Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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慢性関節リウマチ関連抗原蛋白RA-A47 cDNAの単離と構造解析

服部高子, 油谷安孝, 藤沢拓生, 中西徹, 高橋浩二郎, 滝川正春

生化学 70 ( 8 ) 848 - 848 1998.8

Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells Reviewed

T Shimo, T Nakanishi, Y Kimura, T Nishida, K Ishizeki, T Matsumura, M Takigawa

JOURNAL OF BIOCHEMISTRY 124 ( 1 ) 130 - 140 1998.7

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

Previously, we cloned an mRNA predominantly expressed in hypertrophic chondrocytes by differential display-PCR from a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) that is identical to that of connective tissue growth factor (CTGF), In the present study, we investigated the roles of CTGF in the proliferation and migration of vascular endothelial cells using its antisense oligonucleotide and antisense RNA, because angiogenesis into the hypertrophic zone of cartilage occurs at the final step of endochondral ossification. Immunohistochemical and immunofluorescence techniques revealed that not only hypertrophic chondrocytes but also endothelial cells in the cost-chondral junctions of mouse ribs were stained with an anti-CTGF antibody in vivo. Northern blot analysis revealed that CTGF was strongly expressed in chondrocytic cells as well as bovine aorta endothelial (BAE) cells in culture, but not in other types of cells such as osteoblastic cells. Its expression in BAE cells was greater in the growing phase than in the confluent phase. When one-half of a monolayer of a confluent culture of BAE cells had been peeled off, only the cells proliferating and extending into the vacant area were stained with the anti-CTGF antibody. The addition of an antisense oligonucleotide inhibited the proliferation and extension of the BAE cells into the vacant area. The antisense oligonucleotide also inhibited the proliferation of BAE cells in the rapidly proliferating phase. In a Boyden chamber assay, pretreatment with the antisense oligonucleotide markedly inhibited the migration of BAE cells. Furthermore, the abilities to proliferate and migrate of BAE cells, which were stably transfected with expression vectors that generate the antisense RNA of CTGF cDNA, were markedly lower than those of the control. These findings suggest that endogenous CTGF expression is involved in the proliferation and migration of BAE cells.

Demonstration of receptors specific for connective tissue growth factor on a human chondrocytic cell line (HCS-2/8) Reviewed

T Nishida, T Nakanishi, T Shimo, M Asano, T Hattori, T Tamatani, K Tezuka, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 247 ( 3 ) 905 - 909 1998.6

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

The presence of receptors specific for connective tissue growth factor (CTGF) was demonstrated on a human chondrosarcoma-derived chondrocytic cell Line, HCS-2/8. The binding of I-125-labeIed recombinant CTGF to HCS-2/8 cells was inhibited by unlabeled CTGF but not by PDGF-BB or bFGF. Scatchard analysis revealed the presence of two classes of binding sites with Rd values of 18.6 and 259 nM on cells. A cross-linking study revealed the formation of I-12S-CTGF-receptor complex with an apparent molecular weight of 280 kDa, The I-125-CTGF-receptor complex disappeared almost completely on the addition of unlabeled CTGF but not PDGF-BB or bFGF, In addition, the I-125-CTGF-receptor complex was immunoprecipitated with anti-CTGF antiserum but not with anti-PDGF receptor antiserum. These findings suggest that CTGF directly binds to specific receptor molecules on HCS-2/8 cells. (C) 1998 Academic Press.

Stimulatory effects of 4-methylcatechol, dopamine and levodopa on the expression of metallothionein-III (GTF) mRNA in immortalized mouse brain glial cells (VR-2g) Reviewed

C Aoki, T Nakanishi, N Sogawa, K Ishii, N Ogawa, M Takigawa, H Furuta

BRAIN RESEARCH 792 ( 2 ) 335 - 339 1998.5

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

Metallothionein (MT)-III, originally discovered as a growth inhibitory factor (GIF), is a brain specific isomer of MTs and is markedly reduced in the brain of Alzheimer's disease patients (AD) and in several other neurodegenerative diseases. We analyzed the level and regulation of mRNA expression of MT-III in immortalized fetal mouse brain glial cells (VR-2g) by reverse transcriptase-polymerase chain reaction (RT-PCR). The basal expression level of MT-III mRNA is very low in VR-2g cells. 4-Methylcatechol, dopamine (DA) and levodopa (L-3,4-dihydroxyphenylalanine), which stimulate the synthesis of nerve growth factor (NGF), further increased the expression of MT-III mRNA in VR-2g cells. (C) 1998 Elsevier Science B.V.

Isolation and characterization of a rheumatoid arthritis-specific antigen (RA-A47) from a human chondrocytic cell line (HCS-2/8) Reviewed

T Hattori, T Fujisawa, K Sasaki, Y Yutani, T Nakanishi, K Takahashi, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 245 ( 3 ) 679 - 683 1998.4

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

Two types of 47 kDa antigen specifically recognized by sera from rheumatoid arthritis (RA) patients were isolated from the membrane fraction of a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by a 2-step procedure: preparative SDS-PAGE and reverse-phase HPLC. An N-terminal amino acid sequence in one of the 47 kDa antigens, named RA-A47, had 81% homology to that deduced from the DNA sequence of the colligin gene which is reported as human hsp47 gene, and 100% homology to that deduced from the DNA sequence of colligin-2 gene, a homologue of colligin. The RA-A47 cross-reacted with a monoclonal antibody raised against chick heat shock protein (Hsp) 47 and bound to gelatin. The expression of the ra-a47 gene was enhanced by heat shock treatment and TGF-beta stimulation. These findings suggest that RA-A47 is a Hsp47-like protein, presumably the product of the colligin-2 gene, and that a collagen-specific molecular chaperone(s) such as Hsp47 and/or RA-A47 is involved in cartilage destruction in RA. (C) 1998 Academic Press.

Hydrostatic pressure induces expression of interleukin 6 and tumour necrosis factor alpha mRNAs in a chondrocyte-like cell line Reviewed

K Takahashi, T Kubo, Y Arai, Kitajima, I, M Takigawa, J Imanishi, Y Hirasawa

ANNALS OF THE RHEUMATIC DISEASES 57 ( 4 ) 231 - 236 1998.4

Language：English Publishing type：Research paper (scientific journal) Publisher：BRITISH MED JOURNAL PUBL GROUP

Objective-To clarify the effect of pressure on the expressions of proteoglycan core protein and metabolism related cytokines in a chondrocyte-like cell line, HCS-2/8.
Methods-HCS-2/8 cells were exposed to 1, 5, 10, or 50 MPa of hydrostatic pressure (HP) for two hours, and mRNA expressions of interleukin 6 (IL6) and tumour necrosis factor alpha (TNF alpha) were examined by using reverse transcription-polymerase chain reaction (RT-PCR) method with specific primer sets; and mRNA of proteoglycan core protein, stromelysin, and tissue inhibitor of metalloproteinase 1 (TIMP1) were measured with northern blotting.
Results-HP exposure caused temporal morphological changes of the cells, but did not affect cellular viability. IL6 and TNF alpha mRNA expressions were not observed in the control cells under the atmospheric pressure, whereas in the cells treated with HP, pressure dependent enhancement of IL6 mRNA expression was observed between 30 minutes and four hours after the HP release. TNFa mRNA expression also increased 30 minutes after the exposure to 50 MPa of HP and disappeared four hours later. Proteoglycan core protein mRNA levels increased between 30 minutes and four hours after the exposure to 1 or 5 MPa of HP, whereas the levels decreased after 10 or 50 MPa of HP. Stromelysin and TIMP1 mRNA signals did not respond to HP.
Conclusions-HP at excessively high levels induced IL6 and TNF alpha expression and reduced the expression of proteoglycan core protein, while physiological levels of HP increased the expression of proteoglycan core protein. These findings are important when considering the pathology of osteoarthritis.

Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes Reviewed

K Sasaki, T Hattori, T Fujisawa, K Takahashi, H Inoue, M Takigawa

JOURNAL OF BIOCHEMISTRY 123 ( 3 ) 431 - 439 1998.3

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

We recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1 beta (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes, To clarify the mechanism of the IL-l-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-l-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it, Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3, The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release, An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-l-stimulated bFGF release even if increased production of bFGF is related to the release, IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order, N-G-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.

Shikonin, an ingredient of Lithospermum erythrorhizon, inhibits angiogenesis in vivo and in vitro Reviewed

T Hisa, Y Kimura, K Takada, F Suzuki, M Takigawa

ANTICANCER RESEARCH 18 ( 2A ) 783 - 790 1998.3

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

Angiogenesis Is critical for tumor growth and inflammation. Shiunko is a Chinese hel bal ointment used for the treatment of burns in Japan. Its main ingredient is the root of Lithospermum erythrorhizon, which had been used for treating tumors and inflammation in China since the 5th century. We report here that shikonin, the main chemical ingredient of L. erythrorhizon is a novel inhibitor of angiogenesis. It inhibited tumor necrosis factor-alpha-induced and B16 melanoma-induced angiogenesis in mice and normal developmental angiogenesis in the yolk-sac membranes of chick embryos. Shikonin also inhibited proliferation and migration of endothelial cells in culture and network formation by endothelial cells on Matrigel in vitro. The dose-responsive study suggests that the mechanism of this inhibitory effect on angiogenesis involves the prevention of network formation by endothelial cells via blocking integrin alpha(v) beta(3) expression.

Overexpression of c-erbB-3 in various stages of human squamous cell carcinomas Reviewed

T Funayama, T Nakanishi, K Takahashi, S Taniguchi, M Takigawa, T Matsumura

ONCOLOGY 55 ( 2 ) 161 - 167 1998.3

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

Amplification of the c-erbB-3 gene and the expression of its mRNA and protein in human squamous cell carcinomas (SCC) were analyzed. Although no amplification of the c-erbB-3 gene was observed, overexpression of its mRNA was observed in SCC by RT-PCR. The expression of this gene in SCC was 15-50 times higher than in normal fibroblasts. Overexpression of the secreted type of erbB-3 mRNA was also observed in all SCC. Moreover, overproduction of the c-erbB-3 protein was also detected in SCC by dot blot analysis using anti-c-erbB-3 monoclonal antibodies. In nude mice, the expression of c-erbB-3 mRNA was higher in metastatic tumors compared with primary tumors. These results suggest that both the transmembrane and secreted types of c-erbB-3 play a significant role in the formation and development of SCC.

The inhibition of DNA synthesis by prostaglandin E2 in human gingival fibroblasts is independent of the cyclic AMP-protein kinase A signal transduction pathway. Reviewed International journal

Arai H, Nomura Y, Kinoshita M, Nishimura F, Takigawa M, Takahashi K, Washio N, Takashiba S, Murayama Y

Journal of periodontal research 33 ( 1 ) 33 - 39 1998.1

Language：English Publishing type：Research paper (scientific journal) Publisher：1

In this study we attempted to clarify the mechanism of the inhibitory effects of PGE2 on DNA synthesis in Gin-1 (fibroblasts derived from healthy human gingiva) from the aspect of the cyclic AMP-dependent protein kinase signal transduction pathway. PGE2 upregulated intracellular cyclic AMP accumulation and inhibited DNA synthesis in Gin-1 in a dose-dependent manner. When the PGE2-induced intracellular cyclic AMP accumulation was further enhanced by treatment with the cyclic AMP-phosphodiesterase inhibitor, IBMX, the inhibitory effect of PGE2 on DNA synthesis was also enhanced. Furthermore, when we examined the effects of forskolin, an activator of cyclic AMP production, on intracellular cyclic AMP accumulation and DNA synthesis, similar results were obtained. However, inhibitors of cyclic AMP-dependent protein kinase (protein kinase A) such as HA1004 did not diminish the inhibitory effect of PGE2 on DNA synthesis in Gin-1. These results suggest that in Gin-1, PGE2-induced cyclic AMP accumulation may not lead to the activation of protein kinase A or protein kinase A activity may not relate directly to the growth inhibitory effect of PGE2, and that PGE2 does not inhibit DNA synthesis through the cyclic AMP-protein kinase A signal transduction pathway in Gin-1.

Demonstration of receptors for epidermal growth factor on cultured rabbit chondrocytes and regulation of their expression by various growth and differentiation factors. Reviewed

Nishida,T, Nakanishi,T, Shimo,T, Asano,M, Hattori,T, Tamatani,T, Tezuka,K, Takigawa,M

Biochem.Biophys.Res.Commun. 183 14 - 20 1998

Insulin-like growth factors I and II are autocrine factors in stimulating proteoglycan synthesis, a marker of differentiated chondrocytes, acting through their respective receptors on a clonal human chondrosarcoma-derived chondrocyte cell line, HCS-2/8 Reviewed

M Takigawa, T Okawa, HO Pan, C Aoki, K Takahashi, JD Zue, F Suzuki, A Kinoshita

ENDOCRINOLOGY 138 ( 10 ) 4390 - 4400 1997.10

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

Both insulin-like growth factor (IGF)-I and IGF-II increased the synthesis of cartilage-type, large proteoglycan in a human chondrosarcoma-derived chondrocyte cell line, HCS-2/8. In contrast to the stimulatory effects of IGFs on costal chondrocytes of the young rabbit, the stimulatory effect of IGF-II on proteoglycan synthesis in HCS-2/8 cells was more potent than that of IGF-I. IGF-II, but not IGF-I, increased calcium influx into HCS-2/8 cells, and there was a close relation between the stimulation of proteoglycan synthesis and the calcium influx. [I-125]IGF-I bound to HCS-2/8 cells, and this binding was competitively inhibited by low concentrations of unlabeled IGF-I, higher concentrations of IGF-II, and much higher concentrations of insulin. [I-125]IGF-II also bound to the cells, and its binding was competitively inhibited by IGF-II and slightly inhibited by higher concentrations of IGF-I and much higher concentrations of insulin. When radioligand-receptor complexes were separated by SDS-PAGE and subjected to autoradiography, two major bands at 260 and 130 kDa were observed, which correspond to the IGF type II receptor (IGF-IIR) and the or subunit of the IGF type I receptor (IGF-IR), indicating the presence of both receptors. When confluent cultures of HCS-2/8 cells were maintained in serum-free medium, proteoglycan synthesis not decrease unless the medium was repeatedly replaced. Conditioned medium of HCS-2/8 cells stimulated the HCS-2/8 cells to synthesize proteoglycans. RIA revealed that the cells produced both IGF-II and IGF-I. Transcripts of messenger RNAs of both IGF-I and IGF-II and both IGF-IR and IGF-IIR also were detectable by Northern analysis. Both anti-IGF-IR antibody and anti-IGF-II antibody inhibited proteoglycan synthesis. Mannose-6-phosphate, which is known to bind to IGF-IIR, stimulated proteoglycan synthesis, potentiated IGF-II-stimulated proteoglycan synthesis, and enhanced the binding affinity for IGF-II but not for TGF-I. Even in the presence of anti-IGF-LR antibody, IGF-II and mannose-6-phosphate stimulated proteoglycan synthesis in the cells. [Leu(27)]IGF-II, an IGF-II analogue with high affinity only for IGF-IIR, strongly stimulated proteoglycan synthesis in HCS-2/8 cells but [Arg(54), Arg(55)]IGF-II, which binds to only IGF-IR, also stimulated proteoglycan synthesis in the cells. These findings indicate that IGF-I and IGF-II act as autocrine differentiation factors for this chondrocytic permanent cell line, HCS-2/8, mainly via respective receptors.

Chondrocytes are regulated by cellular adhesion through CD44 and hyaluronic acid pathway Reviewed

O Ishida, Y Tanaka, Morimoto, I, M Takigawa, S Eto

JOURNAL OF BONE AND MINERAL RESEARCH 12 ( 10 ) 1657 - 1663 1997.10

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

The articular cartilage consists of resident chondrocytes embedded within the extracellular matrix which contains several components such as collagen and hyaluronic acids (HA), CD44 is a major cell surface receptor for HA and is homologous to cartilage-link proteins, Although CD44 is present in cartilage, it is not clear if chondrocytes adhere to HA through CD44 or whether such adhesion changes the function of chondrocytes, We studied the molecular mechanisms of CD44-related chondrocyte adhesion to HA and the effects of such adhesion on chondrocyte function, Experiments were performed using the human chondrosarcoma-derived chondrocyte-like cell line HCS-2/8. Our results shoved that (a) HCS-2/8 cells highly expressed CD44; (b) HCS-2/8 cells efficiently adhered to HA without any stimuli; (c) monoclonal antibody (mAb)-blocking studies indicated that adhesion of HCS-2/8 cells to HA was mainly mediated by the CD44/HA pathway; (d) cellular adhesion to HA increased the proliferation of HCS-2/8 cells, independent of transforming growth factor-beta (TGF-beta), but this was inhibited by CD44 mAb; (e) the adhesion of chondrocytes to HA also induced c-myc mRNA expression and this was also inhibited by CD44 mAb; and (f) the adhesion of cells to HA augmented TGF-beta mRNA expression, a process also reduced by CD44 mAb, Thus, HCS-2/8 cells effectively adhered to HA through cell surface CD44, The adhesion was also involved in cellular signaling which induced cellular proliferation and expression of c-myc mRNA as well as TGF-beta mRNA expression within the cells, Our results indicate that CD44 on chondrocytes plays an important role in normal and abnormal functions of cartilage through its adhesion to HA, which induces a variety of stimulatory signals to regulate chondrocyte proliferation as well as matrix synthesis in cartilage microenvironment.

Coordinated change between complement C1s production and chondrocyte differentiation in vitro Reviewed

K Nakagawa, H Sakiyama, T Fukazawa, M Matsumoto, M Takigawa, T Toyoguchi, H Moriya

CELL AND TISSUE RESEARCH 289 ( 2 ) 299 - 305 1997.8

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

In vitro synthesis of the first component of complement Cls was examined by using hamster epiphyseal chondrocytes (HAC) and human chondrosarcoma cell line HCS-2/8. Hamster and human Cls produced by the cells were quantified by immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA), respectively. It was possible to measure active and inactive Cls by sandwich ELISA, when we used anti-human Cls monoclonal antibodies, M241 recognizing only active Cls, and M365 and M81 recognizing both active and inactive Cia. Approximately 40% of Cls secreted from HCS-2/8 was found to be activated in the culture medium, whereas Cls from HAC was not. Cls production increased in accordance with chondrocyte differentiation induced by ascorbic acid. In contrast, transforming growth factor-betal and basic fibroblast growth factor, which inhibited differentiation, suppressed Cls production. These results confirmed our previous observation showing that Cls synthesis increased with differentiation into hypertrophic chondrocytes in vivo.

Molecular characterisation of integrin-procollagen C-propeptide interactions Reviewed

D Davies, DS Tuckwell, DA Calderwood, SA Weston, M Takigawa, MJ Humphries

EUROPEAN JOURNAL OF BIOCHEMISTRY 246 ( 2 ) 274 - 282 1997.6

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

The carboxyl-terminal propeptide of type I procollagen (CPP-I) plays a key role in regulation of collagen fibrillogenesis, and may exert feedback control of collagen biosynthesis. We have previously shown that CPP-I is a ligand for the integrin alpha 2 beta 1 [Weston, S. A., Hulmes, D. J. S., Mould, A. P., Watson, R. B. & Humphries, M. J. (1994) Identification of the integrin alpha 2 beta 1 as a cell surface receptor for the C-propeptide of type I procollagen, J. Biol. Chem. 269, 20982-20986] suggesting that some of the phenotypic effects of C-propeptides may be mediated by adhesion receptors. Here we have extended this work to study the molecular basis of this interaction. We have broadened the ligand range by demonstrating that the C-terminal propeptide of type II procollagen supports alpha 2 beta 1-mediated binding of NHS human fibroblasts in cell attachment assays. Also, we have used function-blacking antibodies in cell attachment and solid-phase binding assays with purified integrin to expand the CPP-I receptor family, showing that integrin alpha 1 beta 1 is also a receptor for CPP-I. Integrin alpha-subunit A-domains are known to be major ligand-binding sites and recombinant alpha 1 and alpha 2 subunit A-domains were able to bind CPP-I. Finally we have shown that peptides corresponding to potential integrin-binding sequences in CPP-I do not mediate integrin--CPP-I adhesion. Taken together, these studies indicate that the interactions between C-propeptides and integrins are more numerous than previously reported, that C-propeptides are a new class of molecule which bind to A-domains, and that the integrin--C-propeptide interaction does not utilise established peptide motifs.

Cloning of a mRNA preferentially expressed in chondrocytes by differential display PCR from a human chondrocytic cell line that is identical with connective tissue growth factor (CTGF) mRNA Reviewed

T Nakanishi, Y Kimura, T Tamura, H Ichikawa, Y Yamaai, T Sugimoto, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234 ( 1 ) 206 - 210 1997.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS

Chondrocyte- or chondrosarcoma cell line (HCS)-specific DNA fragments were obtained using differential display-PCR. Nucleotide sequences of 32 species derived from HCS cells were determined. One of the sequence tags (tag no. 24) corresponded to the nucleotide sequence of connective tissue growth factor (CTGF). Northern blot analysis showed that CTGF was highly expressed in HCS cells and rabbit growth cartilage cells in culture but was not expressed in osteoblastic cells in culture. In situ hybridization revealed that CTGF was expressed only in the hypertrophic chondrocytes of costal cartilage and the vertebral column in embryonic mice. The expression of CTGF in HCS cells was up-regulated by the addition of TGF-beta or BMP-2. These findings suggest that CTGF participates in endochondral ossification. (C) 1997 Academic Press.

Hydrostatic pressure influences mRNA expression of transforming growth factor-beta 1 and heat shock protein 70 in chondrocyte-like cell line Reviewed

K Takahashi, T Kubo, K Kobayashi, J Imanishi, M Takigawa, Y Arai, Y Hirasawa

JOURNAL OF ORTHOPAEDIC RESEARCH 15 ( 1 ) 150 - 158 1997.1

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL BONE JOINT SURGERY INC

The present study investigated the influence of hydrostatic pressure on the expression of cytokines and heat shock protein 70 in a chondrocyte-like cell line. Chondrocyte-like cells (HCS-2/8) were exposed to hydrostatic pressure by a special pressure apparatus. Total RNA for cytokines (interleukin-1 beta, basic fibroblast growth factor, insulin-like growth factor-I, and transforming growth factor-beta 1) and for heat shock protein 70 was extracted and was analyzed by a polymerase chain reaction method and Northern blotting. An assay for incorporation of [S-35]sulfate was performed to assess proteoglycan synthesis. The expression of transforming growth factor-beta 1 mRNA was enhanced after exposure to 5 MPa of hydrostatic pressure and was reduced after 50 MPa, whereas the expression of heat shock protein 70 was enhanced following exposure to 50 MPa of hydrostatic pressure. The incorporation of [S-35]sulfate into the cultured cells increased following exposure to 1-5 MPa of hydrostatic pressure and decreased following 10-50 MPa of pressure. These results suggest that hydrostatic pressure at physiologic levels enhances the expression of transforming growth factor-beta 1 mRNA in addition to increasing proteoglycan synthesis in chondrocytes and that excessively high hydrostatic pressure reduces the expression of transforming growth factor-beta 1 mRNA and increases the expression of heat shock protein 70 mRNA while decreasing proteoglycan synthesis.

The basic effect of IGF on chondrocytes. Reviewed

Takigawa,M, Kimura,Y, Takahashi,K

Clin. Pediatr. Endocrinol. 6(suppl10) 169 - 174 1997

A factor in conditioned medium of rabbit costal chondrocytes inhibits the proliferation of cultured entothelial cells and angio-genesis induced by B16 melanoma : its relation with cartilage-derived anti-tumor factor(CATF). Reviewed

Takigawa, M, Shirai, E, Enomoto, M, Pan, H.-O, Suzuki, F, Shiio, T, Yugari, Y

Biochem.Int. 14 357 - 364 1997

Nitric oxide mediates interleukin-1-induced matrix degradation and basic fibroblast growth factor release in cultured rabbit articular chondrocytes: A possible mechanism of pathological neovascularization in arthritis Reviewed

T Tamura, T Nakanishi, Y Kimura, T Hattori, K Sasaki, H Norimatsu, K Takahashi, M Takigawa

ENDOCRINOLOGY 137 ( 9 ) 3729 - 3737 1996.9

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

Prolonged incubation with interleukin-1 beta (IL-1) induced the release of large amounts of NO and subsequently inhibited DNA synthesis and the biosynthesis and accumulation of proteoglycans in cultured rabbit articular chondrocytes (RAC). IL-1 also inhibited DNA synthesis in bovine aortic endothelial cells (BAE). On the other hand, DNA synthesis in BAE cocultured with RAC was not inhibited by prolonged incubation with IL-1. Moreover, conditioned media from RAC incubated for a long period with IL-1 stimulated DNA synthesis in BAE alone. This growth stimulatory activity was mainly due to the release of basic fibroblast growth factor, a heparin-binding growth factor, into RAC culture. Gelatin zymography of the RAC culture medium revealed that IL-1 increased the production of matrix metalloproteinase-a (MMP-2) and MMP-9. N-G-Monomethyl-L-arginine, an inhibitor of NO synthesis, inhibited all of these actions of IL-1. These results indicate that NO from RAC treated with IL-I stimulates MMPs, which, in turn, degrade the extracellular matrix produced by RAG, resulting in the release of large amounts of basic fibroblast growth factor stored in the matrix, which then stimulates adjacent BAE proliferation. Thus, NO produced from RAC treated with IL-1 may modulate angiogenesis in the synovium of arthritic patients.

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