Mechanical Signaling through Osteoblast Focal Adhesions

描述（由申请人提供）：本提案的总体目标是了解介导骨对机械负荷的合成代谢反应的细胞机制。骨的机械载荷诱导骨间隙内的间隙液运动。由流体剪切应力（FSS）引起的成骨细胞和骨细胞的机械刺激被假设在骨对机械负荷的反应中起作用。对这一假设的支持来自于观察，即与在静态培养条件下维持的细胞相比，体外受FSS刺激的成骨细胞和骨细胞表现出更高的代谢活性。本应用的中心焦点是确定介导骨细胞对局部环境的机械敏感性的细胞机制。具体来说，我们建议研究成骨细胞和骨细胞与细胞外基质之间的粘附位点（通常称为局灶接触或局灶粘附）在调节骨细胞对FSS的反应中的作用，这些粘附位点是由整合素细胞粘附分子介导的。本研究的结果应该通过验证局灶黏附作为机械受体通过协调与整合素相互作用的细胞质信号分子的活性而起作用的假设，从而提高对介导骨细胞对机械负荷反应的基本细胞和分子机制的理解。骨细胞对单向和振荡流体的生理相关反应包括增殖活性的改变和向合成代谢或成骨细胞表型分化的增强、基因表达的改变、前列腺素产生的增加、丝裂原活化蛋白激酶（MAPK）和局灶黏着激酶（FAK）的激活以及胰岛素样生长因子受体（IGF-1R）对IGF-1刺激的敏化。提出了三个具体目标。在目的1中，我们将确定破坏局灶粘连的结构完整性对成骨细胞对FSS的生化反应的影响。在Aim 2中，我们将确定FSS激活的focal adhesion kinase （FAK）介导的信号转导通路在机械转导中的作用。在Aim 3中，我们将确定转录因子NMP4/CIZ在响应流体剪切应力时通过局灶粘连激活的信号转导途径中的介导作用。总之，这些研究应该提供新的信息，细胞机制介导骨的机械转导。

DESCRIPTION (provided by applicant): The overall goal of this proposal is understand the cellular mechanisms that mediate the anabolic response of bone to mechanical loading. Mechanical loading of bone induces the movement of interstitial fluid within the spaces inside bone. The resulting mechanical stimulation of osteoblasts and osteocytes caused by fluid shear stress (FSS) is hypothesized to play a role in the response of bone to mechanical loading. Support for this hypothesis comes from the observation that osteoblasts and osteocytes that are stimulated by FSS in vitro exhibit increased metabolic activity compared to cells that are maintained under static culture conditions. The central focus of this application is to determine the cellular mechanisms that mediate the mechanical sensitivity of bone cells to their local environment. Specifically, we propose to examine the role of adhesion sites (commonly referred to as focal contacts or focal adhesions) between osteoblasts and osteocytes to the extracellular matrix that are mediated by integrin cell adhesion molecules in regulating the response of bone cells to FSS. The results of this study should provide an improved understanding of the fundamental cellular and molecular mechanisms that mediate the response of bone cells to mechanical loading by testing the hypothesis that focal adhesions function as mechanoreceptors by coordinating the activity of cytoplasmic signaling molecules that interact with integrins. Physiologically relevant responses by bone cells to unidirectional and oscillatory fluid flow include altered proliferative activity and enhanced differentiation toward an anabolic or osteoblastic phenotype, altered gene expression, increased prostaglandin production, activation of mitogen activated protein kinases (MAPK) and focal adhesion kinase (FAK) and sensitization of the insulin-like growth factor receptor (IGF-1R) to stimulation by IGF-1. Three specific aims are proposed. In Aim 1 we will determine the effect of disrupting the structural integrity of focal adhesions on biochemical response of osteoblasts to FSS. In Aim 2 we will determine the role of focal adhesion kinase (FAK) mediated signal transduction pathways activated by FSS in mechanotransduction. In Aim 3 we will determine the role of the transcription factor NMP4/CIZ in mediating signal transduction pathways activated through focal adhesions in response to fluid shear stress. Together these studies should provide novel information on the cellular mechanisms that mediate mechanotransduction in bone.