley-Liss, Inc.

軟骨由来の成長因子CTGF/Hcs24遺伝子の転写後制御エレメント,CAESARの構造機能連関

久保田聡, 近藤誠二, 江口傑徳, 服部高子, 中西徹, 滝川正春

生化学 72 ( 8 ) 972 - 972 2000.8

Identity of urinary trypsin inhibitor-binding protein to link protein

H Kobayashi, Y Hirashima, GW Sun, M Fujie, T Nishida, M Takigawa, T Terao

JOURNAL OF BIOLOGICAL CHEMISTRY 275 ( 28 ) 21185 - 21191 2000.7

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs). Here we report that the 40-kDa protein (UTI-BP(40)) was purified from the cultured human chondrosarcoma cell line HCS-2/8 by UTI affinity chromatography. Purified UTI-BP(40) was digested with trypsin, and the amino acid sequences of the peptide fragments were determined. The sequences of six tryptic fragments of UTI-BP(40) were identical to subsequences present in human link protein (LP). Authentic bovine LP and UTI-BP(40) displayed identical electrophoretic and chromatographic behavior. The UTI-binding properties of UTI-BP(40) and LP were indistinguishable. Direct binding and competition studies strongly demonstrated that the NH(2)-terminal fragment is the UTI-binding part of the LP molecule, that the COOH-terminal UTI fragment (HI-8) failed to bind the NH(2)-terminal subdomain of the LP molecule, and that LP and UTI-BP(40) exhibited significant hyaluronic acid binding. These results demonstrate that UTI-BP(40) is identical to LP and that the NH(2)-terminal domain of UTI is involved in the interaction with the NH(2)-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.

Expression of neurotrophins and their receptors (TRK) during fracture healing

K Asaumi, T Nakanishi, H Asahara, H Inoue, M Takigawa

BONE 26 ( 6 ) 625 - 633 2000.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

To clarify the roles of neurotrophins and their receptors in bone formation, expression of neurotrophins and their receptors (TRK) in a model of mouse fracture healing was investigated, A total of 120 male ICR mice were studied. The right eighth rib of 70 mice was fractured. For sham operation as a control, the right eighth rib of 50 mice was similarly exposed but not fractured, Localization of TRKA, TRKB, and TRKC in a rectangular region of the rib together with surrounding soft tissues was investigated by immunostaining. Localizations of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) at the fracture callus were also investigated by immunostaining, and their mitochondrial RNA (mRNA) expressions were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), As a result, we observed two types of neurotrophin receptors in the bone forming al ea: immunostaining by anti-TRK was observed in almost all bone forming cells, and staining with anti-TRKC was observed in osteoblast-like cells and hypertrophic chondrocytes, but no staining was observed with anti-TRKB. On the other hand, localization of NGF was observed in almost all bone forming cells, localization of BDNF was observed in osteoblast-like cells, and localization of NT-3 was observed in osteoblast-like cells and hypertrophic chondrocytes at the fracture callus. Expression levels of the mRNA of three neurotrophins in the fractured rib were increased during the process of healing, especially those of NGF and NT-3, which peaked at 2 days after the fracture. The level of BDNF mRNA increased gradually over 8 days, These findings show that neurotrophins and their receptors were expressed in bone forming tells, and suggest that they are involved in the regulation of bone formation as an autocrine and paracrine factor in vivo. (Bone 26:625-633; 2000) (C) 2000 by Elsevier Science Inc. All rights reserved.

軟骨細胞および線維芽細胞株におけるCTGF/Ecogenin遺伝子発現の3'-UTRによる調節

近藤誠二, 久保田聡, 江口傑徳, 服部高子, 中西徹, 菅原利夫, 滝川正春

岡山歯学会雑誌 19 ( 1 ) 205 - 206 2000.6

軟骨由来の成長因子CTGF/Hcs24遺伝子に同定された新たな転写後制御エレメント,CAESAR

久保田聡, 近藤誠二, 江口傑徳, 服部高子, 中西徹, 滝川正春

日本骨代謝学会雑誌 18 ( 2 ) 119 - 119 2000.6

Expression of neurotrophins and their receptors (TRK) during fracture healing.

K Asaumi, T Nakanishi, H Asahara, H Inoue, M Takigawa

Bone 26 ( 6 ) 625 - 633 2000.6

Novel intracellular effects of human connective tissue growth factor expressed in Cos-7 cells

S Kubota, T Hattori, T Shimo, T Nakanishi, M Takigawa

FEBS LETTERS 474 ( 1 ) 58 - 62 2000.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

To clarify the multiple functionality of connective tissue growth factor (CTGF), we examined the effects of nascent CTGF within the cell by transient expression. Tn Cos-7 cells, expression of human CTGF induced an altered cell morphology, It was associated with an increased cellular DNA content and loose attachment, indicating the cells mere in G2/M phase. Overexpression of CTGF did not induce cell growth, whereas recombinant CTGF efficiently stimulated the proliferation extracellularly, These results indicate that intracellular CTGF may act as an antimitotic agent, thus it should also be noted that nascent CTGF was found to accumulate around the central mitotic machinery. (C) 2000 Federation of European Biochemical Societies.

Ex vivo gene delivery using an adenovirus vector in treatment for cartilage defects

T Ikeda, T Kubo, T Nakanishi, Y Arai, K Kobayashi, O Mazda, S Ohashi, K Takahashi, J Imanishi, M Takigawa, Y Hirasawa

JOURNAL OF RHEUMATOLOGY 27 ( 4 ) 990 - 996 2000.4

Language：English Publishing type：Research paper (scientific journal) Publisher：J RHEUMATOL PUBL CO

Objective. To realize local selective gene expression in grafted chondrocytes for cartilage defect, we investigated the usefulness of an ex vivo gene delivery method using an adenovirus vector.
Methods. beta-galactosidase gene (LacZ) was transfected using an adenovirus vector to chondrocytes isolated from rat joints. The cells were then embedded into collagen gel, and LacZ expression in the gel was examined using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining; beta-galactosidase activity was also measured. The collagen gel containing transfected chondrocytes was grafted to the experimental cartilage defects, and the expression of delivered gene was histologically examined after X-gal staining of the tissue containing the grafted area.
Results. X-gal positive chondrocytes in the gel accounted for 82% at one week and 55% at 8 weeks after gene delivery. beta-galactosidase activity decreased with time, but its expression was maintained even at 8 weeks after gene delivery. Chondrocytes used in the allograft maintained their morphology, and the expression of delivered gene continued during the 8 week period.
Conclusion. In this ex vivo method, delivered gene can be expressed efficiently for a long time; this method would be useful in allografts for cartilage defects.

軟骨細胞の基質代謝に及ぼす周期的メカニカルストレスの影響

藤沢拓生, 服部高子, 窪木拓男, 矢谷博文, 山下敦, 滝川正春

日本顎関節学会雑誌 12 ( 1 ) 133 - 134 2000.4

Serum levels of connective tissue growth factor are elevated in patients with systemic sclerosis: Association with extent of skin sclerosis and severity of pulmonary fibrosis

S Sato, T Nagaoka, M Hasegawa, T Tamatani, T Nakanishi, M Takigawa, K Takehara

JOURNAL OF RHEUMATOLOGY 27 ( 1 ) 149 - 154 2000.1

Language：English Publishing type：Research paper (scientific journal) Publisher：J RHEUMATOL PUBL CO

Objective. To determine the serum levels and clinical correlation of connective tissue growth factors (CTGF) in patients with systemic sclerosis (SSc).
Methods. Serum samples from patients with limited cutaneous SSc (lSSc, n = 32), diffuse cutaneous SSc (dSSc, n = 28), systemic lupus erythematosus (SLE, n = 30), polymyositis/dermatomyositis (PM/DM, n = 20), and healthy control subjects (n = 30) were examined by ELISA for detection of CTGF.
Results. Serum CTGF levels in patients with SSc were significantly higher than those in patients with SLE or PM/DM, and in controls, CTGF levels in patients with dSSc were significantly higher than those in patients with lSSc. As for clinical correlation of CTGF, SSc patients with elevated CTGF had pulmonary fibrosis, decreased DLCO, and decreased vital capacity-more frequently than those with normal CTGF levels. Further, DLCO and vital capacity were inversely and directly correlated with serum CTGF levels in patients with SSc. The dSSc patients with disease duration of 1-3 years had significantly elevated levels of CTGF compared with dSSc patients with duration < 1 year or more than 3 years.
Conclusion. Serum CTGF levels were increased in patients with SSc, and correlated with the extent of skin sclerosis and the severity of pulmonary fibrosis. In addition, it appears that production of CTGF is involved in the development or maintenance of fibrosis rather than in initiation of fibrosis in SSc. These data suggest that CTGF plays a critical role in the development of fibrosis in SSc.

Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture

T Nakanishi, T Nishida, T Shimo, K Kobayashi, T Kubo, T Tamatani, K Tezuka, M Takigawa

ENDOCRINOLOGY 141 ( 1 ) 264 - 273 2000.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 func- tion in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs24 effectively promoted the proliferation of HCS-2/8 cells and RGC cells in a dose-dependent manner and also dose dependently increased proteoglycan synthesis in these cells. In addition, these stimulatory effects of CTGF/Hcs24 were neutralized by the addition of anti-CTGF antibodies. Furthermore, the recombinant CTGF/Hcs24 effectively increased alkaline phosphatase activity in RGC cells in culture. Moreover, RT-PCR analysis revealed that the recombinant CTGF/Hcs24 stimulated gene expression of aggrecan and collagen types II and X in RGC cells in culture. These results indicate that CTGF/Hcs24 directly promotes the proliferation and differentiation of chondrocytes.

Rheumatoid arthritis-related antigen 47kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47

T Hattori, K Takahashi, Y Yutani, T Fujisawa, T Nakanishi, M Takigawa

JOURNAL OF BONE AND MINERAL METABOLISM 18 ( 6 ) 328 - 334 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER-VERLAG TOKYO

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of collipin-2. C-504 and G(505) in the cDNA sequences of both cells and C-598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47 kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous finding show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Synovial Fluids of Patients with Temporomandibular Joint Osteoarthritis

Manabu Kanyama, Takuo Kuboki, Shunji Kojima, Takuo Fujisawa, Takako Hattori, Masaharu Takigawa, Atsushi Yamashita

Journal of Orofacial Pain 14 ( 1 ) 20 - 30 2000

Language：English Publishing type：Research paper (scientific journal)

Aims: Imbalance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) may be involved in the breakdown of articular cartilage matrix of the temporomandibular joint (TMJ). Aims: In this study, MMPs, TIMPs, and MMP-1/TIMP-1 complex levels were examined in TMJ synovial fluid samples aspirated from TMJ osteoarthritis (OA) patients (2 males, 8 females
mean age, 29.7 years) and asymptomatic control subjects (2 males, 8 females
mean age, 23.6 years) to determine the likelihood of increased proteolytic activity in the OA joints. Methods: The various types of MMPs and TIMPs were detected by Western blotting with monoclonal antibodies and gelatin zymography. The MMP-1/ TIMP-1 complex level was measured by an enzyme-linked immunosorbent assay kit. All aspirates were first analyzed for total protein content and then individually diluted to make the total protein levels equivalent. Results: The mean MMP-1/TIMP-1 complex concentration in the synovial fluids of the OA patients was 3.92 ± 1.39 ng/mL
this value was significantly lower (P &lt
0.05) than the value from control subjects (5.46 ± 1.32 ng/mL). Matrix metalloproteinase-1 (52 kDa), MMP-3 (57 kDa), TIMP-1 (28 kDa), and TIMP-2 (26 kDa) were detected in all of the normal and the OA samples. However, MMP-1 (28 kDa), MMP-2 (72 kDa), MMP-3 (45 kDa), and MMP-9 (83 kDa) were detected in higher concentration in the OA samples. Conclusion: These findings suggest a strong association between the OA-active joints and the presence of biologically active forms of known tissue degradation enzymes (MMP-1, MMP-3, and MMP-9).

マトリクラインとMMP Invited

服部高子, 滝川正春

現代医療、現代医療社 (4), 909- 915, ( 4 ) 909 - 915 2000

Electrophoretic and serologic characterization of 56 kDa antigen (M56) with autologous serum derived from a chondrosarcoma patient: A shared antigen of immunoresponses in cancer and autoimmune diseases

K Fujiwara, H Udono, T Kunisada, A Kawai, H Inoue, M Takigawa, M Namba, E Nakayama

ELECTROPHORESIS 20 ( 17 ) 3335 - 3342 1999.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-V C H VERLAG GMBH

We investigated whether antibodies specific to autologous cancer cells are produced in the peripheral blood of patients with chondrosarcoma. There have been few reports on the investigation of the immune responses, such as autologous antibody production, to chondrosarcoma. Here, tumor-associated antigens were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and detected by immunoenzymatic amplification. A 56 kDa molecule (M56) was detected in the serum from patients' peripheral blood. M56 is ubiquitously expressed in various kinds of tissue-derived cells. However, the molecule seemed to be retained mostly in the cytosolic compartment of lymphoid cells, while it was expressed on the cell surface of nonlymphoid cancer cells. Furthermore, the antibodies reactive to the 56 kDa molecule were frequently observed in sera derived from patients with other cancers and autoimmune diseases as compared to the sera from healthy control donors, suggesting that M56 is a common target molecule of immune responses in patients with various cancers and autoimmune diseases.

Role and interaction of connective tissue growth factor with transforming growth factor-beta in persistent fibrosis: A mouse fibrosis model

T Mori, S Kawara, M Shinozaki, N Hayashi, T Kakinuma, A Igarashi, M Takigawa, T Nakanishi, K Takehara

JOURNAL OF CELLULAR PHYSIOLOGY 181 ( 1 ) 153 - 159 1999.10

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Skin fibrotic disorders are understood to develop under the influence of some growth factors, such as transforming growth factor-beta (TCF-beta), basic fibroblast growth factor (bFGF), or connective tissue growth factor (CTGF). To establish an appropriate animal model of skin fibrosis by exogenous application of growth factors, we investigated the in vivo effects of growth factors by injecting TCF-beta, CTGF, and bFGF into the subcutaneous tissue of newborn mice. A single application of TGF-beta or bFGF resulted in the formation of transient granulated tissue that disappeared despite 7 days of consecutive injections. A single CTGF injection also caused slight granulation. However, injecting TGF-beta plus CTGF produced long-term fibrotic tissue, which persisted for at least 14 days. Also, fibrotic tissue was observed when CTGF was injected from 4 to 7 days after TGF-beta injections for the first 1-3 days. In situ hybridization analysis revealed the expression of CTGF mRNA in the fibroblasts at least in a few fibrotic conditions. These findings suggest that either CTGF mRNA or an application of exogenous CTGF protein is required for the development of persistent fibrosis. From our study, it appears that interaction of several growth factors is required for persistent fibrotic tissue formation, with TGF-beta causing the induction and CTGF needed for maintenance of skin fibrosis. The animal model on skin fibrosis by exogenous application of growth factors developed in this study may prove useful for future studies on fibrotic disorders. J. Cell. Physiol. 181:153-159, 1999. (C) 1999 Wiley-Liss, Inc.

Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs

T Kuboki, T Nakanishi, M Kanyama, W Sonoyama, T Fujisawa, K Kobayashi, T Ikeda, T Kubo, A Yamashita, M Takigawa

ARCHIVES OF ORAL BIOLOGY 44 ( 9 ) 701 - 709 1999.9

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint using the adenovirus vector is feasible as an effective in vivo method, (C) 1999 Elsevier Science Ltd. All rights reserved.

Control of delivered gene expression in chondrocytes using heat shock protein 70B promoter

Y Arai, T Kubo, K Kobayashi, T Ikeda, K Takahashi, M Takigawa, J Imanishi, Y Hirasawa

JOURNAL OF RHEUMATOLOGY 26 ( 8 ) 1769 - 1774 1999.8

Language：English Publishing type：Research paper (scientific journal) Publisher：J RHEUMATOL PUBL CO

Objective. To investigate whether the expressions of delivered Escherichia coli beta-galactosidase (LacZ) gene and transforming growth factor-beta 1 (TGF-beta 1) gene are regulated by the stress response of human chondrocyte-like cells (HCS-2/8) when heat shock protein 70B (HSP70B) promoter is inserted into the adenovirus vector,
Methods. Two adenovirus vectors that contain either LacZ gene or TGF-beta 1 gene regulated by HSP70B promoter were constructed. One of the adenovirus vectors was added to the culture of HCS-2/8 and gene transduced cells were produced. We applied heat stress (43 degrees C) to the transduced cells for 2 h and examined whether the expression of transduced LacZ and TGF-beta 1 genes is affected by the stress, using 5 bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, measurement of B-galactosidase activity, Northern blotting, and ELISA.
Results. The percentage of X-gal positive stained cells in LacZ gene-delivered cells with heat stress was significantly higher than in controls (no heat stress). With heat stress, beta-galactosidase activity increased significantly, and the band of exogenous TGF-beta 1 mRNA became more apparent and the expression was maintained during the 24 h monitoring period. TGF-beta 1 level in culture supernatant of TGF-beta 1 gene-delivered cells with heat stress (5377.3 +/- 321.1 pg/ml) was significantly higher than in the controls (853.2 +/- 29.2 pg/ml).
Conclusion, HSP70B promoter could regulate the expression of delivered genes according to the intensity of heat stress.

骨・軟骨の発生過程における軟骨由来成長因子CTGF/Hcs24の発現とCbfa1によるその制御

中西徹, 浅野将宏, 縄稚久美子, 山合友一郎, 小守寿文, 西田崇, 吉道玄, 久保田聡, 服部高子, 滝川正春

生化学 71 ( 8 ) 1033 - 1033 1999.8

TNFαによるRA-A47の軟骨細胞内局在の変化と抗原提示

服部高子, 藤沢拓生, 油谷安孝, 中西徹, 滝川正春

生化学 71 ( 8 ) 969 - 969 1999.8

Immunohistochemical localization of connective tissue growth factor in the rat central nervous system

Y Kondo, T Nakanishi, M Takigawa, N Ogawa

BRAIN RESEARCH 834 ( 1-2 ) 146 - 151 1999.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Connective tissue growth factor (CTGF) is an immediate early growth-responsive gene but its distribution and significance in the central nervous system (CNS) are unknown. We investigated the distribution of CTGF-like immunoreactivity (CTGF-IR) in the rat CNS using a specific antiserum against CTGF oligopeptide. The majority of CTGF-IR was observed in astrocytes. Ependymal cells lining the wall of the cerebral ventricle and tanycytes Lining the central canal of the spinal cord showed the strongest CTGF-IR, while there was a diffuse but weak signal in the gray matter of the spinal cord. CTGF-IR was also detected in the cytoplasm of a subpopulation of pyramidal neurons in the cerebral cortex. Our results showed that CTGF-IR is widely distributed in the CNS at bath regional and cellular levels, suggesting a complex functional role in the CNS. (C) 1999 Elsevier Science B.V. All rights reserved.

Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo

T Shimo, T Nakanishi, T Nishida, R Asano, M Kanyama, T Kuboki, T Tamatani, K Tezuka, M Takemura, T Matsumura, M Takigawa

JOURNAL OF BIOCHEMISTRY 126 ( 1 ) 137 - 145 1999.7

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE BIOCHEMICAL SOC

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies, (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process.

Physiological function of connective tissue growth factor (CTGF/Hcs24) - Its roles in the process of endochondral ossification Reviewed

T Nakanishi, M Takigawa

SEIKAGAKU 71 ( 6 ) 429 - 432 1999.6

骨芽細胞様細胞株MC3T3-E1のメカニカルストレスに対する応答性神経栄養因子の役割

稲熊尚広, 山本照子, 中西徹, 山城隆, 山下和夫, 滝川正春

日本骨代謝学会雑誌 17 ( 2 ) 195 - 195 1999.6

アデノウイルスベクター法を用いた顎関節への遺伝子導入の試み

園山亘, 中西徹, 窪木拓男, 完山学, 山下敦, 滝川正春

岡山歯学会雑誌 18 ( 1 ) 283 - 283 1999.6

Detection of specific antibodies against human cultured chondrosarcoma (HCS-2/8) and osteosarcoma (Saos-2) cells in the serum of patients with osteoarthritis of the temporomandibular joint

T Kuboki, T Hattori, T Mizushima, M Kanyama, T Fujisawa, A Yamashita, M Takigawa

ARCHIVES OF ORAL BIOLOGY 44 ( 5 ) 403 - 414 1999.5

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

To find specific humoral antibodies in sera from patients with temporomandibular joint (TMJ) osteoarthritis (OA), an immortal human chondrocyte (HCS-2/8) and osteoblast (Saos-2) cell line derived from a chondrosarcoma and an osteosarcoma, respectively, were used as source proteins of human antigens. Patients with chronically painful TMJ OA (n = 18) but no other joints symptoms were selected from a consecutive series of patients with temperomandibular disorders and sex-matched asymptomatic controls (n = 8) were also recruited. Cellular proteins of the HCS-2/8 and Saos-2 cells were subjected to Western blotting with the OA and control sera as probes. Band-recognition frequency and the peak optical density of the band were compared between groups by chi(2) and t-tests. OA sera recognized various bands for the chondrocytes, and one of these (47-kDa) was specific for the OA sera. In two OA patients whose treatment outcome was less favorable, the,reactivity against the 47-kDa protein was relatively high. In addition, the OA sera clearly cross-reacted with recombinant HSP47. Based on these findings, an autoimmune reaction against chondrocytes could be one of the exaggerating and/or perpetuating mechanisms in the pathophysiology of osteoarthritic TMJs, and the humoral antibody titre against the HSP47-like protein derived from the chondrocytes could be one of the possible markers for the prognosis of the joint pathology. (C) 1999 Elsevier Science Ltd. All rights reserved.

Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1

T Fujisawa, T Hattori, K Takahashi, T Kuboki, A Yamashita, M Takigawa

JOURNAL OF BIOCHEMISTRY 125 ( 5 ) 966 - 975 1999.5

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE BIOCHEMICAL SOC

To clarify the mechanism of cartilage degradation induced by mechanical stress, we investigated the influence of cyclic tension force (CTF) on the metabolism of cultured chondrocytes. The chondrocytes were exposed to CTF using a Flexercell strain unit. Five or 15 kPa of high frequency CTF significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein, Fifteen kPa of high frequency CTF induced the expression of interleukin-1 (IL-1), matrix metalloproteinase (MMP)-2 and -9 mRNA, and increased the production of pro- and active-MMP-9. The degradation of proteoglycan was inhibited by and MMP inhibitor, indicating that MMPs are involved in the degradation of proteoglycans induced by high frequency CTF, Moreover, reducing the frequency of CTF from high to low decreased the inhibition of proteoglycan synthesis, These findings suggest that the CTF frequency is one of the key determinants of chondrocyte metabolism. Low magnitude CTF, whether high or low frequency, did not cause the gene expression of cartilage degradation factors, suggesting that this CTF magnitude causes only minor changes in the cartilage matrix, High magnitude and frequency CTF caused the gene expression of IL-1 and MMP-9, followed by increases in the production of MMP-2 and -9 proteins, suggesting that excessive and continuous cyclic mechanical stress induces the production of IL-1 and MMP-9, resulting in cartilage degradation.

周期的な機械的ストレスを培養軟骨細胞に加え続けると,MMP及びIL-1の発現が誘導され,細胞外マトリックスの分解が引き起こされる

Fujisawa Takuo, Hattori Takako, Takahashi Kojiro, Kuboki Takuo, Yamashita Atsushi, Takigawa Masaharu

The Journal of Biochemistry 125 ( 5 ) 966 - 975 1999.5

Language：English Publisher：(公社)日本生化学会

機械的刺激による軟骨の分解について調べた.Flexercell strain unitを用いて周期的張力(CTF)を培養軟骨細胞に加えたところ,5kPa及び15kPaの高頻度CTFにより,DNA合成,プロテオグリカン合成,コラーゲン合成の低下が見られた.15kPaの高頻度CTFではIL-1,matrix metalloproteinase-2(MMP-2),MMP-9のmRNAの発現と活性型MMP-9の産生が増加した.この時,プロテオグリカンの分解が促進したが,MMPの阻害剤の存在下では分解の促進が抑えられた.張力を小さくすると,軟骨の分解に関わる遺伝子の発現誘導は見られなかった.強度の高頻度CTFはIL-1,MMP-2,MMP-9のmRNAの発現,及び活性型MMP-9の産生を増大させることから,周期的な機械的ストレスが軟骨の分解を引き起こすと考えられる

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=1999&ichushi_jid=J04549&link_issn=&doc_id=19990603080012&doc_link_id=10005463964&url=https%3A%2F%2Fci.nii.ac.jp%2Fnaid%2F10005463964&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_1.gif

Effect of pressure loading on interleukin-8 production in chondrocytes

Toshikazu Kubo, Yuji Arai, Kenji Takahashi, Toshihiro Ishida, Takuo Fujisawa, Masaharu Takigawa, Jiro Imanishi, Yasusuke Hirasawa

Pathophysiology 6 ( 1 ) 35 - 39 1999.4

Language：English Publishing type：Research paper (scientific journal)

The present study investigated the influence of hydrostatic pressure on the expression of interleukin-8 (IL-8) in a human chondrocyte-like cell line (HCS-2/8). The cells were exposed to hydrostatic pressure (1, 5, 10, or 50 MPa) by a special apparatus. Total RNA was extracted, and was analyzed by a polymerase chain reaction method for IL-8 mRNA. IL-8 concentrations in a culture medium were assessed by ELISA. The expression of IL-8 mRNA was enhanced after exposure to 50 MPa of hydrostatic pressure. IL-8 level in a culture supernatant increased following 50 MPa of pressure. Increased IL-8 production in the present study is an important point when we consider the pathology of inflammatory joint diseases, and this point also shows that treatment which reduces mechanical loading could improve, or slow the progression of, joint diseases.

DOI： 10.1016/S0928-4680(98)00032-7

Involvement of cis-acting repressive element(s) in the 3 '-untranslated region of human connective tissue growth factor gene

S Kubota, T Hattori, T Nakanishi, M Takigawa

FEBS LETTERS 450 ( 1-2 ) 84 - 88 1999.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it,vas fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR, (C) 1999 Federation of European Biochemical Societies.

アデノウイルスベクターを用いた顎関節への遺伝子導入の試み

窪木拓男, 中西徹, 完山学, 園山亘, 水島恒尚, 小島俊司, 山下敦, 滝川正春

日本顎関節学会雑誌 11 ( 1 ) 89 - 89 1999.4

Gene delivery to chondrocytes using adenovirus vector Reviewed

T Kubo, Y Arai, K Kobayashi, J Imanishi, M Takigawa, Y Hirasawa

ADVANCES IN OSTEOARTHRITIS 107 - 118 1999

Language：English Publishing type：Research paper (international conference proceedings) Publisher：SPRINGER-VERLAG TOKYO

The objective of this study was to investigate the effects of adenovirus vector (Ax-)mediated gene transduction of E. coli beta-galactosidase (LacZ) and transforming growth factor-beta 1 (TGF-beta 1) into a human chondrocyte-like cell line (HCS-2/8). The expression of transduced genes and their expression periods were examined by 5-bromo-4-chloroindolyl-beta-D-galactoside (X-gal) staining, Northern blotting, ELISA, and Western blotting. To assess the influence of TGF-beta 1 gene transduction, the expression of mRNAs of type II collagen, proteoglycan core protein, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were examined by Northern blotting. Staining with X-gal indicated that the genes were transduced into 99% of the cells. Expression of the transduced genes in the cells was continued for at least 21 days. Transduction of the TGF-beta 1 gene enhanced mRNA expressions of type II collagen and proteoglycan core protein, but suppressed MMP-3 mRNA expression in the cells. These results indicate Ax is useful in chondrocyte gene therapy, and it could be an efficient mediator of TGF-beta 1 gene transduction.

結合組織成長因子CTGF/Hcs24の生理機能 Invited

中西徹, 滝川正春

生化学 71、429-432、 ( 6 ) 429 - 432 1999

骨形成過程における神経栄養因子およびその受容体の発現 Invited

浅海浩二, 中西徹, 浅原弘嗣, 井上一, 滝川正春

骨・関節・靭帯 12、295-297、 ( 3 ) 295 - 297 1999

Adenovirus vector-mediated gene transduction to chondrocytes : In vitro evaluation of therapeutic efficacy of transforming growth factor-β1 and heat shock protein 70 gene transduction. Reviewed

Arai, Y, Kubo, T, Kobayashi, K, Ikeda, T, Takahashi, K, Takigawa, M, Imanishi, J, Hirasawa, Y

J. Rheumatol. 24 1787 - 1795 1999

Cartilaginous differentiation in the joint capsule

Y Yutani, Y Yano, H Ohashi, M Takigawa, Y Yamano

JOURNAL OF BONE AND MINERAL METABOLISM 17 ( 1 ) 7 - 10 1999

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER-VERLAG TOKYO

The proliferation and differentiation of cells are greatly influenced by their environment. Many growth factors and cytokines are reported to be environmental factors that affect the proliferation and differentiation of cells. Mechanical stress is also considered to influence these physiological reactions. The joint capsule, which is a part of the joint tissue, plays a very important role in the stability of the joint and in maintaining the intracapsular phenomenon. In patients with dislocated hip arthropathy, this capsule is involved in the weightbearing function by forming a sliding surface between the capsule and the femoral head articular cartilage. The surface of the tissue macroscopically shows cartilaginous change, which indicates cartilaginous differentiation caused by mechanical stress. We examined the cartilage-specific proteoglycan component, which is composed of cartilaginou matrix at the differentiation site. We investigated proteoglycan production, molecular size, and the gene expression of cartilaginous substrate. At the inner layer of the weightbearing area of the joint capsule, proteoglycan production was significantly higher than that of other noncartilaginous tissue. We also identified the gene expression of cartilaginous proteoglycan using the reverse transcription polymerase chain reaction (RT-PCR) method.

Expression of osteopontin in Meckel's cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis Reviewed

K Ishizeki, S Nomura, M Takigawa, H Shioji, T Nawa

HISTOCHEMISTRY AND CELL BIOLOGY 110 ( 5 ) 457 - 466 1998.11

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

The localization of osteopontin (OP) was examined in Meckel's cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OF-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OF. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix.

Increased expression of connective tissue growth factor in the infarct zone of experimentally induced myocardial infarction in rats

H Ohnishi, T Oka, S Kusachi, T Nakanishi, K Takeda, M Nakahama, M Doi, T Murakami, Y Ninomiya, M Takigawa, T Tsuji

JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 30 ( 11 ) 2411 - 2422 1998.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD

Connective tissue growth factor (CTGF), a 36- to 38-kDa peptide, is selectively induced by transforming growth factor-beta and has been suggested to contribute to tissue repair. To test the hypothesis that CTGF is expressed in myocardial infarct tissue following acute myocardial infarction (AMI), we examined CTGF expression after AMI was experimentally induced in rats. Myocardial infarction was induced by left coronary artery ligation in male Sprague-Dawley rats. Northern blotting demonstrated that the CTGF mRNA expression on days 2, 7 and 14 was increased by 6-, 23- and 8-fold, respectively, compared to that in the pre-ligation hearts. In situ hybridization revealed CTGF mRNA signals on day 2 in myocytes in the infarct marginal zone and spindle-shaped mesenchymal cells (presumably myofibroblasts and fibroblasts) located between surviving myocytes in the infarct peripheral zone. On day 7, the signals were observed in the inner lesion of the infarct around infarct granulation tissue. Western blotting demonstrated that the CTGF protein expression on days 2, 7 and 14 was increased compared to the pre-ligation hearts. Immunopositive staining for CTGF was observed in the inner lesion of the infarct tissue on day 7. In conclusion, the findings demonstrated the increased expression of: CTGF in the infarct tissue. Myocytes in the infarct marginal zone and spindle-shaped mesenchymal cells (presumably myofibroblasts and fibroblasts) were the cells responsible for CTGF production. (C) 1998 Academic Press.

Establishment of the enzyme-linked immunosorbent assay for connective tissue growth factor (CTGF) and its detection in the sera of biliary atresia Reviewed

T Tamatani, H Kobayashi, K Tezuka, S Sakamoto, K Suzuki, T Nakanishi, M Takigawa, T Miyano

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 251 ( 3 ) 748 - 752 1998.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC

Connective tissue growth factor (CTGF) is a mitogenic, chemotactic, and cell matrix-inducing factor for fibroblasts. We generated murine monoclonal antibodies against CTGF and established a sandwich enzyme-linked immunosorbent assay (ELISA) for detection of CTGF. By using the ELISA, we confirmed that CTGF was specifically induced in human fibroblasts by TGF-beta but not by PDGF, FGF, IGF-I, or EGF. We also found that the serum levels of CTGF were significantly correlated with the progression of hepatic fibrosis in biliary atresia. These results indicated that CTGF is potentially a useful parameter for monitoring certain types of fibrotic disorders. (C) 1998 Academic Press.

Adenovirus mediated gene delivery to the joints of guinea pigs Reviewed

T Ikeda, T Kubo, Y Arai, T Nakanishi, K Kobayashi, K Takahashi, J Imanishi, M Takigawa, Y Hirasawa

JOURNAL OF RHEUMATOLOGY 25 ( 9 ) 1666 - 1673 1998.9

Language：English Publishing type：Research paper (scientific journal) Publisher：J RHEUMATOL PUBL CO

Objective. To clarify in vivo applicability of adenovirus mediated gene delivery to examine a gene therapy for human joint diseases.
Methods. We directly injected vectors harbouring beta-galactosidase gene and transforming growth factor (TGF)-beta 1 gene into the joints of Hartley guinea pigs. Expressions of delivered LacZ were examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside staining and reverse transcription-polymerase chain reaction. The levels of TGF-beta 1 that mere delivered to the joint and then transferred to the joint fluid were assessed by ELISA.
Results. LacZ expression was observed in almost all synovial tissue samples and in chondrocytes on the surface of degenerated cartilage. In the other organs, expression of delivered genes was not observed. For 2 weeks following gene delivery TGF-beta 1 levels in joint fluid were significantly higher than the levels in the controls for 2 weeks.
Conclusion. Direct gene delivery into the joint cavity is feasible with the in vivo gene delivery method using adenovirus vector and would be clinically applicable.

Peripheral type benzodiazepine receptor in T lymphocyte rich preparation Reviewed

S Maeda, T Miyawaki, T Nakanishi, M Takigawa, M Shimada

LIFE SCIENCES 63 ( 16 ) 1423 - 1430 1998.9

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Some types of mood disorders and drugs are suggested to affect peripheral type benzodiazepine receptors (PBR), but their mechanisms are unclear. The isolation of pure lymphocytes is requisite for the investigation of the function of PER on lymphocytes, since platelets and monocytes also have many PER. The objective of this study was to establish a method of binding assay for PER using pure T lymphocytes. Mononuclear cells and T lymphocytes were prepared by using a density gradient material and magnetic beads, respectively. The cells were analyzed using Row cytometry and a counting chamber. Binding studies were performed using T lymphocytes from IO normal volunteers. The T lymphocytes were incubated with [H-3]PK11195, harvested on glass fiber filters, and counted with a plate scintillation counter. The binding data were analyzed by the Scatchard method. With the magnetic bead technique, pure T cells were selected that contained only 1.5% monocytes and platelet/cell ratio of 1.4. The Scatchard plot of the data indicated that only one type of specific binding site was involved in the binding. The dissociation constant (Kd) was 3.8+/-1.3nM (mean+/-SD), and the Bmax was 379+/-124 fmol/10(6) cells (mean+/-SD). The density gradient- magnetic beads technique can be used as an appropriate method of preparation of T cells for PBR binding assay.

軟骨細胞の細胞外基質の合成と分解に及ぼす周期的メカニカルストレスの影響

藤沢拓生, 服部高子, 窪木拓男, 高橋浩二郎, 山下敦, 滝川正春

歯科基礎医学会雑誌 40 ( 抄録 ) 392 - 392 1998.9

慢性関節リウマチ関連抗原蛋白RA-A47 cDNAの単離と機能解析

服部高子, 藤沢拓生, 中西徹, 窪木拓男, 山下敦, 滝川正春

歯科基礎医学会雑誌 40 ( 抄録 ) 392 - 392 1998.9

アデノウイルスベクター法を用いた顎関節への遺伝子導入の試み

完山学, 中西徹, 窪木拓男, 園山亘, 山下敦, 滝川正春

歯科基礎医学会雑誌 40 ( 抄録 ) 450 - 450 1998.9

慢性関節リウマチ関連抗原蛋白RA-A47 cDNAの単離と構造解析

服部高子, 油谷安孝, 藤沢拓生, 中西徹, 高橋浩二郎, 滝川正春

生化学 70 ( 8 ) 848 - 848 1998.8

Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells Reviewed

T Shimo, T Nakanishi, Y Kimura, T Nishida, K Ishizeki, T Matsumura, M Takigawa

JOURNAL OF BIOCHEMISTRY 124 ( 1 ) 130 - 140 1998.7

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE BIOCHEMICAL SOC

Previously, we cloned an mRNA predominantly expressed in hypertrophic chondrocytes by differential display-PCR from a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) that is identical to that of connective tissue growth factor (CTGF), In the present study, we investigated the roles of CTGF in the proliferation and migration of vascular endothelial cells using its antisense oligonucleotide and antisense RNA, because angiogenesis into the hypertrophic zone of cartilage occurs at the final step of endochondral ossification. Immunohistochemical and immunofluorescence techniques revealed that not only hypertrophic chondrocytes but also endothelial cells in the cost-chondral junctions of mouse ribs were stained with an anti-CTGF antibody in vivo. Northern blot analysis revealed that CTGF was strongly expressed in chondrocytic cells as well as bovine aorta endothelial (BAE) cells in culture, but not in other types of cells such as osteoblastic cells. Its expression in BAE cells was greater in the growing phase than in the confluent phase. When one-half of a monolayer of a confluent culture of BAE cells had been peeled off, only the cells proliferating and extending into the vacant area were stained with the anti-CTGF antibody. The addition of an antisense oligonucleotide inhibited the proliferation and extension of the BAE cells into the vacant area. The antisense oligonucleotide also inhibited the proliferation of BAE cells in the rapidly proliferating phase. In a Boyden chamber assay, pretreatment with the antisense oligonucleotide markedly inhibited the migration of BAE cells. Furthermore, the abilities to proliferate and migrate of BAE cells, which were stably transfected with expression vectors that generate the antisense RNA of CTGF cDNA, were markedly lower than those of the control. These findings suggest that endogenous CTGF expression is involved in the proliferation and migration of BAE cells.

Demonstration of receptors specific for connective tissue growth factor on a human chondrocytic cell line (HCS-2/8) Reviewed

T Nishida, T Nakanishi, T Shimo, M Asano, T Hattori, T Tamatani, K Tezuka, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 247 ( 3 ) 905 - 909 1998.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC

The presence of receptors specific for connective tissue growth factor (CTGF) was demonstrated on a human chondrosarcoma-derived chondrocytic cell Line, HCS-2/8. The binding of I-125-labeIed recombinant CTGF to HCS-2/8 cells was inhibited by unlabeled CTGF but not by PDGF-BB or bFGF. Scatchard analysis revealed the presence of two classes of binding sites with Rd values of 18.6 and 259 nM on cells. A cross-linking study revealed the formation of I-12S-CTGF-receptor complex with an apparent molecular weight of 280 kDa, The I-125-CTGF-receptor complex disappeared almost completely on the addition of unlabeled CTGF but not PDGF-BB or bFGF, In addition, the I-125-CTGF-receptor complex was immunoprecipitated with anti-CTGF antiserum but not with anti-PDGF receptor antiserum. These findings suggest that CTGF directly binds to specific receptor molecules on HCS-2/8 cells. (C) 1998 Academic Press.

Stimulatory effects of 4-methylcatechol, dopamine and levodopa on the expression of metallothionein-III (GTF) mRNA in immortalized mouse brain glial cells (VR-2g) Reviewed

C Aoki, T Nakanishi, N Sogawa, K Ishii, N Ogawa, M Takigawa, H Furuta

BRAIN RESEARCH 792 ( 2 ) 335 - 339 1998.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Metallothionein (MT)-III, originally discovered as a growth inhibitory factor (GIF), is a brain specific isomer of MTs and is markedly reduced in the brain of Alzheimer's disease patients (AD) and in several other neurodegenerative diseases. We analyzed the level and regulation of mRNA expression of MT-III in immortalized fetal mouse brain glial cells (VR-2g) by reverse transcriptase-polymerase chain reaction (RT-PCR). The basal expression level of MT-III mRNA is very low in VR-2g cells. 4-Methylcatechol, dopamine (DA) and levodopa (L-3,4-dihydroxyphenylalanine), which stimulate the synthesis of nerve growth factor (NGF), further increased the expression of MT-III mRNA in VR-2g cells. (C) 1998 Elsevier Science B.V.

Isolation and characterization of a rheumatoid arthritis-specific antigen (RA-A47) from a human chondrocytic cell line (HCS-2/8) Reviewed

T Hattori, T Fujisawa, K Sasaki, Y Yutani, T Nakanishi, K Takahashi, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 245 ( 3 ) 679 - 683 1998.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC

Two types of 47 kDa antigen specifically recognized by sera from rheumatoid arthritis (RA) patients were isolated from the membrane fraction of a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by a 2-step procedure: preparative SDS-PAGE and reverse-phase HPLC. An N-terminal amino acid sequence in one of the 47 kDa antigens, named RA-A47, had 81% homology to that deduced from the DNA sequence of the colligin gene which is reported as human hsp47 gene, and 100% homology to that deduced from the DNA sequence of colligin-2 gene, a homologue of colligin. The RA-A47 cross-reacted with a monoclonal antibody raised against chick heat shock protein (Hsp) 47 and bound to gelatin. The expression of the ra-a47 gene was enhanced by heat shock treatment and TGF-beta stimulation. These findings suggest that RA-A47 is a Hsp47-like protein, presumably the product of the colligin-2 gene, and that a collagen-specific molecular chaperone(s) such as Hsp47 and/or RA-A47 is involved in cartilage destruction in RA. (C) 1998 Academic Press.

Hydrostatic pressure induces expression of interleukin 6 and tumour necrosis factor alpha mRNAs in a chondrocyte-like cell line Reviewed

K Takahashi, T Kubo, Y Arai, Kitajima, I, M Takigawa, J Imanishi, Y Hirasawa

ANNALS OF THE RHEUMATIC DISEASES 57 ( 4 ) 231 - 236 1998.4

Language：English Publishing type：Research paper (scientific journal) Publisher：BRITISH MED JOURNAL PUBL GROUP

Objective-To clarify the effect of pressure on the expressions of proteoglycan core protein and metabolism related cytokines in a chondrocyte-like cell line, HCS-2/8.
Methods-HCS-2/8 cells were exposed to 1, 5, 10, or 50 MPa of hydrostatic pressure (HP) for two hours, and mRNA expressions of interleukin 6 (IL6) and tumour necrosis factor alpha (TNF alpha) were examined by using reverse transcription-polymerase chain reaction (RT-PCR) method with specific primer sets; and mRNA of proteoglycan core protein, stromelysin, and tissue inhibitor of metalloproteinase 1 (TIMP1) were measured with northern blotting.
Results-HP exposure caused temporal morphological changes of the cells, but did not affect cellular viability. IL6 and TNF alpha mRNA expressions were not observed in the control cells under the atmospheric pressure, whereas in the cells treated with HP, pressure dependent enhancement of IL6 mRNA expression was observed between 30 minutes and four hours after the HP release. TNFa mRNA expression also increased 30 minutes after the exposure to 50 MPa of HP and disappeared four hours later. Proteoglycan core protein mRNA levels increased between 30 minutes and four hours after the exposure to 1 or 5 MPa of HP, whereas the levels decreased after 10 or 50 MPa of HP. Stromelysin and TIMP1 mRNA signals did not respond to HP.
Conclusions-HP at excessively high levels induced IL6 and TNF alpha expression and reduced the expression of proteoglycan core protein, while physiological levels of HP increased the expression of proteoglycan core protein. These findings are important when considering the pathology of osteoarthritis.

Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes Reviewed

K Sasaki, T Hattori, T Fujisawa, K Takahashi, H Inoue, M Takigawa

JOURNAL OF BIOCHEMISTRY 123 ( 3 ) 431 - 439 1998.3

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE BIOCHEMICAL SOC

We recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1 beta (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes, To clarify the mechanism of the IL-l-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-l-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it, Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3, The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release, An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-l-stimulated bFGF release even if increased production of bFGF is related to the release, IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order, N-G-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.

Shikonin, an ingredient of Lithospermum erythrorhizon, inhibits angiogenesis in vivo and in vitro Reviewed

T Hisa, Y Kimura, K Takada, F Suzuki, M Takigawa

ANTICANCER RESEARCH 18 ( 2A ) 783 - 790 1998.3

Language：English Publishing type：Research paper (scientific journal) Publisher：INT INST ANTICANCER RESEARCH

Angiogenesis Is critical for tumor growth and inflammation. Shiunko is a Chinese hel bal ointment used for the treatment of burns in Japan. Its main ingredient is the root of Lithospermum erythrorhizon, which had been used for treating tumors and inflammation in China since the 5th century. We report here that shikonin, the main chemical ingredient of L. erythrorhizon is a novel inhibitor of angiogenesis. It inhibited tumor necrosis factor-alpha-induced and B16 melanoma-induced angiogenesis in mice and normal developmental angiogenesis in the yolk-sac membranes of chick embryos. Shikonin also inhibited proliferation and migration of endothelial cells in culture and network formation by endothelial cells on Matrigel in vitro. The dose-responsive study suggests that the mechanism of this inhibitory effect on angiogenesis involves the prevention of network formation by endothelial cells via blocking integrin alpha(v) beta(3) expression.

Overexpression of c-erbB-3 in various stages of human squamous cell carcinomas Reviewed

T Funayama, T Nakanishi, K Takahashi, S Taniguchi, M Takigawa, T Matsumura

ONCOLOGY 55 ( 2 ) 161 - 167 1998.3

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Amplification of the c-erbB-3 gene and the expression of its mRNA and protein in human squamous cell carcinomas (SCC) were analyzed. Although no amplification of the c-erbB-3 gene was observed, overexpression of its mRNA was observed in SCC by RT-PCR. The expression of this gene in SCC was 15-50 times higher than in normal fibroblasts. Overexpression of the secreted type of erbB-3 mRNA was also observed in all SCC. Moreover, overproduction of the c-erbB-3 protein was also detected in SCC by dot blot analysis using anti-c-erbB-3 monoclonal antibodies. In nude mice, the expression of c-erbB-3 mRNA was higher in metastatic tumors compared with primary tumors. These results suggest that both the transmembrane and secreted types of c-erbB-3 play a significant role in the formation and development of SCC.

The inhibition of DNA synthesis by prostaglandin E2 in human gingival fibroblasts is independent of the cyclic AMP-protein kinase A signal transduction pathway. Reviewed International journal

Arai H, Nomura Y, Kinoshita M, Nishimura F, Takigawa M, Takahashi K, Washio N, Takashiba S, Murayama Y

Journal of periodontal research 33 ( 1 ) 33 - 39 1998.1

Language：English Publishing type：Research paper (scientific journal) Publisher：1

In this study we attempted to clarify the mechanism of the inhibitory effects of PGE2 on DNA synthesis in Gin-1 (fibroblasts derived from healthy human gingiva) from the aspect of the cyclic AMP-dependent protein kinase signal transduction pathway. PGE2 upregulated intracellular cyclic AMP accumulation and inhibited DNA synthesis in Gin-1 in a dose-dependent manner. When the PGE2-induced intracellular cyclic AMP accumulation was further enhanced by treatment with the cyclic AMP-phosphodiesterase inhibitor, IBMX, the inhibitory effect of PGE2 on DNA synthesis was also enhanced. Furthermore, when we examined the effects of forskolin, an activator of cyclic AMP production, on intracellular cyclic AMP accumulation and DNA synthesis, similar results were obtained. However, inhibitors of cyclic AMP-dependent protein kinase (protein kinase A) such as HA1004 did not diminish the inhibitory effect of PGE2 on DNA synthesis in Gin-1. These results suggest that in Gin-1, PGE2-induced cyclic AMP accumulation may not lead to the activation of protein kinase A or protein kinase A activity may not relate directly to the growth inhibitory effect of PGE2, and that PGE2 does not inhibit DNA synthesis through the cyclic AMP-protein kinase A signal transduction pathway in Gin-1.

Demonstration of receptors for epidermal growth factor on cultured rabbit chondrocytes and regulation of their expression by various growth and differentiation factors. Reviewed

Nishida,T, Nakanishi,T, Shimo,T, Asano,M, Hattori,T, Tamatani,T, Tezuka,K, Takigawa,M

Biochem.Biophys.Res.Commun. 183 14 - 20 1998

Insulin-like growth factors I and II are autocrine factors in stimulating proteoglycan synthesis, a marker of differentiated chondrocytes, acting through their respective receptors on a clonal human chondrosarcoma-derived chondrocyte cell line, HCS-2/8 Reviewed

M Takigawa, T Okawa, HO Pan, C Aoki, K Takahashi, JD Zue, F Suzuki, A Kinoshita

ENDOCRINOLOGY 138 ( 10 ) 4390 - 4400 1997.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

Both insulin-like growth factor (IGF)-I and IGF-II increased the synthesis of cartilage-type, large proteoglycan in a human chondrosarcoma-derived chondrocyte cell line, HCS-2/8. In contrast to the stimulatory effects of IGFs on costal chondrocytes of the young rabbit, the stimulatory effect of IGF-II on proteoglycan synthesis in HCS-2/8 cells was more potent than that of IGF-I. IGF-II, but not IGF-I, increased calcium influx into HCS-2/8 cells, and there was a close relation between the stimulation of proteoglycan synthesis and the calcium influx. [I-125]IGF-I bound to HCS-2/8 cells, and this binding was competitively inhibited by low concentrations of unlabeled IGF-I, higher concentrations of IGF-II, and much higher concentrations of insulin. [I-125]IGF-II also bound to the cells, and its binding was competitively inhibited by IGF-II and slightly inhibited by higher concentrations of IGF-I and much higher concentrations of insulin. When radioligand-receptor complexes were separated by SDS-PAGE and subjected to autoradiography, two major bands at 260 and 130 kDa were observed, which correspond to the IGF type II receptor (IGF-IIR) and the or subunit of the IGF type I receptor (IGF-IR), indicating the presence of both receptors. When confluent cultures of HCS-2/8 cells were maintained in serum-free medium, proteoglycan synthesis not decrease unless the medium was repeatedly replaced. Conditioned medium of HCS-2/8 cells stimulated the HCS-2/8 cells to synthesize proteoglycans. RIA revealed that the cells produced both IGF-II and IGF-I. Transcripts of messenger RNAs of both IGF-I and IGF-II and both IGF-IR and IGF-IIR also were detectable by Northern analysis. Both anti-IGF-IR antibody and anti-IGF-II antibody inhibited proteoglycan synthesis. Mannose-6-phosphate, which is known to bind to IGF-IIR, stimulated proteoglycan synthesis, potentiated IGF-II-stimulated proteoglycan synthesis, and enhanced the binding affinity for IGF-II but not for TGF-I. Even in the presence of anti-IGF-LR antibody, IGF-II and mannose-6-phosphate stimulated proteoglycan synthesis in the cells. [Leu(27)]IGF-II, an IGF-II analogue with high affinity only for IGF-IIR, strongly stimulated proteoglycan synthesis in HCS-2/8 cells but [Arg(54), Arg(55)]IGF-II, which binds to only IGF-IR, also stimulated proteoglycan synthesis in the cells. These findings indicate that IGF-I and IGF-II act as autocrine differentiation factors for this chondrocytic permanent cell line, HCS-2/8, mainly via respective receptors.

Chondrocytes are regulated by cellular adhesion through CD44 and hyaluronic acid pathway Reviewed

O Ishida, Y Tanaka, Morimoto, I, M Takigawa, S Eto

JOURNAL OF BONE AND MINERAL RESEARCH 12 ( 10 ) 1657 - 1663 1997.10

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE INC

The articular cartilage consists of resident chondrocytes embedded within the extracellular matrix which contains several components such as collagen and hyaluronic acids (HA), CD44 is a major cell surface receptor for HA and is homologous to cartilage-link proteins, Although CD44 is present in cartilage, it is not clear if chondrocytes adhere to HA through CD44 or whether such adhesion changes the function of chondrocytes, We studied the molecular mechanisms of CD44-related chondrocyte adhesion to HA and the effects of such adhesion on chondrocyte function, Experiments were performed using the human chondrosarcoma-derived chondrocyte-like cell line HCS-2/8. Our results shoved that (a) HCS-2/8 cells highly expressed CD44; (b) HCS-2/8 cells efficiently adhered to HA without any stimuli; (c) monoclonal antibody (mAb)-blocking studies indicated that adhesion of HCS-2/8 cells to HA was mainly mediated by the CD44/HA pathway; (d) cellular adhesion to HA increased the proliferation of HCS-2/8 cells, independent of transforming growth factor-beta (TGF-beta), but this was inhibited by CD44 mAb; (e) the adhesion of chondrocytes to HA also induced c-myc mRNA expression and this was also inhibited by CD44 mAb; and (f) the adhesion of cells to HA augmented TGF-beta mRNA expression, a process also reduced by CD44 mAb, Thus, HCS-2/8 cells effectively adhered to HA through cell surface CD44, The adhesion was also involved in cellular signaling which induced cellular proliferation and expression of c-myc mRNA as well as TGF-beta mRNA expression within the cells, Our results indicate that CD44 on chondrocytes plays an important role in normal and abnormal functions of cartilage through its adhesion to HA, which induces a variety of stimulatory signals to regulate chondrocyte proliferation as well as matrix synthesis in cartilage microenvironment.

Coordinated change between complement C1s production and chondrocyte differentiation in vitro Reviewed

K Nakagawa, H Sakiyama, T Fukazawa, M Matsumoto, M Takigawa, T Toyoguchi, H Moriya

CELL AND TISSUE RESEARCH 289 ( 2 ) 299 - 305 1997.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER VERLAG

In vitro synthesis of the first component of complement Cls was examined by using hamster epiphyseal chondrocytes (HAC) and human chondrosarcoma cell line HCS-2/8. Hamster and human Cls produced by the cells were quantified by immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA), respectively. It was possible to measure active and inactive Cls by sandwich ELISA, when we used anti-human Cls monoclonal antibodies, M241 recognizing only active Cls, and M365 and M81 recognizing both active and inactive Cia. Approximately 40% of Cls secreted from HCS-2/8 was found to be activated in the culture medium, whereas Cls from HAC was not. Cls production increased in accordance with chondrocyte differentiation induced by ascorbic acid. In contrast, transforming growth factor-betal and basic fibroblast growth factor, which inhibited differentiation, suppressed Cls production. These results confirmed our previous observation showing that Cls synthesis increased with differentiation into hypertrophic chondrocytes in vivo.

Molecular characterisation of integrin-procollagen C-propeptide interactions Reviewed

D Davies, DS Tuckwell, DA Calderwood, SA Weston, M Takigawa, MJ Humphries

EUROPEAN JOURNAL OF BIOCHEMISTRY 246 ( 2 ) 274 - 282 1997.6

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER VERLAG

The carboxyl-terminal propeptide of type I procollagen (CPP-I) plays a key role in regulation of collagen fibrillogenesis, and may exert feedback control of collagen biosynthesis. We have previously shown that CPP-I is a ligand for the integrin alpha 2 beta 1 [Weston, S. A., Hulmes, D. J. S., Mould, A. P., Watson, R. B. & Humphries, M. J. (1994) Identification of the integrin alpha 2 beta 1 as a cell surface receptor for the C-propeptide of type I procollagen, J. Biol. Chem. 269, 20982-20986] suggesting that some of the phenotypic effects of C-propeptides may be mediated by adhesion receptors. Here we have extended this work to study the molecular basis of this interaction. We have broadened the ligand range by demonstrating that the C-terminal propeptide of type II procollagen supports alpha 2 beta 1-mediated binding of NHS human fibroblasts in cell attachment assays. Also, we have used function-blacking antibodies in cell attachment and solid-phase binding assays with purified integrin to expand the CPP-I receptor family, showing that integrin alpha 1 beta 1 is also a receptor for CPP-I. Integrin alpha-subunit A-domains are known to be major ligand-binding sites and recombinant alpha 1 and alpha 2 subunit A-domains were able to bind CPP-I. Finally we have shown that peptides corresponding to potential integrin-binding sequences in CPP-I do not mediate integrin--CPP-I adhesion. Taken together, these studies indicate that the interactions between C-propeptides and integrins are more numerous than previously reported, that C-propeptides are a new class of molecule which bind to A-domains, and that the integrin--C-propeptide interaction does not utilise established peptide motifs.

Cloning of a mRNA preferentially expressed in chondrocytes by differential display PCR from a human chondrocytic cell line that is identical with connective tissue growth factor (CTGF) mRNA Reviewed

T Nakanishi, Y Kimura, T Tamura, H Ichikawa, Y Yamaai, T Sugimoto, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234 ( 1 ) 206 - 210 1997.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS

Chondrocyte- or chondrosarcoma cell line (HCS)-specific DNA fragments were obtained using differential display-PCR. Nucleotide sequences of 32 species derived from HCS cells were determined. One of the sequence tags (tag no. 24) corresponded to the nucleotide sequence of connective tissue growth factor (CTGF). Northern blot analysis showed that CTGF was highly expressed in HCS cells and rabbit growth cartilage cells in culture but was not expressed in osteoblastic cells in culture. In situ hybridization revealed that CTGF was expressed only in the hypertrophic chondrocytes of costal cartilage and the vertebral column in embryonic mice. The expression of CTGF in HCS cells was up-regulated by the addition of TGF-beta or BMP-2. These findings suggest that CTGF participates in endochondral ossification. (C) 1997 Academic Press.

Hydrostatic pressure influences mRNA expression of transforming growth factor-beta 1 and heat shock protein 70 in chondrocyte-like cell line Reviewed

K Takahashi, T Kubo, K Kobayashi, J Imanishi, M Takigawa, Y Arai, Y Hirasawa

JOURNAL OF ORTHOPAEDIC RESEARCH 15 ( 1 ) 150 - 158 1997.1

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL BONE JOINT SURGERY INC

The present study investigated the influence of hydrostatic pressure on the expression of cytokines and heat shock protein 70 in a chondrocyte-like cell line. Chondrocyte-like cells (HCS-2/8) were exposed to hydrostatic pressure by a special pressure apparatus. Total RNA for cytokines (interleukin-1 beta, basic fibroblast growth factor, insulin-like growth factor-I, and transforming growth factor-beta 1) and for heat shock protein 70 was extracted and was analyzed by a polymerase chain reaction method and Northern blotting. An assay for incorporation of [S-35]sulfate was performed to assess proteoglycan synthesis. The expression of transforming growth factor-beta 1 mRNA was enhanced after exposure to 5 MPa of hydrostatic pressure and was reduced after 50 MPa, whereas the expression of heat shock protein 70 was enhanced following exposure to 50 MPa of hydrostatic pressure. The incorporation of [S-35]sulfate into the cultured cells increased following exposure to 1-5 MPa of hydrostatic pressure and decreased following 10-50 MPa of pressure. These results suggest that hydrostatic pressure at physiologic levels enhances the expression of transforming growth factor-beta 1 mRNA in addition to increasing proteoglycan synthesis in chondrocytes and that excessively high hydrostatic pressure reduces the expression of transforming growth factor-beta 1 mRNA and increases the expression of heat shock protein 70 mRNA while decreasing proteoglycan synthesis.

The basic effect of IGF on chondrocytes. Reviewed

Takigawa,M, Kimura,Y, Takahashi,K

Clin. Pediatr. Endocrinol. 6(suppl10) 169 - 174 1997

A factor in conditioned medium of rabbit costal chondrocytes inhibits the proliferation of cultured entothelial cells and angio-genesis induced by B16 melanoma : its relation with cartilage-derived anti-tumor factor(CATF). Reviewed

Takigawa, M, Shirai, E, Enomoto, M, Pan, H.-O, Suzuki, F, Shiio, T, Yugari, Y

Biochem.Int. 14 357 - 364 1997

Nitric oxide mediates interleukin-1-induced matrix degradation and basic fibroblast growth factor release in cultured rabbit articular chondrocytes: A possible mechanism of pathological neovascularization in arthritis Reviewed

T Tamura, T Nakanishi, Y Kimura, T Hattori, K Sasaki, H Norimatsu, K Takahashi, M Takigawa

ENDOCRINOLOGY 137 ( 9 ) 3729 - 3737 1996.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

Prolonged incubation with interleukin-1 beta (IL-1) induced the release of large amounts of NO and subsequently inhibited DNA synthesis and the biosynthesis and accumulation of proteoglycans in cultured rabbit articular chondrocytes (RAC). IL-1 also inhibited DNA synthesis in bovine aortic endothelial cells (BAE). On the other hand, DNA synthesis in BAE cocultured with RAC was not inhibited by prolonged incubation with IL-1. Moreover, conditioned media from RAC incubated for a long period with IL-1 stimulated DNA synthesis in BAE alone. This growth stimulatory activity was mainly due to the release of basic fibroblast growth factor, a heparin-binding growth factor, into RAC culture. Gelatin zymography of the RAC culture medium revealed that IL-1 increased the production of matrix metalloproteinase-a (MMP-2) and MMP-9. N-G-Monomethyl-L-arginine, an inhibitor of NO synthesis, inhibited all of these actions of IL-1. These results indicate that NO from RAC treated with IL-I stimulates MMPs, which, in turn, degrade the extracellular matrix produced by RAG, resulting in the release of large amounts of basic fibroblast growth factor stored in the matrix, which then stimulates adjacent BAE proliferation. Thus, NO produced from RAC treated with IL-1 may modulate angiogenesis in the synovium of arthritic patients.

Establishment of an in vitro cell derived from human angiosarcoma Reviewed

T Hisa, S Taniguchi, K Kakudo, M Ichihashi, T Takashima, Y Kato, R Hayakawa, M Takigawa

BULLETIN DU CANCER 83 ( 7 ) 589 - 591 1996.7

Language：English Publishing type：Research paper (scientific journal) Publisher：EDITIONS SCIENTIFIQUES ELSEVIER

We have succeeded in the establishment of a human endothelial cell line derived from a human angiosarcoma. These cells grew as monolayers with a <<cobble stone>> morphology. The cells produced endothelin and showed Weibel-Palade bodies in their cytoplasm. These findings show that the cells have specific differenciated functions, and might be useful for both fundamental endothelial cell biology and biological investigation of angiosarcoma.

Vitamin D inhibits endothelial cell migration Reviewed

T Hisa, S Taniguchi, D Tsuruta, Y Hirachi, S Ishizuka, M Takigawa

ARCHIVES OF DERMATOLOGICAL RESEARCH 288 ( 5-6 ) 262 - 263 1996.5

Mouse Meckel's cartilage chondrocytes evoke bone-like matrix and further transform into osteocyte-like cells in culture Reviewed

K Ishizeki, M Takigawa, T Nawa, F Suzuki

ANATOMICAL RECORD 245 ( 1 ) 25 - 35 1996.5

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Background: we reported that when Meckel's cartilage was transplanted ectopically, chondrocytes transformed into osteocyte-like cells accompanying the extracellular calcified matrix. However, we could not determine whether the osteocyte-like cells were derived from host tissues or from Meckel's cartilage itself. Therefore, we examined whether the Meckel's cartilage chondrocytes, which have a retrogressive ultimate fate, are capable of inducing the observed calcification and further transform into osteocyte-like cells in culture.
Methods: Meckelian chondrocytes isolated enzymatically were plated at a low density and grown in alpha-MEM containing 10% FBS at 37 degrees C under 5% CO2 in air for up to 4 weeks.
Results: Chondrocytes were fibroblast-like cells early in culture, but gradually transformed from polygonal cells into typical chondrocytes showing metachromasia with toluidine blue staining. After an additional week of culture, the chondrocytes transformed from large to small round cells accompanying nodule formations. Small round cells multiple-layered actively, and showed more intense alkaline phosphatase (ALPase) activity. Immunostaining identified type II collagen in the extracellular matrix at 2 weeks of culture, and type I collagen and osteocalcin were later synthesized by round cells. von Kossa's reaction showed extensive precipitation of calcification throughout the flocculent materials. Ultrastructural analysis showed that the cells surrounded by calcified matrix strongly resembled osteocytes.
Conclusions: The present study suggested that the Meckel's cartilage chondrocytes can express the osteocyte-like phenotype in vitro during synthesis of bone-type marker proteins such as osteocalcin or type I collagen. (C) 1996 Wiley-Liss, Inc.

Neurotrophin-3 increases the DNA-binding activities of several transcription factors in a mouse osteoblastic cell line Reviewed

E Iwata, T Nakanishi, N Ogawa, K Ohyama, T Murakami, M Takigawa

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1311 ( 2 ) 85 - 92 1996.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

In the mouse osteoblastic cell line MC3T3-E1, the signaling responses of several DNA-binding proteins induced by the treatment of neurotropin-3 were examined using electrophoretic mobility shift assay. Neurotrophin-3 increased binding activities in nuclear extracts of MC3T3-E1 cells to TPA-responsive element (TRE), cyclic AMP-responsive element (CRE) and serum-responsive element (SRE), but not binding activity in the nuclear extracts to c-Myc binding DNA element. Competition experiments revealed that the binding activity to TRE in the nuclear extracts of neurotrophin-3-treated MC3T3-E1 cells was entirely inhibited by the both unlabeled TRE and CRE probes. On the other hand, the binding activity to CRE was abolished by the unlabeled CRE probe but not by the same amount of unlabeled TRE probe. Moreover, immunodepletion/supershift assay using antibodies directed to Fos, Jun and CREB proteins, showed that the binding activities to TRE and CRE in the nuclear extracts were derived in part from these proteins.

Novel FNR homologues identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans Reviewed

T Hattori, K Takahashi, T Nakanishi, H Ohta, K Fukui, S Taniguchi, M Takigawa

FEMS MICROBIOLOGY LETTERS 137 ( 2-3 ) 213 - 220 1996.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Based upon DNA sequence data and positive immunochemical reactivity of expressed protein, novel homologues of the FNR family were identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans. The similarity to E. coli FNR and to HlyX (itself 71% similar to E. coli FNR, while regulating expression of hemolysin operon in Actinobacillus pleuropneumoniae) was estimated from the deduced partial amino acid sequence to be, in the above order of tested species, 98, 98, 86, and 85%, and 75, 75, 88, and 88%, respectively. The phylogenetic relatedness indicates a rather closer link of HlyX to the FNR homologues from both pathogens, H. aphrophilus and A. actinomycetemcomitans. The possibility that the A. actinomycetemcomitans FNR homologue functions as a redox-sensing transcriptional factor to regulate, in addition to anaerobic respiration, microaerobic expression of the leukotoxin operon (Itx gene) is suggested.

Expression of c-fos gene inhibits proteoglycan synthesis in transfected chondrocyte Reviewed

M Tsuji, S Funahashi, M Takigawa, M Seiki, K Fujii, T Yoshida

FEBS LETTERS 381 ( 3 ) 222 - 226 1996.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The effect of expression of c-fos gene on proteoglycan synthesis, one of the important markers of cartilage metabolism, was examined by introducing the c-fos DIVA into HCS 2/8 chondrocytes. The [S-35]sulfate incorporation into proteoglycan was decreased in the c-fos transfectants expressing exogenous c-fos mRNA, when compared to a control transfectant. A significant increase in transcription of MMP-3 with the suppressed transcription of aggrecan and TIMP-1 were also observed in the c-fos transfectants, Moreover, analysis of the effect of AP-1 proteins on the collagenase and TIMP-1 promoters in gastric carcinoma KKLS cells revealed that c-Fos combined with any of the Jun-related proteins failed to stimulate the TIMP-1 promoter, though collagenase promoter was effectively activated by any Fos/Jun-related protein heterocomplex, These findings indicate that the c-fos expression may govern the cartilage metabolism and hence may play an important role in the pathogenesis of joint destruction in arthritis.

Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression Reviewed

K Ishizeki, M Takigawa, Y Harada, F Suzuki, T Nawa

ANATOMY AND EMBRYOLOGY 193 ( 1 ) 61 - 71 1996.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER VERLAG

We examined whether Meckel's cartilage of embryonic mice, 17 days in utero, undergo the cellular transformation into the osteocyte-like phenotype under organ culture conditions. Explants were grown by our original pithole method modified Trowell-type cultures for up to 4 weeks at 37 degrees C under 5% CO2 in air. Specimens were examined using histological procedures including immunostaining and electron microscopy. In addition, the effects of beta-glycerophosphate on matrix calcification were also examined in cultures with or without beta-glycerophosphate. Addition of beta-glycerophosphate induced calcification at a higher level, but calcium mineral deposition occurred regardless of the addition of beta-glycerophosphate to the culture medium. Light and electron microscopic analyses showed that freshly isolated chondrocytes prior to cell culture had typical hypertrophic morphology, but shortly after commencement of culture, they showed morphological modifications. The cells showing chondrocytic phenotypes became basophilic elliptical cells, and eventually transformed into flattened osteocyte-like cells. Bone-like features for cellular elements were characterized by spindle-shaped cells with elongated processes accompanying bone-specific thick-banded collagen fibrils. Immunostaining showed that at 2 weeks in culture, type I and type II collagens coexisted in the matrix, but subsequently type II collagen synthesis ceased and was replaced by type I collagen synthesis. Immunofluorescent labeling for osteocalcin was noted first in the peripheral cells by 1 week, but at 3 weeks this reaction spread to the central zone in explants. Alkaline phosphatase activity (ALPase) was expressed on the cells in the central zone prior to calcium mineral deposition as shown by von Kossa's reaction at 3 weeks in culture. These results showed that Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression.

Influence of hydrostatic pressure on expression of heat shock protein 70 and matrix synthesis in chondrocytes Reviewed

K Takahashi, T Kubo, Y Arai, Y Hirasawa, J Imanishi, K Kobayashi, M Takigawa

HIGH PRESSURE BIOSCIENCE AND BIOTECHNOLOGY 13 79 - 82 1996

Language：English Publishing type：Research paper (international conference proceedings) Publisher：ELSEVIER SCIENCE PUBL B V

To investigate the influence of hydrostatic pressure (HP) on the expressions of heat shock protein 70 (HSP70), and to know the relationship between HSP70 expression and matrix synthesis, we subjected chondrocyte-like cells to HP. HSP70 were enhanced after exposure to 10 to 50 MPa of HP. S-35 sulfate incorporation into the cultured cells increased after exposure to 5 MPa of HP and decreased after 50 MPa of HP.

Specific serum antibodies against membranous proteins of a human immortal chondrocytic cell line (HCS-2/8) in rheumatoid arthritis and their relationship to the natural history of this disease Reviewed

Akira Sakawa, Yasutaka Yutani, Kentaro Inui, Akira Shimazu, Yoshiki Yamano, Akira Kinosita, Fujio Suzuki, Takako Hattori, Masaharu Takigawa

Journal of Bone and Mineral Metabolism 14 ( 3 ) 146 - 152 1996

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Japan

An immortal human chondrocytic cell line (HCS-2/8) derived from a chondrosarcoma was used as a source of human antigens to find humoral antibodies to cell surface proteins of human chondrocytes in sera from patients with rheumatoid arthritis (RA). Membrane fractions prepared from the cell line were subjected to Western blot analysis using RA and normal sera as probes. RA sera recognized about a dozen bands, but three of these bands, with molecular weights of 105 kDa, 68 kDa, and 47 kDa, were found to be specific for the RA sera (P &lt
0.05). These bands disappeared following V8 protease digestion, indicating that they were proteins. Among patients with 4 years or more of RA disease activity, reactivity against 105-kDa and 68-kDa proteins was relatively high in those whose joints showed a high degree of erosion. We suspect that levels of these two antibodies are suggestive of changes associated with the natural course of RA.

DOI： 10.1007/BF02239482

Interleukin-1 induces Matrix degradation and release of basic fibroblast growth factor in clutured rabbit articular chondrocytes via nitric oxide production : A possible mechanism of pathological neovascularization in arthritis.

Tamura, T, Makanishi, T, Kimura, Y, Hattori, T, Sasaki, K, Norimatsu, H, Takahashi, K, Takigawa, M, Nitric oxide, mediates interleukin, induced matrix degradation, basic fibroblast growth factor release in cultured rabbit articular chondrocytes, A possible mechanism of, pathological neovascularization in arthritis

Endocrinology 137 ( 9 ) 3729 - 3737 1996

APOPTOSIS IN NORMAL SKIN

T HISA, S TANIGUCHI, H KOBAYASHI, Y SHIGENAGA, S NOMURA, M TAKIGAWA

ACTA DERMATO-VENEREOLOGICA 75 ( 5 ) 412 - 413 1995.9

PURIFICATION OF AN ANGIOGENESIS INHIBITOR FROM CULTURE-MEDIUM CONDITIONED BY A HUMAN CHONDROSARCOMA-DERIVED CHONDROCYTIC CELL-LINE, HCS-2/8 Reviewed

Y OHBA, Y GOTO, Y KIMURA, F SUZUKI, T HISA, K TAKAHASHI, M TAKIGAWA

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1245 ( 1 ) 1 - 8 1995.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

We previously reported that a novel human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, produced an anti-tumor angiogenesis factor and secreted it into the culture medium [Takigawa et al.: Anticancer Res., 10, 311-316, 1990]. In the present study, we purified the inhibitor by monitoring gelatinase inhibitory activity from the conditioned medium (CM) of HCS-2/8 cells. By a simple three-step procedure, gel filtration chromatography, ion-exchange chromatography, and reverse-phase HPLC, 200 mu g of the inhibitor was obtained from 6 liters of CM with 239-fold enrichment. Purified inhibitor, named hCHIAMP (human chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity), showed a single protein band with a molecular mass (M(r)) of 24 000 (24K) under reducing conditions and M(r) 22K under nonreducing conditions on SDS-PAGE. On reverse-zymography, purified hCHIAMP showed a single band of 22K M(r) under nonreducing conditions. Its NH2-terminal amino acid sequence determined up to the 11th amino acid residue was identical with that of the tissue inhibitor of metalloproteinases-2 (TIMP-2). On Western blotting, anti-human TIMP-2 antibody cross-reacted with hCHIAMP. hCHIAMP at a dose of as little as 0.45 mu g significantly inhibited angiogenesis in the yolk sac of chick embryos induced by 0.25 mu mol of spermine. Because HCS-2/8 is a clonal cell line, these findings clearly showed for the first time that chondrocytes themselves produce a potent inhibitor of angiogenesis, which is also an inhibitor of gelatinase. The findings also indicate that hCHIAMP is a TIMP-2-like molecule. Because the HCS-2/8 cells are an immortal cell line and of human origin, hCHIAMP could be useful for therapy of angiogenic diseases including solid tumors.

DEMONSTRATION OF ENDOTHELIN (ET) RECEPTORS ON CULTURED RABBIT CHONDROCYTES AND STIMULATION OF DNA-SYNTHESIS AND CALCIUM INFLUX BY ET-1 VIA ITS RECEPTORS Reviewed

A KINOSHITA, T TAMURA, C AOKI, T NAKANISHI, S SOBUE, F SUZUKI, K TAKAHASHI, M TAKIGAWA

CELL BIOLOGY INTERNATIONAL 19 ( 8 ) 647 - 654 1995.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS (LONDON) LTD

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with I-125-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K-d values of 1 x 10(-10) M and 5 x 10(-9) M. The numbers of high- and low- affinity receptors were 1 x 10(4) and 2 x 10(5) per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-beta 1, IL-1 beta RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.

Expression of trkC in a mouse osteoblastic cell line and its response to neurotrophin-3. Reviewed

Nakanishi T, Ohyama K, Aoki C, Kudo A, Hattori T, Takahashi K, Taniguchi S, Takigawa M

Biochemical and biophysical research communications 203 ( 2 ) 1268 - 1274 1994.9

CHANGES IN PARATHYROID-HORMONE RECEPTORS DURING CHONDROCYTE CYTODIFFERENTIATION Reviewed

M IWAMOTO, A JIKKO, H MURAKAMI, A SHIMAZU, K NAKASHIMA, M IWAMOTO, M TAKIGAWA, H BABA, F SUZUKI, Y KATO

JOURNAL OF BIOLOGICAL CHEMISTRY 269 ( 25 ) 17245 - 17251 1994.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The purpose of this study was to investigate the relationship between changes in parathyroid hormone (PTH) receptor levels and chondrocyte maturation during endochondral ossification. Chondrocytes were isolated from the growth plate of rabbit ribs and maintained in the presence of 10% serum in mass cultures. Treatment with PTH-(1-84) and a PTH-(1-34) fragment suppressed the increases in alkaline phosphatase activity and in type X collagen and 1 alpha,25-dihydroxyvitamin D-3 receptor levels and abolished Ca-45 incorporation into mineral, all of which occurred in parallel untreated cultures in the hypertrophic (terminal) stage. These effects of PTH were observed at low concentrations (10(-10) to 10(-9) M) and within 24-48 h of treatment. PTH-(1-84) and PTH-(1-34) also increased [S-36]sulfate incorporation into newly synthesized proteoglycans. In contrast, the middle and carboxyl-terminal fragments of PTH tested had little effect on proteoglycan synthesis or terminal differentiation. The binding of I-125-PTH-(1-34) to cells in the growth plate was greater than that to cells in liver, skin, muscle, brain, or kidney. When the correlation between binding levels and stage of maturation was examined, we found that I-125-PTH-(134) binding to its 72-kDa receptor was low in resting and proliferating chondrocytes, increased 10 fold in matrix-forming chondrocytes, and thereafter decreased in hypertrophic chondrocytes both in vitro and in situ. Scatchard analysis revealed that the changes in PTH binding were due to changes in the number, and not in the affinity, of the receptor. The changes in PTH-(1-34) binding paralleled those in [S-35]sulfate incorporation into proteoglycans. These findings suggest that stage dependent increases in PTH/PTH-related peptide receptor levels localize the hormone stimulation of proteoglycan synthesis and inhibition of precocious hypertrophy in the matrix-forming zone of growth plates.

PROTOONCOGENE EXPRESSION IN A HUMAN CHONDROSARCOMA CELL-LINE - HCS-2/8 Reviewed

J ZHU, HO PAN, F SUZUKI, M TAKIGAWA

JAPANESE JOURNAL OF CANCER RESEARCH 85 ( 4 ) 364 - 371 1994.4

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE CANCER ASSOCIATION

HCS-2/8 is a stable human chondrosarcoma cell line with many chondrocytic characteristics and has the capacity to form chondrosarcomas in nude mice. The cells display both biochemically and morphologically definable changes in sparse, subconfluent, confluent and over-confluent phases of in vitro culture. Such features of HCS-2/8 cells may reflect the processes of both proliferation and differentiation of chondrocytes in vivo. We examined the correlations of these changes of HCS-2/8 cells with their transcript levels of 21 proto-oncogenes by Northern analysis. We found no detectable transcripts of 9 proto-oncogenes (c-sis, c-met, c-src, c-lyn, c=fgr, c-ros, c-pim, Blym and N-myc), but detected transcripts of 12 other proto-oncogenes (int-2, erbB, c-abl, c-raf-l, c=fyn, K-ras, H-ras, c-mos, c-myc, c-myb, c-fos, and c-jun). In the over-confluent phase, the levels of c=fos and c-raf-1 were increased several dozen times and about 5 times, respectively, while the level of c-abl was about 1/5th of that in the sparse, subconfluent and confluent phases of culture. The level of int 2 increased about 10-fold in the confluent and overconfluent phases of in vitro culture. The transcript levels of c-mos and K-ras were high in the sparse phase, low in the subconfluent and confluent phases and high in the over-confluent phase. The levels of the other 6 proto-oncogenes in HCS-2/8 cells were constant in all phases of in vitro culture.

CONFORMATION DEPENDENCE OF INTEGRIN-TYPE-II COLLAGEN-BINDING - INABILITY OF COLLAGEN PEPTIDES TO SUPPORT ALPHA(2)BETA(1) BINDING, AND MEDIATION OF ADHESION TO DENATURED COLLAGEN BY A NOVEL ALPHA(5)BETA(1)-FIBRONECTIN BRIDGE Reviewed

DS TUCKWELL, S AYAD, ME GRANT, M TAKIGAWA, MJ HUMPHRIES

JOURNAL OF CELL SCIENCE 107 993 - 1005 1994.4

Language：English Publishing type：Research paper (scientific journal) Publisher：COMPANY OF BIOLOGISTS LTD

The mechanism of interaction of chondrocytic cells with cartilage-specific type II collagen has been examined using HCS-2/8 human chondrosarcoma cells as a model system. By the criteria of specific collagen secretion and integrin expression profile, HCS-2/8 have a similar differentiated phenotype to normal chondrocytes and are therefore a good model system. HCS-2/8 cells were able to attach and spread on both native and heat-denatured pepsinised type II collagen, and assays using denatured cyanogen bromide fragments apparently localised the major cell binding site to the CB10 fragment. However, when they were used as soluble inhibitors, cyanogen bromide fragments were found to block adhesion to denatured collagen, but had no effect on either attachment or spreading on the native molecule. The inability of cyanogen bromide fragments to reproduce the cell binding site of native collagen demonstrated a strict dependence on collagen conformation. This was also reflected in the receptors that were employed by HCS-2/8 cells for binding to type II collagen: binding to native collagen was mediated by the integrin alpha(2) beta(1) while binding to denatured collagen was mediated by a novel alpha(5) beta(1)-fibronectin bridge. The identification of this bridge adds to the mechanisms by which cells can bind to denatured collagens. The previously characterised KDGEA active site peptide from type I collagen was found to be inactive as an inhibitor of type II collagen-mediated adhesion. The implications of these findings for the strategies used to identify adhesive active sites within collagens are discussed. In particular, these data suggest that, unlike other integrin ligands, a synthetic peptide-based approach is not suitable for the identification of collagen active sites.

HISTATIN AS A SYNERGISTIC STIMULATOR WITH EPIDERMAL GROWTH-FACTOR OF RABBIT CHONDROCYTE PROLIFERATION Reviewed

Y MURAKAMI, H NAGATA, S SHIZUKUISHI, K NAKASHIMA, T OKAWA, M TAKIGAWA, A TSUNEMITSU

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 198 ( 1 ) 274 - 280 1994.1

DEMONSTRATION OF RECEPTORS FOR EPIDERMAL GROWTH-FACTOR ON CULTURED RABBIT CHONDROCYTES AND REGULATION OF THEIR EXPRESSION BY VARIOUS GROWTH AND DIFFERENTIATION FACTORS Reviewed

A KINOSHITA, M TAKIGAWA, F SUZUKI

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 183 ( 1 ) 14 - 20 1992.2

EFFECTS OF VARIOUS GROWTH AND DIFFERENTIATION FACTORS ON EXPRESSION OF PARATHYROID-HORMONE RECEPTORS ON RABBIT COSTAL CHONDROCYTES IN CULTURE Reviewed

M TAKIGAWA, A KINOSHITA, M ENOMOTO, A ASADA, F SUZUKI

ENDOCRINOLOGY 129 ( 2 ) 868 - 876 1991.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

In our preliminary report we demonstrated PTH receptors on rabbit costal chondrocytes in culture. In the present study regulation of expression of PTH receptors of the chondrocytes by various growth and differentiation factors and its relation to the synthesis of glycosaminoglycan (GAG), a differentiated phenotype of chondrocytes, were investigated. Treatment with retinoic acid decreased GAG synthesis in cultured chondrocytes and the number of PTH receptors, measured by binding of [I-125]-[Nle8,18,Tyr34]bovine PTH-(1-34) amide to the cells. However, the affinity of the receptors did not change. The decrease in the number of PTH receptors was dose and time dependent and parallel to the decrease in GAG synthesis. When chondrocytes that had been treated with retinoic acid were cultured in the absence of retinoic acid for 3 days, both their GAG synthesis and their number of PTH receptors were restored. Epidermal growth factor and fibroblast growth factor also decreased both the number of PTH receptors and GAG synthesis in the cells. However, these treatments did not change their affinity. Treatment with insulin-like growth factor-I, (Bu)2cAMP, and transforming growth factor-beta resulted in increases in GAG synthesis as well as in the number of PTH receptors without any change in their affinity. In addition, the PTH-stimulated cAMP level in chondrocytes pretreated with retinoic acid, epidermal growth factor, and fibroblast growth factor was lower than that in control cells. On the other hand, the PTH-stimulated cAMP level in chondrocytes pretreated with insulin-like growth factor-I and transforming growth factor-beta was higher than that in control cells. These observations suggest that the increase in the number of PTH receptors on chondrocytes is closely related to expression of the differentiated phenotype of chondrocytes and that the number of the receptors is a good marker of the differentiated phenotype of chondrocytes.

ESTABLISHMENT FROM A HUMAN CHONDROSARCOMA OF A NEW IMMORTAL CELL-LINE WITH HIGH TUMORIGENICITY INVIVO, WHICH IS ABLE TO FORM PROTEOGLYCAN-RICH CARTILAGE-LIKE NODULES AND TO RESPOND TO INSULIN INVITRO Reviewed

M TAKIGAWA, H PAN, A KINOSHITA, K TAJIMA, Y TAKANO

INTERNATIONAL JOURNAL OF CANCER 48 ( 5 ) 717 - 725 1991.7

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

The human chondrosarcoma cell line (HCS-2/8) established by our group expresses cartilage phenotypes such as production of cartilage-type proteoglycans and collagen type II, but its tumorigenicity is low. To develop an in vitro experimental system for studies of human chondrosarcomas, a new immortal cell line of human chondrosarcoma, named HCS-2/A, was established from the same tumor. HCS-2/A cells proliferated with a doubling time of 3 1/2 days in a medium containing 20% fetal bovine serum (FBS). This growth rate was comparable to that of HCS-2/8 cells. However, HCS-2/A cells proliferated more rapidly than HCS-2/8 cells in the presence of 2-10% FBS. Like HCS-2/8 cells, HCS-2/A cells had a polygonal shape in sparse cultures and became spherical as they reached confluence, after which they formed nodules composed of multilayered cells and a large quantity of extracellular matrix showing strong metachromasia. The nodules formed by HCS-2/A cells were thicker and also larger in diameter than those formed by HCS-2/8 cells. Electron microscopically, the cells in the nodules resembled chondrocytes in vivo, but each cell had an irregular-shaped nucleus which is a characteristic of tumor cells. The cells actively synthesized "cartilage-specific" large proteoglycans and their level of proteoglycan synthesis was comparable to that of HCS-2/8 cells. Insulin, which stimulates proteoglycan and DNA syntheses in cultured chondrocytes, markedly increased proteoglycan synthesis in HCS-2/A cells. On the other hand, the hormone only slightly increased proteoglycan synthesis in HCS-2/8 cells. Insulin also stimulated DNA synthesis in cultured HCS-2/A cells, but not in HCS-2/8 cells. Immunostaining revealed that HCS-2/A cells produced type-II collagen but not type-I collagen. However, the level of collagen synthesis of HCS-2/A cells was lower than that of HCS-2/8 cells. Inoculation of HCS-2/A cells into athymic mice resulted in the formation of chondrosarcomas that grew faster than those arising from HCS-2/8 cells.

Establishment from a human chondrosarcoma of a new immortal cell line with abilities to form proteoglycan-rich cartilage nodules and to respond to insulin in vitro and high tumorigenicity in vivo. Reviewed

Takigawa, M, Pan, H-O, Kinoshita, A, Tajima, K, Takano, Y

Int. J. Cancer 48 717 - 725 1991

INDUCTION OF ANGIOGENESIS IN CHICK YOLK-SAC MEMBRANE BY POLYAMINES AND ITS INHIBITION BY TISSUE INHIBITORS OF METALLOPROTEINASES (TIMP AND TIMP-2) Reviewed

M TAKIGAWA, Y NISHIDA, F SUZUKI, J KISHI, K YAMASHITA, T HAYAKAWA

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 171 ( 3 ) 1264 - 1271 1990.9

EFFECTS OF TUMOR NECROSIS FACTOR-ALPHA ON PROLIFERATION AND EXPRESSION OF DIFFERENTIATED PHENOTYPES IN RABBIT COSTAL CHONDROCYTES IN CULTURE Reviewed

M ENOMOTO, HO PAN, A KINOSHITA, Y YUTANI, F SUZUKI, M TAKIGAWA

CALCIFIED TISSUE INTERNATIONAL 47 ( 3 ) 145 - 151 1990.9

TUMOR ANGIOGENESIS AND POLYAMINES - ALPHA-DIFLUOROMETHYLORNITHINE, AN IRREVERSIBLE INHIBITOR OF ORNITHINE DECARBOXYLASE, INHIBITS B16 MELANOMA-INDUCED ANGIOGENESIS INOVO AND THE PROLIFERATION OF VASCULAR ENDOTHELIAL-CELLS INVITRO Reviewed

M TAKIGAWA, M ENOMOTO, Y NISHIDA, HO PAN, A KINOSHITA, F SUZUKI

CANCER RESEARCH 50 ( 13 ) 4131 - 4138 1990.7

PHYSIOLOGICAL-ROLE OF VITAMIN-A IN GROWTH CARTILAGE CELLS - LOW CONCENTRATIONS OF RETINOIC ACID STRONGLY PROMOTE THE PROLIFERATION OF RABBIT COSTAL GROWTH CARTILAGE CELLS IN CULTURE Reviewed

M ENOMOTO, H PAN, F SUZUKI, M TAKIGAWA

JOURNAL OF BIOCHEMISTRY 107 ( 5 ) 743 - 748 1990.5

A CLONAL HUMAN CHONDROSARCOMA CELL-LINE PRODUCES AN ANTI-ANGIOGENIC ANTITUMOR FACTOR Reviewed

M TAKIGAWA, HO PAN, M ENOMOTO, A KINOSHITA, Y NISHIDA, F SUZUKI, K TAJIMA

ANTICANCER RESEARCH 10 ( 2A ) 311 - 315 1990.3

Insulin-like growth factors (]G0001[) and (]G0002[) are autocrine factors in stimulating proteoglycan synthesis, a marker of differentiated chondrocytes, acting through their respective receptors on a clonal human. chondrosarcoma-derived chondrocyte ce・・・ Reviewed

Takigawa, M, Kinoshita, A, Enomoto, M, Asada, A, Suzuki, F

Endocrinology 138 4390 - 4400 1990

POLYAMINES AND ANGIOGENESIS - INHIBITION OF TUMOR ANGIOGENESIS BY THE IRREVERSIBLE INHIBITOR OF ORNITHINE DECARBOXYLASE, ALPHA-DIFLUOROMETHYLORNITHINE Reviewed

M TAKIGAWA, M ENOMOTO, Y NISHIDA, HO PAN, A KINOSHITA, F SUZUKI

BIOLOGY AND CHEMISTRY OF POLYAMINES 12 203 - 211 1990

ESTABLISHMENT FROM MOUSE GROWTH CARTILAGE OF CLONAL CELL-LINES WITH RESPONSIVENESS TO PARATHYROID-HORMONE, ALKALINE-PHOSPHATASE ACTIVITY, AND ABILITY TO PRODUCE AN ENDOTHELIAL-CELL GROWTH INHIBITOR Reviewed

M TAKIGAWA, E SHIRAI, M ENOMOTO, A KINOSHITA, HO PAN, F SUZUKI, Y YUGARI

CALCIFIED TISSUE INTERNATIONAL 45 ( 5 ) 305 - 313 1989.11

DEMONSTRATION OF RECEPTORS FOR PARATHYROID-HORMONE ON CULTURED RABBIT COSTAL CHONDROCYTES Reviewed

M ENOMOTO, A KINOSHITA, HO PAN, F SUZUKI, YAMAMOTO, I, M TAKIGAWA

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 162 ( 3 ) 1222 - 1229 1989.8

ESTABLISHMENT OF A CLONAL HUMAN CHONDROSARCOMA CELL-LINE WITH CARTILAGE PHENOTYPES Reviewed

M TAKIGAWA, K TAJIMA, HO PAN, M ENOMOTO, A KINOSHITA, F SUZUKI, Y TAKANO, Y MORI

CANCER RESEARCH 49 ( 14 ) 3996 - 4002 1989.7

The distribution of differentiated phenotypes of chondrocytes in osteoarthritic hip. Reviewed

Yutani, Y, Asada, K, Takigawa, M, Omori, K, Shimazu, A, The distribution of, differentiated phenotypes of chondrocytes in osteoarthritic hip

Jpn.J.Rheum.Joint Surg. 8 179 - 186 1989

PARATHYROID HORMONE-RESPONSIVE CLONAL CELL-LINES FROM MOUSE GROWTH CARTILAGE Reviewed

M TAKIGAWA, E SHIRAI, M ENOMOTO, A KINOSHITA, F SUZUKI

NEW ACTIONS OF PARATHYROID HORMONE 423 - 428 1989

Cartilage-derived anti-tumor factor (CATF) - Partial purification and correlation of inhibitory activity against tumor growth with anti-angiogenic activity Reviewed

Masaharu Takigawa, Eiji Shirai, Motomi Enomoto, Yuji Hiraki, Fujio Suzuki, Tsuyoshi Shiio, Yasumi Yugari

Journal of Bone and Mineral Metabolism 6 ( 2 ) 29 - 38 1988.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer-Verlag

Cartilage-derived anti-tumor factor (CATF) inhibits the proliferation and DNA synthesis of bovine pulmonary artery endothelial (BPAE) cells in culture (Takigawa, M. et al. Cell. Biol. Inn. Rep., 9, 619-625, 1985). In the present study, we partially purified CATF by monitoring inhibition of DNA synthesis in BPAE cells and tested the effects of the purified materials on the growth of solid tumors and tumor-induced angiogenesis. Crude CATF (CATF20-300k), the fraction of 20 k to 300 k daltons, separated by ultrafiltration was further separated into three fractions by ultrafiltration. The fraction of 100 k to 300 k daltons (CATF100-300k) caused slightly more inhibition than CATF20-300k of DNA synthesis in BPAE cells. On the other hand, the fraction of 20 k to 50 k daltons had only a slight effect, and the fraction of 50 k to 100 k had even less effect on DNA synthesis in BPAE cells. CATF100-300k caused slightly more inhibition than CATF20-300k of the growth of solid tumors of B16 melanoma, while the fraction of 20 k to 100 k daltons did not inhibit the growth of tumors at all. CATF100-300k also inhibited B16 melanoma-induced angiogenesis in chick embryo chorioallantoic membranes (CAM), whereas the fraction of 20 k to 100 k daltons had little effect on the angiogenesis. CATF100-300k was further purified by DEAE-Sepharose CL-6B chromatography. The main peak with activity on DNA synthesis in BPAE cells was eluted with 0.3 to 0.35 M NaCl at pH 8.0. The activity of this peak on DNA synthesis in BPAE cells was about 70 fold that of CATF100-300k. The purified CATF also inhibited the growth of B16 melanoma and B16 melanoma-induced angiogenesis in CAM. On the other hand, the inactive fraction on DNA synthesis in BPAE cells obtained by DEAE-Sepharose chromatography was also inactive in inhibiting the growth of B16 melanoma and B16 melanoma-induced angiogenesis in CAM. These findings strongly suggest that CATF is an anionic macromolecule(s) and has anti-angiogenic activity, thereby inhibiting the growth of solid tumors. © 1988 Japanese Society of Bone Metabolism Research.

DOI： 10.1007/BF02375643

DIFFERENTIAL-EFFECTS OF 1-ALPHA,25-DIHYDROXYCHOLECALCIFEROL AND 24R,25-DIHYDROXYCHOLECALCIFEROL ON THE PROLIFERATION AND THE DIFFERENTIATED PHENOTYPE OF RABBIT COSTAL CHONDROCYTES IN CULTURE Reviewed

M TAKIGAWA, M ENOMOTO, E SHIRAI, Y NISHII, F SUZUKI

ENDOCRINOLOGY 122 ( 3 ) 831 - 839 1988.3

HYDROCORTISONE STIMULATION OF PROLIFERATION AND GLYCOSAMINOGLYCAN SYNTHESIS IN RABBIT CRANIOFACIAL CHONDROCYTES INVITRO Reviewed

M TAKIGAWA, T TAKANO, K NAKAGAWA, M SAKUDA, F SUZUKI

ARCHIVES OF ORAL BIOLOGY 33 ( 12 ) 893 - 899 1988

Enhanced responsiveness to parathyroid hormone and induction of functional differentiation of cultured rabbit costal chondrocytes by a pulsed electromagnetic field

Yuji Hiraki, Naoto Endo, Masaharu Takigawa, Akira Asada, Hideaki Takahashi, Fujio Suzuki

BBA - Molecular Cell Research 931 ( 1 ) 94 - 100 1987.10

Language：English Publishing type：Research paper (scientific journal)

Pulsed electromagnetic fields promote healing of delayed united and ununited fractures by triggering a series of events in fibrocartilage. We examined the effects of a pulsed electromagnetic field (recurrent bursts, 15.4 Hz, of shorter pulses of an average of 2 gauss) on rabbit costal chondrocytes in culture. A pulsed electromagnetic field slightly reduced the intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) level in the culture. However, it significantly enhanced cAMP accumulation in response to parathyroid hormone (PTH) to 140% of that induced by PTH in its absence, while it did not affect cAMP accumulation in response to prostaglandin E1 or prostaglandin I2. The effect on cAMP accumulation in response to PTH became evident after exposure of the cultures to the pulsed electromagnetic field for 48 h, and was dependent upon the field strength. cAMP accumulation in response to PTH is followed by induction of ornithine decarboxylase, a good marker of differentiated chondrocytes, after PTH treatment for 4 h. Consistent with the enhanced cAMP accumulation, ornithine decarboxylase activity induced by PTH was also increased by the pulsed electromagnetic field to 170% of that in cells not exposed to a pulsed electromagnetic field. Furthermore, stimulation of glycosaminoglycan synthesis, a differentiated phenotype, in response to PTH was significantly enhanced by a pulsed electromagnetic field. Thus, a pulsed electromagnetic field enhanced a series of events in rabbit costal chondrocytes in response to PTH. These findings show that exposure of chondrocytes to a pulsed electromagnetic field resulted in functional differentiation of the cells. © 1987.

DOI： 10.1016/0167-4889(87)90054-1

ENHANCED RESPONSIVENESS TO PARATHYROID-HORMONE AND INDUCTION OF FUNCTIONAL-DIFFERENTIATION OF CULTURED RABBIT COSTAL CHONDROCYTES BY A PULSED ELECTROMAGNETIC-FIELD Reviewed

Y HIRAKI, N ENDO, M TAKIGAWA, A ASADA, H TAKAHASHI, F SUZUKI

BIOCHIMICA ET BIOPHYSICA ACTA 931 ( 1 ) 94 - 100 1987.10

CHONDROCYTES DEDIFFERENTIATED BY SERIAL MONOLAYER-CULTURE FORM CARTILAGE NODULES IN NUDE-MICE Reviewed

M TAKIGAWA, E SHIRAI, K FUKUO, K TAJIMA, Y MORI, F SUZUKI

BONE AND MINERAL 2 ( 6 ) 449 - 462 1987.9

A FACTOR IN CONDITIONED MEDIUM OF RABBIT COSTAL CHONDROCYTES INHIBITS THE PROLIFERATION OF CULTURED ENDOTHELIAL-CELLS AND ANGIOGENESIS INDUCED BY B-16 MELANOMA - ITS RELATION WITH CARTILAGE-DERIVED ANTITUMOR FACTOR (CATF) Reviewed

M TAKIGAWA, E SHIRAI, M ENOMOTO, HO PAN, F SUZUKI, T SHIIO, Y YUGARI

BIOCHEMISTRY INTERNATIONAL 14 ( 2 ) 357 - 363 1987.2

THE EFFECT OF PARATHYROID-HORMONE (1-34) ON CYCLIC-AMP LEVEL, ORNITHINE DECARBOXYLASE ACTIVITY, AND GLYCOSAMINOGLYCAN SYNTHESIS OF CHRONDROCYTES FROM MANDIBULAR CONDYLAR CARTILAGE, NASAL SEPTAL CARTILAGE, AND SPHENO-OCCIPITAL SYNCHONDROSIS IN CULTURE Reviewed

T TAKANO, M TAKIGAWA, E SHIRAI, K NAKAGAWA, M SAKUDA, F SUZUKI

JOURNAL OF DENTAL RESEARCH 66 ( 1 ) 84 - 87 1987.1

Effects of various tumor promoters on expression of cartilage phenotypes in rabbit costal chondrocytes in culture Reviewed

Masaharu Takigawa, Koji Tajima, Keisuke Fukuo, Hirota Fujiki, Fujio Suzuki

Journal of Biochemistry 101 ( 2 ) 397 - 404 1987

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press

12-O-Tetradecanoylphorbol-13-acetate (TPA), a skin tumor-promoting phorbol ester, and teleocidin and aplysiatoxin, which are potent tumor promoters in mouse skin but are chemically unrelated to phorbol esters, induced change of cultured rabbit costal chondrocytes from a polygonal to a fibroblastic shape and inhibited glycosaminoglycan (GAG) synthesis and metachromatic matrix formation in these cells. The potencies of teleocidin and aplysiatoxin to inhibit GAG synthesis were almost the same as that of TPA. On the other hand, Tween 60 and cantharidin, weak mouse skin tumor promoters, phenobarbital, a liver tumor promoter, and saccharin, a bladder tumor promoter, had no effect on the morphology or GAG synthesis of cultured chondrocytes. Like TPA, teleocidin and aplysiatoxin increased DNA and RNA syntheses of chondrocytes. Parathyroid hormone (PTH) and dibutyryl cyclic AMP reversed the morphological and histochemical changes caused by a 4-day treatment with teleocidin or aplysiatoxin as well as with TPA, reversal being apparent after 2 days. PTH increased intracellular cyclic AMP after 2 min in chondrocytes pretreated with teleocidin or aplysiatoxin as well as with TPA. PTH also increased ornithine decarboxylase [ODC
EC 4.1.1.17] activity in these chondrocytes after 4 h. These results show that retention of responsiveness to PTH is a typical characteristic of chondrocytes dedifferentiated by treatment with TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin. The results also suggest that ODC induction mediated by elevation of cyclic AMP plays an important role in re-differentiation of teleocidin- and aplysiatoxin-treated chondrocytes. © 1987 The Journal of Biochemistry.

DOI： 10.1093/oxfordjournals.jbchem.a121924

COMPARISON OF INHIBITION BY A TUMOR PROMOTER (12-O-TETRADECANOYLPHORBOL-13-ACETATE) OF EXPRESSION OF THE DIFFERENTIATED PHENOTYPE OF CHONDROCYTES IN RABBIT COSTAL CHONDROCYTES IN CULTURE WITH INHIBITION BY RETINOIC ACID Reviewed

K FUKUO, M TAKIGAWA, K TAJIMA, M ENOMOTO, Y KUMAHARA, F SUZUKI

JOURNAL OF BIOCHEMISTRY 99 ( 2 ) 385 - 396 1986.2

TUMOR PROMOTER-INDUCED REFRACTORY STATE AGAINST ORNITHINE DECARBOXYLASE INDUCTION BY 12-O-TETRADECANOYLPHORBOL-13-ACETATE IN MOUSE EPIDERMIS Reviewed

M TAKIGAWA, RC SIMSIMAN, RK BOUTWELL

CANCER RESEARCH 46 ( 1 ) 106 - 112 1986.1

Differential effects of parathyroid hormone and somatomedin-like growth factors on the sizes of proteoglycan monomers and their synthesis in rabbit costal chondrocytes in culture

Yuji Hiraki, Yasutaka Yutani, Masaharu Takigawa, Yukio Kato, Fujio Suzuki

BBA - Molecular Cell Research 845 ( 3 ) 445 - 453 1985.6

Language：English Publishing type：Research paper (scientific journal)

In the proteoglycans extracted from rabbit costal chondrocytes in culture, two populations of proteoglycans were distinguished by density gradient centrifugation under dissociative conditions. The major component was the faster sedimenting population (proteoglycan I), the putative 'cartilage-specific' proteoglycans, and the minor component was the slower sedimenting population (proteoglycan II). The monomeric size of proteoglycan I was closely related to the differentiation-state of chondrocytes and was a good marker of the differentiated chondrocytes. Treatment of the cultures with parathyroid hormone (PTH) induced an increase in the monomeric size of proteoglycan I. This increase was ascribed to an increase in the molecular size of the glycosaminoglycan chain in proteoglycan I. On the other hand, somatomedin-like growth factors, such as multiplication-stimulating activity (MSA) and cartilage-derived factor (CDF), did not affect the size of proteoglycan I, while they markedly stimulated the synthesis of proteoglycan I. In contrast, treatment with nonsomatomedin growth factors, such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), resulted in not only a decrease in glycosaminoglycan synthesis but also a slight decrease in size of proteoglycan I. However, synthesis and size of proteoglycan II were little affected by these agents. Thus, the present study clearly shows that PTH and somatomedin-like growth factors have differential functions in bringing about the expression of the differentiated phenotype of chondrocytes: PTH influences chain elongation and termination of glycosaminoglycans in proteoglycan I, while somatomedin-like growth factors affect primarily the synthesis and secretion of proteoglycan I. © 1985.

DOI： 10.1016/0167-4889(85)90210-1

INDUCTION OF SPERMIDINE SPERMINE N1-ACETYLTRANSFERASE BY PARATHYROID-HORMONE IN RABBIT COSTAL CHONDROCYTES IN CULTURE Reviewed

MATSUIYUASA, I, S OTANI, S MORISAWA, M TAKIGAWA, M ENOMOTO, F SUZUKI

JOURNAL OF BIOCHEMISTRY 97 ( 1 ) 387 - 390 1985

CARTILAGE-DERIVED ANTI-TUMOR FACTOR (CATF) INHIBITS THE PROLIFERATION OF ENDOTHELIAL-CELLS IN CULTURE Reviewed

M TAKIGAWA, E SHIRAI, M ENOMOTO, Y HIRAKI, M FUKUYA, F SUZUKI, T SHIIO, Y YUGARI

CELL BIOLOGY INTERNATIONAL REPORTS 9 ( 7 ) 619 - 625 1985

DIFFERENTIAL-EFFECTS OF PARATHYROID-HORMONE AND SOMATOMEDIN-LIKE GROWTH-FACTORS ON THE SIZES OF PROTEOGLYCAN MONOMERS AND THEIR SYNTHESIS IN RABBIT COSTAL CHONDROCYTES IN CULTURE Reviewed

Y HIRAKI, Y YUTANI, M TAKIGAWA, Y KATO, F SUZUKI

BIOCHIMICA ET BIOPHYSICA ACTA 845 ( 3 ) 445 - 453 1985

STIMULATION BY GLUCOCORTICOIDS OF THE DIFFERENTIATED PHENOTYPE OF CHONDROCYTES AND THE PROLIFERATION OF RABBIT COSTAL CHONDROCYTES IN CULTURE Reviewed

T TAKANO, M TAKIGAWA, F SUZUKI

JOURNAL OF BIOCHEMISTRY 97 ( 4 ) 1093 - 1100 1985

DIFFERENTIATION AND DE-DIFFERENTIATION OF CULTURED CHONDROCYTES - INCREASE IN MONOMERIC SIZE OF CARTILAGE-SPECIFIC PROTEOGLYCANS BY DIBUTYRYL CYCLIC-AMP AND COMPLETE INHIBITION OF THEIR SYNTHESIS BY RETINOIC ACID Reviewed

Y HIRAKI, Y YUTANI, M FUKUYA, M TAKIGAWA, F SUZUKI

BIOCHEMISTRY INTERNATIONAL 10 ( 2 ) 267 - 272 1985

EFFECTS OF SYNTHETIC ANALOGS AND FRAGMENTS OF BOVINE PARATHYROID-HORMONE ON ADENOSINE-3',5'-MONOPHOSPHATE LEVEL, ORNITHINE DECARBOXYLASE ACTIVITY, AND GLYCOSAMINOGLYCAN SYNTHESIS IN RABBIT COSTAL CHONDROCYTES IN CULTURE - STRUCTURE-ACTIVITY RELATIONS Reviewed

T TAKANO, M TAKIGAWA, E SHIRAI, F SUZUKI, M ROSENBLATT

ENDOCRINOLOGY 116 ( 6 ) 2536 - 2542 1985

STUDIES ON CHONDROCYTES FROM MANDIBULAR CONDYLAR CARTILAGE, NASAL SEPTAL CARTILAGE, AND SPHENO-OCCIPITAL SYNCHONDROSIS IN CULTURE .1. MORPHOLOGY, GROWTH, GLYCOSAMINOGLYCAN SYNTHESIS, AND RESPONSIVENESS TO BOVINE PARATHYROID-HORMONE (1-34) Reviewed

M TAKIGAWA, M OKADA, T TAKANO, H OHMAE, M SAKUDA, F SUZUKI

JOURNAL OF DENTAL RESEARCH 63 ( 1 ) 19 - 22 1984

Cartilage derived anti-tumor factor(CATF) : A high morecular weight in cartilage extract inhibits tumor growth. Reviewed

Suzuki, F, Takigawa, M, Hiraki, Y, Kato, Y, Fukuo, K. Shiio, T, Yugari, Y

J.Bone Mineral Metab. 2 231 - 235 1984

CYTOSKELETON AND DIFFERENTIATION - EFFECTS OF CYTOCHALASIN B AND COLCHICINE ON EXPRESSION OF THE DIFFERENTIATED PHENOTYPE OF RABBIT COSTAL CHONDROCYTES IN CULTURE Reviewed

M TAKIGAWA, T TAKANO, E SHIRAI, F SUZUKI

CELL DIFFERENTIATION 14 ( 3 ) 197 - 204 1984

INHIBITION OF POLYAMINE SYNTHESIS AND PROLIFERATION IN MOUSE L-CELLS BY DL-ALPHA-HYDRAZINO-DELTA-AMINOVALERIC ACID, AN INHIBITOR OF ORNITHINE DECARBOXYLASE Reviewed

E GOHDA, M TAKIGAWA, H INOUE, Y KATO, Y DAIKUHARA, Y TAKEDA

JOURNAL OF BIOCHEMISTRY 94 ( 1 ) 97 - 106 1983

PURIFICATION OF 12-O-TETRADECANOYLPHORBOL-13-ACETATE-INDUCED ORNITHINE DECARBOXYLASE FROM MOUSE EPIDERMIS Reviewed

FW PERRELLA, M TAKIGAWA, RK BOUTWELL

INTERNATIONAL JOURNAL OF BIOCHEMISTRY 15 ( 7 ) 885 - 889 1983

A bioassay for parathyroid hormone using rabbit costal chondrocytes in culture. Reviewed

Takano, T, Takigawa, M, Suzuki, F

Jpn.J.Oral.Biol. 25 394 - 397 1983

RESTORATION BY PARATHYROID-HORMONE AND DIBUTYRYL-CYCLIC-AMP OF EXPRESSION OF THE DIFFERENTIATED PHENOTYPE OF CHONDROCYTES INHIBITED BY A TUMOR PROMOTER, 12-0-TETRADECANOYLPHORBOL-13-ACETATE Reviewed

M TAKIGAWA, K FUKUO, T TAKANO, F SUZUKI

CELL DIFFERENTIATION 13 ( 4 ) 283 - 291 1983

ROLE OF POLYAMINES IN EXPRESSION OF THE DIFFERENTIATED PHENOTYPE OF CHONDROCYTES - EFFECT OF DL-ALPHA-HYDRAZINO-DELTA-AMINOVALERIC ACID (DL-HAVA), AN INHIBITOR OF ORNITHINE DECARBOXYLASE, ON CHONDROCYTES TREATED WITH PARATHYROID-HORMONE Reviewed

T TAKANO, M TAKIGAWA, F SUZUKI

JOURNAL OF BIOCHEMISTRY 93 ( 2 ) 591 - 598 1983

THE DIFFERENCE BETWEEN THE EFFECTS OF SINGLE AND DOUBLE APPLICATIONS OF 12-O-TETRADECANOYLPHORBOL-13-ACETATE, A POTENT TUMOR PROMOTER, ON POLYAMINE METABOLISM AND NUCLEIC-ACID SYNTHESIS IN MOUSE EPIDERMIS Reviewed

M TAKIGAWA, RC SIMSIMAN, RK BOUTWELL

CARCINOGENESIS 4 ( 1 ) 5 - 7 1983

INHIBITION OF MOUSE SKIN TUMOR PROMOTION AND OF PROMOTER-STIMULATED EPIDERMAL POLYAMINE BIOSYNTHESIS BY ALPHA-DIFLUOROMETHYLORNITHINE Reviewed

M TAKIGAWA, AK VERMA, RC SIMSIMAN, RK BOUTWELL

CANCER RESEARCH 43 ( 8 ) 3732 - 3738 1983

POLYAMINE BIOSYNTHESIS AND SKIN TUMOR PROMOTION - INHIBITION OF 12-O-TETRADECANOYLPHORBOL-13-ACETATE-PROMOTED MOUSE SKIN TUMOR-FORMATION BY THE IRREVERSIBLE INHIBITOR OF ORNITHINE DECARBOXYLASE ALPHA-DIFLUOROMETHYLORNITHINE Reviewed

M TAKIGAWA, AK VERMA, RC SIMSIMAN, RK BOUTWELL

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 105 ( 3 ) 969 - 976 1982

RESTORATION BY CYCLIC-AMP OF THE DIFFERENTIATED PHENOTYPE OF CHONDROCYTES FROM DEDIFFERENTIATED CELLS PRETREATED WITH RETINOIDS Reviewed

M TAKIGAWA, T TAKANO, F SUZUKI

MOLECULAR AND CELLULAR BIOCHEMISTRY 42 ( 3 ) 145 - 153 1982

SIMULATION OF THE INITIAL-STAGE OF ENDOCHONDRAL OSSIFICATION - INVITRO SEQUENTIAL CULTURE OF GROWTH CARTILAGE CELLS AND BONE-MARROW CELLS Reviewed

F SUZUKI, T TAKASE, M TAKIGAWA, A UCHIDA, Y SHIMOMURA

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES 78 ( 4 ) 2368 - 2372 1981

Effects of parathyroid hormone and cyclic AMP analogues on the activity of ornithine decarboxylase and expression of the differentiated phenotype of chondrocytes in culture Reviewed

M. Takigawa, T. Takano, F. Suzuki

Journal of Cellular Physiology 106 ( 2 ) 259 - 268 1981

Language：English Publishing type：Research paper (scientific journal)

Parathyroid hormone (PTH) greatly increased the level of adenosine 3′,5′ cyclic monophosphate (cAMP) in rabbit costal chondrocytes in culture 2 minutes after its addition. PTH, as well as N6 O2' dibutyryl adenosine 3′,5′ cyclic monophosphate (DBcAMP) and 8 Bromo adenosine 3′,5′ cyclic monophosphate (8 Br‐cAMP) induced ornithine decarboxylase (ODC
L‐ornithine carboxylyase
EC 4.1.1.17), which reached a maximum 4 hours after their addition. Neither cAMP, N6 O2′ dibutyryl guanosine 3′,5′ cyclic monophosphate (DBcGMP), nor sodium butyrate increased the activity of the enzyme. PTH had no effect on DNA synthesis, while DBcAMP and 8 Br‐cAMP decreased DNA synthesis. Expression of the differentiated phenotype of chondrocytes in culture was also induced by PTH, DBcAMP, and 8 Br‐cAMP, but not by cAMP, DBcGMP, or sodium butyrate, as judged by morphological change. Glycosaminoglycan synthesis, a characteristic of the cartilage phenotype, began to increase 8 hours after addition of PTH or DBcAMP, reaching a plateau 32 hours after their addition. These findings suggest that PTH induces increase of ODC activity and expression of the differentiated phenotype of chondrocytes through increase of cAMP and that induction of ODC is closely related to expression of the differentiated phenotype of chondrocytes. Copyright © 1981 Wiley‐Liss, Inc.

DOI： 10.1002/jcp.1041060212

ROLE OF POLYAMINES IN EXPRESSION OF THE DIFFERENTIATED PHENOTYPE OF CHONDROCYTES IN CULTURE Reviewed

T TAKANO, M TAKIGAWA, F SUZUKI

MEDICAL BIOLOGY 59 ( 5-6 ) 423 - 427 1981

POLYAMINE AND DIFFERENTIATION - INDUCTION OF ORNITHINE DECARBOXYLASE BY PARATHYROID-HORMONE IS A GOOD MARKER OF DIFFERENTIATED CHONDROCYTES Reviewed

M TAKIGAWA, H ISHIDA, T TAKANO, F SUZUKI

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES 77 ( 3 ) 1481 - 1485 1980

Changes in polyamine metabolism of rat liver after administration of D-galactosamine : Favorable effects of putrescine administration on galactosmaine-induced hepatic injury. Reviewed

Daikuhara, Y, Tamada, F, Takigawa, M, Takeda, Y, Mori, Y

Gastroenterology 77 123 - 132 1980

Induction by parathyroid hormone of ornithine decarboxylase in rabbit costal chondrocytes in culture. Reviewed

Takigawa, M, Watanabe, R, Ishida, H, Asada, A, Suzuki, F

J.Biochem. 85 311 - 314 1979

The role of putrescine in cell proliferation on the skin of mice induced by ethyl-phenylpropiolate. Reviewed

Takigawa, M, Inoue, H, Gohda, E, Asada, A, Takeda, Y, Mori, Y

Exp.Mol.Pathol. 27 183 - 196 1977

Effect of DL-α-hydrazino-δ-aninovalerric acid,an inhibitor of ornithine decarboxlase,on polyamine metabolism in isoproterenol-stimulated mouse parotid glands. Reviewed

Inoue, H, Kato, Y, Takigawa, M, Adachi K, Takeda, Y

J.Biochem. 77 879 - 893 1975

軟骨培養細胞株の樹立。 Invited

滝川正春

組織培養 15 165 - 169 1900

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