Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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アデノウイルスベクター法を用いた顎関節への遺伝子導入の試み

園山亘, 中西徹, 窪木拓男, 完山学, 山下敦, 滝川正春

岡山歯学会雑誌 18 ( 1 ) 283 - 283 1999.6

Detection of specific antibodies against human cultured chondrosarcoma (HCS-2/8) and osteosarcoma (Saos-2) cells in the serum of patients with osteoarthritis of the temporomandibular joint

T Kuboki, T Hattori, T Mizushima, M Kanyama, T Fujisawa, A Yamashita, M Takigawa

ARCHIVES OF ORAL BIOLOGY 44 ( 5 ) 403 - 414 1999.5

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

To find specific humoral antibodies in sera from patients with temporomandibular joint (TMJ) osteoarthritis (OA), an immortal human chondrocyte (HCS-2/8) and osteoblast (Saos-2) cell line derived from a chondrosarcoma and an osteosarcoma, respectively, were used as source proteins of human antigens. Patients with chronically painful TMJ OA (n = 18) but no other joints symptoms were selected from a consecutive series of patients with temperomandibular disorders and sex-matched asymptomatic controls (n = 8) were also recruited. Cellular proteins of the HCS-2/8 and Saos-2 cells were subjected to Western blotting with the OA and control sera as probes. Band-recognition frequency and the peak optical density of the band were compared between groups by chi(2) and t-tests. OA sera recognized various bands for the chondrocytes, and one of these (47-kDa) was specific for the OA sera. In two OA patients whose treatment outcome was less favorable, the,reactivity against the 47-kDa protein was relatively high. In addition, the OA sera clearly cross-reacted with recombinant HSP47. Based on these findings, an autoimmune reaction against chondrocytes could be one of the exaggerating and/or perpetuating mechanisms in the pathophysiology of osteoarthritic TMJs, and the humoral antibody titre against the HSP47-like protein derived from the chondrocytes could be one of the possible markers for the prognosis of the joint pathology. (C) 1999 Elsevier Science Ltd. All rights reserved.

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Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1

T Fujisawa, T Hattori, K Takahashi, T Kuboki, A Yamashita, M Takigawa

JOURNAL OF BIOCHEMISTRY 125 ( 5 ) 966 - 975 1999.5

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

To clarify the mechanism of cartilage degradation induced by mechanical stress, we investigated the influence of cyclic tension force (CTF) on the metabolism of cultured chondrocytes. The chondrocytes were exposed to CTF using a Flexercell strain unit. Five or 15 kPa of high frequency CTF significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein, Fifteen kPa of high frequency CTF induced the expression of interleukin-1 (IL-1), matrix metalloproteinase (MMP)-2 and -9 mRNA, and increased the production of pro- and active-MMP-9. The degradation of proteoglycan was inhibited by and MMP inhibitor, indicating that MMPs are involved in the degradation of proteoglycans induced by high frequency CTF, Moreover, reducing the frequency of CTF from high to low decreased the inhibition of proteoglycan synthesis, These findings suggest that the CTF frequency is one of the key determinants of chondrocyte metabolism. Low magnitude CTF, whether high or low frequency, did not cause the gene expression of cartilage degradation factors, suggesting that this CTF magnitude causes only minor changes in the cartilage matrix, High magnitude and frequency CTF caused the gene expression of IL-1 and MMP-9, followed by increases in the production of MMP-2 and -9 proteins, suggesting that excessive and continuous cyclic mechanical stress induces the production of IL-1 and MMP-9, resulting in cartilage degradation.

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周期的な機械的ストレスを培養軟骨細胞に加え続けると,MMP及びIL-1の発現が誘導され,細胞外マトリックスの分解が引き起こされる

Fujisawa Takuo, Hattori Takako, Takahashi Kojiro, Kuboki Takuo, Yamashita Atsushi, Takigawa Masaharu

The Journal of Biochemistry 125 ( 5 ) 966 - 975 1999.5

Language：English Publisher：(公社)日本生化学会

機械的刺激による軟骨の分解について調べた.Flexercell strain unitを用いて周期的張力(CTF)を培養軟骨細胞に加えたところ,5kPa及び15kPaの高頻度CTFにより,DNA合成,プロテオグリカン合成,コラーゲン合成の低下が見られた.15kPaの高頻度CTFではIL-1,matrix metalloproteinase-2(MMP-2),MMP-9のmRNAの発現と活性型MMP-9の産生が増加した.この時,プロテオグリカンの分解が促進したが,MMPの阻害剤の存在下では分解の促進が抑えられた.張力を小さくすると,軟骨の分解に関わる遺伝子の発現誘導は見られなかった.強度の高頻度CTFはIL-1,MMP-2,MMP-9のmRNAの発現,及び活性型MMP-9の産生を増大させることから,周期的な機械的ストレスが軟骨の分解を引き起こすと考えられる

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=1999&ichushi_jid=J04549&link_issn=&doc_id=19990603080012&doc_link_id=10005463964&url=https%3A%2F%2Fci.nii.ac.jp%2Fnaid%2F10005463964&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_1.gif

Effect of pressure loading on interleukin-8 production in chondrocytes

Toshikazu Kubo, Yuji Arai, Kenji Takahashi, Toshihiro Ishida, Takuo Fujisawa, Masaharu Takigawa, Jiro Imanishi, Yasusuke Hirasawa

Pathophysiology 6 ( 1 ) 35 - 39 1999.4

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

The present study investigated the influence of hydrostatic pressure on the expression of interleukin-8 (IL-8) in a human chondrocyte-like cell line (HCS-2/8). The cells were exposed to hydrostatic pressure (1, 5, 10, or 50 MPa) by a special apparatus. Total RNA was extracted, and was analyzed by a polymerase chain reaction method for IL-8 mRNA. IL-8 concentrations in a culture medium were assessed by ELISA. The expression of IL-8 mRNA was enhanced after exposure to 50 MPa of hydrostatic pressure. IL-8 level in a culture supernatant increased following 50 MPa of pressure. Increased IL-8 production in the present study is an important point when we consider the pathology of inflammatory joint diseases, and this point also shows that treatment which reduces mechanical loading could improve, or slow the progression of, joint diseases.

DOI： 10.1016/S0928-4680(98)00032-7

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Involvement of cis-acting repressive element(s) in the 3 '-untranslated region of human connective tissue growth factor gene

S Kubota, T Hattori, T Nakanishi, M Takigawa

FEBS LETTERS 450 ( 1-2 ) 84 - 88 1999.4

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

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it,vas fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR, (C) 1999 Federation of European Biochemical Societies.

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アデノウイルスベクターを用いた顎関節への遺伝子導入の試み

窪木拓男, 中西徹, 完山学, 園山亘, 水島恒尚, 小島俊司, 山下敦, 滝川正春

日本顎関節学会雑誌 11 ( 1 ) 89 - 89 1999.4

Gene delivery to chondrocytes using adenovirus vector Reviewed

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

ADVANCES IN OSTEOARTHRITIS 107 - 118 1999

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

The objective of this study was to investigate the effects of adenovirus vector (Ax-)mediated gene transduction of E. coli beta-galactosidase (LacZ) and transforming growth factor-beta 1 (TGF-beta 1) into a human chondrocyte-like cell line (HCS-2/8). The expression of transduced genes and their expression periods were examined by 5-bromo-4-chloroindolyl-beta-D-galactoside (X-gal) staining, Northern blotting, ELISA, and Western blotting. To assess the influence of TGF-beta 1 gene transduction, the expression of mRNAs of type II collagen, proteoglycan core protein, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were examined by Northern blotting. Staining with X-gal indicated that the genes were transduced into 99% of the cells. Expression of the transduced genes in the cells was continued for at least 21 days. Transduction of the TGF-beta 1 gene enhanced mRNA expressions of type II collagen and proteoglycan core protein, but suppressed MMP-3 mRNA expression in the cells. These results indicate Ax is useful in chondrocyte gene therapy, and it could be an efficient mediator of TGF-beta 1 gene transduction.

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結合組織成長因子CTGF/Hcs24の生理機能 Invited

中西徹, 滝川正春

生化学 71、429-432、 ( 6 ) 429 - 432 1999

骨形成過程における神経栄養因子およびその受容体の発現 Invited

浅海浩二, 中西徹, 浅原弘嗣, 井上一, 滝川正春

骨・関節・靭帯 12、295-297、 ( 3 ) 295 - 297 1999

Adenovirus vector-mediated gene transduction to chondrocytes : In vitro evaluation of therapeutic efficacy of transforming growth factor-β1 and heat shock protein 70 gene transduction. Reviewed

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

J. Rheumatol. 24 1787 - 1795 1999

Cartilaginous differentiation in the joint capsule

Y Yutani, Y Yano, H Ohashi, M Takigawa, Y Yamano

JOURNAL OF BONE AND MINERAL METABOLISM 17 ( 1 ) 7 - 10 1999

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

The proliferation and differentiation of cells are greatly influenced by their environment. Many growth factors and cytokines are reported to be environmental factors that affect the proliferation and differentiation of cells. Mechanical stress is also considered to influence these physiological reactions. The joint capsule, which is a part of the joint tissue, plays a very important role in the stability of the joint and in maintaining the intracapsular phenomenon. In patients with dislocated hip arthropathy, this capsule is involved in the weightbearing function by forming a sliding surface between the capsule and the femoral head articular cartilage. The surface of the tissue macroscopically shows cartilaginous change, which indicates cartilaginous differentiation caused by mechanical stress. We examined the cartilage-specific proteoglycan component, which is composed of cartilaginou matrix at the differentiation site. We investigated proteoglycan production, molecular size, and the gene expression of cartilaginous substrate. At the inner layer of the weightbearing area of the joint capsule, proteoglycan production was significantly higher than that of other noncartilaginous tissue. We also identified the gene expression of cartilaginous proteoglycan using the reverse transcription polymerase chain reaction (RT-PCR) method.

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Expression of osteopontin in Meckel's cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis Reviewed

K Ishizeki, S Nomura, M Takigawa, H Shioji, T Nawa

HISTOCHEMISTRY AND CELL BIOLOGY 110 ( 5 ) 457 - 466 1998.11

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

The localization of osteopontin (OP) was examined in Meckel's cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OF-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OF. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix.

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Increased expression of connective tissue growth factor in the infarct zone of experimentally induced myocardial infarction in rats

H Ohnishi, T Oka, S Kusachi, T Nakanishi, K Takeda, M Nakahama, M Doi, T Murakami, Y Ninomiya, M Takigawa, T Tsuji

JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 30 ( 11 ) 2411 - 2422 1998.11

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

Connective tissue growth factor (CTGF), a 36- to 38-kDa peptide, is selectively induced by transforming growth factor-beta and has been suggested to contribute to tissue repair. To test the hypothesis that CTGF is expressed in myocardial infarct tissue following acute myocardial infarction (AMI), we examined CTGF expression after AMI was experimentally induced in rats. Myocardial infarction was induced by left coronary artery ligation in male Sprague-Dawley rats. Northern blotting demonstrated that the CTGF mRNA expression on days 2, 7 and 14 was increased by 6-, 23- and 8-fold, respectively, compared to that in the pre-ligation hearts. In situ hybridization revealed CTGF mRNA signals on day 2 in myocytes in the infarct marginal zone and spindle-shaped mesenchymal cells (presumably myofibroblasts and fibroblasts) located between surviving myocytes in the infarct peripheral zone. On day 7, the signals were observed in the inner lesion of the infarct around infarct granulation tissue. Western blotting demonstrated that the CTGF protein expression on days 2, 7 and 14 was increased compared to the pre-ligation hearts. Immunopositive staining for CTGF was observed in the inner lesion of the infarct tissue on day 7. In conclusion, the findings demonstrated the increased expression of: CTGF in the infarct tissue. Myocytes in the infarct marginal zone and spindle-shaped mesenchymal cells (presumably myofibroblasts and fibroblasts) were the cells responsible for CTGF production. (C) 1998 Academic Press.

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Establishment of the enzyme-linked immunosorbent assay for connective tissue growth factor (CTGF) and its detection in the sera of biliary atresia Reviewed

T Tamatani, H Kobayashi, K Tezuka, S Sakamoto, K Suzuki, T Nakanishi, M Takigawa, T Miyano

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 251 ( 3 ) 748 - 752 1998.10

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

Connective tissue growth factor (CTGF) is a mitogenic, chemotactic, and cell matrix-inducing factor for fibroblasts. We generated murine monoclonal antibodies against CTGF and established a sandwich enzyme-linked immunosorbent assay (ELISA) for detection of CTGF. By using the ELISA, we confirmed that CTGF was specifically induced in human fibroblasts by TGF-beta but not by PDGF, FGF, IGF-I, or EGF. We also found that the serum levels of CTGF were significantly correlated with the progression of hepatic fibrosis in biliary atresia. These results indicated that CTGF is potentially a useful parameter for monitoring certain types of fibrotic disorders. (C) 1998 Academic Press.

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Adenovirus mediated gene delivery to the joints of guinea pigs Reviewed

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

JOURNAL OF RHEUMATOLOGY 25 ( 9 ) 1666 - 1673 1998.9

Language：English Publishing type：Research paper (scientific journal) Publisher：J RHEUMATOL PUBL CO

Objective. To clarify in vivo applicability of adenovirus mediated gene delivery to examine a gene therapy for human joint diseases.
Methods. We directly injected vectors harbouring beta-galactosidase gene and transforming growth factor (TGF)-beta 1 gene into the joints of Hartley guinea pigs. Expressions of delivered LacZ were examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside staining and reverse transcription-polymerase chain reaction. The levels of TGF-beta 1 that mere delivered to the joint and then transferred to the joint fluid were assessed by ELISA.
Results. LacZ expression was observed in almost all synovial tissue samples and in chondrocytes on the surface of degenerated cartilage. In the other organs, expression of delivered genes was not observed. For 2 weeks following gene delivery TGF-beta 1 levels in joint fluid were significantly higher than the levels in the controls for 2 weeks.
Conclusion. Direct gene delivery into the joint cavity is feasible with the in vivo gene delivery method using adenovirus vector and would be clinically applicable.

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Peripheral type benzodiazepine receptor in T lymphocyte rich preparation Reviewed

S Maeda, T Miyawaki, T Nakanishi, M Takigawa, M Shimada

LIFE SCIENCES 63 ( 16 ) 1423 - 1430 1998.9

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Some types of mood disorders and drugs are suggested to affect peripheral type benzodiazepine receptors (PBR), but their mechanisms are unclear. The isolation of pure lymphocytes is requisite for the investigation of the function of PER on lymphocytes, since platelets and monocytes also have many PER. The objective of this study was to establish a method of binding assay for PER using pure T lymphocytes. Mononuclear cells and T lymphocytes were prepared by using a density gradient material and magnetic beads, respectively. The cells were analyzed using Row cytometry and a counting chamber. Binding studies were performed using T lymphocytes from IO normal volunteers. The T lymphocytes were incubated with [H-3]PK11195, harvested on glass fiber filters, and counted with a plate scintillation counter. The binding data were analyzed by the Scatchard method. With the magnetic bead technique, pure T cells were selected that contained only 1.5% monocytes and platelet/cell ratio of 1.4. The Scatchard plot of the data indicated that only one type of specific binding site was involved in the binding. The dissociation constant (Kd) was 3.8+/-1.3nM (mean+/-SD), and the Bmax was 379+/-124 fmol/10(6) cells (mean+/-SD). The density gradient- magnetic beads technique can be used as an appropriate method of preparation of T cells for PBR binding assay.

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軟骨細胞の細胞外基質の合成と分解に及ぼす周期的メカニカルストレスの影響

藤沢拓生, 服部高子, 窪木拓男, 高橋浩二郎, 山下敦, 滝川正春

歯科基礎医学会雑誌 40 ( 抄録 ) 392 - 392 1998.9

慢性関節リウマチ関連抗原蛋白RA-A47 cDNAの単離と機能解析

服部高子, 藤沢拓生, 中西徹, 窪木拓男, 山下敦, 滝川正春

歯科基礎医学会雑誌 40 ( 抄録 ) 392 - 392 1998.9

アデノウイルスベクター法を用いた顎関節への遺伝子導入の試み

完山学, 中西徹, 窪木拓男, 園山亘, 山下敦, 滝川正春

歯科基礎医学会雑誌 40 ( 抄録 ) 450 - 450 1998.9

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