Mechanical Signaling through Osteoblast Focal Adhesions

描述(由申请人提供)：本方案的总体目标是了解调节骨骼对机械负荷的合成代谢反应的细胞机制。骨的机械负荷导致间质液体在骨内间隙内的运动。流体剪应力(FSS)引起的对成骨细胞和骨细胞的机械刺激被认为在骨对机械载荷的反应中发挥作用。对这一假说的支持来自于观察到，与在静态培养条件下保持的细胞相比，在体外受到FSS刺激的成骨细胞和骨细胞表现出更高的代谢活性。这一应用的中心焦点是确定调节骨细胞对其局部环境的机械敏感性的细胞机制。具体地说，我们建议研究整合素细胞黏附分子介导的成骨细胞和骨细胞与细胞外基质之间的黏附部位(通常称为灶性接触或灶性黏附)在调节骨细胞对FSS的反应中所起的作用。这项研究的结果应该能更好地理解骨细胞对机械负荷的反应的基本细胞和分子机制，通过检验局部粘连作为机械受体的假设，通过协调与整合素相互作用的细胞质信号分子的活动来实现。骨细胞对单向和振荡流体流动的生理反应包括：增殖活性改变和向合成代谢或成骨表型分化增强，基因表达改变，前列腺素合成增加，丝裂原活化蛋白激酶(MAPK)和粘着斑激酶(FAK)激活，胰岛素样生长因子受体(IGF-1R)对IGF-1刺激敏感。提出了三个具体目标。在目标1中，我们将确定破坏局灶性粘连结构完整性对成骨细胞对FSS的生化反应的影响。在目标2中，我们将确定粘着斑激酶(FAK)介导的信号转导通路在FSS激活的机械转导中的作用。在目标3中，我们将确定转录因子NMP4/CIZ在介导局部粘连激活的信号转导通路中的作用。总而言之，这些研究应该为骨中介导机械转导的细胞机制提供新的信息。

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.