VITAMIN D HORMONE MECHANISM OF ACTION IN CULTURED CELLS

本申请建议继续调查 维生素D激素（1，25（OH）2D3）的分子和生物学功能 在培养的细胞系统中。 1，25（OH）2D3将成为新的焦点 在成骨细胞样骨细胞和上皮细胞中的作用 肠道，维生素D在骨骼重塑中发挥主要作用 和矿物质吸收。 在上一个赠款期间，我们 完成了分子克隆、过表达和免疫细胞化学 定位的人维生素D受体（hVDR），并阐明了 维生素D反应元件（VDRE）在大鼠骨钙素基因。 这些 研究结果将扩展到探测hVDR的磷酸化， 与其与VDRE结合并增强 转染细胞中的骨钙素启动子/报告基因构建体。 将采用定点诱变来确定 磷酸化的hVDR和相关蛋白的刺激 将影响完整细胞中的激酶以测试体内显著性。 接下来，hVDR将在昆虫杆状病毒系统中过表达，并在昆虫细胞中表达。 e.大肠杆菌，以产生足够数量的生化研究。 纯化表达的hVDR用于研究蛋白质磷酸化 激酶C和酪蛋白激酶II。 hVDR的磷酸肽图谱 将使用在COS-7细胞中过表达的基因来阐明 1，25（OH）2D3依赖性，hVDR磷酸化。 初步数据显示， 核受体辅助因子（RAF）是高亲和力VDR所必需的 与VDRE的结合将通过表征、纯化和 在CV-1细胞中克隆主要RAF。 RAF将通过筛选 用纯化的hVDR/[32 p] VDRE构建表达载体文库。 最终 上述组分将在体外重组为纯化的因子， 转录系统，旨在确定机制， 1，25（OH）2D3刺激骨钙素的组织特异性表达。 最后，从分子水平比较了1，25（OH）_2D_3/VDR对骨钙素的作用 将与钙结合蛋白-D28k（CaBP）诱导的影响， 培养的胚状体因为它被放线菌酮抑制， 1，25（OH）2D3对CaBP的调节可能是独特的，因为中间体 诱导的蛋白质可以结合到CaBP启动子中的非VDRE增强子 和/或CaBP mRNA的稳定化。 总的来说，这些实验 应该定义了维生素D 控制着关键的骨骼和肠道基因的表达， 结果可能为临床维生素D的分子基础提供线索 抵抗力以及维生素D在预防 骨质疏松

This application proposes the continuation of our investigation of the molecular and biological functions of the vitamin D hormone (1,25(OH)2D3) in cultured cell systems. There will be a new focus on 1,25(OH)2D3 action in osteoblast-like bone cells and epithelial cells of the intestine, where vitamin D exerts its major effects upon bone remodeling and mineral absorption, respectively. In the last grant period we accomplished the molecular cloning, overexpression and immunocytochemical localization of the human vitamin D receptor (hVDR), and elucidated the vitamin D response element (VDRE) in the rat osteocalcin gene. These findings will be extended to probe the phosphorylation of hVDR in relation to its ability to bind to the VDRE and enhance transcription of an osteocalcin promoter/reporter construct in transfected cells. Site-directed mutagenesis will be employed to determine the residues in hVDR that are phosphorylated and stimulation of the relevant protein kinases in intact cells will be effected to test in vivo significance. Next, hVDR will be overexpressed in the insect baculovirus system and in E. coli to generate sufficient quantities for biochemical study. Expressed hVDR will be purified for study of phosphorylation by protein kinase C and casein kinase II, in vitro. Phosphopeptide mapping of hVDR overexpressed in COS-7 cells will be used to elucidate the site of 1,25(OH)2D3-dependent, hVDR phosphorylation. Preliminary data that a nuclear receptor auxiliary factor (RAF) is required for high-affinity VDR binding to the VDRE will be pursued by characterizing, purifying and cloning the major RAF in CV-1 cells. RAF will be cloned by screening an expression vector library with purified hVDR/[32p]VDRE. Ultimately, the above components will be reassembled as purified factors in an in vitro transcription system designed to determine the mechanism whereby 1,25(OH)2D3 stimulates the tissue specific expression of osteocalcin. Finally, a molecular comparison of 1,25(OH)2D3/VDR action on osteocalcin will be made with the effect on calbindin-D28k (CaBP) induction in cultured embryonic duodena. Because it is inhibited by cycloheximide, CaBP regulation by 1,25(OH)2D3 may be unique in that an intermediary induced protein may bind to a non-VDRE enhancer in the CaBP promoter and/or stabilization of CaBP mRNA may occur. In toto, these experiments should define the precise signal transduction pathways whereby vitamin D controls the expression of crucial 'bone and intestinal genes, and these results may provide clues to the molecular basis of clinical vitamin D resistance as well as to the role of vitamin D in the prevention of osteoporosis.