RhoA signaling controls mesenchymal stem cell lineage commitment via Lef-1 in asthma

摘要 哮喘是一种严重的慢性疾病，并且日益受到临床和公共卫生的关注。的主要障碍 哮喘的预防和治疗一直是其病因的多样性和我们的认识不足， 生物机制。我们的长期目标是阐明基本的潜在机制， 确定新的哮喘治疗靶点。最近的研究表明，过敏原引起上皮损伤 允许环境过敏原进入气道组织， 重塑许多研究表明，间充质干细胞（MSC），多能干细胞， 可以迁移到损伤部位并分化成上皮细胞或成纤维细胞/肌成纤维细胞， 气道修复/重塑然而，这些研究是在培养的MSC或转移的MSC中进行的。 外源性MSC在小鼠肺损伤模型中的作用，以及控制MSC的主要内源性因素 分化仍不清楚。在这个建议中，我们的目标是直接跟踪血统承诺/分化， MSC在小鼠哮喘模型中随时间的推移使用MSC谱系追踪小鼠模型并阐明其潜在的 机制等在试验阶段，我们的小组作出了重大贡献，揭示了一个重要的联系， 间充质干细胞和哮喘之间的关系我们观察到哮喘小鼠模型气道中的MSC增加， 证明活性TGFβ1对于MSC向受损气道的募集是必需的。但 我们最近的一项重大突破是发现TGFβ1激活的RhoA作为一种分子开关， MSC在动脉修复/重塑过程中的命运。这一发现提出了RhoA信号可能 在确定用于气道修复/重塑的MSC的谱系命运中也是关键的。事实上，我们已经利用 MSC谱系追踪模型追踪RhoA介导的MSC分化，发现RhoA抑制 信号转导抑制MSC分化为肌成纤维细胞，但促进MSC分化为上皮细胞， 在我们的小鼠模型中。此外，我们鉴定了Wnt信号淋巴增强结合因子1（Lef 1）， 是骨髓间充质干细胞中RhoA激活的最上调基因。有趣的是，Lef 1的敲低诱导MSC 分化远离成纤维细胞/肌成纤维细胞，而是朝向气道上皮细胞。这些令人兴奋的新数据 引导我们检验RhoA信号传导在气道过程中控制MSC谱系定型/分化的假设， 通过Lef 1在哮喘中的修复/重塑。提出了三个独立但相关的具体目标。要求1 提出了实验来确定RhoA信号通路在谱系定型/分化中的作用， 使用已建立的他莫昔芬诱导的MSC谱系追踪小鼠，随时间推移在哮喘小鼠模型中的MSC 模型目的二是阐明RhoA信号通路调控的分子机制 MSC的定型/分化。目的3提出了研究以确定RhoA激活的Lef 1在 MSC的谱系定型/分化。总的来说，这些研究对于理解 环境过敏原诱导的哮喘的发生和哮喘新的治疗靶点的发展。

ABSTRACT Asthma is a serious chronic illness, and a growing clinical and public health concern. A major obstacle to the prevention and treatment of asthma has been its diverse etiologies and our inadequate understanding of the biological mechanisms. Our long-term goals are to elucidate the fundamental underlying mechanisms and identify novel therapeutic targets for asthma. Recent studies suggest that allergen caused epithelial damage allows environmental allergens access to the airway tissue and may lead to the development of airway remodeling. Numerous studies have suggested that mesenchymal stem cells (MSCs), multipotent stem cells, can migrate to injured sites and differentiate into epithelial cells or fibroblasts/myofibroblasts that may contribute to airway repair/remodeling. However, these studies were performed either in cultured MSCs or transferred exogenous MSCs in mouse model of lung injury, and the primary endogenous factors that control MSC differentiation remain unclear. In this proposal, we aim to directly track the lineage commitment/differentiation of MSCs in mouse model of asthma over time using MSC lineage tracing mouse model and elucidate the underlying mechanisms. During the pilot stage, our group has made significant contributions to uncovering an important link between MSCs and asthma. We observed increased MSCs in the airways of asthma mouse model and demonstrated that active TGFβ1 is essential for the recruitment of MSCs to the damaged airways. However, a major breakthrough came with our recent finding that TGFβ1-activated RhoA functions as a molecular switch for the fate of MSCs during arterial repair/remodeling. This finding raises the possibility that RhoA signaling may also be critical in determining the lineage fate of MSCs for airway repair/remodeling. Indeed, we have utilized MSC lineage tracing model to track the RhoA-mediated MSC differentiation and found that inhibition of RhoA signaling suppressed MSC differentiation into myofibroblasts, but promoted MSC differentiation into epithelial cells in our mouse model. Furthermore, we identified the Wnt signaling lymphoid enhancer-binding factor 1 (Lef1) as the most up-regulated gene of RhoA-activation in MSCs. Intriguingly, knockdown of Lef1 induced MSC differentiation away from fibroblasts/myofibroblasts but towards airway epithelial cells. These exciting new data led us to test the hypothesis that RhoA signaling controls MSC lineage commitment/differentiation during airway repair/remodeling in asthma through Lef1. Three independent yet related specific aims are proposed. Aim 1 proposes experiments to define the role of RhoA signaling pathway in the lineage commitment/differentiation of MSCs in asthma mouse model over time using an established tamoxifen inducible MSC lineage tracing mouse model. Aim 2 will elucidate the molecular mechanisms underlying the RhoA signaling regulated lineage commitment/differentiation of MSCs. Aim 3 proposes studies to determine the role of RhoA-activated Lef1 in the lineage commitment/differentiation of MSCs. Collectively, these studies are significant in understanding of how environmental allergen-induced asthma occurs and in the development of new therapeutic targets for asthma.