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

项目 1 - TGF-β1 直接调节气道平滑肌中的兴奋收缩信号传导以引起哮喘气道高反应性

• 批准号: 10465060
• 负责人: Reynold Alexander Panettieri
• 金额: $ 51.51万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10465060
• 关键词: Actins; Agonist; Asthma; Binding; Bronchoconstriction; Bronchodilation; Calcium; Cells; Characteristics; Coin; Collaborations; Coupling; Cytometry; Cytoskeleton; Data; Disease; Exposure to; Functional disorder; G Actin; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Generations; Goals; Human; Inflammation; Interleukin-13; Lung; Magnetism; Measures; Mediating; Microscopy; Modeling; Molecular; Muscle Contraction; Myosin Light Chains; Pathogenesis; Pathway interactions; Pattern; Phenotype; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Play; Process; Proteins; ROCK1 gene; Regulation; Reporting; Rho-associated kinase; Role; Severities; Signal Pathway; Signal Transduction; Slice; Smad Proteins; Small Interfering RNA; Smooth Muscle Myocytes; TGFB1 gene; Techniques; Testing; Transforming Growth Factor beta; Transforming Growth Factors; Viral; airway hyperresponsiveness; airway obstruction; airway remodeling; alpha Actin; asthma exacerbation; cofilin; human model; inhibitor; injured airway; injury and repair; knock-down; latrunculin A; myosin phosphatase; new therapeutic target; novel; polymerization; prevent; respiratory smooth muscle; response

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.

项目概要 频繁的恶化会引起气道损伤和重塑，并确定严重哮喘的严重程度。 也表现为不可逆气道阻塞的表型。然而，气道重塑在其中的作用。 不可逆气道阻塞的发病机制仍存在争议。 TGF-β1 是否调节人气道平滑肌在协调气道重塑中发挥关键作用； (HASM) 缩短和气道高反应性 (AHR) 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 我们的首要目标是定义调节支气管运动张力的分子转导过程。 TGF-β1 对哮喘中 HASM 兴奋-收缩 (EC) 耦合的影响 我们已经证明了收缩性。 激动剂通过激活 G12、RhoA、PI3Kδ 和 Rho 激酶来诱发 HASM 缩短。 我们提出一个中心假设：TGF-β1 介导 TGF-β1 对 EC 偶联的影响。 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 为了检验这一假设，我们开发了人类单细胞力产生的新技术。 在目标 1 中，我们将进行精确切割肺切片 (hPCLS)、靶向蛋白质敲除和 AHR 人体模型。 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 使用哮喘和非哮喘来源的 HASM 细胞和 hPCLS PI3Kδ-/Rho 激酶依赖性方式， 疾病状态对 TGF-β1 诱导的 PI3Kδ 和 Rho 激酶激活的作用将被确定。 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 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存在。 介导 TGF-β1 对 HASM 收缩的影响。 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 与 Project 3、Cores A 和 B 合作，我们将表征关键的蛋白丝切蛋白。 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。