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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-2/8¹

Department of Biochemistry and Molecular Dentistry (M.T., K.T.) and Biochemical Research Center (C.A.), Okayama University Dental School, Okayama 700, Japan; Department of Orthopaedic Surgery (T.O.), Osaka City University Medical School, Osaka 545, Japan; Departments of Biochemistry,(H.-O.P., F.S.) and Pedodontics (A.K.), Osaka University Faculty of Dentistry, Suita, Osaka 565, Japan; and Department of Medical Oncology (J.Z.), Glasgow University, Glasgow G61 1BD, United Kingdom

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

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. [¹²⁵I]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. [¹²⁵I]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 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 did 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 IGF-I. Even in the presence of anti-IGF-IR antibody, IGF-II and mannose-6-phosphate stimulated proteoglycan synthesis in the cells. [Leu²⁷]IGF-II, an IGF-II analogue with high affinity only for IGF-IIR, strongly stimulated proteoglycan synthesis in HCS-2/8 cells but [Arg⁵⁴, Arg⁵⁵]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.

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