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

• 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
• 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

 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(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骨髓间充质干细胞的β-连环蛋白活性抑制体内外脂肪生成。细胞间充质干细胞的潜能是由一个信号通路保持的，该通路在局部粘连(FAs)时启动，引发↑Fyn/FAK、↑mTORC2、↑Akt、↓Gsk3、β和↑β-catenin的级联反应。我们已经证明，机械应变将mTORC2和Akt招募到FAs；然而，负责这种细胞内信号分割的机制和mTORC2亚基的特定激活位置尚不清楚。初步工作表明，该菌株诱导了mTORC2与肌球蛋白马达的关联。就像其他细胞内的“货物”，包括β-整合素，附着在肌球蛋白上被运输到FA，肌球蛋白可能会在机械力的作用下将mTORC2重新招募到FA。我们还发现Fyn和mTORC2/Akt参与了RhoA的机械调节激活；然而，初步的工作表明，在RhoA诱导的细胞骨架重组和成脂抑制过程中，可能需要环境基金或GAP中介机构。这项提案的重点将是研究mTORC2特异性亚基的监管修改，并确定菌株如何招募mTORC2到FA被激活。此外，我们还将探讨mTORC2/Akt如何调节环境基金和GAP RhoA效应器，以自动调节细胞骨架以响应物理压力。提出的假设将通过以下具体目标进行检验：1)确定机械应变如何激活mTORC2；2)确定mTORC2调节细胞骨架重组的机制。将使用原代骨髓间充质干细胞进行药理抑制/基因敲除研究，以检查肌球蛋白介导的mTORC2 FA募集，确定mTORC2亚单位上机械响应的磷酸化位点，并确定这些修饰如何影响细胞骨架重构。此外，还将使用RhoA、Gap和Gap下拉试验来研究力对细胞骨架适应调节器的影响。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，这一过程对于适当调节机械感觉和MSC谱系命运至关重要。因此，这项工作将为设计未来利用运动影响脂肪和骨骼发育的策略提供信息。

项目成果

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.

• DOI: 10.1007/s11914-020-00647-7
• 发表时间: 2021-04
• 期刊: Current osteoporosis reports
• 影响因子: 4.3
• 作者: Wright CS;Robling AG;Farach-Carson MC;Thompson WR
• 通讯作者: Thompson WR