CYTOSKELETAL FUNCTION IN OSTEOBLAST MECHANOTRANSDUCTION

此R03提案的长期目标是定义如何应用 对成骨细胞的流体剪切力促进基因转录和新的 骨形成。骨的机械刺激诱导新骨 在形成之前，必需基因的表达增加 用于骨基质蛋白的沉积。骨细胞体液剪切力 培养，导致肌动蛋白细胞骨架的重组和刺激 关于基因表达的。我们认为成骨细胞可以检测并响应 使用涉及整合素的传感装置在骨骼中的机械刺激 在连接到内部肌动蛋白细胞骨架的细胞表面。这 传感装置可以将机械刺激转化为肌动蛋白的变化 增加内力发展的纤维组织， 反过来，向细胞核发出信号，增加必要基因的表达 以形成新的骨基质。将使用MC 3T3-E1成骨细胞 作为一个模型来检验假说，即骨骼中的机械转导 涉及由于以下原因导致的细胞内张力增加：1) 在骨内将肌动蛋白细丝重组为收缩应力纤维 细胞，以及2)将应力纤维中的肌动蛋白细丝固定在 质膜的内面。提出了两个具体目标，以 检验这一假设。在目标1中，我们将确定是否扰乱 肌动蛋白细丝和整合素之间的连接抑制了流体剪切- 诱导信号。这一目标将决定肌动蛋白-膜锚定 是导致应力纤维的机械转导途径的一部分 环氧合酶-2(COX-2)和c-选择素的形成和表达增加 FOS。在目标2中，我们将研究调节应力纤维的机制。 对流体剪切作出响应的地层。具体来说，我们将确定 肌球蛋白轻链磷酸化及其与GTP结合的作用 Rho蛋白在流体剪切诱导信号中的作用。这一目标将考验 肌球蛋白轻链激酶的作用，以及激活 GTP酶Rho在应激纤维形成及COX-2表达中的作用 和c-fos。这些研究旨在提高我们对 机械转导促进新陈代谢形成的机制 骨头。

The long range goal of this R03 proposal is to define how application of fluid shear forces to osteoblasts promotes gene transcription and new bone formation. Mechanical stimulation of bone induces new bone formation that is preceded by increased expression of genes necessary for deposition of bone matrix proteins. Fluid shear of bone cells, in culture, causes reorganization of the actin cytoskeleton and stimulation of gene expression. We propose that osteoblasts detect and respond to mechanical stimuli in bone using a sensing apparatus involving integrins at the cell surface that link to the internal actin cytoskeleton. This sensing apparatus may translate mechanical stimuli into changes in actin filament organization that increase internal force development which, in turn, signals the nucleus to increase expression of genes necessary for new bone matrix formation. MC 3T3-E1 osteoblasts cells will be used as a model to test the hypothesis that mechanotransduction in bone involves increased intracellular tension that occurs as a result of: 1) reorganizing actin filaments into contractile stress fibers inside bone cells, and 2) anchoring the actin filaments in stress fibers to the inner face of the plasma membrane. Two specific aims are proposed to test this hypothesis. In aim 1 we will determine if disrupting the linkage between actin filaments and integrins inhibits fluid shear- induced signaling. This aim will determine if actin-membrane anchorage is part of the mechanotransduction pathway leading to stress fiber formation, and increased expression of cyclooxygenase-2 (Cox-2) and c- fos. In aim 2 we will examine the mechanisms that regulate stress fiber formation in response to fluid shear. Specifically, we will determine the role of myosin light chain phosphorylation and the GTP-binding protein rho in fluid shear-induced signaling. This aim will test the role of the enzyme myosin light chain kinase, and activation of the GTPase rho, in the formation of stress fibers and expression of Cox-2 and c-fos. These studies are designed to improve our understanding of the mechanisms of mechanotransduction in promoting formation of new bone.