ESTROGEN REGULATION OF THE OSTEOBLAST INSP3 RECEPTOR

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The proposed study will compare the effects of 17 beta-estradiol on inositol trisphosphate receptor (InsP3R) expressioin and IL-6 secretion from G-292 cells . G-292 cells are from an established human osteoblastic osteosarcoma cell line. Studies of primary cultures of human osteoblasts derived from oral (maxillary tuberosities) and non- oral (spongy bone) bone will also be carried out to substantiate the cell culture model. The objective of the proposed study is to test the hypothesis that estrogens down regulate INSP3R expressioin in osteoblasts and, as a consequence, reduce the capacity of this population of cells to secrete osteoclst-activating cytokines, including IL-6. A positive outcome would be of substantial significance because it would identify a site of estrogen action in osteoblasts that affects signal transduction and secretory capacity. The studies will achieve simultaneously the objective of comparing the estrogen-responsiveness of oral and non-oral derived human osteoblasts and thus establish a basis for investigative relationships between osteoporosis and oral bone loss. Biochemical and molecular markers of the derived osteoblasts, including growth and differentiation phenotype related genes as well as estrogen receptor status, will be assessed and used in the stage specific evaluation of estrogen responsiveness. Our preliminary observations indicate that 17 beta-estradiol down regulates type I INSP3R gene expression in human G-292 osteosarcoma cells and rat primary calvarial osteoblasts. These observations will be extended by examining potential regulation of other INSP3R types and by determining the effects of altered InsP3R expression on IL-6 secretion. The effects of estrogen on InsP3R expression and IL-6 secretion will be determined using RNase protection assays to quantitate type-specific mRNA levels, ligand binding studies and wester blotting techniques to determine InsP3R density, fluorescent Ca2plus release studies to assess funcitonal status, and sandwich ELISA protocols for quantitation of IL-6 secrettion. Depletion of InsP3-sensitive secretory calcicum will substantiate the requirement for this pool in elicited IL-6 secretion. The testing of the stated hypothesis could provide compelling preliminary data for extensive future studies and identify potential sites for therapeutic intervention in this area.