Mechanical Partitioning of mTORC2 to Direct Mesenchymal Stem Cell Fate

 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（MSC）的谱系分配。当MSC的命运倾向于脂肪生成而远离骨生成时，如卸载，老化或雌激素缺乏，骨质量降低，骨折风险增加。动态骨骼负荷通过增强骨髓间充质干细胞中β-连环蛋白活性抑制体内外脂肪形成。MSC的潜能通过信号传导途径得以保持，该信号传导途径起始于粘着斑（FA），引发↑Fyn/FAK → ↑ mTORC 2 → ↑Akt → ↓ GSK 3 β和↑β-连环蛋白的级联反应。我们已经表明，机械应变招聘mTORC 2和Akt的脂肪酸，然而，负责这种细胞内信号分配和mTORC 2亚基的特定激活位点的机制是未知的。初步工作表明，应变诱导mTORC 2与肌球蛋白马达的关联。正如其他细胞内“货物”（包括β-整联蛋白）附着于肌球蛋白以转运至FA，肌球蛋白可使mT 0 RC 2响应于机械力而募集至FA。我们还表明，Fyn和mTORC 2/Akt参与机械调节激活RhoA，然而，初步工作表明，GEF或GAP中介可能需要RhoA诱导的细胞骨架重组和脂肪形成抑制。该提案的重点将是检查mTORC 2特异性亚基的调节修饰，并确定菌株如何将mTORC 2招募到待激活的FA。此外，我们将询问mTORC 2/Akt如何调节GEF和GAP RhoA效应子以响应物理力而自动调节细胞骨架。提出的假设将通过以下具体目标进行检验：1）确定mTORC 2如何被机械应变激活; 2）确定mTORC 2调节细胞骨架重组的机制。将使用原代骨髓来源的MSC进行药理学抑制/敲低研究，以检查肌球蛋白介导的mTORC 2 FA募集，鉴定mTORC 2亚基上的机械响应磷酸化位点，并确定这些修饰如何影响细胞骨架重塑。此外，RhoA，GEF和GAP下拉试验将用于研究力对细胞骨架适应调节剂的影响。这些研究对于理解机械负荷调节细胞骨架组装和强化的机制具有意义，这是机械感觉和MSC谱系命运的适当调节所必需的过程。因此，这项工作将为未来利用运动影响脂肪和骨骼发育的策略的设计提供信息。

 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 β-catenin activity in MSCs. MSC potential is preserved by a signaling pathway, which is initiated at focal adhesions (FAs) setting off a cascade of ↑Fyn/FAK to ↑mTORC2 to ↑Akt to ↓GSK3β and ↑β-catenin. We have shown that mechanical strain recruits mTORC2 and Akt to FAs; however, the mechanisms responsible for this intracellular signal partitioning and the specific activation sites of mTORC2 subunits are unknown. Preliminary work suggests that strain induces an association of mTORC2 with myosin motors. Just as other intracellular "cargo", including β- integrins, attach to myosins to be transported to FAs, myosins may enable recruitment of mTORC2 to FAs in response to mechanical force. We have also shown that Fyn and mTORC2/Akt participate in mechanically regulated activation of RhoA; however, preliminary work suggests that GEF or GAP intermediaries may be required for RhoA-induced cytoskeletal reorganization and adipogenic repression. The focus of this proposal will be to examine the regulatory modifications of mTORC2-specific subunits and to determine how strain recruits mTORC2 to FAs to be activated. Additionally, we will ask how mTORC2/Akt regulate GEF and GAP RhoA effectors to auto-regulate the cytoskeleton in response to physical force. The proposed hypotheses will be examined through the following specific aims: 1) determine how mTORC2 is activated by mechanical strain; 2) identify mechanisms by which mTORC2 regulates cytoskeletal reorganization. Pharmacological inhibition/knockdown studies will be performed using primary marrow-derived MSCs to examine myosin- mediated mTORC2 FA recruitment, to identify mechanically responsive phosphorylation sites on mTORC2 subunits, and to determine how these modifications influence cytoskeletal remodeling. Additionally, RhoA, GEF, and GAP pull down assays will be employed to study the effects of force on modulators of cytoskeletal adaptation. These studies have implications for understanding the mechanisms by which mechanical loading regulates cytoskeletal assembly and reinforcement, a process essential for proper regulation of mechanosensation and MSC lineage fate. As such, this work will inform the design of future strategies for using exercise to affect development of fat and bone.