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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

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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

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DOI： 10.1007/978-1-0716-2744-0_7

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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

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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

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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

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DOI： 10.1007/978-1-0716-2744-0_15

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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

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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

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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

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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

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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

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DOI： 10.1002/jcp.30348

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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

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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

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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

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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

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© 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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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DOI： 10.1007/s12079-018-0487-x

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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DOI： 10.1016/j.bone.2015.11.007

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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

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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

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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

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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

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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

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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

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DOI： 10.1002/jbmr.2502

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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

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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

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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

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DOI： 10.1002/jcb.24728

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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.

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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

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DOI： 10.1016/j.bone.2013.11.010

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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

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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

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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

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

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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

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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

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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

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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

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DOI： 10.1371/journal.pone.0083545

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DOI： 10.1016/j.biochi.2012.10.016

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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

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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

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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

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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

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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

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DOI： 10.1242/jcs.101956.

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DOI： 10.1007/s12079-012-0177-z.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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

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

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Language：Japanese   Publisher：先端医学社  

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Other Link： http://search.jamas.or.jp/link/ui/2011089282

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

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

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

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

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

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Language：Japanese   Publisher：(公社)日本生化学会  

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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

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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

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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

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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

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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

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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

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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

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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

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Language：Japanese   Publisher：(公社)日本生化学会  

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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

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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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

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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

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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

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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.

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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

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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

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Language：Japanese   Publisher：岡山歯学会  

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Language：Japanese   Publisher：岡山歯学会  

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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

DOI： 10.1007/s12079-007-0002-2

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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)

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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

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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

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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.

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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.

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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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

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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)

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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

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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.

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：日本癌学会  

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Language：English   Publisher：(公社)日本生化学会  

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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

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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

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Language：English   Publisher：WILEY-BLACKWELL  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：English   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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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

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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. © 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2004.10.015

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Language：English   Publisher：AMER SOC BONE & MINERAL RES  

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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

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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

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Language：English   Publisher：WILEY-LISS  

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Language：Japanese   Publisher：日本癌学会  

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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

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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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

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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.

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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

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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

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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

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Language：Japanese   Publisher：(公社)日本生化学会  

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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

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Language：Japanese   Publisher：中山書店  

CiNii Article

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Other Link： http://search.jamas.or.jp/link/ui/2004224953

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

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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

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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

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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.

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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

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Language：Japanese   Publisher：(公社)日本矯正歯科学会  

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