MECHANICAL LOADING OF BONE AND PROSTAGLANDINS

骨和前列腺素的机械装载

• 批准号: 6719007
• 负责人: CAROL C. PILBEAM
• 金额: $ 27.55万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-11 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6719007
• 关键词: biological signal transduction; bone metabolism; cell differentiation; cell proliferation; enzyme activity; enzyme induction /repression; fluid flow; gel mobility shift assay; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; green fluorescent proteins; homeostasis; laboratory mouse; mechanical stress; mitogen activated protein kinase; northern blottings; osteoblasts; osteogenesis; phosphorylation; prostaglandin endoperoxide synthase; prostaglandins; site directed mutagenesis; western blottings

DESCRIPTION (Adapted from the Investigator's Abstract): The adaptive response of bone to mechanical loading is a fundamental tenet of bone biology. The importance of mechanical loading for the homeostasis of bone turnover and the maintenance of skeletal integrity are emphasized in osteoporosis prevention programs. Mechanical stimuli are essential to therapies in orthodontics and orthopedics. Nevertheless, very little is known about the pathways leading from mechanical stimuli to altered gene transcription. A popular hypothesis is that mechanical strains generate interstitial fluid flow within the mineralized bone matrix, which subsequently exerts shear stress on cellular membranes. Previous studies suggest that prostaglandins (PGs) can mediate some of the anabolic effects of mechanical loading on bone, and fluid shear stress (FSS) can stimulate production of PGs via induction of cyclooxygenase-2 (COX-2) expression. The goal of this application is to elucidate pathways by which FSS induces COX-2 in osteoblastic cells and to examine the role of COX-2 in mediating effects of FSS. This research application takes advantage of an unusual large-scale flow chamber and mice transgenic for the COX-2 promoter-fused to a luciferase reporter (Pluc). We will characterize the induction of COX-2 by FSS in osteoblastic MC3T3-E1 cells and in primary osteoblasts. Transcriptional regulation will be studied in MC3T3-E1 cells stably transfected with Pluc constructs and in primary osteoblasts from Pluc transgenic mice. We will test the hypothesis that the FSS induction of COX-2 transcription in osteoblasts occurs via protein kinase C (PKC)-mediated activation of the extracellular regulated kinase (ERK) signaling pathway. Using site-directed mutagenesis and 5'-sequential deletion analysis, we will examine the roles of putative cis-acting sites, including a "shear stress response element" and an AP-1 binding site, in mediating the FSS induction of COX-2 promoter activity. We will look for effects of FSS on primary osteoblast proliferation and differentiation and expression of factors supporting osteoclastogenesis. To assess the role of COX-2 in these effects, we will use primary osteoblasts from mice with the COX-2 gene disrupted. Finally, we will develop mice transgenic for the COX-2 promoter fused to a green fluorescent protein (GFP). These mice will be subjected to brief periods of loading following hind limb unloading (tail suspension) and histology for GFP fluorescence done to identify bone cells expressing COX-2 in response to loading.

描述（改编自研究者摘要）：适应性反应 骨对机械负荷的耐受性是骨生物学的基本原则。的 机械负荷对骨转换稳态的重要性以及 预防骨质疏松症强调保持骨骼完整性 程序.机械刺激对于正畸治疗至关重要， 整形外科尽管如此，我们对这些信号的传导途径知之甚少。 机械刺激改变基因转录。一个流行的假设是， 机械应变在矿化骨内产生间质流体流动 基质，其随后对细胞膜施加剪切应力。先前 研究表明，前列腺素（PGs）可以介导一些合成代谢， 机械负荷对骨的影响和流体剪切应力（FSS）可以 通过诱导环氧合酶-2（考克斯-2）刺激PG产生 表情本申请的目的是阐明FSS 诱导成骨细胞中的考克斯-2，并检测考克斯-2在 FSS的介导作用。此研究应用程序利用了 不寻常的大规模流动室和考克斯-2转基因小鼠 与荧光素酶报告基因（Pluc）融合的启动子。我们将描述 FSS对成骨细胞MC 3 T3-E1和原代培养细胞中考克斯-2诱导作用 成骨细胞将在MC 3 T3-E1细胞中研究转录调控 用Pluc构建体稳定转染，并在来自Pluc的原代成骨细胞中 转基因小鼠。我们将检验FSS诱导考克斯-2表达的假设， 成骨细胞中的转录通过蛋白激酶C（PKC）介导的 细胞外调节激酶（ERK）信号通路的激活。使用 定点突变和5 '-顺序缺失分析，我们将检查 假定的顺式作用位点的作用，包括“剪切应力反应 在介导考克斯-2的FSS诱导中， 启动子活性我们将观察FSS对原代成骨细胞的影响 增殖和分化以及支持因子的表达 破骨细胞生成为了评估考克斯-2在这些效应中的作用，我们将使用 来自考克斯-2基因被破坏的小鼠的原代成骨细胞。最后我们将 开发融合绿色荧光标记的考克斯-2启动子的转基因小鼠 蛋白（GFP）。这些小鼠将接受短暂的负荷 在后肢卸载（尾部悬浮）和GFP的组织学检查后， 进行荧光检测以鉴定骨细胞表达考克斯-2， 加载中