VITAMIN D HORMONE MECHANISM OF ACTION IN CULTURED CELLS

描述：本申请提出了设计用于表征 维生素D激素1，25-二羟维生素D3的作用机制 （1，25（OH）2D 3）在调节与细胞凋亡相关的基因表达中的作用 骨矿物质稳态的病理生理学。计划研究的重点 是核维生素D受体（VDR），维生素D受体超家族的成员。 作为1，25（OH）2D 3发挥功能的类固醇/类维生素A/甲状腺激素受体 配体激活转录因子与类维生素A X协同作用 受体（RXR）。在第一个具体目标中，利用培养的人类细胞， 计划是评估：1）L/S基因的功能意义 人VDR（hVDR）mRNA的3 '-非翻译区的多态性，2） Cdx-2位点5 '端非翻译区多态性; 3）细胞特异性 由新的选择性剪接产生的N-末端变体hVDR蛋白 5 '-启动子外显子。通过检测hVDR表达和同种型对 1，25（OH）2D 3在各种细胞中的作用，可以深入了解 1，25（OH）_2D_3的非经典生物学作用的分子基础及其在生物学中的应用 药物类似物，如上皮细胞分化。第二特定 目的是继续确定hVDR的位点和意义 磷酸化，聚焦于蛋白激酶A催化的翻译后 受体的修饰。第三个具体目标涉及表征 激素配体、靶基因启动子和VDR相互作用的作用 蛋白质在转导转录调节信号中的作用。控制 实验将包括超活性20-epi-1，25（OH）2D 3类似物和类维生素A 特异性结合RXR的天然启动子构建体， 从骨钙素和骨桥蛋白维生素D调节基因，以评估 DNA序列对1，25（OH）2D 3激素反应影响。组成型 将通过定点诱变产生活性RXR和VDR， 进一步研究了受体配体和RXR伴侣所起的作用。最后 本申请提出从成骨细胞cDNA克隆新的VDR共调节子 利用噬菌体展示方法的文库。这些研究，针对 VDR基因、亚型和磷酸化受体蛋白及其共调控 合作伙伴，预计将有助于详细了解分子 1，25（OH）2D 3激素作用的基础，对临床治疗的意义 骨质疏松症等骨骼疾病。

DESCRIPTION: This application proposes experiments designed to characterize the mechanism of action of the vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in regulating the expression of genes relevant to the pathophysiology of bone mineral homeostasis. The focus of the planned studies is the nuclear vitamin D receptor (VDR), a member of the superfamily of steroid/retinoid/thyroid hormone receptors that functions as a 1,25(OH)2D3 ligand-activated transcription factor in cooperation with the retinoid X receptor (RXR). In the first Specific Aim, utilizing cultured human cells, the plan is to evaluate the functional significance of the: 1) L/S gene polymorphism in the 3'-untranslated region of the human VDR (hVDR) mRNA, 2) Cdx-2 site polymorphism in the 5'-untranslated region, and 3) cell specific N-terminal variant hVDR proteins resulting from alternative splicing of novel 5'-promoter exons. By examining the impact of hVDR expression and isoforms on 1,25(OH)2D3 action in a variety of cells, insight may be gained into the molecular basis for the nonclassical biological actions of 1,25(OH)2D3 and its drug analogs, such as in epithelial cell differentiation. The second Specific Aim is to continue to determine the sites and significance of hVDR phosphorylation, focusing on protein kinase A catalyzed posttranslational modification of the receptor. The third Specific Aim involves characterizing the role of hormonal ligands, target gene promoters, and VDR-interacting proteins in transducing the signal for transcriptional regulation. Controlled experiments will include a superactive 20-epi-1,25(OH)2D3 analog and a retinoid that binds specifically to RXR, as well as natural promoter constructs derived from the osteocalcin and osteopontin vitamin D regulated genes to evaluate the influence of DNA sequence on 1,25(OH)2D3 hormonal responses. Constitutively active RXRs and VDR will be generated by site-directed mutagenesis to dissect further the roles played by receptor ligands and the RXR partner. Finally, the application proposes to clone a novel VDR co-regulator(s) from osteoblast cDNA libraries utilizing phage display methodology. These studies, directed at the VDR gene, isoform and phosphorylated receptor proteins, and their co-regulatory partners, are expected to facilitate a detailed understanding of the molecular basis of 1,25(OH)2D3 hormone action, with therapeutic implications for clinical disorders of bone such as osteoporosis.