Skeletal tissue homeostasis is balanced by bone resorption and bone formation. The physiological integration of these processes is called remodeling, whereas the mechanisms that determine balance are called coupling. Insulin-like growth factor I (IGF-I) appears to be one coupling factor since it is produced by bone cells or released during resorption, and enhances precursor cell replication and new bone matrix production. IGF-I synthesis is regulated by systemic and locally produced factors, and bone cells also produce abundant levels of IGF-II. The high amount of endogenous IGFs in bone predicts the need to regulate their actions by mechanisms beyond modulating their synthesis. In this regard, bone cells produce several of the IGF binding proteins (IGFBPs), and IGFBP expression is also under hormone and local factor control. The various IGFBPs may have independent or overlapping effects by which they localize, sequester, inhibit or potentiate IGF activity, probably by influencing interactions between IGFs and IGF receptors. Furthermore, changes in IGF receptor number and affinity, and the relative amount of type 1 (high affinity IGF- I; signal transducing) and type 2 (high affinity IGF-II; mannose-6- phosphate transferase) receptors may control IGF actions. The studies proposed here will examine the synthesis and localization of IGFBPs produced by primary cell cultures that represent less differentiated and more differentiated (osteoblast-enriched; Ob) populations from fetal rat parietal bone, and the expression of type 1 and type 2 IGF receptors in these cultures. The studies will focus specifically on agents that induce cAMP (and thereby activate protein kinase A; PKA), and that increase protein kinase C (PKC) activity. These agents include three forms of prostaglandin, forskolin, and phorbol ester, which independently or simultaneously increase cAMP and PKC dependent cellular events. These two classes of agents potently and differentially influence IGF-I and IGFBP expression, and IGF receptor binding in Ob cultures, and produce distinct metabolic differences in less differentiated and Ob cultures. These studies will systematically examine expression of IGFBPs at the levels of mRNA (by Northern analysis) and protein (by Western ligand and antibody probing) in less differentiated and Ob cultures in response to both classes of agents. Type 1 and type 2 IGF receptor profiles in each culture will be assessed at the levels of mRNA (by RNase protection analysis) and protein (by Scatchard analysis, polyacrylamide gel electrophoresis, and phosphorylation state) to distinguish specific alterations in each. Many calciotropic hormones or locally produced factors that increase resorption activate cAMP or PKC dependent events, and several of these factors act indirectly by way of cells other than osteoclasts in bone. Therefore, a careful characterization of IGFBP and IGF receptor expression in non-osteoclastic bone cells, with attention to agents that activate cAMP or PKC, is critical in order to understand the biochemical and molecular mechanisms that regulate IGF activity in skeletal tissue. Debilitating bone diseases such as osteoporosis exact a heavy burden on individuals and on society due to pain, limitations in mobility, and high cost medical care. Although the etiology of osteoporosis varies, the underlying feature is loss of bone mass and increased fracture incidence. The information collected from the work proposed here could help to design appropriate intervention methods to enhance bone formation or minimize bone loss in this and other metabolic bone diseases.
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