Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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ウサギ変形性顎関節症モデルにおける軟骨細胞のアポトーシスの関与

藤沢拓生, 笠井照夫, 窪木拓男, 矢谷博文, 服部高子, 滝川正春

日本顎関節学会雑誌 14 ( 1 ) 110 - 110 2002.4

Kunitz-type protease inhibitor bikunin disrupts phorbol ester-induced oligomerization of CD44 variant Isoforms containing epitope v9 and subsequently suppresses expression of urokinase-type plasminogen activator in human chondrosarcoma cells

M Suzuki, H Kobayashi, M Fujie, T Nishida, M Takigawa, N Kanayama, T Terao

JOURNAL OF BIOLOGICAL CHEMISTRY 277 ( 10 ) 8022 - 8032 2002.3

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

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses phorbol ester (PMA)-stimulated expression of urokinase-type plasminogen activator (uPA). In the present study, we tried to answer this mechanism using human chondrosarcoma HCS-2/8 cells. Our results showed the following novel findings: (a) the standard form of CD44 (CD44s; 85 kDa) is expressed in both unstimulated and PMA-stimulated cells, while CD44v isoforms containing epitope v9 (110 kDa) are strongly up-regulated in response to treatment with PMA, (b) CD44v isoforms containing epitope v9 present on the same cell exclusively form aggregates in stimulated cells; (c) induction of uPA mRNA expression could be achieved by using a second cross-linker antibody to cross-link Fab monomers of anti-CD44; (d) co-treatment of stimulated cells with anti-CD44 mAb alone or anti-CD44v9 mAb alone suppresses PMA-induced clustering of CD44, which results in inhibition of uPA overexpression; (e) bikunin efficiently disrupts PMA-induced clustering of CD44, but does not prevent PMA-induced up-regulation of CD44v isoforms containing epitope v9; and (f) after exposure to bik, similar to150-kDa band is mainly detected with immunoprecipitation and this band is shown to be a heterodimer composed of the 110-kDa v9-containing CD44v isoforms and a 45-kDa bik receptor (bik-R). In conclusion, we provide, for the first time, evidence that the bik-R can physically interact with the CD44v isoforms containing epitope v9 and function as a repressor to down-regulate PMA-stimulated uPA expression, at least in part, by preventing clustering of CD44v isoforms containing epitope v9.

DOI： 10.1074/jbc.M108545200

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骨格成長と骨折治癒過程におけるCTGF/Hcs24の遺伝子発現

滝川正春, 中西徹, 西田崇, 縄稚久美子, 中田英二

厚生労働省特定疾患対策研究事業研究報告書脊柱靭帯骨化症に関する調査研究班平成13年度 69 - 76 2002.3

Language：Japanese Publisher：厚生労働省特定疾患対策研究班

肥大軟骨細胞由来の成長因子CTGF/Hcs24の生理的,病理的役割を解明するため,胎生10,15,17日齢,生後1日齢のマウスを用いて骨格成長過程と骨折治癒過程におけるCTGF/Hcs24の遺伝子発現について検討した.マウス胎生期における発現は,胎生7日の初期に高く,胎生10日で一旦低下した後,胎生15日〜17日で再び増強した.マウスでは多くの内軟骨性骨化が胎生13日で始まることから,後期の遺伝子発現は内軟骨性骨化と関連すると考えられるが,初期の遺伝子発現の亢進は幼若細胞の分化と関連すると考えられた.また,CTGF/Hcs24は骨折治癒過程においても肥大軟骨細胞から産生され肥大軟骨細胞層を中心として軟骨細胞層全体に存在することが明らかとなり,CTGF/Hcs24は軟骨細胞にとってオートクリン・パラクリン因子であることが明らかとなった.更に,骨折後初期において,増殖しつつある骨膜細胞や血管内皮細胞にも発現していることから,組織再生の際には肥大軟骨細胞だけでなく種々の細胞が発現・産生する因子であることが示唆された

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Development of Dentin Regeneration Therapy : Expression of Type I Collagen and Alkaline Phosphatase Induced by CTGF in Human Cultured Dental Pulp

Hirotoshi SHIMIZU, Yoshihiro NISHITANI, Tomiko YAMADA, Takashi NISHIDA, Masaharu TAKIGAWA, Masahiro YOSHIYAMA, Department of Operative Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Operative Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Operative Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Biochemistry and Molecular Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Biochemistry and Molecular Dentistry Okayama University Graduate School of Medicine and Dentistry, Department of Operative Dentistry Okayama University Graduate School of Medicine and Dentistry

Journal of hard tissue biology = Journal of hard tissue biology 11, ( 2 ) 62 - 67 2002

High pressure effects on cellular expression profile and mRNA stability. A cDNA array analysis

RK Sironen, HM Karjalainen, K Torronen, MA Elo, K Kaarniranta, M Takigawa, HJ Helminen, MJ Lammi

BIORHEOLOGY 39 ( 1-2 ) 111 - 117 2002

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

Hydrostatic pressure has a profound effect on cartilage tissue and chondrocyte metabolism. Depending on the type and magnitude of pressure various responses can occur in the cells. The mechanisms of mechanotransduction at cellular level and the events leading to specific changes in gene expression are still poorly understood. We have previously shown that induction of stress response in immortalized chondrocytes exposed to high static hydrostatic pressure increases the stability of heat shock protein 70 mRNA. In this study, our aim was to examine the effect of high pressure on gene expression profile and to study whether stabilization of mRNA molecules is a general phenomenon under this condition. For this purpose a cDNA array analysis was used to compare mRNA expression profile in pressurized vs. non-pressurized human chondrosarcoma cells (HCS 2/8). mRNA stability was analyzed using actinomycin-treated and nontreated samples collected after pressure treatment. A number of immediate-early genes, and genes regulating cell cycle and growth were up-regulated due to high pressure. Decrease in osteonectin, fibronectin, and collagen types VI and XVI mRNAs was observed. Also bikunin, cdc37 homologue and Tiam1, genes linked with hyaluronan metabolism, were down-regulated. In general, stability of down-regulated mRNA species appeared to increase. However, no increase in mRNA above control level due to stabilization was noticed in the genes available in the array. On the other hand, mRNAs of certain immediate-early genes, like c-jun, jun-B and c-myc, became destabilized under pressure treatment. Increased accumulation of mRNA on account of stabilization under high pressure conditions seems to be a tightly regulated, specific phenomenon.

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CTGF/Hcs24, a hypertrophic chondrocyte specific gene product, stimulates the proliferation and expression of the cartilage phenotype but not hypertrophy or calcification or articular cartilage in culture.

Nishida T, Kubota S, Nakanishi T, Kuboki T, Yosimichi G, Kondo S, Takigawa M

J Cell Physiol 192, 55 - 63 2002

岡山大学歯学部におけるチュートリアル教育(2) －5年次生に導入された「Evidence-based medicine(EBM)に基づく歯科医療」の解析と評価－

宮本学, 窪木拓男, 高務朋将, 西谷佳浩, 鷲尾憲文, 水口一, 若狭享, 多田徹, 原哲也, 高木慎, 福永城司, 真野隆充, 山田庸介, 尾形小霧, 松尾龍二, 永井教之, 矢谷博文, 滝川正春, 山本照子

岡山歯学会雑誌 21 ( 2 ) 247 - 253 2002

Connective tissue growth factor as a major angiogenic agent that is induced by hypoxia in a human breast cancer cell line

T Shimo, S Kubota, S Kondo, T Nakanishi, A Sasaki, H Mese, T Matsumura, M Takigawa

CANCER LETTERS 174 ( 1 ) 57 - 64 2001.12

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

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here, we present the evidence that the hypoxic induction of angiogenesis by human breast cancer cells (MDA-231) can be ascribed at least in part to CTGF, Our results indicate that (i) CTGF is abundantly present in MDA-231 cells in vitro and in vivo, (ii) its secretion is up-regulated by hypoxia, and (iii) its gene expression is enhanced in MDA-231 cells cultured under hypoxic conditions. These data suggest CTGF may stimulate angiogenesis by paracrine mechanisms, thereby contributing to the invasion of breast cancer cells. This is the first evidence that human cancer cells differentially express CTGF protein and mRNA under the control of hypoxic conditions. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

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CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK) Reviewed

G Yosimichi, T Nakanishi, T Nishida, T Hattori, T Takano-Yamamoto, M Takigawa

EUROPEAN JOURNAL OF BIOCHEMISTRY 268 ( 23 ) 6058 - 6065 2001.12

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE LTD

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24) promotes proliferation and differentiation of chondrocytes in culture. We investigated the roles of two major types of mitogen activated protein kinase (MAPK) in the promotion of proliferation and differentiation by CTGF/Hcs24. Here we report the effects of the MAPKK/MEK 1/2 inhibitor, PD098059, and p38 MAPK inhibitor, SB203580, in a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) and rabbit growth cartilage (RGC) cells treated with CTGF/Hcs24. In the proliferation phase, CTGF/Hcs24 induced a approximate to fivefold increase in the phosphorylation of p44/42 MAPK/ERK and a approximate to twofold increase in that of p38 MAPK in an in vivo kinase assay. These inhibitors of MAPKK and MAPK suppressed phosphorylation of ets-like gene-1 (Elk-1) and nuclear activating transcription factor-2 (Atf-2) induced by CTGF/Hcs24 in a dose-dependent manner, respectively. Western blot analysis showed that phosphorylation of ERK was induced from 30 to 60 min and phosphorylation of p38 MAPK from 10 to 15 min after the addition of CTGF/Hcs24 in confluence HCS-2/8 cells. PD098059 suppressed the DNA synthesis of HCS-2/8 cells and RGC cells, while SB203580 did not. On the other hand, the p38 MAPK inhibitor, SB203580, completely inhibited the CTGF/Hcs24-induced synthesis of proteoglycans in HCS-2/8 cells and RGC cells but the MEK1/2 inhibitor, PD098059, did not. These results suggest that ERK mediates the CTGF/Hcs24-induced proliferation of chondrocytes, and that p38 MAPK mediates the CTGF/Hcs24-induced differentiation of chondrocytes.

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遺伝子発現の抑圧的調節を仲介するマウスctgf3'-UTR segmentの性質

近藤誠二, 久保田聡, 江口傑徳, 服部高子, 中西徹, 菅原利夫, 滝川正春

日本口腔科学会雑誌 50 ( 6 ) 568 - 568 2001.11

Cell-type-specific trans-activation of herpes simplex virus thymidine kinase promoter by the human T-cell leukemia virus type I Tax protein

S Kubota, Y Mukudai, T Hattori, T Eguchi, S Kondo, M Takigawa

DNA AND CELL BIOLOGY 20 ( 9 ) 563 - 568 2001.9

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

The human T-cell leukemia virus type I Tax protein (HTLV-I Tax) is known as a trans-activating factor for a variety of genes, including those of cytokines. Here, we show that Tax is capable of activating the herpes simplex virus thymidine kinase (HSV-TK) promoter in certain mammalian cell lines. In murine NIH 3T3 fibroblasts and human HeLa cells, trans-activation by Tax was remarkably strong, whereas in human chondrocytic HCS-2/8 and monkey kidney Cos-7 cells, the responsiveness of the TK promoter to Tax was poor. Deletion analysis revealed that one of the two previously described Sp1 sites is required for the Tax responsiveness, whereas the CTF binding site is not. The results suggest possible interactions between the oncogenic Tax protein and the viral TK in coinfected cells in vivo. Care should be taken in the context of HTLV-I research, as the HSV-TK promoter has been widely used in molecular biology and gene therapeutics.

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Novel mode of processing and secretion of connective tissue growth factor/ecogenin (CTGF/Hcs24) in chondrocytic HCS-2/8 cells

S Kubota, T Eguchi, T Shimo, T Nishida, T Hattori, S Kondo, T Nakanishi, M Takigawa

BONE 29 ( 2 ) 155 - 161 2001.8

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

The synthesis, processing, and secretion of human connective tissue growth factor (CTGF/Hcs24) in a human chondrocytic cell line, HCS-2/8, were analyzed immunochemically. By metabolic pulse-labeling, chasing, and subsequent immunoprecipitation analyses, active synthesis of CTGF was observed not only in growing HCS-2/8 cells, but also in confluent cells. However, secretion and processing of CTGF were found to be regulated differentially, depending upon the growth status. During phases of growth, HCS-2/8 cells released CTGF molecules immediately without sequestering them within the cell layer. In contrast, after the cells reached confluence, the secretion slowed, resulting in an accumulation of CTGF in the cells or extracellular matrices (ECMs). Also, in confluent cell layers, a 10 kDa protein that was reactive to an anti-CTGF serum was observed. This CTGF-related small protein was not detected immediately after labeling, but gradually appeared within 6 h after chase, which suggests its entity as a processed subfragment of CTGF. Surprisingly, the 10 kDa protein was stable even 48 h after synthesis, and was not released by ECM digestion, suggesting an intracellular maintenance and function. Taken together, the behavior of CTGF in HCS-2/8 cells is remarkably different from that reported in fibroblasts, which may represent unique roles for CTGF in the growth and differentiation of chondrocytes. (C) 2001 by Elsevier Science Inc. All rights reserved.

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多機能成長因子CTGF/Hcs24遺伝子の転写後制御エレメント,CAESARの作用機序

久保田聡, 近藤誠二, 椋代義樹, 江口傑徳, 服部高子, 中西徹, 滝川正春

生化学 73 ( 8 ) 710 - 710 2001.8

Tumor necrosis factor alpha induces expression of genes for matrix degradation in human chondrocyte-like HCS-2/8 cells through activation of NF-kappa B: Abrogation of the tumor necrosis factor alpha effect by proteasome inhibitors

T Sakai, F Kambe, H Mitsuyama, N Ishiguro, K Kurokouchi, M Takigawa, H Iwata, H Seo

JOURNAL OF BONE AND MINERAL RESEARCH 16 ( 7 ) 1272 - 1280 2001.7

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

Tumor necrosis factor alpha (TNF-alpha) has been suggested to induce chondrocytic chondrolysis in both inflammatory and degenerative joint diseases. However, its intracellular signaling pathway leading to the chondrolysis has not been studied in detail. Thus, we investigated whether TNF-alpha activates a transcription factor nuclear factor KB (NF-KB) in human chondrocyte-like cells (HCS-2/8) and induces the expression of genes involved in the degradation of cartilage matrix. Treatment of the cells with TNF-alpha markedly increased the levels of matrix metalloproteinase 1 (MMP-1), MMP3, intercellular adhesion molecule 1 (ICAM-1), and cyclo-oxygenase 2 (COX-2) messenger RNAs (mRNAs). The increase in the mRNAs was associated with the activation of p65/p50 heterodimer NF-kappaB. I kappaB-alpha and I kappaB-beta, cytoplasmic molecules preventing the nuclear translocation of NF-kappaB, were degraded rapidly by TNF-alpha followed by their synthesis to the basal level. Treatment with proteasome inhibitors inhibited the degradation of both IKB-alpha and IKB-beta and prevented the TNF-alpha -dependent nuclear translocation of p65. Furthermore, the inhibitors completely prevented the TNF-alpha -dependent induction of MMP-1, MMP3, ICAM-1, and COX-2 mRNAs. Thus, it is shown that the activation of p65/p50 NF-KB by TNF-a! plays a cardinal role in inducing the expression of MMP-1, MMP-3, ICAM-1, and COX-2 genes, which are involved in matrix degradation and inflammatory reaction in chondrocytes, leading to chondrocytic chondrolysis.

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ヒト軟骨肉腫由来軟骨様細胞株HCS-2/8における結合組織成長因子CTGF-Hcs24の遺伝子発現制御機構(Regulatory Mechanism of Human Connective Tissue Growth Factor (CTGF/Hcs24) Gene Expression in a Human Chondrocytic Cell Line, HCS-2/8)

江口傑徳, 久保田聡, 近藤誠二, 志茂剛, 服部高子, 中西徹, 窪木拓男, 矢谷博文, 滝川正春

The Journal of Biochemistry 130 ( 1 ) 79 - 87 2001.7

Language：English Publisher：(公社)日本生化学会

多機能成長因子CTGF/Hcs24の軟骨における遺伝子発現制御機構を明らかにするために,軟骨細胞様HCS-2/8細胞を用い分子生物学的検討を行った.HCS-2/8細胞はCTGF-Hcs24を高発現しており,これはプロモーターのTGF-β応答領域を介した,TGF-βによる転写活性の上昇が関与していることが示された.しかし一方で,別の転写因子の関与も同時に推察された

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In vitro及びin vivoにおける肥大軟骨細胞由来の成長因子CTGF/Hcs24の関節軟骨細胞に対する作用

西田崇, 中西徹, 久保田聡, 吉道玄, 近藤誠二, 滝川正春

日本骨代謝学会雑誌 19 ( 2 ) 30 - 30 2001.7

CTGF/Hcs24軟骨強制発現トランスジェニックマウスの解析

縄稚久美子, 中西徹, 吉道玄, 中田英二, 服部高子, 小守壽文, 滝川正春

日本骨代謝学会雑誌 19 ( 2 ) 30 - 30 2001.7

Type II alveolar epithelial cells and interstitial fibroblasts express connective tissue growth factor in IPF

LH Pan, K Yamauchi, M Uzuki, T Nakanishi, M Takigawa, H Inoue, T Sawai

EUROPEAN RESPIRATORY JOURNAL 17 ( 6 ) 1220 - 1227 2001.6

Language：English Publishing type：Research paper (scientific journal) Publisher：EUROPEAN RESPIRATORY SOC JOURNALS LTD

Connective tissue growth factor (CTGF) is a growth and chemotactic factor for fibroblasts encoded by an immediate early gene that is transcriptionally activated by transforming growth factor-beta, Previous studies have shown that both CTGF messenger ribonuclear acid (mRNA) and protein are expressed in renal fibrosis and bleomycin-induced pulmonary fibrosis in mice. The aim of the present study,ras to investigate the localization of CTGF protein and its mRNA expression in the fibrotic lung tissue of patients with idiopathic pulmonary fibrosis (IPF),
Using human fibrotic lung tissue obtained from eight autopsy cases and four biopsy cases with IPF, immunohistochemical staining, ib situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) were performed.
The cellular immunoreactivity for CTGF was markedly increased in the lung tissue of patients with IPF, compared to normal lungs. The immunolocalization of CTGF was confined predominantly to proliferating type II alveolar epithelial cells and activated fibroblasts, In the normal lung, type II alveolar epithelial cells stained for CTGF were sparsely distributed. CTGF mRNA was localized in proliferating type II alveolar epithelial cells and activated fibroblasts in the interstitium of fibrotic lung tissues. RT-PCR analysis showed that CTGF mRNA was expressed at a higher level in fibrotic lungs than in normal lungs.
In both an autocrine and a paracrine manner, type II alveolar epithelial cells and activated fibroblasts may play a critical role in pulmonary fibrosis by producing connective tissue growth factor which modulates fibroblast proliferation and extracellular matrix production.

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Characterization of binding properties of urinary trypsin inhibitor to cell-associated binding sites on human chondrosarcoma cell line HCS-2/8

Y Hirashima, H Kobayashi, M Suzuki, Y Tanaka, N Kanayama, M Fujie, T Nishida, M Takigawa, T Terao

JOURNAL OF BIOLOGICAL CHEMISTRY 276 ( 17 ) 13650 - 13656 2001.4

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

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness, It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP(40)), which:is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP(40)). Here we characterize binding properties of UTI-BPs UTI complexes in the cells, In vitro ligand blot, cell binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP(40) and UTP-BP(45) bind (125)I-UTL A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP(40) Additional experiments, using various reagents to block binding of (125)I-UTI and NG-UTI to the UTI-BP(40) and UTI-BP(45) confirm that the chondroitin sulfate Side chain of UTI is required for its binding to UTI-BP(45) analysis of binding of (125)I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP(40) (which can bind NG-UTI), and the high affinity sites are the UTI-BP(45), In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.

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Overexpression of connective tissue growth factor hypertrophic chondrocyte-specific gene product 24 decreases bone density in adult mice and induces dwarfism

T Nakanishi, T Yamaai, M Asano, K Nawachi, M Suzuki, T Sugimoto, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 281 ( 3 ) 678 - 681 2001.3

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

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for fibroblasts, chondrocytes, and vascular endothelial cells. In the present study, we established transgenic (Tg) mice that overproduce CTGF/Hcs24 under the control of mouse type XI collagen promoter. Tg mice could develop and their embryonic and neonatal growth occurred normally. But they showed dwarfism within a few months of birth. X-ray analysis revealed that their bone density was decreased compared with normal mice. The femurs in the hindlimbs in particular showed an apparent low density. These results indicated that overexpression of CTGF/Hcs24 affects certain steps of endochondral ossification. In addition, the testes were much smaller than normal and fertility was affected in Tg mice, indicating that CTGF/Hcs24 may also regulate the embryonic development of the testis. (C) 2001 Academic Press.

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