Mechanical Partitioning of mTORC2 to Direct Mesenchymal Stem Cell Fate

mTORC2 的机械分区指导间充质干细胞的命运

基本信息

批准号：
9099270
负责人：
William Roy Thompson
金额：
$ 46.64万
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：
2016
资助国家：
美国
起止时间：
2016-06-10 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9099270
关键词：
Actins Adipocytes Adolescent Affect Aging American Biological Assay Bone Marrow Cardiovascular Diseases Cell Lineage Chemicals Child Complement Complex Cytoskeletal Modeling Cytoskeleton Data Development Disease Elderly Equilibrium Estrogens Event Exercise FRAP1 gene Fatty acid glycerol esters Focal Adhesions Fracture Future Guanine Guanine Nucleotide Exchange Factors Health In Vitro Integrin beta Chains Integrins Malignant Neoplasms Marrow Mechanics Mediating Mesenchymal Stem Cells Modification Molecular Motors Motor Movement Myosin ATPase Non-Insulin-Dependent Diabetes Mellitus Nuclear Obesity Osteogenesis Osteoporosis Overweight PTK2 gene Phosphorylation Phosphorylation Site Phosphotransferases Process Proteins Psychological reinforcement Recruitment Activity Regulation Repression Ribosomal Protein S6 Kinase Risk Role Signal Pathway Signal Transduction Site Site-Directed Mutagenesis Skeleton Small Interfering RNA Stress Fibers Structure United States Walking Western Blotting Work base beta catenin bone bone cell bone health bone quality design disability exercise intervention in vivo knock-down lifetime risk lipid biosynthesis obesity in children osteogenic prevent public health relevance response rho sedentary lifestyle skeletal stem cell differentiation stem cell fate upstream kinase

项目摘要

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.

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 的命运在诸如卸载、衰老、或雌激素缺乏，骨质量下降，骨折风险增加，动态骨骼负荷通过增强 MSC 中的 β-连环蛋白活性来抑制体外和体内脂肪形成。 MSC 潜力通过信号通路得以保留，该信号通路在粘着斑 (FA) 处启动，引发 ↑Fyn/FAK 至 ↑mTORC2 至 ↑Akt 至 ↓GSK3β 和 ↑β-连环蛋白的级联。我们已经表明，机械应变招募 mTORC2。然而，负责这种细胞内信号分配的机制和 mTORC2 的特定激活位点初步研究表明，菌株诱导 mTORC2 与肌球蛋白马达的关联，就像其他细胞内“货物”（包括 β-整联蛋白）附着在肌球蛋白上并转运至 FA 一样，肌球蛋白可能使 mTORC2 募集到 FA 上。我们还表明 Fyn 和 mTORC2/Akt 参与 RhoA 的机械调节激活；然而，初步工作表明 GEF RhoA 诱导的细胞骨架重组和脂肪形成抑制可能需要 GAP 中介体，该提案的重点是检查 mTORC2 特异性亚基的调节修饰，并确定菌株如何将 mTORC2 招募到 FA 中以被激活。询问 mTORC2/Akt 如何调节 GEF 和 GAP RhoA 效应器来自动调节细胞骨架以响应物理力。以下具体目标：1) 确定 mTORC2 如何被机械应变激活；2) 确定 mTORC2 调节细胞骨架重组的机制，将使用原代骨髓来源的 MSC 来检查肌球蛋白介导的 mTORC2 FA 募集，从而确定 mTORC2 调节细胞骨架重组的机制。识别 mTORC2 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、 GAP Pull Down 测定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重要意义，这是正确调节机械感觉和 MSC 谱系命运的过程。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设计提供信息。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Differential Iron Requirements for Osteoblast and Adipocyte Differentiation.

DOI：
10.1002/jbm4.10529
发表时间：
2021-09
期刊：
JBMR plus
影响因子：
3.8
作者：
Edwards DF 3rd;Miller CJ;Quintana-Martinez A;Wright CS;Prideaux M;Atkins GJ;Thompson WR;Clinkenbeard EL
通讯作者：
Clinkenbeard EL

Skeletal Functions of Voltage Sensitive Calcium Channels.