This application proposes experiments designed to characterize the mechanism of action of the vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in regulating the expression of genes relevant to the pathophysiology of bone mineral homeostasis. The focus of the planned studies is the nuclear vitamin D receptor (VDR), a member of the superfamily of steroid/retinoid/thyroid hormone receptors that functions as a 1,25(OH)2D3 ligand-activated transcription factor in cooperation with the retinoid X receptor (RXR). In the first Specific Aim, utilizing cultured human cells, the plan is to evaluate the functional significance of the: 1) L/S gene polymorphism in the 3'-untranslated region of the human VDR (hVDR) mRNA, 2) Cdx-2 site polymorphism in the 5'-untranslated region, and 3) cell specific N-terminal variant hVDR proteins resulting from alternative splicing of novel 5'-promoter exons. By examining the impact of hVDR expression and isoforms on 1,25(OH)2D3 action in a variety of cells, insight may be gained into the molecular basis for the nonclassical biological actions of 1,25(OH)2D3 and its drug analogs, such as in epithelial cell differentiation. The second Specific Aim is to continue to determine the sites and significance of hVDR phosphorylation, focusing on protein kinase A catalyzed posttranslational modification of the receptor. The third Specific Aim involves characterizing the role of hormonal ligands, target gene promoters, and VDR-interacting proteins in transducing the signal for transcriptional regulation. Controlled experiments will include a superactive 20-epi-1,25(OH)2D3 analog and a retinoid that binds specifically to RXR, as well as natural promoter constructs derived from the osteocalcin and osteopontin vitamin D regulated genes to evaluate the influence of DNA sequence on 1,25(OH)2D3 hormonal responses. Constitutively active RXRs and VDR will be generated by site-directed mutagenesis to dissect further the roles played by receptor ligands and the RXR partner. Finally, the application proposes to clone a novel VDR co-regulator(s) from osteoblast cDNA libraries utilizing phage display methodology. These studies, directed at the VDR gene, isoform and phosphorylated receptor proteins, and their co-regulatory partners, are expected to facilitate a detailed understanding of the molecular basis of 1,25(OH)2D3 hormone action, with therapeutic implications for clinical disorders of bone such as osteoporosis.
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