Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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

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

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

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

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

DOI： 10.1002/jcp.20251

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

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

BONE 36 ( 5 ) 884 - 892 2005.5

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

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

DOI： 10.1016/j.bone.2004.10.015

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

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

Cell Communication and Signaling 3 6 - e-Pub 2005.4

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

Background: The chondrosarcoma-derived HCS-2/8 has been known to be an excellent model of human articular chondrocytes. By mimicking the arthritic conditions through the treatment of HCS-2/8 cells with cytokines, we estimated the gene expression response of ccn1 and ccn2 during the course of joint inflammation in vitro. Results: In order to mimic the initiation of inflammation, HCS-2/8 cells were treated with tumor necrosis factor (TNF)-α. To induce pro-inflammatory or reparative responses, TGF-β was employed. Effects of an anti-inflammatory glucocorticoid were also evaluated. After stimulation, expression levels of ccn1 and ccn2 were quantitatively analyzed. Surprisingly, not only ccn2, but also ccn1 expression was repressed upon TNF-α stimulation, whereas both mRNAs were uniformly induced by transforming growth factor (TGF)-β and a glucocorticoid. Conclusion: These results describing the same response during the course of inflammation suggest similar and co-operative roles of these 2 ccn family members in the course of arthritis. © 2005 Moritani et al
licensee BioMed Central Ltd.

DOI： 10.1186/1478-811X-3-6

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Methylation status of EXT1 and EXT2 promoters and two mutations of EXT2 in chondrosarcoma

T Tsuchiya, T Osanai, A Ogose, G Tamura, T Chano, Y Kaneko, A Ishikawa, H Orui, T Wada, T Ikeda, M Namba, M Takigawa, H Kawashima, T Hotta, A Tsuchiya, T Ogino, T Motoyama

CANCER GENETICS AND CYTOGENETICS 158 ( 2 ) 148 - 155 2005.4

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

Germline mutation and functional loss of EXT1 or EXT2 are commonly found in multiple osteochondrornas and predispose to the development of chondrosarcoma. Mutations of EXT1 and EXT2 have rarely been detected in sporadic secondary chondrosarcomas from osteochondrorna; these frequently display loss of heterozygosity at the EXT1 and EXT2 loci, but primary chondrosarcomas typically do not. To evaluate promoter methylation (which is an epigenetic gene silencing mechanism) of EXT1 and EXT2, we performed methylation-specific polymerase chain reaction (PCR) for 20 chondrosarcoma cases (12 primary, 3 secondary to osteochondroma, 2 secondary to enchondromatosis, 2 extraskeletal ordinary. and I clear cell) and in five cell lines. In addition, mutation analysis of the EXT1 and EXT2 coding regions was performed using PCR-single-strand conformation polymorphism and sequencing analysis for 12 of the 20 chondrosarcoma cases (8 primary, I secondary to enchondromatosis, I secondary to osteochondroma, and 2 extraskeletal ordinary) and five cell lines. Promoter methylation of EXT1 and EXT2 was not detected in any of the cases, and both EXT1 and EXT2 were expressed in all cell lines. Two missense mutations in EXT2 (D227E and R299H) were detected among the chondrosarcoma cases. When considering tumor development in primary chondrosarcoma, we should include mutations in EXT2, along with the status of other members of the EXT gene family. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cancergentyto.2004.08.031

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Dexamethasone induces apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt-phosphatidylinositol 3 '-kinase signaling pathway

D Chrysis, F Zaman, AS Chagin, M Takigawa, L Savendahl

ENDOCRINOLOGY 146 ( 3 ) 1391 - 1397 2005.3

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

Although glucocorticoids are known to induce apoptosis in chondrocytes, the mechanisms for this effect and the potential antiapoptotic role of IGF-I are unknown. To address this, we studied the effects of dexamethasone ( Dexa) on apoptosis in the HCS-2/8 chondrocytic cell line. Dexa (25 muM) increased apoptosis (cell death ELISA) by 39% and 45% after 48 and 72 h, respectively (P < 0.01 and P < 0.05, respectively). IGF-I (100 ng/ml) decreased Dexa-induced apoptosis to levels similar to control cells. Apoptosis was associated with cleavage of poly-ADP-ribose polymerase (PARP) and alpha-fodrin and activation of caspases-8, -9, and -3 ( Western), an effect that was counteracted when chondrocytes were cocultured with Dexa + IGF-I. Inhibitors for caspases-8, -9, and -3 (50 muM each) equally suppressed Dexa-induced apoptosis (P < 0.01). Time-response experiments showed that caspase-8 was activated earlier ( at 12 h) than caspase-9 ( at 36 h). We studied the phosphatidylinositol 3'-kinase (PI3K) pathway to further investigate the mechanisms of Dexa-induced apoptosis. Dexa decreased Akt phosphorylation by 93% (P<0.001) without affecting total Akt and increased the p85alpha subunit 4-fold. The Akt inhibitor SH-6 (10 muM) increased apoptosis by 54% (P < 0.001). When combining Dexa with SH-6, apoptosis was not further increased, showing that Dexa-induced apoptosis is mediated through inhibition of the PI3K pathway. Addition of IGF-I to SH-6- or Dexa + SH-6-treated cells decreased apoptosis by 21.2% (P < 0.001) and 20.6% ( P < 0.001), respectively. We conclude that Dexa-induced apoptosis is caspase dependent with an early activation of caspase-8. IGF-I can rescue chondrocytes from Dexa-induced apoptosis partially through the activation of other pathways than the PI3K signaling pathway. Based on our in vitro data, we speculate that in vivo treatment with glucocorticoids may diminish longitudinal growth by increasing apoptosis of proliferative growth plate chondrocytes.

DOI： 10.1210/en.2004-1152

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Regulation of chicken ccn2 gene by interaction between RNA cis-element and putative trans-factor during differentiation of chondrocytes

Y Mukudai, S Kubota, T Eguchi, S Kondo, K Nakao, M Takigawa

JOURNAL OF BIOLOGICAL CHEMISTRY 280 ( 5 ) 3166 - 3177 2005.2

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

CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3'-untranslated region (3'-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3'-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.

DOI： 10.1074/jbc.M411632200

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Downregulation of rheumatoid arthritis-related antigen RA-A47 (HSP47/colligin-2) in chondrocytic cell lines induces apoptosis and cell-surface expression of RA-A47 in association with CD9

T Hattori, K von der Mark, H Kawaki, Y Yutani, S Kubota, T Nakanishi, H Eberspaecher, B de Crombrugghe, M Takigawa

JOURNAL OF CELLULAR PHYSIOLOGY 202 ( 1 ) 191 - 204 2005.1

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

Previously, we showed that gene expression of the rheurnatoid arthritis-related antigen RA-A47, which is identical to human heat shock protein (HSP)47, was downregulated in chondrocytes by inflammatory cytokines such as TNFalpha. Associated with this phenomenon, RA-A47 appeared on the cell surface concomitant with upregulation of metabolic factors related to cartilage destruction. The upregulation of the metabolic factors could be achieved by clownregulation of RA-A47 expression with ra-a47-specific anti-senseoligonucleotide. Here, we show that the enhanced surface expression of RA-A47 on a chondrocytic cell line, HCS-2/ 8 was also a direct result of RA-A47 downregulation by ra-a47 anti-sense oligonucleoticle, independent of the cytokine effects. Moreover, cell-surface expression of CD9, a l integrin-associated transmembrane protein that is involved in cell adhesion and cell motility events, was enhanced in the ra-a47 anti-sense oligonucleoticle-treated cells. The CD9 was colocalized with RA-A47 on the cell surface, where it may have affected integrin signaling. Furthermore, Annexin-V binding to the cell surface and the level of a number of apoptosis-related genes including caspase-9 were increased after ra-a47 anti-sense oligonucleoticle treatment, suggesting that enhanced surface expression of RA-A47 and CD9 may be initiating apoptosis. Differential screening using a cDNA gene array showed induction of metal lothionein-III and chemokine receptor CXCR4 and of factors of the Notch signaling pathway by the anti-sense treatment, but not by TNFalpha.. Thus, here we show for the first time an alternative mechanism of inducing apoptosis by downregulating molecular chaperones, independent of the action of TNFalpha. The surface-exposed RA-A47 may induce autoantibodies and inflammatory reactions in autoimmune disease situations such as rheurnatoid arthritis. J. Cell. Physiol. 202: 191 -204, 2005. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/jcp.20112

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Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis

H Okada, T Kikuta, T Kobayashi, T Inoue, Y Kanno, M Takigawa, T Sugaya, JB Kopp, H Suzuki

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 16 ( 1 ) 133 - 143 2005.1

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

Connective tissue growth factor (CTGF) is one of the candidate factors that are thought to mediate the downstream profibrotic action of TGF-beta. However, its precise role in renal interstitial fibrogenesis has not yet been clarified. It was demonstrated previously that CTGF was expressed in tubular epithelial cells that had been engulfed by interstitial fibrosis in the remnant kidney of the subtotal nephrectomy (SNx) model. In the present study, co-cultures of tubular epithelial cells (mProx24) and tubulointerstitial fibroblasts (TFB) that mimic the subepithelial mesenchyme in the kidney were used to study the profibrotic effects of TGF-beta1-induced CTGF. In these co-cultures, TGF-beta1 treatment resulted in significantly increased mRNA levels of type I collagen and fibronectin in the TFB. These effects were both direct and indirect, with the latter being mediated by CTGF derived from the co-cultured mProx24. Then TGF-beta1 transgenic mice were subtotally nephrectomized and treated with CTGF antisense oligodeoxynucleotide, and their kidneys were analyzed for fibrosis. Intravenous administration of CTGF antisense oligodeoxynucleotide significantly blocked CTGF expression in the proximal tubular epithelial cells in the remnant kidney of these animals despite the sustained level of TGF-beta1 mRNA. This reduction in CTGF mRNA level paralleled a reduction in mRNA levels of matrix molecules as well as proteinase inhibitors plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase-1, suppressing renal interstitial fibrogenesis. In conclusion, tubular CTGF acts as a downstream mediator of the profibrotic effects of TGF-beta1 in the remnant kidney, which is a promising target for antifibrotic drugs designed to treat TGF-beta1- dependent interstitial fibrosis.

DOI： 10.1681/ASN.2004040339

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Introduction for CCN proteins.

Perbal, B, Takigawa, M

In CCN Proteins: A New Family of Cell Growth and Differentiation Regulators (Perbal B. & Takigawa M. eds.) 1 - 18 2005

Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage

M Gebauer, J Saas, J Haag, U Dietz, M Takigawa, E Bartnik, T Aigner

ARTHRITIS RESEARCH & THERAPY 7 ( 2 ) R274 - R284 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：BIOMED CENTRAL LTD

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal ( n = 9) and osteoarthritic ( n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1 beta and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage ( P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression.

DOI： 10.1186/ar1479

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Increased levels of CTGF mRNA expression in a murine model of allergic airway inflammation

Hong Mei Piao, Kohei Yamauchi, Li-Hua Pan, Toshihide Nakadate, Harumasa Ito, Takashi Mouri, Hitoshi Kobayashi, Takashi Sawai, Tohru Nakanishi, Masaharu Takigawa, Hiroshi Inoue

Allergology International 54 ( 1 ) 107 - 115 2005

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

Background: Connective tissue growth factor (CTGF) is known to play a direct role in fibrosis in various organs as a downstream mediator of TGF-β. Objective: To evaluate a role in subepithelial fibrosis in the asthmatic airway, we investigated CTGF mRNA expression and CTGF producing cells in the airways of a murine asthma model with allergic inflammation. Methods: After repetitive inhalation challenges with ovalbumin (OVA), cell numbers and TGF-β1 concentrations in bronchoalveolar lavage fluid from immunized mice were measured. Collagen deposition in lung tissue was estimated by measuring hydroxyproline content. CTGF mRNA and GAPDH mRNA levels were determined by quantitative RT-PCR method. Immunohistochemistry for CTGF with anti-CTGF antibody was performed. Results: Numbers of eosinophils and TGF-β1 concentration increased markedly in BALF on the 7th day and 14 th day after inhalation challenge with OVA. Hydroxyproline content in lung tissue increased significantly on the 14th day after inhalation challenge of OVA compared to control. The ratio of CTGF mRNA /GAPDH mRNA in lung tissue in mice exposed to OVA increased 10-fold compared to those exposed to saline. Immunohistochemistry revealed that the number of CTGF-positive cells increased in bronchial submucosa after inhalation challenge of OVA. Conclusions: Our results suggested that CTGF might be one of the potential molecules involved in subepithelial fibrosis in murine airways with allergic inflammation. ©2005 Japanese Society of Allergology.

DOI： 10.2332/allergolint.54.107

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CCNファミリー：その構造と機能. Invited

滝川正春

細胞 37,462-465 2005

Roles of CCN2/CTGF in the control of growth and regeneration.

Takigawa, M, Nishida, T, Kubota, S

In CCN Proteins: A New Family of Cell Growth and Differentiation Regulators (Perbal B. & Takigawa M. eds.) 19 - 59 2005

Cartducin, a Paralog of acrp30/adiponectin, is induced during chondrogenic differentiation and promotes proliferation of chondrogenic precursors and chondrocytes.

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

J. Cell. Physiol. 206(2), 537 - 544, 2005

Molecular phenotyping of HCS-2/8 cells as an in vitro model of human chondrocytes

J Saas, K Lindauer, B Bau, M Takigawa, T Aigner

OSTEOARTHRITIS AND CARTILAGE 12 ( 11 ) 924 - 934 2004.11

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

Objective: Cultures of primary articular chondrocytes for studying chondrocyte biology are notoriously difficult to handle. One alternative is the use of chondrocytic cell lines. Because the HCS-2/8 cells are the most widely used cell line in cartilage research, we investigated the molecular phenotype of these cells by mRNA-expression profiling.
Design: Monolayers of HCS-2/8 cells were cultured to sub-confluence, confluence and over-confluence; primary human chondrocytes were grown in monolayer culture and alginate-bead cultures and several other chondrocytic cell lines were cultured as monolayers. RNA was isolated and analyzed by cDNA array profiling using Affymetrix GeneChips (U95A/U95Av2) and quantitative PCR.
Results: Important similarities, but also remarkable differences between the HCS-2/8 cells and adult human articular chondrocytes were detected: Aggrecan and several cartilage typical collagens as well as SOX9 transcripts were strongly expressed in HCS-2/8 cells, whereas HCS-2/8 cells expressed hardly any chondrocyte-typical cartilage matrix degrading enzymes. Of all culturing conditions, clustering analysis showed that HCS-2/8 cultured at confluence are most closely related to primary chondrocytes.
Conclusion: Our study confirms how careful one needs to be in choosing in vitro model systems for investigating effects of interest. The major issue of chondrocyte cell lines appears to be that they mainly proliferate and show less expression of genes of matrix synthesis and turnover. A successful approach will have to select suitable chondrocyte cell lines and to validate findings obtained using primary chondrocytes. This allows to establish a reproducible in vitro model showing the property of interest and subsequently to relate back the obtained results to the physiologic situation. (C) 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2004.08.002

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Implication of prostaglandin E-2 in TNF-alpha-induced release of m-calpain from HCS-2/8 chondrocytes. Inhibition of m-calpain release by NSAIDs

K Fushimi, S Nakashima, Y Banno, A Akaike, M Takigawa, K Shimizu

OSTEOARTHRITIS AND CARTILAGE 12 ( 11 ) 895 - 903 2004.11

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

Objective: Calpains are known as Ca2+-dependent intracellular neutral cysteine proteases. However, m-calpain is detected in synovial fluid of arthritic joints and is shown to possess the proteoglycanase activity in vitro. The mechanism of m-calpain release into the extracellular spaces during arthritis has not yet been well characterized. In the present study, we have analyzed m-calpain release from cultured chondrocytes stimulated by a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on m-calpain release were also examined.
Methods: Human chondrocytic HCS-2/8 cells were stimulated by TNF-alpha in the presence or absence of an NSAID. m-Calpain in the cells and culture medium was quantified by Western blot analysis using an anti-m-calpain antibody. Western blots were subjected to densitometric analysis and band intensities were determined.
Results: TNF-alpha (10 ng/ml) stimulated m-calpain release with transient increase in cellular m-calpain in HCS-2/8 cells. NSAIDs examined (aspirin, loxoprofen-SRS, diclofenac sodium, indomethacin and NS398) inhibited m-calpain release and production of prostaglandin E-2 (PGE(2)) induced by 10 ng/ml TNF-alpha. Exogenously added PGE(2) accelerated the release of m-calpain in response to a lower concentration of TNF-alpha (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-alpha-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-alpha.
Conclusions: These results suggest that TNF-alpha stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE(2) receptor by produced PGE(2) is deeply involved in this process. (C) 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2004.08.001

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Expression and localization of connective tissue growth factor (CTGF/Hcs24/CCN2) in osteoarthritic cartilage

S Omoto, K Nishida, Y Yamaai, M Shibahara, T Nishida, T Doi, H Asahara, T Nakanishi, H Inoue, M Takigawa

OSTEOARTHRITIS AND CARTILAGE 12 ( 10 ) 771 - 778 2004.10

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

Objective: The investigation of the expression and localization of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24/CCN family member 2 (CTGF/Hcs24/CCN2) in normal and osteoarthritic (OA) cartilage, and quantification of CTGF/Hcs24-positive cells.
Methods: Cartilage samples of patients (n = 20) with late stage CA were obtained at total joint replacement surgery. Morphologically normal cartilage was harvested for comparison purposes from the femoral heads of 6 other patients with femoral neck fracture. Paraffin -embedded sections were stained by Safranin O. The severity of the OA lesions was divided into four stages (normal, early, moderate, and severe). The localization of protein and mRNA for CTGF/Hcs24 was investigated by immunohistochemistry and in situ hybridization, respectively. The population of CTGF/Hcs24-positive chondrocytes in CA cartilage and chondro-osteophyte was quantified by counting the number of the cells under light microscopy.
Results: Signals for CTGF/Hcs24 were detected in a small percentage of chondrocytes throughout the layers of normal cartilage. In early stage CA cartilage, the CTGF/Hcs24-positive chondrocytes were localized mainly in the superficial layer. In moderate to severe OA cartilage, intense staining for CTGF/Hcs24 was observed in proliferating chondrocytes forming cell clusters next to the cartilage surface. In chondro-osteophyte, strong signals were found in the chondrocytes of the proliferative and hypertrophic zones.
Conclusion: CTGF/Hcs24 expression was detected in both normal and OA chondrocytes of human samples. The results of the current study suggested that expression of CTGF/Hcs24 was concomitant with development of CA lesions and chondrocyte differentiation in chondro-osteophyte. (C) 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2004.06.009

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Regeneration of defects in the articular cartilage in rat knee joints by connective tissue growth factor hypertrophic chondrocyte-specific gene product 24 CCN family member 2 (CTGF/Hcs24/CCN2).

T Nishida, S Kubota, T Kuboki, K Nakao, T Kushibiki, Y Tabata, M Takigawa

JOURNAL OF BONE AND MINERAL RESEARCH 19 S216 - S216 2004.10

Abundant retention and release of connective tissue growth factor (CTGF/CCN2) by platelets

S Kubota, K Kawata, T Yanagita, H Doi, T Kitoh, M Takigawa

JOURNAL OF BIOCHEMISTRY 136 ( 3 ) 279 - 282 2004.9

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

Wound healing and tissue regeneration are usually initiated by coagulation followed by fibrous tissue formation. In the present study, we discovered an abundance of connective tissue growth factor (CTGF/CCN2) in human platelets, which was released along with the coagulation process. The CTGF/CCN2 content in platelets was 10-fold higher than that in arterial tissue. Furthermore, the CTGF/CCN2 content in a single platelet was computed to be more than 20-fold higher than that of any other growth factor reported. Considering that CTGF/CCN2 promotes angiogenesis, cartilage regeneration, fibrosis and platelet adhesion, it may be now regarded as one of the major functional components of platelets.

DOI： 10.1093/jb/mvh126

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Connective tissue growth factor causes persistent proa2(I) collagen gene expression induced by transforming growth factor-b in a mouse fibrosis model

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

ARTHRITIS AND RHEUMATISM 50 ( 9 ) S624 - S625 2004.9

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