Role of Src Kinase in Mechanically-Induced Bone Formation

项目摘要：我们寻求了解指导骨形成和骨形成的分子机制 响应机械负荷的吸收。这些机械转导的药理学操作 骨细胞中的 (MTD) 信号传导过程具有治疗潜力。我们提出了一种新颖的策略 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）。根本目标是操纵 MTD 因此，如果机制能够实现，即使是适度的运动也会产生巨大的合成代谢效应 抑制负荷诱导的骨形成受到药物抑制。骨细胞（OCY），最 骨骼中丰富的细胞类型，协调骨骼对机械负荷的反应。我们建议 酪氨酸激酶 Src 在 OCY 中发挥作用，作为负载诱导的骨形成的新型抑制剂。全球源 无效小鼠具有高骨量（HBM）。这部分是由于破骨细胞中 Src 依赖性缺陷所致。 介导的骨吸收。然而，我们可能会忽视酪氨酸激酶在以下方面可能发挥的重要作用： 如果我们将 Src KO 小鼠的 HBM 表型完全归因于骨骼 MTD 的合成代谢臂 骨吸收中的破骨细胞缺陷。我们建议 Src 还有一个未被充分重视的角色： 抑制机械诱导的合成代谢信号的成骨细胞/骨细胞（OB/OCY）群体。 具体来说，我们建议在机械刺激激活后，Src 与整合素解离 （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 富含脯氨酸的激酶 2 (Pyk2) 和甲基化 DNA 结合蛋白甲基 CpG 结合域 Protein-2 (MBD2)，调节关键骨基因的表观遗传学。因此，OCY 可以利用 SrcPyk2-MBD2 “mechanosome”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 使用体内和体外方法抑制骨形成，长期目标是更好地 了解 Src 抑制剂增强骨密度和预防骨折的临床和转化潜力 易感性。提出了三个目标： 目标 1 将确定目标 Src 删除的效果 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 显微镜在体外承受流体剪切应力。

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.