The broad objective of proposed continuing research is to elucidate the molecular role of the vitamin D hormone [1,25(OH)2D3] and its nuclear receptor (VDR) in affecting normal calcium and phosphorus metabolism, thereby providing insight into how 1,25(OH)2D3 and its clinically efficacious analogs may prevent debilitating bone diseases such as osteoporosis. Effort for the next renewal period will be concentrated on understanding the structure/function of VDR, specifically its conformational states as influenced by 1,25(OH)2D3 ligand congeners, heterodimeric protein partners like retinoid X receptors (RXRs), and vitamin D responsive element targets in DNA promoters. Conformational analysis of VDR will include protease sensitivity and antibody epitope accessibility experiments, combined with overexpression of full-length and a ligand-binding domain fragment of VDR that binds side-chain modified analogs of 1,25(OH)2D3. This information will be extended to isolating cell-specific co-modulator (co-activator and co-intergrator) proteins that interact with VDR and RXR-VDR to transduce the 1,25(OH)2D3 signal and activate the transcription of vitamin D regulated genes. Co-modulators and co-repressors/chaperones will be identified and cloned using yeast two-hybrid and one-hybrid methodology, fortified with direct biochemical and transfection strategies to prove the functional relevance of VDR- and RXR-VDR-interacting proteins in the transcription pathway. Finally, these findings will be integrated and probed in the natural setting of cultured human fibroblasts, in order to characterize the biologic impact of VDR gene polymorphisms on the function of VDR, and the mediation of the action of its ligand to enhance DNA transcription. Several polymorphic forms of the VDR gene have been linked in population studies to determine bone mineral density and possibly osteoporosis susceptibility, but fundamental knowledge of the molecular basis whereby allelic variations in VDR confer altered sensitivity to 1,25(OH)2D3 is lacking. Therefore, by investigating VDR structure/function, specifically ligand-generated conformations, interaction with co-activators, and the affect of genotype on transcriptional activation phenotype, it should be possible to clarify the participation of this nuclear receptor system in the pathophysiology and treatment of demineralizing bone disorders.
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