INSULIN-LIKE GROWTH-FACTOR-I AND GROWTH-FACTOR-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/

Both insulin-like growth factor (IGF)-I and IGF-II increased the synth esis of cartilage-type, large proteoglycan in a human chondrosarcoma-d erived chondrocyte cell line, HCS-2/8. In contrast to the stimulatory effects of IGFs on costal chondrocytes of the young rabbit, the stimul atory 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 calci um influx into HCS-2/8 cells, and there was a close relation between t he stimulation of proteoglycan synthesis and the calcium influx. [I-12 5]IGF-I bound to HCS-2/8 cells, and this binding was competitively inh ibited by low concentrations of unlabeled IGF-I, higher concentrations of IGF-II, and much higher concentrations of insulin. [I-125]IGF-II a lso bound to the cells, and its binding was competitively inhibited by IGF-II and slightly inhibited by higher concentrations of IGF-I and m uch higher concentrations of insulin. When radioligand-receptor comple xes were separated by SDS-PAGE and subjected to autoradiography, two m ajor bands at 260 and 130 kDa were observed, which correspond to the I GF type II receptor (IGF-IIR) and the or subunit of the IGF type I rec eptor (IGF-IR), indicating the presence of both receptors. When conflu ent cultures of HCS-2/8 cells were maintained in serum-free medium, pr oteoglycan synthesis not decrease unless the medium was repeatedly rep laced. Conditioned medium of HCS-2/8 cells stimulated the HCS-2/8 cell s to synthesize proteoglycans. RIA revealed that the cells produced bo th IGF-II and IGF-I. Transcripts of messenger RNAs of both IGF-I and I GF-II and both IGF-IR and IGF-IIR also were detectable by Northern ana lysis. Both anti-IGF-IR antibody and anti-IGF-II antibody inhibited pr oteoglycan synthesis. Mannose-6-phosphate, which is known to bind to I GF-IIR, stimulated proteoglycan synthesis, potentiated IGF-II-stimulat ed proteoglycan synthesis, and enhanced the binding affinity for IGF-I I 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 c ells. [Leu(27)]IGF-II, an IGF-II analogue with high affinity only for IGF-IIR, strongly stimulated proteoglycan synthesis in HCS-2/8 cells b ut [Arg(54), Arg(55)]IGF-II, which binds to only IGF-IR, also stimulat ed proteoglycan synthesis in the cells. These findings indicate that I GF-I and IGF-II act as autocrine differentiation factors for this chon drocytic permanent cell line, HCS-2/8, mainly via respective receptors .