VITAMIN D HORMONE MECHANISM OF ACTION IN CULTURED CELLS

这份申请书建议我们继续调查 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。在上一次授权期内，我们 完成了分子克隆、高效表达和免疫细胞化学 人维生素D受体(HVDR)的定位，并阐明了 大鼠骨钙素基因中的维生素D反应元件(VDRE)。这些 这些发现将被扩展到探索hVDR的磷酸化 与其与VDRE结合并增强转录能力的关系 转基因细胞中骨钙素启动子/报告基因的构建。 将采用定点突变技术来检测 被磷酸化的hVDR和相关蛋白的刺激 完整细胞中的激酶将受到影响，以检验体内意义。 接下来，hvdr将在昆虫杆状病毒系统和 以产生足够的量进行生化研究。 表达的hVDR将被提纯，用于蛋白质磷酸化的研究 在体外，激酶C和酪蛋白激酶II。HVDR的磷酸肽图 在COS-7细胞中过表达将被用来阐明 依赖1，25(OH)2D3，hVDR磷酸化。初步数据显示， 核受体辅助因子是高亲和力VDR所必需的 对VDRE的绑定将通过表征、纯化和 克隆了CV-1细胞中主要的RAF。RAF将通过筛选和克隆 纯化的hVDR/[32P]VDRE表达载体文库。归根结底， 上述成分将在体外以纯化因子的形式重组 转录系统被设计用来确定 1，25(OH)2D3刺激骨钙素的组织特异性表达。 1，25(OH)2D3/VDR对骨钙素作用的分子比较 将对Calbindin-D28k(CABP)的诱导产生影响 培养的胚胎十二指肠。因为它被环己亚胺抑制， 1，25(OH)2D3对CABP的调节可能是唯一的，因为中间体 诱导蛋白可能与CABP启动子中的非VDRE增强子结合 和/或可能发生CABP mRNA的稳定。总之，这些实验 应该确定准确的信号转导途径，维生素D 控制关键的骨骼和肠道基因的表达，这些 结果可能为临床维生素D的分子基础提供线索 抗药性以及维生素D在预防糖尿病中的作用 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.