OSTEOBLAST PHENOTYPE SUPPRESSION BY PARATHYROID HORMONE

描述：(改编自调查人员摘要)申请 建议研究慢性甲状旁腺激素的观察基础 给药抑制成骨细胞分化。为了获得洞察力 关于这种荷尔蒙效应的分子机制， 申请者选择将注意力集中在骨桥蛋白基因上。 骨桥蛋白是骨基质的一种成分，基于体外实验 使用分化成骨细胞的细胞培养研究，表达在 前成骨细胞向完全分化的成骨细胞的转变。 以往的研究表明，骨桥蛋白基因转录是 甲状旁腺素、福司可林和cAMP类似物对其有抑制作用。骨桥蛋白启动子 对骨化三醇(维生素D3)和佛波酯有反应成分， 但不包含阵营响应元素。这一假设认为 申请人建议测试的是骨桥蛋白的下调 甲状旁腺激素的基因表达是通过诱导早期可诱导的cAMP介导的 抑制物(冰块)。这种基因已被证明可以下调佛波尔。 响应要素。为了检验这一假设，申请人 建议使用一种特性良好的大鼠颅骨成骨细胞系， IRC 10/30-myc1，以显示佛波酯反应元件和 ICER的表达是必要和充分的，以提供 甲状旁腺素抑制骨桥蛋白基因表达的分子信号。 应用程序的具体目标是：1)显示AP-1站点 (佛波尔反应元件)对于PTH来说是必要和充分的 抑制骨桥蛋白基因转录；2)证明甲状旁腺素和骨桥蛋白 CAMP诱导成骨细胞系ICER蛋白的形成 3)证明ICERs的过表达抑制转录 来自骨桥蛋白启动子。

DESCRIPTION: (Adapted from investigator's abstract) The application proposes to study the basis of the observation that chronic PTH administration suppresses osteoblast differentiation. To gain insights into the molecular mechanism involved in this hormone effect, the applicant has chosen to focus his attention on the osteopontin gene. Osteopontin is a component of the bone matrix that, based on in vitro cell culture studies using differentiating osteoblasts, is expressed at the transition of pre-osteoblasts into fully differentiated osteoblasts. Previous studies have shown that osteopontin gene transcription is inhibited by PTH, forskolin and cAMP analogues. The Osteopontin promoter has response elements for calcitriol (vitamin D3) and phorbol esters, but does not contain a cAMP response element. The hypothesis that the applicant proposes to test is that the downregulation of osteopontin gene expression by PTH is mediated via induction of inducible cAMP early repressors (ICERS). This gene has been shown to downregulate phorbol response elements. In order to test the hypothesis, the applicant proposes to use a well-characterized rat calvarial osteoblast cell line, IRC 10/30-myc1, to show that the phorbol ester response elements and the expression of ICERs are necessary and sufficient to provide the molecular signals for PTH suppression of osteopontin gene expression. The Specific Aims of the application are: 1) to show that AP-1 sites (phorbol response elements) are necessary and sufficient for PTH to suppress osteopontin gene transcription; 2) to demonstrate that PTH and cAMP induce the formation of ICER proteins in osteoblast cell lines; and 3) to demonstrate that overexpression of ICERs suppresses transcription from the osteopontin promoter.