CHARACTERIZATION OF AN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-5 PROTEASE PRODUCED BY RAT ARTICULAR CHONDROCYTES AND A NEUROBLASTOMA CELL-LINE

Both articular cartilage and the central nervous system are target org ans for insulin-like growth th factors (IGFs), We have previously desc ribed the hormonal regulation of IGF binding proteins (IGFBPs) in the conditioned media (CM) of rat articular chondrocytes and in a rat neur oblastoma cell line (B104), In the studies presented here, we have inv estigated the role of IGFBP-5 proteases in these complex systems, Prot eolysis of [I-125] IGFBP-5 was maximal after 2-3 h incubation with CM of either cell type and did not further increase, even with an incubat ion of 12 h, Assessment of the effect of pH on protease activity showe d that proteolysis was active between pH 6 and pH 9, but not at more a cidic pH. Among the various protease inhibitors, serine protease inhib itors [benzamidine (100 mM), aprotinin (1 mg/ml), PMSF (10 mM)] and me talloprotease inhibitors [EDTA (1 mM), 1,10-phenanthroline (10 mM)] we re the most effective in inhibiting the proteolysis of IGFBP-5, wherea s aspartic and cysteine protease inhibitors were ineffective, These re sults indicate that the IGFBP-5 protease in the conditioned medium of rat articular chondrocytes and B104 cells belongs to a family of serin e-metallo proteases, Interestingly, divalent cations, such as Zn++(1 m M) and Ca++(10 mM) also inhibited the IGFBP-5 proteolysis. This effect was not observed with monovalent ions, such as Na+ and K+. We also ex amined the effect of IGFs on IGFBP-5 protease activity, and found that IGF-I and -II inhibited the proteolysis in cell-free conditioned medi um, while des(1-3) IGF-I was less effective, The IGFs may act to prote ct [I-125] IGFBP-5 from the proteases in the CM, although the precise mechanism remains unknown, Thus, IGFBP-5 protease activity produced by both rat articular cartilage and B104 cells is a serine-metallo prote ase, that is inhibited by divalent cations and in the presence of IGF.