(from the application): Cell renewal is essential for bone remodeling and repair processes which occur throughout life. Impairment in cell renewal has been identified as a molecular mechanism responsible for the pathogenesis of senile (type II) osteoporosis. The importance of insulin-like growth factors (IGFs) in skeletal cell renewal is emphasized by the following key findings: 1) IGFs are the most abundant mitogens produced by bone cells, 2) IGFs are important stimulaters of bone cell proliferation, and 3) IGFs contribute approximately 40% to basal bone cell proliferation in vitro. Although the actions of IGFs in skeletal cell renewal can be modulated by a number of IGF system components, this proposal is focused on one IGF system component, namely IGF binding protein-4 (IGFBP-4). Our recent findings emphasize that changes in IGFBP-4 expression may in part contribute to the impaired skeletal cell renewal in diseases such as osteoporosis: First, IGFBP-4 levels in the conditioned medium of bone cells correlate negatively with the bone cell proliferation rate. Second, inhibition of endogenous IGFBP-4 production has been shown to stimulate bone cell proliferation. Third, serum IGFBP-4 levels are elevated with age and in type II osteoporosis patients with hip fracture. Based on the premise that molecular mechanisms which regulate IGFBP-4 expression are crucial to our understanding of the role of IGFBP-4 in skeletal cell renewal, we have proposed studies to characterize a negative regulatory mechanism which involves a specific negative regulatory element (NRE) present in the first exon of IGFBP-4 gene. In the proposed studies, the precise sequence(s) in the IGFBP-4 NRE which interact with protein(s) in a cell specific manner will be determined by footprint analysis, electrophoretic mobility shift analysis and Southwestern analysis. Subsequently, the sites of DNA-protein interaction in the NRE will be systematically mutated and included in the IGFBP-4- promoter-reporter constructs used for functional studies. The differences in IGFBP-4 promoter activity using wild type and mutant NRE containing promoter constructs will be evaluated after transient transfection of bone cells which express varying levels of IGFBP-4 mRNA and protein. It is anticipated that confirmation of the hypothesis that interaction between the NRE and specific binding proteins contribute to negative transcriptional regulation of IGFBP-4 gene expression in various cell types could lead to a better understanding of the pathogenesis of senile osteoporosis and to the development of novel therapies to increase cell renewal by suppressing IGFBP-4 expression in diseases such as osteoporosis.
