NUCLEAR EVENTS IN PTH ACTION ON BONE CELLS

说明（改编自申请人的摘要）：甲状旁腺激素 (PTH)在骨中具有多种作用，刺激骨吸收， 阵 本提案的总体目标是调查 PTH刺激胶原酶表达的机制， 成骨细胞样骨肉瘤细胞（UMR 106-01）。 在这场竞争性的复兴中， 应用程序，研究人员将继续研究转录 介导PTH对胶原酶基因作用的因子。 的 研究者已经证明，通过 甲状旁腺素需要新的蛋白质合成， PTH对胶原酶基因转录至151 bp 5 '侧翼DNA的影响 转录起始位点的上游。 总的假设是， 甲状旁腺激素通过一种新的转录方式刺激胶原酶基因表达 直接或间接的因素。 初步数据显示， 没有已知的顺式作用元件或反式激活因子， PTH对胶原酶基因的特异性影响。 的具体目标 建议是：1）确定核蛋白结合的暂定 通过筛选已知的胶原酶基因的PTH反应元件 使用凝胶迁移率变动分析和分离新的反式激活因子 通过用寡核苷酸筛选表达cDNA文库， 顺式作用区的探针或通过使用 寡核苷酸亲和柱，随后进行肽测序和cDNA 用简并寡核苷酸探针进行文库筛选; 2）检查 调节（即，PTH反应性，cAMP信号转导依赖性， 和蛋白质合成需要）的PTH反应元件结合 蛋白质使用凝胶迁移分析，蛋白质印迹（新的因素将需要 多克隆抗体的产生）、免疫沉淀和北方 印迹; 3）鉴定PTH应答元件的功能结构域 使用缺失或突变构建体和瞬时转录因子 用胶原酶启动子/CAT表达共转染UMR细胞 载体;和4）确定PTH应答元件转录的作用 体外因子（通过测量正义和反义表达的影响 胶原酶基因启动子/CAT载体表达的构建体）和 在体内，通过开发过表达或“敲除” PTH应答元件结合蛋白基因。

DESCRIPTION (Adapted from the Applicant's Abstract): Parathyroid hormone (PTH) has multiple actions in bone and stimulates both bone resorption and formation. The overall goal of this proposal is to investigate the mechanisms by which PTH stimulates the expression of collagenase in osteoblast-like osteosarcoma cells (UMR 106-01). In this competing renewal application, the investigator will continue studies on the transcription factor(s) which mediate the effects of PTH on the collagenase gene. The investigator has demonstrated that stimulation of collagenase expression by PTH requires new protein synthesis and has localized the indirect effects of PTH on transcription of the collagenase gene to the 151 bp 5'-flanking DNA upstream to the transcription start site. The overall hypothesis is that PTH stimulates collagenase gene expression by means of a novel transcription factor either directly or indirectly. Preliminary data suggests that there is no known cis-acting element or trans-activating factor which accounts for the PTH-specific effects on the collagenase gene. The Specific Aims of the proposal are to: 1) identify nuclear proteins which bind to the tentative PTH response element(s) of the collagenase gene by screening known trans-activating factors using gel mobility shift assays and isolating novel factor(s) by screening an expression cDNA library with an oligonucleotide probe to the cis-acting region(s) or by protein purification using an oligonucleotide affinity column followed by peptide sequencing and cDNA library screening with degenerate oligonucleotide probes; 2) examine the regulation (i.e, PTH responsiveness, cAMP signal transduction dependence, and protein synthesis requirement) of the PTH response element binding proteins using gel shift assays, Western blots (novel factors will require production of polyclonal antibodies), immunoprecipitation, and Northern blots; 3) identify the functional domains of the PTH response element transcription factors using deletion or mutation constructs and transient co-transfection of UMR cells with the collagenase promoter/CAT expression vectors; and 4) determine the role of PTH response element transcription factor in vitro (by measuring the effects of sense and antisense expression constructs on expression of the collagenase gene promoter/CAT vectors) and in vivo by developing transgenic mice with overexpression or "knock out" of the PTH response element binding protein gene.