Role of Src Kinase in Mechanically-Induced Bone Formation

Src 激酶在机械诱导骨形成中的作用

基本信息

批准号：
9174915
负责人：
Fredrick M Pavalko
金额：
$ 51.71万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9174915
关键词：
Alleles Antibody Therapy Binding Bone Density Bone Resorption Botox Cell Culture Techniques Cell Nucleus Cells Clinical Conceptions CpG Islands Cytoplasm DNA DNA Methylation DNA-Binding Proteins Defect Dissection Epigenetic Process Exercise Fluorescence Resonance Energy Transfer Fracture Gene Targeting Genes Genetic Recombination Goals Health Human In Vitro Integrins Knockout Mice Liquid substance LoxP-flanked allele Mechanical Stimulation Mechanics Mediating Membrane Methylation Microscopy Molecular Movement Multiprotein Complexes Mus Osteoblasts Osteoclasts Osteocytes Osteogenesis Pathway interactions Pharmacology Phase III Clinical Trials Phenotype Phosphotransferases Play Population Predisposition Process Proline Protein Binding Domain Protein Tyrosine Kinase Reaction Reagent Reporting Research Resistance Risk Role Signal Pathway Signal Transduction Skeleton Stimulus TNFSF11 gene Tail Suspension Tamoxifen Therapeutic Time Translating Visual Work arm bone bone cell bone loss bone mass bone quality bone strength cell type dentin matrix protein 1 epigenetic regulation experimental study fluorescence lifetime imaging gene product improved in vivo inhibitor/antagonist long bone mechanical load mechanotransduction novel novel strategies osteogenic prevent promoter response shear stress skeletal src-Family Kinases

项目摘要

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.

项目摘要：我们寻求了解指导骨形成和骨形成的分子机制对这些机械负荷的药物操作的吸收。骨细胞中的 (MTD) 信号传导过程具有治疗潜力。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）的根本目标是操纵 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是药物抑制骨细胞（OCY）的最有效方法。骨骼中丰富的细胞类型，协调骨骼对机械负荷的反应。酪氨酸激酶 Src 在 OCY 中作为负载诱导的整体 Src 的新型抑制剂发挥作用。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。然而，我们可能会忽视酪氨酸激酶在骨吸收中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 的合成代谢臂我们认为 Src 在骨吸收中还有一个未被充分认识的作用。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我们建议通过实验剖析相关的 CpG 岛的分子机制。 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提出了三个目标：目标 1 将确定定向 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 3 将确定 Src 在机械敏感骨基因的表观遗传调控中的作用。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。