The object of this proposal is to obtain a better understanding of the actions of the active form of vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], both in vivo and at the molecular level. This renewal application proposes to continue studies related to the functional significance of calbindin, whose synthesis by 1,25(OH)2D3 represents one of the most pronounced in vivo effects of 1,25(OH)2D3 known. Proposed studies include the use of calbindin-D28k knock-out mice and will focus on effects of mammalian calbindin-D28k in the nephron's distal tubule, on modulation of insulin secretion, and on protection against apoptotic cell death. In the previous grant period, the investigators completed the characterization of calbindin's response to 1,25(OH)2D3, which they found to be modestly transcriptional. To obtain further insight into the molecular mechanism of 1,25(OH)2D3 action, regulation of the 24(OH)ase gene, the gene in addition to calbindin which is markedly induced by 1,25(OH)2D3 in the intestine and kidney, will be examined. Cell type -specific mechanisms by which 1,25(OH)2D3 and other hormones and signaling pathways coordinately regulate 24(OH)ase will be examined, including: 1) regulation of VDR and RXR; 2) effects on regulatory regions in the promoter; and 3) effects on DNA binding to transcription factors. In addition, novel transcription factors that interact with VDR to mediate transcription of the 24(OH)ase gene and the osteopontin gene (for comparison) will be identified and characterized (candidates are YY1, SRC-1, and TAFII110). Cell type and gene -specific requirements of ligand independent modulation of VDR-mediated transcription will be examined. Experiments will also be done related to the functional significance of 24(OH)ase, using null mutant mice, which will focus on the proposed role of 24(OH)ase in vitamin D metabolism. It is likely that an increased understanding of the functional significance of target proteins, combined with increased understanding of cell- and gene- specific interactions of 1,25(OH)2D3 and VDR with other hormones and transcription factors, will play an increasingly important role in the elucidation of the mechanisms by which 1,25(OH)2D3 mediates its biological effect and how aberrant regulation may be involved in diseases such as osteoporosis and perturbations of parathyroid function.
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