Mechanical Loading of Bone and Prostaglandins

骨和前列腺素的机械负荷

• 批准号: 7148285
• 负责人: CAROL C. PILBEAM
• 金额: $ 32.56万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7148285
• 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 (provided by applicant): Mechanical loading of bone maintains bone mass and skeletal integrity. Loading stimulates new bone formation and unloading causes bone loss. Cyclooxygenase (COX)-2, the major enzyme for prostaglandin (PG) responses in bone, can enhance both bone formation and resorption and may mediate some effects of either loading or unloading. Our overall goal is to understand how mechanical signals are transmitted into biochemical signals and the role of COX-2 in these processes. Mechanical strains in bone can generate interstitial fluid flow that exerts fluid shear stress (FSS) on cells and induces COX-2. Because in vivo studies suggest that effects of short duration loading are not improved by longer duration, we believe the signaling cascade after removal of FSS will be important for determining outcomes of loading. In vitro, we will examine responses of COX-2, and selected other genes important for bone turnover, in osteoblastic and osteocytic cells during and after stopping FSS. We will determine if the protein kinase D (PKD) pathway mediates the FSS induction of COX-2 via activation of ERK after stopping FSS; if the transcription factors Cbfa1 (Runx2) and NFAT are mediators of the FSS induction of COX-2 transcription; and if the development of COX-2 expression after FSS also depends on regulation of mRNA stability. We will assess the role of COX-2 produced PGs, induced by FSS, in FSS effects on other genes using nonsteroidal anti-inflammatory drugs (NSAIDs) and cells derived from Cox-2+/+ and Cox-2-/- mice. In vivo, we will study effects of dynamic axial loading of the ulnae and effects of unloading of hind limbs by tail suspension. We will use mice with Cox-2 deletion conditionally targeted to osteoblastic and osteocytic cells with the 2.3-kb Co1a1 promoter driving Cre recombinase (Col1a1-Cre) or the 2.3-kb Col1a1 promoter driving Cre recombinase fused to a mutated ligand binding domain of the estrogen receptor (Col1a1-CreERT2), which becomes active only after administration of the synthetic estrogen antagonist 4-hydroxy-tamoxifen. We will examine potential compensation by cyclooxygenase (COX)-1 for absent COX-2 by comparing targeted Cox-2 deletion in Cox- 1+/+ and Cox-1-/- mice. We will measure bone gain or loss and gene expression at intervals after loading or unloading to determine effects of Cox-2 deletion (+/- Cox-1 deletion) on the response of other genes that may mediate effects of loading and unloading. Public health relevance: Physical activity that mechanically loads bone can increase bone mass and decrease bone loss. Osteoporosis, associated with bone loss and leading to debilitating skeletal fractures, increases with age and physical inactivity. A better understanding of how mechanical loading regulates bone may help us to develop better therapies for prevention and treatment of osteoporosis.

描述（由申请方提供）：骨的机械负荷可维持骨量和骨骼完整性。负荷刺激新骨形成，卸载导致骨丢失。环氧合酶（考克斯）-2是骨中前列腺素（PG）反应的主要酶，可促进骨形成和骨吸收，并可能介导某些负荷或卸载作用。我们的总体目标是了解机械信号是如何传递到生化信号和考克斯-2在这些过程中的作用。骨中的机械应变可产生间质液流，其对细胞施加流体剪切应力（FSS）并诱导考克斯-2。由于体内研究表明，短期负荷的影响并没有改善较长的持续时间，我们相信信号级联后，去除FSS将是重要的，以确定结果的负荷。在体外，我们将检查考克斯-2的反应，并选择其他重要的基因骨转换，在成骨细胞和骨细胞细胞的过程中和停止FSS后。我们将确定蛋白激酶D（PKD）途径是否在停止FSS后通过激活ERK介导FSS对考克斯-2的诱导;转录因子Cbfa 1（Runx 2）和NFAT是否是FSS诱导考克斯-2转录的介导剂;以及FSS后考克斯-2表达的发展是否也取决于mRNA稳定性的调节。我们将使用非甾体抗炎药（NSAID）和来自考克斯-2+/+和考克斯-2-/-小鼠的细胞，评估FSS诱导的考克斯-2产生的PG在FSS对其他基因的影响中的作用。在体内，我们将研究尺骨的动态轴向负荷的影响和尾部悬吊对后肢的卸载影响。我们将使用具有考克斯-2缺失的小鼠，其条件性靶向成骨细胞和骨细胞细胞，所述成骨细胞和骨细胞具有驱动Cre重组酶的2.3-kb Co 1a 1启动子（Col 1a 1-Cre）或驱动Cre重组酶的2.3-kb Col 1a 1启动子（Col 1a 1-CreERT 2），所述Cre重组酶与雌激素受体的突变配体结合结构域融合，所述雌激素受体仅在施用合成雌激素拮抗剂4-羟基-他莫昔芬后才变得活跃。我们将通过比较考克斯- 1+/+和考克斯-1-/-小鼠中的靶向考克斯-2缺失来检查环氧合酶（考克斯）-1对缺失考克斯-2的潜在补偿。我们将在加载或卸载后间隔测量骨增加或损失以及基因表达，以确定考克斯-2缺失（+/-考克斯-1缺失）对可能介导加载和卸载效应的其他基因的反应的影响。与公共卫生的相关性：对骨骼施加机械负荷的体力活动可以增加骨量，减少骨质流失。骨质疏松症与骨质流失有关，并导致骨骼骨折，随着年龄的增长和身体不活动而增加。更好地了解机械负荷如何调节骨可能有助于我们开发更好的治疗方法来预防和治疗骨质疏松症。