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

项目摘要 频繁的加重会导致呼吸道损伤和重塑，并定义哮喘的严重程度。重度哮喘 也表现为不可逆的呼吸道阻塞。然而，气道重塑在动脉粥样硬化中的作用 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(转化生长因子β1) 在调节气道重塑中起关键作用；转化生长因子-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。我们最近发现转化生长因子-β-1 单独引起收缩并增强激动剂诱导的HASM缩短，HASM是关键细胞调节 支气管运动音调。我们的首要目标是定义调节 转化生长因子-β-1对哮喘大鼠肺泡巨噬细胞兴奋-收缩偶联的影响我们已经证明了可缩性 激动剂通过激活G12、RhoA、PI3Kδ和Rho激动剂引起HASM缩短。这些过程是否 转化生长因子-β-1对EC偶联的中介作用尚不清楚。我们假设一个中心假设转化生长因子-β1 通过激活PI3Kδ、Rho Kinase和RhoA依赖的肌动蛋白聚合来调节HASM的缩短 得了哮喘。为了验证这一假设，我们开发了单细胞力量产生的新技术，人类 精确切割肺切片(HPCLS)、靶向蛋白敲除和AHR人体模型。在目标1中，我们将 明确转化生长因子-β-1是否增加了激动剂诱导的大鼠肺间质膜收缩和支气管运动张力 PI3Kδ/Rho激酶依赖方式。使用哮喘和非哮喘来源的HASM细胞和hPCLS， 疾病状态对转化生长因子-β1诱导的PI3Kδ和Rho Kinase激活的影响将被确定。的作用 Smad蛋白在介导转化生长因子-β1诱导的单细胞收缩和AHR中的作用将在Smad3/4之后被表征 击倒对手。在目标2中，我们将确定G12和RhoA的缺失是否调节RhoA，PI3Kδ， 转化生长因子-β-1作用后，岩层活性和骨质膜缩短。在目标3中，我们将探索肌动蛋白动力学 介导转化生长因子-β-1对血管平滑肌收缩的影响。Cofilin和丝状/球状肌动蛋白的磷酸化水平 肌动蛋白聚合的关键调节因子比率将在存在和不存在乳房菌素A(AN)的情况下进行测量 肌动蛋白干扰物)或siRNA转化为粘连蛋白。在与项目3、核心A和B的合作中，我们将描述 转化生长因子-β-1对支气管运动张力和肾上腺皮质激素受体影响的信号转导途径 治疗靶点和分子，以防止或消除这些影响，其特点是在 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.