Vitamin D Hormone Signaling in Bone Mineral Homeostasis

描述（由申请人提供）：本项目的目的是表征肾脏维生素D激素1，25-二羟维生素D3（1，25（OH）2D 3）在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方面的分子作用机制。待检验的假设是，1，25（OH）2D 3通过配体化核维生素D受体（VDR）发挥其支持矿化骨骼的作用，所述核维生素D受体（VDR）继而募集其类维生素A X受体（RXR）异二聚体配偶体以识别骨相关靶基因启动子中的维生素D反应元件（VDRE），其中1，25（OH）2D 3-VDR-RXR吸引共调节蛋白/酶复合物，通过染色质重塑和与RNA聚合酶II的连接抑制或诱导DNA转录。培养的1，25（OH）2D 3靶细胞将用作特定目的的模型系统，旨在阐明1，25（OH）2D 3信号传导中的VDR介导事件。目的1将确定1，25（OH）2D 3-VDR是否通过影响Wnt/LRP 5/b-连环蛋白和BMP/SMAD/Runx 2信号级联来调节成骨细胞发育/骨形成，并可能通过控制成骨细胞中新识别的磷酸盐调节系统FGF 23和PHEX来限制骨过度矿化。目的2探讨1，25（OH）2D 3-VDR通过增强上皮钙转运蛋白1（TRPV 6）和钠-磷酸盐协同转运蛋白2c（Npt 2c）的表达，促进小肠和肾脏钙磷转运的作用。方法学将包括基因组学和染色质免疫沉淀（ChIP）分析，以扫描候选VDRE，加上DNA凝胶迁移率变化，真实的时间PCR和启动子解剖，以确定VDR控制的基因。通过比较1，25（OH）2D 3与其超活性类似物，ChIP测定将评估完整细胞VDR-RXR-VDRE结合，并表征差异控制骨矿物质靶基因表达的转录共调节因子的顺序募集。最后，在目标3中，ChIP展示将用于揭示新的上游VDR控制的基因，如果任何提出的VDR靶点通过介导的转因子作为二级或三级诱导/抑制而出现。拟议研究的意义在于，1，25（OH）2D 3维持适当骨矿化的精确分子机制，以及在骨（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;理解这些途径应增强我们开发维生素D模拟物以预防和治疗骨质疏松症的能力。因此，尽管公认维生素D在促进钙吸收和骨重塑以防止骨质疏松症的发展和导致的脊柱、髋部和腕部骨折方面具有相关性，但维生素D激素实现这种有益效果的方式尚未完全阐明。定义维生素D作用的分子途径以增强和保护矿化骨骼不仅可以揭示用骨合成代谢1，25（OH）2D 3类似物预防和治疗骨质疏松症的新治疗策略，而且还可以揭示维生素D在诸如结肠、皮肤、乳房和前列腺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