Role of mechanically activated Src/mTORC2 signaling on cytoskeletal adaptation

描述(申请人提供)：空间、化学和机械信号都有助于多潜能间充质干细胞(MSCs)的谱系分配。当MSCs的命运倾向于脂肪生成而不是成骨时，在卸载、老化或雌激素缺乏的情况下，骨质量降低，骨折风险增加。动态骨骼负荷通过增强MSCs的连环蛋白活性来抑制体内外脂肪的形成。MSC的潜势是通过信号级联来保持的，该信号级联是在局部粘连(FAs)时启动的，引发了mTORC2-Akt-GSK3？和-catenin的级联。MTORC2是如何在足总被力激活的尚不清楚。我们的初步数据表明，mTORC2需要Src才能激活。SRC激酶参与机械调节的信号事件，尤其是激活新FA形成所必需的RhoA。我们的数据表明，RhoA的激活需要mTORC2，这表明RhoA激活对Src的要求可能是通过mTORC2的激活间接实现的。这项建议的重点将是研究Src激酶在依赖应变的mTORC2激活中的作用以及这些信号事件对细胞骨架适应的贡献。这些问题将通过以下具体目标来研究：1)确定src家族激酶的机械激活如何导致mTORC2的激活；2)确定mTORC2在力依赖的RhoA激活中的作用；3)确定src的机械激活是否通过细胞骨架适应而增强。药理抑制/基因敲除研究将使用原代骨髓来源的MSC来检查与细胞骨架重塑的机械调节相关的Src激酶和其他信号分子。这些研究有助于理解机械负荷调节细胞骨架组装和增强的机制，这一过程对于适当调节机械感觉和细胞骨架适应是必不可少的。这项建议中概述的研究培训，再加上一个出色的指导委员会和北卡罗来纳大学充足的资源，为促进必要的科学发展提供了完美的环境，从而启动了富有成效的独立学术研究事业。

DESCRIPTION (provided by applicant): Spatial, chemical, and mechanical signals all contribute to lineage allocation of pluripotent mesenchymal stem cells (MSCs). When the fate of MSCs tips in favor of adipogenesis and away from osteogenesis in conditions such as unloading, aging, or estrogen deficiency, bone quality is diminished and risk of fracture increases. Dynamic skeletal loading inhibits adipogenesis in vitro and in vivo by enhancing ¿-catenin activity in MSCs. MSC potential is preserved by a signaling cascade, which is initiated at focal adhesions (FAs) setting off a cascade of mTORC2 to Akt to GSK3¿ and ¿-catenin. How mTORC2 is activated by force at the FA is unknown. Our preliminary data suggests mTORC2 requires Src for activation. Src kinases participate in mechanically regulated signaling events notably in activating RhoA necessary for new FA formation. Our data indicates that mTORC2 is required for RhoA activation, suggesting that the requirement of Src in RhoA activation may be indirect via the activation of mTORC2. The focus of this proposal will be to examine the role of Src kinases in strain-dependent mTORC2 activation and the contribution of those signaling events to cytoskeletal adaptation. These questions will be examined through the following specific aims: 1) determine how mechanical activation of Src-family kinases causes mTORC2 activation; 2) define the role of mTORC2 in force-dependent RhoA activation; 3) determine if mechanical activation of Src is enhanced by cytoskeletal adaptation. Pharmacological inhibition/knockdown studies will be performed using primary marrow-derived MSC to examine Src kinases and other signaling molecules associated with mechanical regulation of cytoskeletal remodeling. These studies have implications for understanding the mechanism by which mechanical loading regulates cytoskeletal assembly and reinforcement, a process essential for proper regulation of mechanosensation and cytoskeletal adaptation. The research training outlined in this proposal, combined with an outstanding mentoring committee and the ample resources of UNC provide the perfect environment to foster the necessary scientific growth to launch a productive, independent academic research career.