Osteoporosis is a disease characterized by low bone density and deterioration of mineralized bone leading to enhanced susceptibility to fracture. Several growth factors have been implicated in the coupling of formation to resorption during the bone remodeling process. The net bone forming activity of many growth factors has prompted numerous studies focused on the regulation of osteoblast cell proliferation, differentiation and activity. The current study focuses on the insulin-like growth factor (IGF) system, an important growth factor system involved in the regulation o f bone formation and bone resorption.

IGF binding protein —5 (IGFBP-5), the most abundant IGF binding protein in bone, has been shown to play a key role in the regulation of bone formation. IGFBP-5 actions in bone involve IGF-dependent and IGF-independent mechanisms. Regulation of IGFBP-5 levels in bone can be achieved in part by specific proteolysis of this IGF binding protein by IGFBP-5 specific and non-specific proteases. Although it is now known that a variety of cell types produce protease/s capable o f degrading IGFBP-5, the structural identity of few IGFBP-5 specific proteases have been established to date. Thus, the specific aims of this research were to 1) purify and characterize the IGFBP-5 specific protease from U2 osteosarcoma conditioned medium, 2) generate rhIGFBP-5 Nterminal and C-temainal fragments based on the sites cleaved by the IGFBP-5 protease, and 3) generate rhIGFBP-5 analogs resistant to IGFBP-5 protease activity, that otherwise retain the same biological characteristics of native IGFBP-5.

Toward the purification of the IGFBP-5 protease, we have identified a novel hydrophobic IGF-independent aspartic protease that binds zinc ions capable of complexing with a 2-macroglobulin, a known protease inhibitor. Furthermore, we have obtained several unique peptide sequences, however, the identity o f this protease remains elusive. In addition, we have constructed recombinant N-terminal and C-terminal IGFBP-5 peptides to evaluate the activity of proteolytic fragments in vitro and in vivo. A particular rhIGFBP-5 deletion mutant was generated that retains IGF-II binding affinity but does not confer resistance to the protease, probably due to a conformational change exposing alternative cleavage site/s.