This renewal application proposes to continue the investigation of vitamin D hormone (1,25(OH)2D3) action as mediated by its nuclear receptor, with a new emphasis on the functional interplay between 1,25(OH)2D3 and sex steroids in regulating bone remodeling. In the last grant period we succeeded in the molecular cloning of cDNAs to the avian and human vitamin D receptors (cVDR and hVDR) and we plan to expand upon these finding by: i) isolating the natural gene for cVDR and using intronic probes to study its nutritional and developmental control in relation to the major vitamin D induced protein in chickens, CaBP28k and ii) expressing hVDR both by in vitro transcription/translation and in E. coli in order to probe its zinc content,phosphorylation on serine residues, potential dimerization, and specific association with vitamin D responsive elements (VDREs) in genes such as osteocalcin/bone gla protein (BGP), vitamin D-24-OHase and type I collagen. Candidate 1,25(OH)2D3 hormone binding mutant VDRs from LLC-MK2 cells and a kindred with vitamin D resistance will also be characterized at the molecular biological level employing polymerase chain reaction. Concurrently, we plan to extend our published observation of estrogen receptors and effects on TGF-beta and collagen mRNA levels in osteoblast- like cells. Putative estrogen receptors (ER) in normal human osteoblasts will be further analyzed by immunochemical visualization and antagonist affinity labeling with tamoxifen aziridine. Recent collaborative experiments indicate that cotransfected hVDR confers 1,25(OH)2D3 responsiveness to a reporter-linked estrogen responsive element (ERE) palindrome. Therefore, estrogen and 1,25(OH)2D3 bind to a functionally overlapping subfamily of the steroid, thyroid and retinoid superfamily of receptors. We propose to exploit this new insight and utilize normal human bone cells to elucidate the mechanism whereby sex steroids and 1,25(OH)2D3 modulate autocrine and paracrine growth factor (i.e. TGF-beta, IGFI, IGFII, etc.) influence on osteoblasts and their coupling to osteoclasts and bone marrow/thymus derived cells of the immune system in the concerted process of bone remodeling. The relationship of VDR and ER to their respective DNA recognition sites, VDRE and ERE, will be probed in bone cell expressed genes such as type I collagen, BGP and several relevant growth factors. These investigations should not only lead us to the VDRE switch in controlled genes, but they may also unveil the basic mechanisms of certain vitamin D resistant bone and mineral diseases as well as possibly clarify the molecular pathogenesis of postmenopausal osteoporosis.
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