NUCLEAR EVENTS IN PTH ACTION ON BONE CELLS

描述(摘自申请人摘要)：甲状旁腺激素 甲状旁腺素(PTH)在骨骼中有多种作用，刺激骨吸收和 队形。这项提案的总体目标是调查 甲状旁腺激素刺激胶原酶表达的机制 成骨样骨肉瘤细胞(UMR 106-01)。在这场相互竞争的更新中 申请后，研究人员将继续对转录进行研究 介导甲状旁腺素对胶原酶基因影响的因子(S)。这个 研究人员已经证明，胶原酶的刺激表达 甲状旁腺激素需要新的蛋白质合成，并已局部化了间接影响 甲状旁腺素对胶原酶基因转录到151bp5‘-侧翼DNA的影响 位于转录起始点的上游。总体假设是 甲状旁腺素通过一种新的转录方式刺激胶原酶基因的表达 直接或间接的因素。初步数据显示， 是不是已知的顺式作用元件或反式激活因子 甲状旁腺素对胶原酶基因的特异性影响。《公约》的具体目标 建议是：1)识别与暂定的 胶原酶基因甲状旁腺素反应元件(S)的筛选已知 凝胶迁移率移位分析反式激活因子及其分离新方法 利用寡核苷酸筛选表达文库对S因子的影响 顺式作用区的探索(S)或通过蛋白质纯化 寡核苷酸亲和层析柱后的多肽测序和cDNA 用简并寡核苷酸探针筛选文库；2)检测 调节(即甲状旁腺素反应性、cAMP信号转导依赖性、 和蛋白质合成要求)的甲状旁腺素反应元件结合 使用凝胶移位分析的蛋白质，Western blotts(新的因素需要 多克隆抗体的生产)、免疫沉淀和Northern Blots；3)确定PTH反应元件的功能结构域 使用缺失或突变结构和瞬时转录因子 胶原酶启动子/CAT共表达对UMR细胞的影响 载体；4)确定PTH反应元件转录的作用 体外因子(通过测量正义和反义表达的效果 胶原酶基因启动子/CAT载体表达的构建)和 在体内通过培育过表达或“敲除”的转基因小鼠 甲状旁腺素反应元件结合蛋白基因。

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.