The applicants' long-term objective is to identify the molecular mechanisms responsible for the pathogenesis of senile (type II) osteoporosis. Because impaired bone formation (BF) is a major contributor to this disease, and because insulin-like growth factors (IGFs) have been shown to be important simulators of BF in vitro and in vivo, their studies have been focused towards identifying the role of the IGF system in regulation of BF. This renewal application is focused on one of the IGF system components, IGF binding protein-5 (IGFBP-5). Recent in vitro and in vivo findings support the premise that IGFBP-5 is a key component of the IGF system in bone: first, IGFBP-5 stimulates both basal and IGF-induced osteoblast (Ob) cell proliferation in vitro. Second, IGFBP-5 appears to function as a traditional IGFBP to promote IGF action, and, in addition, appears to act independently of IGF action through its own signal transduction pathway. Third, IGFBP-5 treatment in vivo, in both rats and mice, causes stimulation of BF, as indicated by increases in serum osteocalcin. Fourth, the serum level of IGFBP-5 is significantly decreased in hip fracture patients, compared to control patients, which raises the possibility that a deficiency in IGFBP-5 could be a contributing factor to senile osteoporosis. The first goal of this application is to understand the role of an Ob-derived IGFBP-5-specific protease in regulating the effective concentration of IGFBP-5 in local areas of bone. Towards this goal, the applicants propose to purify the IGFBP-5 protease produced by human Obs and clone the full-length cDNA of the IGFBP-5-specific protease. IGFBP-5 protease will be expressed by recombinant means for biochemical and functional characterization. The functional significance of IGFBP-5 protease will be studied by comparing the biological activity of protease-resistant IGFBP-5 analogs with that of native IGFBP-5 both in vitro, using serum-free cultures of human Obs, and in vivo, using a mouse model system, or alternatively by transgenic approach involving antisense and overexpression of IGFBP-5 protease in human Obs. The function of IGFBP-5-specific protease will also be evaluated by testing the activity of recombinant IGFBP-5 fragments which are identical to those generated by IGFBP-5-specific protease. The application's second goal is to evaluate the IGF-independent effects of IGFBP-5. Towards this end, the investigators propose to determine the in vitro and in vivo effects of IGFBP-5 on BF parameters in transgenic mice lacking functional genes for IGF-I, IGF-II, or type I IGF receptor. The applicants anticipate that these studies will lead to an increased understanding of the role of IGFBP-5 and its protease in the regulation of BF, and in the pathogenesis of osteoporosis. In addition, if the hypothesis that IGFBP-5 increases BF in vivo is confirmed, this would raise the possibility that IGFBP-5 or its analogs could serve as therapeutic agents in the treatment of osteoporosis.
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