DEGRADATION OF INSULIN-LIKE GROWTH-FACTOR (IGF)-BINDING PROTEIN-3 (IGFBP-3) BY A METAL-DEPENDENT PROTEASE PRODUCED BY HUMAN FIBROBLASTS - EFFECTS OF IGFS ON PROTEASE ACTIVITY

Insulin-like growth factor binding protein-3 (IGFBP-3) is the major IG F-binding protein in adult human plasma and is produced by numerous ce ll lines in vitro. The concentrations of IGFBP-3 in human fibroblast c onditioned media are increased by IGFs via poorly defined post-transla tional events. To determine whether IGFs increase IGFBP-3 levels by pr otecting IGFBP-3 from proteolytic degradation, we examined the process ing of native IGFBP-3 produced by human fibroblasts and recombinant hu man IGFBP-3 (nIGFBP-3 and rhIGFBP-3, respectively) in human fibroblast conditioned media. When cell-free conditioned media collected from hu man fibroblasts were incubated at 37 degrees C for various times, a ti me-dependent decrease in intact nIGFBP-3 was noted. Likewise, rhIGFBP- 3 was also degraded by human fibroblast conditioned media into several immunoreactive fragments with M, 14-26 kD. Serine protease inhibitors had no effect on degradation of IGFBP-3, while chelating agents preve nted degradation. The addition of Zn2+ to conditioned media pre-treate d with a chelating agent caused the reappearance of immunoreactive IGF BP-3 fragments, suggesting that IGFBP-3 is degraded by a Zn2+- depende nt protease. The addition of IGF-I, IGF-II or des(1-3) IGF-I inhibited the proteolytic degradation of IGFBP-3 in a dose-dependent fashion. I n contrast, insulin, which has no affinity for IGFBPs, had no effect o n IGFBP-3 degradation, suggesting that protection of IGFBP-3 is relate d to the ability of the ligand to bind IGFBP-3. These studies demonstr ate that IGFs protect IGFBP-3 from degradation by a Zn2+-dependent pro tease(s) produced by human fibroblasts in culture, providing a mechani sm by which IGFs can increase IGFBP-3 levels independent of cellular p rotein synthesis and secretion.