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

项目摘要 频繁的急性发作会引起气道损伤和重塑，并确定哮喘的严重程度。重度哮喘 也表现为具有不可逆气道阻塞的表型。然而，气道重塑在气道炎症中的作用 不可逆的气道阻塞的发病机制仍然存在争议。转化生长因子β1（TGF-β1） 在气道重塑中起着关键作用; TGF-β1是否调节人气道平滑肌 （HASM）缩短和气道高反应性（AHR）未知。我们最近发现TGF-β1 单独引起收缩并增强激动剂诱导的HASM缩短，HASM是调节HASM的关键细胞。 支气管张力我们的首要目标是确定调节蛋白质的分子转导过程， TGF-β1对哮喘HASM兴奋-收缩偶联的影响我们已经证明，收缩 激动剂通过激活G12、RhoA、PI 3 K δ和Rho激酶诱导HASM缩短。这些过程是否 介导TGF-β1对EC偶联的影响尚不清楚。我们提出了一个中心假设，即TGF-β1 通过激活PI 3 K δ、Rho激酶和RhoA依赖的肌动蛋白聚合调节HASM缩短 哮喘病为了验证这一假设，我们开发了单细胞力产生的新技术， 精确切割肺切片（hPCLS）、靶向蛋白质敲除和AHR的人类模型。在目标1中，我们 确定TGF-β1是否增加支气管张力并增强激动剂诱导的HASM缩短， PI 3 K δ-/Rho激酶依赖性方式。使用哮喘和非哮喘衍生的HASM细胞和hPCLS， 将确定疾病状态对TGF-β1诱导的PI 3 K δ和Rho激酶活化的影响。的作用 Smad蛋白介导TGF-β1诱导的单细胞收缩和AHR将在Smad 3/4后表征 击倒。在目标2中，我们将确定G12和RhoA消耗是否调节RhoA，PI 3 K δ， 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收缩的作用。丝切蛋白和丝状/球状肌动蛋白的磷酸化水平 比率，肌动蛋白聚合的关键调节剂，将在存在和不存在latrunculin A的情况下进行测量（一种 肌动蛋白破坏物）或siRNA。在与项目3、核心A和B的合作中，我们将描述关键的 研究TGF-β1对支气管张力和AHR作用的信号通路，并鉴定新的 治疗靶点和分子，以防止或消除这些作用，这是典型的观察后， 哮喘恶化和/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.