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：我们试图了解指导骨形成和的分子机制响应机械负荷的回应。这些机械转导的药理操作（MTD）骨细胞中的信号传导过程具有治疗潜力。我们提出了一种新颖的策略研究抑制负载刺激作用的信号传导机制（而不是聚焦）在刺激新骨形成的信号通路上）。基本目标是操纵MTD 途径，即使是适度的运动水平也会产生代谢作用，如果抑制负荷诱导的骨形成被药物抑制。骨细胞（OCY），最多丰富的细胞类型在骨骼中，协调骨头对机械负荷的响应。我们建议酪氨酸激酶SRC在OCY中起作用，是负荷诱导的骨形成的新型抑制剂。全球SRC 空小鼠的骨骼质量高（HBM）。这部分归因于破骨细胞中的SRC依赖性缺陷介导的骨骼分辨率。但是，我们冒着失去酪氨酸激酶可能发挥的重要作用的风险如果我们将SRC KO小鼠的HBM表型完全归因于骨骼MTD的合成代谢组骨骼分辨率的破骨细胞缺陷。我们建议SRC在抑制机械诱导的合成代谢信号的成骨细胞/骨细胞（OB/OCY）种群。具体而言，我们建议在通过机械刺激激活后，SRC从整合素分离出来（膜机械传感器）并将其易位到核，作为多蛋白质复合物的一部分富含脯氨酸的激酶-2（PYK2）和甲基化的DNA结合蛋白甲基-CPG结合结构域蛋白-2（MBD2），用于调节关键骨基因的表观遗传学。那就是OCY可以使用SRCPYK2-MBD2 通过改变启动子 - 相关的CPG群岛。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，其长期目标是更好了解SRC抑制剂增强骨密度和断裂的临床和翻译潜力敏感性。提出了三个目的：AIM 1将确定目标SRC删除的影响在基本和负载诱导的骨形成以及小鼠骨质损失的骨细胞上。 AIM 2意志确定SRC在机械敏感骨基因的表观遗传调节中的作用。目标3意志确定SRC在成骨细胞和骨细胞的细胞质和核中的分子相互作用使用FRET-FLIM显微镜在体外经过液体剪切应力。