Role of Src Kinase in Mechanically-Induced Bone Formation

项目概述：我们试图了解指导骨形成的分子机制， 对机械负荷的再吸收。这些机械传导的药理学操作 (MTD)骨细胞中的信号传导过程具有治疗潜力。我们提出了一种新的策略， 研究抑制负荷刺激效应的信号机制（而不是聚焦 刺激新骨形成的信号通路）。基本目标是操纵MTD 因此，即使是适度的运动水平也会产生巨大的合成代谢效应， 受抑制负荷诱导的骨形成受到抑制。骨细胞（OCY）， 骨中丰富的细胞类型，协调骨对机械载荷的响应。我们建议 酪氨酸激酶Src在OCY中作为负荷诱导的骨形成的新抑制剂发挥作用。全球来源 无效小鼠具有高骨量（HBM）。这部分是由于破骨细胞中的Src依赖性缺陷， 介导的骨吸收。然而，我们有可能忽略了酪氨酸激酶可能在 骨骼MTD的合成代谢臂，如果我们将Src KO小鼠的HBM表型完全归因于 破骨细胞骨吸收缺陷。我们认为，Src在以下方面还有一个未被充分认识的作用： 成骨细胞/骨细胞（OB/OCY）群体，其抑制机械诱导的合成代谢信号。 具体地说，我们提出，在机械刺激激活后，Src从整合素中解离 （膜机械传感器）并作为多蛋白复合物的一部分易位到细胞核， 富含脯氨酸激酶2（Pyk2）与甲基化DNA结合蛋白甲基化CpG结合结构域 蛋白-2（MBD2），调节关键骨基因的表观遗传学。因此，OCY可以利用SrcPyk2-MBD2 - "机械体"，以通过改变启动子来促进或抑制合成代谢或抗分解代谢骨基因， 相关的CpG岛我们建议通过实验剖析分子机制， src抑制骨形成使用在体内和体外的方法与更好的长期目标 了解Src抑制剂增强骨密度和骨折的临床和转化潜力 易感性提出了三个目标：目标1将确定靶向Src缺失的效果， 骨细胞对小鼠基础和负荷诱导的骨形成以及废用诱导的骨丢失的影响。目标2将 确定Src在机械敏感性骨基因的表观遗传调控中的作用。目标3将 确定Src在成骨细胞和骨细胞的细胞质和细胞核中的分子相互作用 使用FRET-FLIM显微镜在体外经受流体剪切应力。

Project summary: We seek to understand the molecular mechanisms that direct bone formation and resorption in response to mechanical loading. Pharmacologic manipulation of these mechanotransduction (MTD) signaling processes in bone cells has therapeutic potential. We propose a novel strategy that investigates signaling mechanisms that suppress the stimulatory effects of loading (rather than focusing on signaling pathways that stimulate new bone formation). The fundamental goal is to manipulate MTD pathways so that even modest levels of exercise can have outsized anabolic effects if mechanisms that inhibit load-induced bone formation are pharmacologically suppressed. Osteocytes (OCY), the most abundant cell type in bone, coordinate the response of bone to mechanical load. We propose that the tyrosine kinase Src functions in OCY as a novel suppressor of load-induced bone formation. Global Src null mice have high bone mass (HBM). This is due in part to Src-dependent defects in osteoclast- mediated bone resorption. However, we risk missing an important role that tyrosine kinases may play in the anabolic arm of skeletal MTD if we attribute the HBM phenotype of Src KO mice entirely to an osteoclast defect in bone resorption. We suggest there is an additional underappreciated role for Src in the osteoblast/osteocyte (OB/OCY) population that inhibits mechanically-induced anabolic signals. Specifically, we propose that upon activation by mechanical stimuli, Src dissociates from integrins (membrane mechanosensors) and translocates to the nucleus as part of a multi-protein complex with Proline-rich Kinase-2 (Pyk2) and the methylated DNA binding protein Methyl-CpG Binding Domain Protein-2 (MBD2), to regulate epigenetics of key bone genes. Thus, OCY may utilize a SrcPyk2-MBD2 “mechanosome” to promote or suppress anabolic or anti-catabolic bone genes by altering promoter- associated CpG islands. We propose to experimentally dissect the molecular mechanisms through which Src inhibits bone formation using in vivo and in vitro approaches with the long term goal of better understanding the clinical and translational potential of Src inhibitors to enhance bone density and fracture susceptibility. Three aims are proposed: Aim 1 will determine the effect of targeted Src deletion from osteocytes on basal and load-induced bone formation and on disuse-induced bone loss in mice. Aim 2 will determine the role of Src in epigenetic regulation of mechanically sensitive bone genes. Aim 3 will determine the molecular interactions of Src in the cytoplasm and nucleus of osteoblasts and osteocytes subjected to fluid shear stress in vitro using FRET-FLIM microscopy.