Vitamin D Hormone Signaling in Bone Mineral Homeostasis

描述（由申请人提供）：该项目的目标是表征肾脏维生素D激素1,25-二羟基维生素D3 （1,25(OH)2D3）调节骨矿物质离子以预防佝偻病/骨软化症和骨质疏松症等疾病的分子机制。需要验证的假设是，1,25(OH)2D3通过核维生素D受体（VDR）的配体发挥其作用来支持矿化骨架，而核维生素D受体反过来又招募其类维生素a受体（RXR）异二聚体伴侣来识别骨骼相关靶基因启动子中的维生素D响应元件（VDREs）。1,25(OH)2D3-VDR-RXR吸引调节蛋白/酶复合物，通过染色质重塑和RNA聚合酶II的链接抑制或诱导DNA转录。培养的125 (OH)2D3靶细胞将被用作特异性目的的模型系统，旨在阐明125 (OH)2D3信号传导中vdr介导的事件。目的1将确定1,25(OH)2D3-VDR是否通过影响Wnt/LRP5/b-catenin和BMP/SMAD/Runx2信号级联来调节成骨细胞的发育/骨形成，并可能通过控制成骨细胞中新发现的磷酸盐调节系统FGF23和PHEX来限制骨过度矿化。Aim 2将探讨1,25(OH)2D3-VDR分别通过增强上皮钙转运蛋白1 （TRPV6）和磷酸钠共转运蛋白2c （Npt2c）的表达，在小肠和肾脏中刺激钙和磷酸盐转运的作用。方法学将包括基因组学和染色质免疫沉淀（ChIP）分析来扫描候选VDREs，加上DNA凝胶迁移位移，实时PCR和启动子解剖来鉴定vdr控制的基因。将1,25(OH)2D3与其超活性类似物进行比较，ChIP分析将评估完整细胞VDR-RXR-VDRE结合，并表征转录调节剂的顺序募集，这些转录调节剂可以不同地控制骨矿物质靶基因的表达。最后，在Aim 3中，ChIP显示将用于揭示新的上游VDR控制基因，如果任何提出的VDR靶点通过介导的转录因子出现二级或三级诱导/抑制。所提出的研究的意义在于，1,25(OH)2D3维持适当骨矿化的精确分子机制，以及在骨骼（Aim 1）和小肠和肾脏（Aim 2）中表达的新型维生素d调节基因的相对重要性尚不清楚；理解这些途径将提高我们开发维生素D模拟物的能力，以预防和治疗骨质减少症。因此，尽管人们认识到维生素D在促进钙吸收和骨骼重塑方面的相关性，以防止骨质疏松症的发展以及由此导致的脊柱、髋关节和手腕骨折，但维生素D激素实现这种有益效果的方式尚未得到充分阐明。确定维生素D作用增强和保存矿化骨骼的分子途径，不仅可以揭示用骨合成代谢125 (OH)2D3类似物预防和治疗骨质疏松症的新治疗策略，还可以揭示维生素D在结肠、皮肤、乳房和血管等部位的新抗癌作用

DESCRIPTION (provided by applicant): 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)2D3 target 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/b-catenin and BMP/SMAD/Runx2 signal cascades, and possibly limits bone over mineralization 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