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

描述(由申请人提供)： 在退伍军人中，由于衰老、类固醇治疗或受伤导致的行动不便导致的骨质疏松症是一个日益严重的问题。骨质疏松症通常是由破骨细胞性骨吸收增加引起的，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前体细胞的谱系决定的因素决定。我们有令人兴奋的新发现，LMP-1是我们实验室发现的一种细胞内蛋白，它抑制人骨髓间充质干细胞(HMSCs)的成脂作用，并刺激成骨途径。这项建议将证明LMP-1通过阻断S-1的作用来发挥这些作用，并在最终的翻译目标中，将为使用我们实验室开发的小分子治疗骨质疏松症的新的合成代谢治疗策略的概念提供证据。研究计划：我们的初步数据表明，LMP-1通过快速增强β-连环蛋白活性而对血统承诺产生影响。Wnt10b是一种分泌因子，结合表面受体并激活β-连环蛋白；我们的初步研究表明，Wnt10b的活性是LMP-1激活β-连环蛋白所必需的。在成脂培养条件下，我们已经确定，β-连环蛋白的LMP-1激活也需要肿瘤坏死因子-α的活性。这项提议试图揭示这些分子中的每一个在LMP-1诱导的有利于MSCs向成骨细胞谱系承诺的事件中的作用。我们将证明：1)激活β-连环蛋白是LMP-1抑制骨髓间充质干细胞成脂和诱导成骨细胞向成骨细胞分化的核心机制；2)在成脂和成骨培养条件下，SMurf1抑制通过不同途径导致MSX2表达增加；3)体内改变骨髓间充质干细胞的向成骨细胞定向分化对骨质疏松动物的骨稳态具有合成代谢作用。方法：通过测量成熟成骨细胞标志物的表达和计数骨结节来评价成骨；通过测量脂肪细胞标志物的表达和培养体内的脂肪积累来评价成脂作用。β-连环蛋白(TCF/Lef)、NFkB或PPARγ转录活性的激活将使用报告构建体来测量。A)LMP-1在成骨和成脂作用中增强β-catenin活性的必要性将通过用具有结构性活性的GSK3β结构来阻断β-catenin的活性来测试；b)MSX2和Wnt10b的表达增加将通过应用siRNA沉默它们的表达来测试。为了探索导致这些变化的最初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S-1，并应用LMP-1模拟小分子来阻断S-1的作用。LMP-1与SMurf1的相互作用被预测为抑制包括Smad1、Smad5、Traf2在内的信号分子的降解，使它们更容易介导导致Wnt10b表达增强的事件。我们将验证旨在模拟LMP-1对S-1的阻断作用的小分子，方法是证明它可以增加靶蛋白的水平，并通过增加Wnt10b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SMurf1封闭的小分子在体内驱动MSCs对成骨细胞谱系的承诺，并通过促进骨形成和减少脂肪生成来改变骨质疏松大鼠的骨稳态。

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.