Insulin-like growth factor binding protein-5 proteolytic activity in ovinearticular chondrocyte culture

We have previously demonstrated that ovine articular chondrocytes synthesis e and release insulin-like growth factor binding protein-5 (IGFBP-5) which subsequently undergoes proteolysis in the tissue culture medium. The IGFBP- 5 proteolytic activity has now been characterised and its substrate specifi city analysed using recombinant IGFBP-5 and purified chondrocyte-derived IG FBPs. Iodinated human recombinant IGFBP-5 was incubated with chondrocyte cu lture or conditioned medium in the presence or absence of various inhibitor s. Serine protease inhibitors aprotinin and heparin effectively inhibited t he breakdown of IGFBP-5. Furthermore, insulin-like growth factor-I (IGF-I) but not its structural analogues with reduced affinity for IGFBP-5, was als o able to partially protect IGFBP-5 from degradation indicating that the as sociation of IGF with the binding protein was required for the inhibition o f the proteolytic activity. The inflammatory cytokine interleukin-l did not have any effect on IGFBP-5 proteolysis. The proteolytic activity appears t o be IGFBP-5-specific since the incubation of chondrocyte-derived IGFBPs wi th chondrocyte conditioned medium resulted in the loss of IGFBP-5 while the levels of the other two IGFBPs (IGFBP-2 and a 24 kDa IGFBP) remained uncha nged. In conclusion, we show that IGFBP-5 is specifically cleaved by a seri ne protease released by primary cultures of ovine articular chondrocytes an d also demonstrate the ability of IGF-I to inhibit the proteolytic activity both in cell culture and in cell-free conditions. (C) 1998 Elsevier Scienc e B.V. All rights reserved.