Mechanical regulation of cytoskeleton guides beta-catenin effect on MSC fate

细胞骨架的机械调节引导β-连环蛋白对MSC命运的影响

• 批准号: 9252230
• 负责人: Janet E Rubin
• 金额: $ 33.44万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9252230
• 关键词: AKT inhibition; Actins; Adhesions; Adipocytes; Adipose tissue; Adult; Bone Formation Stimulation; Bone Marrow; Cell Lineage; Cell Nucleus; Cells; Complex; Cytoskeletal Modeling; Cytoskeleton; Data; Exercise; F-Actin; Fatty acid glycerol esters; Focal Adhesions; Goals; Grant; Health; Histologic; In Vitro; Knock-out; Knockout Mice; Lamins; Marrow; Measures; Mechanics; Mesenchymal Stem Cells; Methods; Mus; Nuclear; Obesity; Osteogenesis; Pathway interactions; Periodicity; Pharmacology; Phosphotransferases; Physicians; Physiological; Population; Proteins; Recruitment Activity; Regulation; Regulatory Pathway; Resistance; Role; Running; Scientist; Signal Pathway; Signal Transduction; Stem cells; Stress Fibers; Structure; Tamoxifen; Testing; Time; Work; base; beta catenin; bone; bone cell; bone fragility; bone health; bone strength; improved; in vivo; innovation; lipid biosynthesis; mechanical force; mechanical load; mouse model; osteogenic; osteoprogenitor cell; prevent; public health relevance; response; sedentary; skeletal; synergism; trafficking

 DESCRIPTION (provided by applicant): Exercise builds skeletal strength by inducing new bone formation, which is accompanied by decreased marrow adipose tissue (MAT). A lack of exercise enhances MSC adipogenesis, and increased MAT has been shown to be associated with bone fragility. Understanding mechanisms by which exercise regulates mdMSC lineage allocation to increase osteogenesis and decrease adipogenesis is thus significant for managing skeletal health in an increasingly sedentary population. We have shown that mechanical activation and nuclear trafficking of ßcatenin prevents adipogenesis and promotes MSC entry into osteoprogenitor lineage. ßcatenin signaling is initiated by recruitment of Fyn kinase to foca adhesions during strain, followed by Fyn activation of the mTORC2->Akt->GSK3ß inhibition cascade leading to increased ßcatenin. ßcatenin effects depend on cellular context: in mdMSC, increased cytoskeletal structure improves osteogenesis and cytoskeletal deficiency promotes adipogenesis. The Fyn/mTORC2 proximal signal also activates RhoA to build complexity and connectivity of the cytoskeleton. We propose that mechanically induced cytoskeletal reorganization promotes nuclear entry of ßcatenin, underwriting its anti-adipogenic, pro-osteogenic effect. Importantly, the role of focal adhesion-based Fyn in initiating ßcatenin and RhoA effectors may be unique to the mdMSC, potentially providing a specific pharmacologic target to enhance MSC response to exercise. To test this, we will delineate the synergistic contributions of Fyn/mTORC2's two downstream effectors, RhoA and ßcatenin, to MSC lineage fate allocation in vitro and in vivo. We will find if RhoA generated cytoskeletal reorganization is necessary or sufficient to alter mdMSC cell fate and define how mechanical load activates RhoA (SA1). In SA2, we ask if activation of ßcatenin can alter cell fate in the face of cytoskeletal deficiency, inquiring whether the cytoskeleton drives nuclear/cytoplasmic partitioning of ßcatenin and considering if the actin-nesprin-LINC-nuclear tether might guide ßcatenin into the nucleus. In SA3, using an innovative method to quantify and localize MAT, combined with measures of bone formation, we will investigate mechanical control of mdMSC lineage in vivo, asking if the Fyn/mTORC2 signaling unique to mdMSC is critical during exercise stimulation of bone formation; for this we will generate a Tamoxifen driven Prx1-Cre knock out of Fyn.

 描述（由申请人提供）：运动通过诱导新骨形成来增强骨骼强度，同时伴有骨髓脂肪组织（MAT）减少。缺乏运动会增强MSC脂肪生成，并且MAT增加已被证明与骨脆性相关。因此，了解运动调节mdMSC谱系分配以增加骨生成和减少脂肪生成的机制对于管理越来越多久坐人群的骨骼健康具有重要意义。我们已经证明，β连环蛋白的机械活化和核运输阻止脂肪形成并促进MSC进入骨祖细胞谱系。β连环蛋白信号传导通过在应变期间Fyn激酶募集至福卡粘附而启动，随后Fyn激活mTORC 2->Akt-> GSK 3 β抑制级联，导致β连环蛋白增加。β连环蛋白的作用取决于细胞环境：在mdMSC中，增加的细胞骨架结构改善骨生成，细胞骨架缺乏促进脂肪生成。Fyn/mT 0 RC 2近端信号还激活RhoA以建立细胞骨架的复杂性和连接性。我们认为，机械诱导的细胞骨架重组促进β连环蛋白进入细胞核，从而承担其抗脂肪形成、促成骨作用。重要的是，基于局灶性粘附的Fyn在启动β连环蛋白和RhoA效应物中的作用可能是mdMSC所独有的，从而潜在地提供了增强MSC对运动的反应的特定药理学靶点。为了验证这一点，我们将描述Fyn/mTORC 2的两个下游效应物RhoA和β连环蛋白对体外和体内MSC谱系命运分配的协同作用。我们将发现RhoA引起的细胞骨架重组是否 需要或足以改变mdMSC细胞命运并定义机械负荷如何激活RhoA（SA 1）。在SA 2中，我们询问β连环蛋白的激活是否可以在细胞骨架缺陷时改变细胞命运，询问细胞骨架是否驱动β连环蛋白的核/胞质分配 并考虑肌动蛋白-nesprin-LINC-核系链是否可能引导β连环蛋白进入细胞核。在SA 3中，使用一种创新的方法来量化和定位MAT，结合骨形成的测量，我们将研究体内mdMSC谱系的机械控制，询问mdMSC特有的Fyn/mTORC 2信号传导在骨形成的运动刺激期间是否至关重要;为此，我们将产生他莫昔芬驱动的Prx 1-Cre敲除Fyn。