MECHANICAL LOADING OF BONE AND PROSTAGLANDINS

骨和前列腺素的机械装载

• 批准号: 6322141
• 负责人: CAROL C. PILBEAM
• 金额: $ 27.36万
• 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/6322141
• 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《调查者摘要》)：适应性反应 从骨到机械载荷是骨生物学的一个基本原则。这个 机械负荷对骨转换动态平衡的重要性 在预防骨质疏松症中强调保持骨骼完整性 程序。机械刺激在正畸治疗中是必不可少的 整形外科。尽管如此，人们对从地球上 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液体流动 然后在细胞膜上施加剪切力。上一首 研究表明，前列腺素(PGs)可以调节某些合成代谢 机械载荷对骨骼和流体剪应力(FSS)的影响 通过诱导环氧合酶-2(COX-2)刺激前列腺素的产生 表情。此应用程序的目标是阐明FSS通过的途径 诱导成骨细胞表达环氧合酶-2，探讨环氧合酶-2在成骨细胞中的作用 FSS的中介作用。此研究应用程序利用了 不寻常的大规模流室与转COX-2基因小鼠 启动子-融合到荧光素酶报告基因(PLUC)。我们将描述 FSS对成骨细胞MC3T3-E1和原代细胞COX-2的诱导作用 成骨细胞。将研究MC3T3-E1细胞的转录调控 稳定表达Pluc基因及其在原代成骨细胞中的表达 转基因小鼠。我们将检验FSS对COX-2的诱导 成骨细胞的转录是通过蛋白激酶C(PKC)介导的 激活细胞外调节激酶(ERK)信号通路。vbl.使用 定点突变和5‘-序列缺失分析，我们将检查 假定的顺式作用部位的作用，包括“剪应力反应” 元件“和AP-1结合位点，介导COX-2诱导FSS 启动子活性。我们将观察FSS对原代成骨细胞的影响 增殖分化与支持因子的表达 破骨细胞生成。为了评估COX-2在这些效应中的作用，我们将使用 携带COX-2基因的小鼠的原代成骨细胞被破坏。最后，我们会 绿色荧光融合环氧合酶-2启动子转基因小鼠的建立 蛋白质(GFP)。这些小鼠将经历短暂的负荷期 后肢卸载(尾部悬吊)后GFP的组织学观察 荧光检测骨细胞对COX-2的表达 正在装车。