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病，也是一个日益严重的临床和公共卫生问题。一个主要的障碍是 预防和治疗哮喘的病因多样，我们对哮喘的认识还不够充分。 生物机制。我们的长期目标是阐明基本的潜在机制和 确定哮喘的新治疗靶点。最近的研究表明，过敏原导致上皮损伤 允许环境过敏原进入呼吸道组织，并可能导致呼吸道的发展 改建。大量研究表明，间充质干细胞(MSCs)、多潜能干细胞、 可以迁移到损伤部位并分化为上皮细胞或成纤维细胞/肌成纤维细胞，这可能有助于 到呼吸道修复/重塑。然而，这些研究要么是在培养的MSCs中进行的，要么是在转移的 外源性间充质干细胞在小鼠肺损伤模型中的作用及其主要内源性调控因素 分化仍不清楚。在本提案中，我们的目标是直接跟踪 间充质干细胞在小鼠哮喘模型中随时间的变化采用MSC示踪小鼠模型并阐明其潜在的机制 机械装置。在试点阶段，我们小组为揭示一个重要的环节做出了重大贡献 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和哮喘模型小鼠呼吸道中MSCs的增加 结果表明，活化的转化生长因子β-1对MSCs在受损的呼吸道中的募集是必不可少的。然而，a 我们最近的发现取得了重大突破，转化生长因子β1激活的RhoA起着分子开关的作用 骨髓间充质干细胞在动脉修复/重建过程中的去向。这一发现增加了RhoA信号可能 在决定MSCs在呼吸道修复/重建中的谱系命运方面也是至关重要的。事实上，我们已经利用了 MSC谱系追踪模型追踪RhoA介导的MSC分化并发现抑制RhoA 信号转导抑制MSC向肌成纤维细胞分化，但促进MSC向上皮细胞分化 我们老鼠模型中的细胞。此外，我们还鉴定了Wnt信号增强因子1(Lef1)。 作为骨髓间充质干细胞中最上调的RhoA激活基因。耐人寻味的是，Lef1基因的敲除诱导了MSC 分化方向不是成纤维细胞/肌成纤维细胞，而是呼吸道上皮细胞。这些令人兴奋的新数据 导致我们测试了RhoA信号在呼吸道过程中控制MSC谱系承诺/分化的假设 Lef1在哮喘中的修复/重塑提出了三个既相互独立又相互联系的具体目标。目标1 建议进行实验，以确定RhoA信号通路在血统承诺/分化中的作用 利用已建立的三苯氧胺可诱导的MSC谱系示踪小鼠建立哮喘小鼠模型中的MSCs 模特。目标2将阐明RhoA信号调控谱系背后的分子机制 MSCs的承诺/分化。目的3提出研究以确定RhoA激活的Lef1在 间充质干细胞的谱系承诺/分化。总而言之，这些研究对于理解 环境过敏原诱发的哮喘发生在哮喘新的治疗靶点的开发中。

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.