OSTEOBLAST PHENOTYPE SUPPRESSION BY PARATHYROID HORMONE

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DESCRIPTION: (Adapted from investigator's abstract) The application proposes to study the basis of the observation that chronic PTH administration suppresses osteoblast differentiation. To gain insights into the molecular mechanism involved in this hormone effect, the applicant has chosen to focus his attention on the osteopontin gene. Osteopontin is a component of the bone matrix that, based on in vitro cell culture studies using differentiating osteoblasts, is expressed at the transition of pre-osteoblasts into fully differentiated osteoblasts. Previous studies have shown that osteopontin gene transcription is inhibited by PTH, forskolin and cAMP analogues. The Osteopontin promoter has response elements for calcitriol (vitamin D3) and phorbol esters, but does not contain a cAMP response element. The hypothesis that the applicant proposes to test is that the downregulation of osteopontin gene expression by PTH is mediated via induction of inducible cAMP early repressors (ICERS). This gene has been shown to downregulate phorbol response elements. In order to test the hypothesis, the applicant proposes to use a well-characterized rat calvarial osteoblast cell line, IRC 10/30-myc1, to show that the phorbol ester response elements and the expression of ICERs are necessary and sufficient to provide the molecular signals for PTH suppression of osteopontin gene expression. The Specific Aims of the application are: 1) to show that AP-1 sites (phorbol response elements) are necessary and sufficient for PTH to suppress osteopontin gene transcription; 2) to demonstrate that PTH and cAMP induce the formation of ICER proteins in osteoblast cell lines; and 3) to demonstrate that overexpression of ICERs suppresses transcription from the osteopontin promoter.