The goal of this project is to characterize the molecular mechanism of action of the renal vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in regulating bone mineral ions to prevent diseases such as rickets/osteomalacia and osteoporosis. The hypothesis to be tested is that 1,25(OH)2D3 exerts its actions to support the mineralized skeleton via liganding of the nuclear vitamin D receptor (VDR), which in turn recruits its retinoid X receptor (RXR)heterodimeric partner to recognize vitamin D responsive elements (VDREs) in bone relevant target gene promoters, with 1,25(OH)2D3-VDR-RXR attracting comodulator protein/enzyme complexes that either repress or induce DNA transcription by chromatin remodeling and linkage to RNA polymerase II. Cultured 1,25(OH)2D3target cells will be used as model systems for Specific Aims designed to elucidate the VDR-mediated events in signaling by 1,25(OH)2D3.
Aim 1 will determine if 1,25(OH)2D3-VDR modulates osteoblast development/bone formation by impacting the Wnt/LRP5/p-catenin and BMP/SMAD/Runx2 signal cascades, and possibly limits bone overmineralization by controlling a newly recognized phosphate regulatory system, FGF23 and PHEX, in osteoblasts.
Aim 2 will probe the role of 1,25(OH)2D3-VDR to stimulate calcium and phosphate translocation in small intestine and kidney through the enhanced expression of epithelial calcium transporter 1 (TRPV6) and sodium-phosphate cotransporter 2c (Npt2c), respectively. Methodology will include genomics and chromatin immunoprecipitation (ChIP) assays to scan for candidate VDREs, plus DNA gel mobility shift, real time PCR and promoter dissection to identify VDR-controlled genes. Comparing 1,25(OH)2D3 to its superactive analogs, ChIP assays will assess intact cell VDR-RXR-VDRE binding and characterize the sequential recruitment of transcriptional comodulators that differentially control the expression of bone mineral target genes. Finally, in Aim 3 ChIP display will be used to reveal novel upstream VDR-controlled genes should any of the proposed VDR targets emerge as secondarily or tertiarily induced/repressed via mediating transfactors. The significance of the proposed studies is that the precise molecular mechanisms whereby 1,25(OH)2D3 maintains proper bone mineralization, as well as the relative importance of novel vitamin D-regulated genes expressed in bone (Aim 1) and small intestine and kidney (Aim 2), are not understood;comprehending these pathways should enhance our ability to develop vitamin D mimetics to prevent and treat osteopenic disorders. Thus, despite the recognized relevance of vitamin D in promoting calcium absorption and bone remodeling to preclude the development of osteoporosis and resulting fractures of the spine, hip and wrist, the manner through which the vitamin D hormone accomplishes this beneficial effect has not been fully elucidated. Defining the molecular pathway of vitamin D action to enhance and preserve the mineralized skeleton may not only reveal new therapeutic strategies to prevent and treat osteoporosis with bone anabolic 1,25(OH)2D3 analogs, but could also shed light on the novel anticancer effects of vitamin D at sites such as the colon, skin, breast and
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