Mechanisms by which LMP-1 Regulates Lineage Commitment in Mesenchymal Stem Cells

LMP-1 调节间充质干细胞谱系定型的机制

• 批准号: 8449476
• 负责人: Frances Louisa Titus
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8449476
• 关键词: Adipocytes; Adverse effects; Aging; Animals; Automobile Driving; Binding; Bone Marrow; Bone Resorption; Cells; Communities; Data; Disease; Dose; Enhancers; Estrogens; Event; Fatty acid glycerol esters; Femoral Fractures; Fracture; General Population; Growth; Homeostasis; Human; Immobilization; Implant; Injury; Laboratories; Lead; Lipids; Malignant Neoplasms; Marrow; Measures; Mediating; Mesenchymal Stem Cells; Nodule; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis prevention; PPAR gamma; Pathway interactions; Peroxisome Proliferator-Activated Receptors; Pharmaceutical Preparations; Population; Proteins; Rattus; Relative (related person); Reporter; Repression; Risk; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Stem cells; Steroids; Stimulus; Surface; Testing; Therapeutic; Therapeutic Agents; Tumor Necrosis Factor-alpha; Ubiquitin; Veterans; Work; beta catenin; bisphosphonate; bone; bone loss; bone mass; bone morphogenetic protein 2; cell type; cost; design; human TNF protein; in vivo; innovation; lipid biosynthesis; mimetics; mineralization; novel; novel therapeutics; osteogenic; osteosarcoma; prevent; progenitor; receptor; small molecule; transcription factor

DESCRIPTION (provided by applicant): Osteoporosis due to aging, steroid treatment or immobility due to injury is an increasing problem within the Veteran population. Osteoporosis, often caused by increased osteoclastic bone resporption, is also associated with increased adipocytes and decreased osteoblasts in the bone marrow. The relative proportion of each cell type is determined by factors that regulate lineage determination of the marrow progenitor cell they share in common. We have exciting new observations that LMP-1, an intracellular protein discovered in our laboratory, inhibits the adipogenic and stimulates the osteogenic pathways in human Mesenchymal Stem Cells (hMSCs). This proposal will demonstrate that LMP-1 exerts these effects by blocking Smurf1 action, and in the final translational aim, will provide proof of concept of a novel anabolic therapeutic strategy for treating osteoporosis using a small molecule developed in our laboratory. RESEARCH PLAN: Our preliminary data suggest that LMP-1 exerts effects on lineage commitment by rapidly enhancing beta-catenin activity. Wnt10b is a secreted factor that binds surface receptors and activates beta- catenin; our preliminary studies show that Wnt10b activity is required for LMP-1 to activate beta-catenin. Under adipogenic culture conditions, we have determined that TNF-alpha activity is also required for LMP-1 activation of beta-catenin. This proposal seeks to unveil the role of each of these molecules in LMP-1-induced events favoring commitment of MSCs to the osteoblast lineage. We will demonstrate that 1) activation of beta- catenin is central to the mechanism by which LMP-1 inhibits adipogenesis and induces the osteoblast lineage commitment in MSCs, 2) that Smurf1 inhibition leads to increased expression of Msx2 via different pathways under adipogenic and osteogenic culture conditions, and 3) that altering lineage commitment in MSCs in vivo has an anabolic effect on bone homeostasis in osteoporotic animals. METHODS: Osteogenesis will be assessed by measuring expression of markers of mature osteoblasts and counting bone nodules; adipogenesis will be assessed by measuring expression of adipocyte markers and lipid accumulation within cultures. Activation of beta-catenin (TCF/LEF), NFkB, or PPARgamma transcriptional activity will be measured using reporter constructs. The necessity for a) enhanced activation of beta-catenin for the effect of LMP-1 on osteogenesis and adipogenesis will be tested by blocking beta-catenin activity with a constituitively active GSK3beta construct; b) increased expression of Msx2 and Wnt10b will be tested by applying siRNA to silence their expression. To probe the initial LMP-1 interaction responsible for these changes, we will apply siRNA to Smurf1 under adipogenic and osteogenic growth conditions and also apply an LMP-1 mimetic small molecule that blocks Smurf1 action. LMP-1 interaction with Smurf1 is predicted to inhibit degradation of signaling molecules including Smad1, Smad5, Traf2, making them more available to mediate events leading to enhanced expression of Wnt10b. We will validate the small molecule designed to mimic the LMP-1 blocking effect on Smurf1 by showing that it increases levels of the target proteins and stabilizes beta- catenin by increasing the expression of Wnt10b. In the final translational aim, we will demonstrate that the Smurf1-blocking small molecule drives commitment of MSCs to the osteoblast lineage in vivo and can alter bone homeostasis in osteoporotic rats by favoring increased bone formation and decreased adipogenesis.

描述（由申请人提供）： 骨质疏松症由于老化，类固醇治疗或不动，由于受伤是一个日益严重的问题，在退伍军人人口。骨质疏松症通常由骨细胞骨吸收增加引起，也与骨髓中脂肪细胞增加和成骨细胞减少有关。每种细胞类型的相对比例由调节它们共有的骨髓祖细胞的谱系确定的因素决定。我们有令人兴奋的新观察，LMP-1，在我们的实验室发现的细胞内蛋白，抑制成脂和刺激成骨途径在人类间充质干细胞（hMSCs）。该提案将证明LMP-1通过阻断Smurf 1作用发挥这些作用，并在最终的翻译目标中，将提供使用我们实验室开发的小分子治疗骨质疏松症的新型合成代谢治疗策略的概念证明。研究：我们的初步数据表明，LMP-1通过快速增强β-连环蛋白活性对谱系定型产生影响。Wnt 10 b是一种分泌因子，可结合表面受体并激活β-连环蛋白;我们的初步研究表明，LMP-1激活β-连环蛋白需要Wnt 10 b活性。在成脂培养条件下，我们已经确定TNF-α活性也是LMP-1激活β-连环蛋白所必需的。该提案旨在揭示这些分子中的每一个在LMP-1诱导的有利于MSC向成骨细胞谱系的承诺的事件中的作用。我们将证明：1）β-连环蛋白的激活是LMP-1抑制脂肪形成并诱导MSC中成骨细胞谱系定型的机制的核心; 2）Smurf 1抑制导致在脂肪形成和成骨培养条件下通过不同途径增加Msx 2的表达; 3）改变体内MSC中的谱系定型对骨质疏松动物的骨稳态具有合成代谢作用.方法：将通过测量成熟成骨细胞标志物的表达和计数骨结节来评估骨生成;将通过测量培养物内脂肪细胞标志物的表达和脂质蓄积来评估脂肪生成。将使用报告构建体测量β-连环蛋白（TCF/LEF）、NF κ B或PPARgamma转录活性的激活。a）通过用组成型活性GSK 3 β构建体阻断β-连环蛋白活性来测试β-连环蛋白活化增强对LMP-1对骨生成和脂肪生成的作用的必要性; B）通过应用siRNA沉默Msx 2和Wnt 10 b的表达来测试Msx 2和Wnt 10 b表达的增加。为了探索导致这些变化的初始LMP-1相互作用，我们将在成脂和成骨生长条件下将siRNA应用于Smurf 1，并应用LMP-1模拟小分子阻断Smurf 1作用。LMP-1与Smurf 1的相互作用预计会抑制信号分子的降解，包括Smad 1，Smad 5，Traf 2，使它们更容易介导导致Wnt 10 b表达增强的事件。我们将验证设计用于模拟LMP-1对Smurf 1阻断作用的小分子，通过显示其增加靶蛋白的水平并通过增加Wnt 10 b的表达来稳定β-连环蛋白。在最终的翻译目标中，我们将证明Smurf 1阻断小分子驱动骨髓间充质干细胞在体内向成骨细胞谱系的承诺，并通过促进骨形成增加和脂肪生成减少来改变骨质疏松大鼠的骨稳态。