Project 1 - TGF-beta1 directly modulates excitation-contraction signaling in airway smooth muscle to evoke airway hyperresponsiveness in asthma

项目概要 频繁的恶化会引起气道损伤和重塑，并确定哮喘的严重程度。严重哮喘 也表现为不可逆气道阻塞的表型。然而，气道重塑在 不可逆性气道阻塞的发病机制仍存在争议。转化生长因子β1（TGF-β1） 在协调气道重塑中发挥关键作用； TGF-β1是否调节人气道平滑肌 (HASM) 缩短和气道高反应性 (AHR) 尚不清楚。我们最近发现TGF-β1 单独引起收缩并增强激动剂诱导的 HASM（关键细胞调节）的缩短 支气管运动音。我们的首要目标是定义调节的分子转导过程 TGF-β1 对哮喘中 HASM 兴奋收缩 (EC) 耦合的影响。我们已经证明收缩 激动剂通过激活 G12、RhoA、PI3Kδ 和 Rho 激酶引起 HASM 缩短。是否有这些过程 介导 TGF-β1 对 EC 偶联的影响仍不清楚。我们提出一个中心假设：TGF-β1 通过激活 PI3Kδ、Rho 激酶和 RhoA 依赖性肌动蛋白聚合来调节 HASM 缩短 在哮喘中。为了检验这一假设，我们开发了单细胞力产生的新技术，人类 精密切割肺切片 (hPCLS)、靶向蛋白质敲除和 AHR 人体模型。在目标 1 中，我们将 定义 TGF-β1 是否会增加支气管运动张力并增强激动剂诱导的 HASM 缩短 PI3Kδ-/Rho 激酶依赖性方式。使用哮喘和非哮喘来源的 HASM 细胞和 hPCLS， 将确定疾病状态对 TGF-β1 诱导的 PI3Kδ 和 Rho 激酶激活的影响。的作用 Smad 蛋白介导 TGF-β1 诱导的单细胞收缩和 AHR 将在 Smad3/4 之后进行表征 击倒。在目标 2 中，我们将确定 G12 和 RhoA 消耗是否会调节 RhoA、PI3Kδ、 TGF-β1 暴露后 ROCK 活性和 HASM 缩短。在目标 3 中，我们将探讨肌动蛋白动力学是否 介导 TGF-β1 对 HASM 收缩的影响。丝切蛋白和丝状/球状肌动蛋白的磷酸化水平 比率是肌动蛋白聚合的关键调节剂，将在存在和不存在 latrunculin A（一种 肌动蛋白干扰物）或丝切蛋白的 siRNA。与项目 3、核心 A 和 B 合作，我们将描述关键的特征 介导 TGF-β1 对支气管运动张力和 AHR 影响的信号通路，并鉴定出新的 预防或消除这些效应的治疗靶点和分子，这些效应在治疗后观察到 哮喘加重和/或严重哮喘。

Project Summary Frequent exacerbations induce airway injury and remodeling, and define the severity of asthma. Severe asthma also manifests as a phenotype with irreversible airway obstruction. However, the role of airway remodeling in the pathogenesis of irreversible airway obstruction remains controversial. Transforming growth factor β1 (TGF-β1) plays a pivotal role in orchestrating airway remodeling; whether TGF-β1 modulates human airway smooth muscle (HASM) shortening and airway hyperresponsiveness (AHR) is unknown. We recently discovered that TGF-β1 alone evokes contraction and augments agonist-induced shortening of HASM, the pivotal cell regulating bronchomotor tone. Our overarching goal is to define the molecular transduction processes that regulate TGF-β1 effects on HASM excitation-contraction (EC) coupling in asthma. We have shown that contractile agonists evoked HASM shortening by activating G12, RhoA, PI3Kδ and Rho kinase. Whether these processes mediate TGF-β1 effects on EC coupling remain unknown. We posit a central hypothesis that TGF-β1 modulates HASM shortening by activating PI3Kδ, Rho Kinase and RhoA-dependent actin polymerization in asthma. To test this hypothesis, we developed novel techniques of single cell force generation, human precision cut lung slices (hPCLS), targeted protein knockdown and, human models of AHR. In Aim 1, we will define whether TGF-β1 increases bronchomotor tone and augments agonist-induced HASM shortening in a PI3Kδ-/Rho kinase-dependent manner. Using asthma- and non-asthma-derived HASM cells and hPCLS, disease-state effects on TGF-β1-induced activation of PI3Kδ and Rho Kinase will be determined. The role of Smad proteins in mediating TGF-β1-induced single cell contraction and AHR will be characterized after Smad3/4 knockdown. In Aim 2, we will determine whether G12 and RhoA depletion modulates activation of RhoA, PI3Kδ, ROCK activity and HASM shortening after TGF-β1 exposure. In Aim 3, we will explore whether actin dynamics mediates TGF-β1 effects on HASM contraction. Phosphorylation levels of cofilin and filamentous/globular actin ratios, key modulators of actin polymerization, will be measured in the presence and absence of latrunculin A (an actin disruptor) or siRNA to cofilin. In collaboration with Project 3, Cores A, and B, we will characterize the pivotal signaling pathways mediating the effects of TGF-β1 on bronchomotor tone and AHR, and identify novel therapeutic targets and molecules to prevent or abrogate these effects, which are characteristically observed after asthma exacerbations and/or in severe asthma.