Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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Differential regulation of biglycan and decorin synthesis by connective tissue growth factor in cultured vascular endothelial cells

T Kaji, C Yamamoto, M Oh-I, T Nishida, M Takigawa

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 322 ( 1 ) 22 - 28 2004.9

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

It is possible that connective tissue growth factor (CTGF) serves as either an independent regulator or a downstream effector of transforming growth factor-beta (TGF-beta) on the proteoglycan synthesis in vascular endothelial cells. Since TGF-beta regulates endothelial proteoglycan synthesis in a cell density-dependent manner, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [S-35]sulfate or S-35-labeled amino acids in the presence of CTGF, and the labeled proteoglycans were characterized by biochemical techniques. The results indicate that CTGF suppresses the synthesis of biglycan but newly induced that of decorin in the cells when the cell density is low; in addition, no change was observed in the hydrodynamic size and the glycosaminoglycan chain length of these two small chondroitin/dermatan sulfate proteoglycans. The regulation of endothelial proteoglycan synthesis by CTGF is completely different from that by TGF-beta, suggesting that CTGF is not a downstream effector of TGF-beta but an independent regulator in vascular endothelial cells with respect to the proteoglycan synthesis. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.07.078

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低酸素におけるCTGF mRNAの安定化機構核内タンパク質結合を介した3'-非翻訳領域(UTR)の関与

近藤誠二, 久保田聡, 森谷徳文, 滝川正春

日本癌学会総会記事 63回 400 - 400 2004.9

Regeneration of defects in articular cartilage in rat knee joints by CCN2 (connective tissue growth factor) Reviewed

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

JOURNAL OF BONE AND MINERAL RESEARCH 19 ( 8 ) 1308 - 1319 2004.8

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

CTGF/CCN2, a hypertrophic chondrocyte-specific gene product, possessed the ability to repair damaged articular cartilage in two animal models, which were experimental osteoarthritis and full-thickness defects of articular cartilage. These findings suggest that CTGF/CCN2 may be useful in regeneration of articular cartilage.
Introduction: Connective tissue growth factor (CTGF)/CCN2 is a unique growth factor that stimulates the proliferation and differentiation, but not hypertrophy, of articular chondrocytes in vitro. The objective of this study was to investigate the therapeutic use of CTGF/CCN2.
Materials and Methods: The effects of recombinant CTGF/CCN2 (rCTGF/CCN2) on repair of damaged cartilage were evaluated by using both the monoiodoacetic acid (MIA)-induced experimental rat osteoarthritis (OA) model and full-thickness defects of rat articular cartilage in vivo.
Results: In the MIA-induced OA model, quantitative real-time RT-PCR assays showed a significant increase in the level of CTGF/CCN2 mRNA, and immunohistochemical analysis and in situ hybridization revealed that the clustered chondrocytes, in which Clustering indicates an attempt to repair the damaged cartilage, produced CTGF/CCN2. Therefore, CTGF/CCN2 was suspected to play critical roles in cartilage repair. In fact, a single injection of rCTGF/CCN2 incorporated in gelatin hydrogel (rCTGF/CCN2-hydrogel) into the joint cavity of MIA-induced OA model rats repaired their articular cartilage to the extent that it became histologically similar to normal articular cartilage. Next, to examine the effect of rCTGF/CCN2 on the repair of articular cartilage, we created defects (2 mm in diameter) on the surface of articular cartilage in situ and implanted rCTGF/CCN2-hydrogel or PBS-hydrogel therein with collagen sponge. In the group implanted with rCTGF/CCN2-hydrogel collagen, new cartilage filled the defect 4 weeks postoperatively. In contrast, only soft tissue repair occurred when the PBS-hydrogel collagen was implanted. Consistent with these in vivo effects, rCTGF/CCN2 enhanced type II collagen and aggrecan mRNA expression in mouse bone marrow-derived stromal cells and induced chondrogenesis in vitro.
Conclusion: These findings suggest the utility of CTGF/CCN2 in the regeneration of articular cartilage.

DOI： 10.1359/JBMR.040322

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二次骨化中心形成過程における結合組織成長因子CTGF/CCN2の発現血管新生因子としての関与

岡森彦, 久保田聡, 江口傑徳, 河田かずみ, 黒田知沙, 皆木省吾, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 22回 199 - 199 2004.8

ニワトリ軟骨細胞の分化過程におけるCCN2/CTGF遺伝子の転写後発現調節機構の解析

椋代義樹, 久保田聡, 江口傑徳, 近藤誠二, 中尾匡志, 滝川正春

日本骨代謝学会学術集会プログラム抄録集 22回 159 - 159 2004.8

Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) during distraction osteogenesis

H Kadota, T Nakanishi, K Asaumi, T Yamaai, E Nakata, S Mitani, K Aoki, A Aiga, H Inoue, M Takigawa

JOURNAL OF BONE AND MINERAL METABOLISM 22 ( 4 ) 293 - 302 2004.7

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

To investigate the localization and expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24/CCN family member 2 (CTGF/Hcs24/CCN2) during distraction osteogenesis in the rat femur, we studied a total of 54 male rats (11 weeks old). We performed osteotomy in the midshaft of the right femur. After 7 days (lag phase), distraction was started, at the rate of 0.25 mm/12 h for 21 days (distraction phase) by using a small external fixator, and this was followed by a 7-day consolidation phase. Localization and expression of CTGF/Hcs24 during distraction osteogenesis in the femur were examined by immunostaining, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). Immunostaining showed the localization of CTGF/Hcs24 in various cells located in the bone-forming area around the osteotomy site. During the distraction phase, in situ hybridization showed that CTGF/Hcs24 mRNA was expressed not only in hypertrophic chondrocytes and osteoblasts but also in fibroblast-like cells and mesenchymal cells at sites of end-ochondral ossification, and not only in osteoblasts but also in pre-osteoblasts and fibroblast-like cells at sites of intramembranous ossification. RT-PCR showed higher level expression of CTGF/Hcs24 mRNA in the distracted group than in the nondistracted group. These results revealed an elevated pattern of CTGF/Hcs24 mRNA expression during distraction osteogenesis, and suggest that CTGF/Hcs24 may play some roles in the endochondral and intramembranous ossification processes that occur during distraction osteogenesis.

DOI： 10.1007/s00774-004-0486-2

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[Cartilage and mechanical stress from the point of the view of development, growth, pathology and therapeutic aspects].

Takuo Fujisawa, Masaharu Takigawa, Takuo Kuboki

Clinical calcium 14 ( 7 ) 29 - 35 2004.7

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

Articular cartilage is always subjected to various types, magnitudes and cycles of mechanical stress. While it has been recognized that these stresses regulate the chondrocyte growth and differentiation, the mechanism is still unclear. Here, we summarize the effect of mechanical stress on cartilage metabolism from the point of view of development, growth, pathology and therapeutic aspect.

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Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis

H Yokoi, M Mukoyama, T Nagae, K Mori, T Suganami, K Sawai, T Yoshioka, M Koshikawa, T Nishida, M Takigawa, A Sugawara, K Nakao

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 15 ( 6 ) 1430 - 1440 2004.6

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

Connective tissue growth factor (CTGF/CCN2) is one of the candidate factors mediating fibrogenic activity of TGF-beta. It was shown previously that the blockade of CTGF by antisense oligonucleotide (ODN) inhibits TGF-beta-induced production of fibronectin and type I collagen in cultured renal fibroblasts. The in vivo contribution of CTGF in renal interstitial fibrosis, however, remains to be clarified. With the use of a hydrodynamics-based gene transfer technique, the effects of CTGF antisense ODN are investigated in rat kidneys with unilateral ureteral obstruction (UUO). FITC-labeled ODN injection via the renal vein showed that the ODN was specifically introduced into the interstitium. At day 7 after UUO, the gene expression of CTGF, fibronectin, fibronectin ED-A, and alphal(l) collagen in untreated or control ODN-treated obstructed kidneys was prominently upregulated. CTGF antisense ODN treatment, by contrast, markedly attenuated the induction of CTGF, fibronectin, fibronectin ED-A, and alphal(l) collagen genes, whereas TGF-beta gene upregulation was not affected. The antisense treatment also reduced interstitial deposition of CTGF, fibronectin ED-A, and type I collagen and the interstitial fibrotic areas. The number of myofibroblasts determined by the expression of alpha-smooth muscle actin was significantly decreased as well. Proliferation of tubular and interstitial cells was not altered with the treatment. These findings indicate that CTGF expression in the interstitium plays a crucial role in the progression of interstitial fibrosis but not in the proliferation of tubular and interstitial cells during UUO. CTGF may become a potential therapeutic target against tubulointerstitial fibrosis.

DOI： 10.1097/01.ASN.0000130565.69170.85

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Gene expression profile of human chondrocyte HCS-2/8 cell line by EST sequencing analysis

YK Jung, JH Jeong, HM Ryoo, HN Kim, YJ Kim, EK Park, HJ Si, SY Kim, M Takigawa, BH Lee, RW Park, IS Kim, JY Choi

GENE 330 85 - 92 2004.4

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

Large-scale single-pass sequencing of randomly selected cDNA clones from cell type specific libraries has proven to be a powerful approach for the discovery of novel gene functions, identification of novel gene family members, and definition of gene expression profiles. HCS-2/8 chondrocyte has been used as a cell culture model to study chondrocyte differentiation. Here we performed 3350 single-pass sequencing reactions obtained from the 5' ends of cDNAs from HCS-2/8 cells. To define the expression profiles of HCS-2/ 8 chondrocytes, we analyzed the identity of these representative cDNA sequences using database searches (BLAST). The sequences represent 1927 unique genes with known function (i.e., unigene clusters), 38 transcripts that are similar to genes with known function, 739 expressed genes with unknown function (i.e., expressed sequence tags), and 18 cDNAs which have not previously been sequenced. Interestingly, many transcripts,were expressed from chromosome 12 compared with total genes, while the fewer numbers of cDNAs were derived front genes on chromosomes 14, IS and Y. The chondrocytic phenotype of HCS-2/8 cells is reflected by abundant expression of,genes related to cell structure and motility and the 20 most frequently expressed unigenes reflect a chondrocyte-related gene expression signature. Thus. our data establish a representative set of more than 2000 genes expressed in a chondrocytic cell line. This finding provides a framework for understanding cell growth and differentiation of chondrocytes and their metabolic function in the formation and remodeling of cartilage. C (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2004.01.007

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Module-specific antibodies against human connective tissue growth factor: Utility for structural and functional analysis of the factor as related to chondrocytes

M Minato, S Kubota, H Kawaki, T Nishida, A Miyauchi, H Hanagata, T Nakanishi, T Takano-Yamamoto, M Takigawa

JOURNAL OF BIOCHEMISTRY 135 ( 3 ) 347 - 354 2004.3

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

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1. antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.

DOI： 10.1093/jb/mvh042

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Connective tissue growth factor(CTGF)の軟骨細胞特異的な転写調節機構の探索

江口傑徳, 久保田聡, 椋代義樹, 森谷徳文, 中尾匡志, 滝川正春

生化学 76 ( 3 ) 303 - 303 2004.3

軟骨とメカニカルストレス－発生、成長、病態、治療などとの観点から－。 Invited

藤澤拓生, 窪木拓男, 滝川正春

Clinical Calcium 14, 1049-1055 ( 7 ) 1049 - 1055 2004

Language：Japanese Publisher：(株)医薬ジャーナル社

関節はさまざまな種類,大きさ,頻度のメカニカルストレスにさらされている.メカニカルストレスは軟骨細胞に対してアナボリックにもカタボリックにも作用し,軟骨細胞の増殖,分化を調節していることが明らかになっているが,その詳細は不明である.そこで,軟骨の発生,成長,病態,治療などとの観点からメカニカルストレスの作用について考察し述べた

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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.

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

J. Cell. Physiol. 202, 191 - 204, 2004

CCNファミリー． Invited

滝川正春

Molecular Medicine 41, 756-758 ( 6 ) 756 - 758 2004

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

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

J. Am. Soc. Nephrol. 16(1), 133 - 143, 2004

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

Mukudai, Y, Kubota, S, Takanori, E, Kondo, S, Nakao, K, Takigawa, M

J. Biol. Chem. 280, 3166 - 3177, 2004

Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression

T Fukunaga, T Yamashiro, S Oya, N Takeshita, M Takigawa, T Takano-Yamamoto

BONE 33 ( 6 ) 911 - 918 2003.12

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

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2003.07.010

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Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction

M Kanyama, T Kuboki, K Akiyama, K Nawachi, FM Miyauchi, H Yatani, S Kubota, T Nakanishi, M Takigawa

ARCHIVES OF ORAL BIOLOGY 48 ( 10 ) 723 - 730 2003.10

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

Objective: To understand bone regeneration process after tooth extraction could be a clue to develop a new strategy for alveolar bone reconstruction. Recently, accumulated evidences support that connective tissue growth factor (CTGF) is implicated in tissue repair of many tissues. In this study, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets. Design: Five weeks old wild type mate rats (weighing 120 g) were used for this experiment. Expression of CTGF was determined by immunohistochemistry and in situ hybridization in the rat upper molar tooth extraction sockets at 2, 4, 7, 10 and 14 days after tooth extraction. Results: CTGF was expressed strongly in the endothelial. cells migrating into the granulation tissue at the bottom of the sockets during 4 days after tooth extraction. During the reparative process, no apparent chondrocyte-like cell appeared in the sockets, while osteoblast-like cells proliferated in the sockets with low CTGF expression at 7, 10, 14 days after extraction. As expected, no staining was observed with the preimmune rabbit IgG and CTGF sense probe. CTGF may play an important rote in angiogenesis and granulation tissue formation specifically at early heating stage after tooth extraction to initiate alveolar bone repair. Conclusion: CTGF was expressed at early heating stage of the rat tooth extraction wound. (C) 2003 Elsevier Ltd. All. rights reserved.

DOI： 10.1016/S0003-9969(03)00153-5

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Novel enzyme-linked immunosorbent assay systems for the quantitative analysis of connective tissue growth factor (CTGF/Hcs24/CCN2): Detection of HTLV-I tax-induced CTGF from a human carcinoma cell line

H Kawaki, S Kubota, M Minato, NH Moritani, T Hattori, H Hanagata, M Kubota, A Miyauchi, T Nakanishi, M Takigawa

DNA AND CELL BIOLOGY 22 ( 10 ) 641 - 648 2003.10

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

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24/CCN2) is known as a multifunctional growth factor. It stimulates proliferation, migration, and extracellular matrix production of mesenchymal cells, and is highly expressed in hypertrophic chondrocytes. In this study, we constructed useful ELISA systems for the analysis of CTGF and its modular fragments. For this objective we prepared four different antihuman CTGF monoclonal antibodies. One, specific for the VWC module, was utilized as the detecting antibody, and the other three, recognizing CT, IGFBP, and VWC modules, respectively, were employed as capture antibodies. Then we established three novel quantitative analysis systems for CTGF. The first system recognizing CT and VWC modules was useful to measure full-length CTGF with improved sensitivity. Utilizing this system, we found significant enhancement of CTGF production from a human carcinoma cell line transduced by HTLV-I tax gene, where the finding indicates the possible involvement of Tax in carcinogenesis. The second system, seeing IGFBP and VWC modules, could quantify not only CTGF, but also may be useful to analyze processed N-terminal fragments. The third system, utilizing capture and detection antibodies against the VWC module, was able to quantify the VWC module only, while it did not recognize full-length CTGF. Since CTGF is actually processed into subfragments, and functional assignment of each module is of interest, these systems are expected to contribute to the progress of CTGF investigations.

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Transcriptional induction of connective tissue growth factor/hypertrophic chondrocyte-specific 24 gene by dexamethasone in human chondrocytic cells Reviewed

S Kubota, NH Moritani, H Kawaki, H Mimura, M Minato, M Takigawa

BONE 33 ( 4 ) 694 - 702 2003.10

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

Connective tissue growth factor (CTGF/Hcs24) is a critical growth factor for chondrocytic growth and differentiation. In this report, we describe for the first time glucocorticoid-mediated induction of the CTGF/Hcs24 gene in a chondrocytic cell line, HCS-2/8. Steady-state mRNA levels of CTGF/Hcs24 were remarkably increased after treatment with 50 nM dexamethasone, as confirmed by Northern blotting and quantitative real-time polymerase chain reaction (PCR) analysis. Corresponding to the increase in mRNA, production of CTGF/Hcs24 protein was remarkably enhanced, following a time course of up to 6 h. The observed increase in mRNA can be ascribed to transcriptional enhancement, since the stability of CTGF/Hcs24 mRNA was not affected by the same concentration of dexamethasone, which was indicated by the results of an mRNA degradation assay. However, unexpectedly, the prototypic ctgf/hcs24 promoter was not responsible for the dexamethasone stimulation, suggesting the glucocorticoid receptor binding site(s) to be elsewhere in the CTGF/Hcs24 gene. Enhancement of the prototypic promoter activity by dexamethasone was observed in murine fibroblastic cells, demonstrating the complexity of the regulatory mechanism of ctgf/hcs24 gene expression. Of importance, dexamethasone at the same concentration significantly stimulated proteoglycan synthesis in HCS-2/8 cells up to the same levels as exogenously added CTGF/Hcs24. These findings represent a novel effect of glucocorticoid on the production of CTGF/Hcs24 by chondrocytic cells, and indicate that CTGF/Hcs24 may mediate the stimulative effect of dexamethasone on chondrocytic phenotypes. Also, our results shed light on the complex mechanism of CTGF/Hcs24 induction by glucocorticoids. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S8756-3282(03)00227-8

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Papers - TAKIGAWA Masaharu