Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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

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

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

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

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

DOI： 10.1007/978-90-481-3779-4_19

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

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

Annick Perbal, Masaharu Takigawa, Bernard Perbal

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

Language：English Publisher：Springer Netherlands

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

DOI： 10.1007/978-90-481-3779-4

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

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

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

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

Yuichi Hikichi, Koji Yoshimura, Masaharu Takigawa

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

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

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

DOI： 10.1016/j.bbrc.2009.06.052

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

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

BIOCHEMICAL JOURNAL 420 413 - 420 2009.6

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

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

DOI： 10.1042/BJ20081991

CTGF and Apoptosis in Mouse Osteocytes Induced by Tooth Movement

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

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

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

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

DOI： 10.1177/0022034509334649

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

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

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

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

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

DOI： 10.1111/j.1600-0765.2008.01093.x

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

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

FEBS LETTERS 583 ( 6 ) 1006 - 1010 2009.3

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

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

DOI： 10.1016/j.febslet.2009.02.025

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

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

JOURNAL OF BIOCHEMISTRY 145 ( 2 ) 207 - 216 2009.2

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

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

DOI： 10.1093/jb/mvn159

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

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

BONE 44 ( 1 ) 24 - 31 2009.1

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

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

DOI： 10.1016/j.bone.2008.08.125

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

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

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

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

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

DOI： 10.1359/JBMR.080615

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

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 皆木省吾, 滝川正春

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

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

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

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

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

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

DOI： 10.1128/MCB.00495-08

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

河田かずみ, 久保田聡, 江口傑徳, 青山絵理子, 皆木省吾, 滝川正春

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

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

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

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

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

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

DOI： 10.1016/j.bbrc.2008.06.089

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

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

ARTHRITIS AND RHEUMATISM 58 ( 9 ) 2798 - 2808 2008.9

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

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

DOI： 10.1002/art.23761

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

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

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

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

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

DOI： 10.1002/jeb.21724

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

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

ANTICANCER RESEARCH 28 ( 4C ) 2343 - 2348 2008.7

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

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

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

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

OSTEOARTHRITIS AND CARTILAGE 16 ( 7 ) 787 - 795 2008.7

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

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

DOI： 10.1016/j.joca.2007.11.001

Promotion of bone regeneration by CCN2 incorporated into gelatin hydrogel

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

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

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

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

DOI： 10.1089/ten.tea.2007.0167

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