The object of this proposal is to obtain a better understanding of the actions of vitamin D, a principal factor required for the development and maintenance of bone as well as for maintenance of calcium homeostasis. This renewal application proposes in specific aim I to examine the functional significance of the recently identified vitamin D responsive intestinal calcium channel TRPV6, as well as the functional significance of intestinal calbindin, using recently generated TRPV6 and calbindin-D9k null mice. Studies are proposed to examine the effect of complete ablation of TRPV6 or calbindin-D9k on 1,25(OH)2D3 regulated intestinal calcium absorption and on active calcium transport under low dietary calcium conditions. Compensatory and reciprocal mechanisms involved in the alterations in calcium homeostasis observed in the TRPV6 knockout (KO) mouse or the calbindin-D9k KO mouse will be examined. Studies are also proposed to examine the mechanisms by which 1,25(OH)2D3 regulates intestinal TRPV6 gene expression. Mice lacking both TRPV6 and calbindin-D9k will be generated to determine whether double KO mice will display a more pronounced phenotype. Studies with these KO mice will result in new insight into basic mechanisms involved in intestinal calcium transport that have remained incomplete since the discovery of calbindin over 30 years ago.
In specific aim II studies begun in the last grant period related to the regulation of 24(OH)ase, the other major target of 1,25(OH)2D3, will be continued. In preliminary studies we noted that SWI/SNF, which uses the energy of ATP hydrolysis to remodel chromatin, is an important determinant of 1,25(OH)2D3 induced 24(OH)ase transcription. Studies are proposed to examine the role of SWI/SNF in VDR mediated transcription, the kinetics of the molecular events in the cross talk between VDR, CBP, C/EBP B and SWI/SNF as well as cell type and VDR target gene specificity related to the kinetics of the molecular events. These proposed studies complement studies begun in the previous grant period related to the factors that affect 24(OH)ase transcription (C/EBP (3, CBP, YY1 and DRIP205). This proposal, which combines studies related to the functional significance of target proteins with studies related to the molecular mechanism of 1,25(OH)2D3 action, will provide new insight into the mechanisms by which vitamin D mediates its biological effects and how aberrant regulation may be involved in diseases such as osteoporosis.
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