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Effects of CTGF/Hcs24, a Product of a Hypertrophic Chondrocyte-Specific Gene, on the Proliferation and Differentiation of Chondrocytes in Culture¹

Department of Biochemistry and Molecular Dentistry (T.N., T.N., T.S., M.T.) and Biodental Research Center (T.N., T.N., M.T.), Okayama University Dental School, Okayama 700-8525; Departments of Orthopedic Surgery (T.K.) and Microbiology (K.K.), Kyoto Prefectural University of Medicine, Kyoto 602-8566; and Pharmaceutical Frontier Research Laboratories, Japan Tobacco, Inc. (T.T., K.T.), Yokohama 236-0004, Japan

Address all correspondence and requests for reprints to: Masaharu Takigawa, D.D.S., Ph.D., Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, 2–5-1 Shikata-cho, Okayama 700-8525, Japan. E-mail: takigawa{at}dent.okayama-u.ac.jp

Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 function in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs24 effectively promoted the proliferation of HCS-2/8 cells and RGC cells in a dose-dependent manner and also dose dependently increased proteoglycan synthesis in these cells. In addition, these stimulatory effects of CTGF/Hcs24 were neutralized by the addition of anti-CTGF antibodies. Furthermore, the recombinant CTGF/Hcs24 effectively increased alkaline phosphatase activity in RGC cells in culture. Moreover, RT-PCR analysis revealed that the recombinant CTGF/Hcs24 stimulated gene expression of aggrecan and collagen types II and X in RGC cells in culture. These results indicate that CTGF/Hcs24 directly promotes the proliferation and differentiation of chondrocytes.

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