EVIDENCE THAT HUMAN BONE-CELLS IN CULTURE PRODUCE INSULIN-LIKE GROWTHFACTOR-BINDING PROTEIN-4 AND PROTEIN-5 PROTEASES

Previous studies have shown that the actions of insulin-like growth fa ctor-II (IGF-II) in bone are determined by both its concentration and the concentrations of the IGF-binding proteins (IGFBPs). As IGFBP conc entrations may be regulated not only at the level of production, but a lso at the level of degradation, IGFBP proteases may be an important c omponent of the IGF-II regulatory system. In this study, we have ident ified IGFBP-4 and IGFBP-5 protease activity in the conditioned medium (CM) of the human osteosarcoma U2 cell line (U20S) and untransformed n ormal human bone cell (HBC) derived from skull. Proteolysis of the 29- kilodalton (kDa) [I-125]IGFBP-5 produced an 18- to 20-kDa fragment of IGFBP-5, and 25 kDa [I-125]IGFBP-4 yielded two lower mol wt fragments in the presence of IGF-II. CM from IGF-II-treated U20S and normal HBC cultures exhibited decreased IGFBP-5 proteolytic activity compared to control cultures. In contrast, CM from IGF-II-treated HBC cultures had increased proteolytic activity against IGFBP-4. To determine the mech anisms by which IGF-II modulates IGFBP-4 and -5 proteolytic activity, CM from control U20S cell culture was incubated with [I-125]IGFBP-4 or -5 in the presence of Various concentrations of IGF-II and IGF analog s under cell-free conditions. It was found that exogenous IGF-II stimu lated IGFBP-4 proteolysis, but IGF analogs that had no or extremely lo w affinity to IGFBP-4 failed to induce IGFBP-4 proteolysis. On the con trary, exogenous IGF-II had no effect on IGFBP-5 proteolysis in cell-f ree U20S CM. Both IGFBP-4 and IGFBP-5 proteolytic activities were inhi bited by aprotinin, zinc chloride, and EDTA and eluted as a single maj or peak between mol wt markers of 160 and 67 kDa upon gel filtration. Based on the findings that HBCs in culture produce a protease(s) capab le of cleaving both IGFBP-4 and IGFBP-5 and that IGF-II can promote or inhibit proteolytic degradation of IGFBP-4 and IGFBP-5, respectively, it is proposed that IGFBP protease(s) may be an important modulator o f IGF activity in bone.