Role of mechanically activated Src/mTORC2 signaling on cytoskeletal adaptation

描述（由申请人提供）：空间、化学和机械信号都有助于多能间充质干细胞（MSCs）的谱系分配。当MSCs的命运倾向于脂肪形成，而在卸载、衰老或雌激素缺乏等情况下远离成骨时，骨质量降低，骨折风险增加。动态骨骼负荷通过增强MSCs中的¿-catenin活性抑制体外和体内脂肪生成。MSC电位通过信号级联保存，该级联始于局灶粘连（FAs），引发mTORC2到Akt到GSK3¿和¿-catenin级联。mTORC2如何在FA上被强力激活尚不清楚。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，特别是激活新FA形成所需的RhoA。我们的数据表明，mTORC2是RhoA激活所必需的，这表明Src在RhoA激活中的要求可能是通过mTORC2的激活间接实现的。本研究的重点是研究Src激酶在菌株依赖性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进行研究：1)确定src家族激酶的机械激活如何导致mTORC2激活；2)明确mTORC2在力依赖性RhoA激活中的作用；3)确定Src的机械活化是否因细胞骨架适应而增强。将使用原代骨髓来源的MSC进行药理学抑制/敲除研究，以检查Src激酶和其他与细胞骨架重塑机械调节相关的信号分子。这些研究对理解机械负荷调节细胞骨架组装和增强的机制具有重要意义，这是机械感觉和细胞骨架适应的适当调节过程。本提案中概述的研究培训，结合优秀的指导委员会和北卡罗来纳大学丰富的资源，为培养必要的科学成长提供了完美的环境，以启动一个富有成效的、独立的学术研究生涯。

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.