The Role of Caveolins in Lung Inflammation

小窝蛋白在肺部炎症中的作用

• 批准号: 8431776
• 负责人: Christina Maria Pabelick
• 金额: $ 32.04万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8431776
• 关键词: Address; Affect; Agonist; Airway Resistance; Area; Asthma; Biochemical; Caveolae; Caveolins; Cell membrane; Cells; Cellular Structures; Chronic Bronchitis; Cyclic ADP-Ribose; Data; Development; Disease; Fluorescence; Foundations; Generations; Goals; Health; Human; Imaging Techniques; In Vitro; Inflammation; Inflammatory; Knock-out; Knockout Mice; Lead; Link; Lipids; Lung; Lung Inflammation; MAP Kinase Gene; Maintenance; Measures; Mediating; Membrane Proteins; Mitogen-Activated Protein Kinases; Modeling; Molecular Biology; Mus; Muscarinic M3 Receptor; Ovalbumin; Pathway interactions; Pharmacology; Phosphorylation; Physiological; Proteins; Regulation; Reporting; Research; Rho-associated kinase; Role; STIM1 gene; Sarcoplasmic Reticulum; Second Messenger Systems; Shapes; Shortness of Breath; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Structural Protein; Structure; TNF gene; TNFRSF1A gene; Techniques; Tissues; Transfection; Tumor Necrosis Factor Receptor; Tumor Necrosis Factor-Beta; Tumor Necrosis Factor-alpha; Viral; Work; airway hyperresponsiveness; airway inflammation; base; caveolin 1; caveolin-2; cell type; cytokine; flasks; genetic regulatory protein; in vivo; inhibitor/antagonist; intravital imaging; laser capture microdissection; methacholine; migration; mouse model; new therapeutic target; protein expression; receptor; respiratory smooth muscle; response; rho; scaffold; second messenger

DESCRIPTION (provided by applicant): Exaggerated airway narrowing and inflammation are hallmarks of clinically-important diseases such as asthma. Airway tone, largely determined by intracellular Ca2+ ([Ca2+]i) (mediated by Ca2+ influx and sarcoplasmic (SR) Ca2+ release) and Ca2+ sensitivity for force generation of airway smooth muscle (ASM), is enhanced by pro-inflammatory cytokines such as TNFa. In this regard, the role of caveolae and caveolins in the lung is an exciting and emerging area of research. Caveolae are small, uncoated flask-shaped invaginations of the plasma membrane (PM) of most cell types, and contain structural proteins called caveolins (caveolin-1, -2 and/or -3). Recent studies including our own have demonstrated that normal human ASM expresses caveolae and caveolin-1, with specific proteins involved in [Ca2+]i and force regulation co-localizing with caveolin-1. Preliminary studies show that airway inflammation (either in vitro following TNFa exposure or in vivo in a mouse model) increases caveolin-1 expression and enhances caveolar regulation of ASM contractility, suggesting that overall, caveolin-1 is associated with or promotes ASM contractility. The long term goal of the proposed studies is to understand the mechanisms by which caveolae and caveolins regulate ASM contractility under normal conditions and with airway disease. Ca2+ The overall hypothesis is that caveolae facilitate PM- SR interactions, thus influencing [Ca2+]i and force in ASM. In addition, we hypothesize that airway inflammation increases caveolae and caveolin-1 expression, and thus enhances PM-SR interactions, leading to increased [Ca2+]i and force in ASM. The novelty of our studies lies in linking previous anatomical and biochemical evidence for caveolin-1 in ASM to a physiological role in regulation of ASM contractility. Overall Approach: In vitro studies with human ASM cells and tissues in Aim 1 will examine mechanisms that can be modulated by caveolin-1 with agonist (ACh) stimulation under normal conditions, focusing on agonist receptors, second messengers (PLC/IP3, cADPR), regulation of store-operated Ca2+ entry (SOCE) by (STIM1/Orai1), and RhoA (Ca2+ sensitization). Mechanisms underlying altered caveolin-1 expression (Aim 2) and caveolin-1 contribution to enhanced ASM contractility (Aim 3) with inflammation (induced by TNFa) will then be examined, focusing on specific mechanisms (TNFR1, PTRF, MAPK, NF-?B). Finally in Aim 4, in vitro data will be integrated at the in vivo level using an ovalbumin (OVA) model of airway hyperresponsiveness in wildtype (WT) and caveolin-1 knockout (KO) mice. The Specific Aims of this proposal are: 1) To determine mechanisms by which caveolae/caveolin-1 regulate [Ca2+]i and force responses of human ASM to agonist 2 ) To determine mechanisms by which cytokine (TNFa) stimulation alters caveolin-1 expression in human ASM 3) To determine the mechanisms by which caveolin-1 contributes to TNFa-induced enhancement of [Ca2+]i and force in human ASM; 4) To determine the role of caveolin-1 in airway hyperresponsiveness in vivo.

描述（由申请人提供）：气道过度狭窄和炎症是哮喘等临床重要疾病的标志。气道张力主要由细胞内 Ca2+ ([Ca2+]i)（由 Ca2+ 流入和肌浆 (SR) Ca2+ 释放介导）和气道平滑肌 (ASM) 产生力的 Ca2+ 敏感性决定，并通过促炎细胞因子（如 TNFa）增强。在这方面，小窝和小窝在肺部的作用是一个令人兴奋的新兴研究领域。小凹是大多数细胞类型质膜 (PM) 的小型、无涂层烧瓶状内陷，并含有称为小凹蛋白 (caveolin-1、-2 和/或 -3) 的结构蛋白。最近的研究（包括我们自己的研究）表明，正常人类 ASM 表达小凹和小凹蛋白-1，其中特定蛋白涉及 [Ca2+]i 和与小凹蛋白-1 共定位的力调节。初步研究表明，气道炎症（无论是 TNFa 暴露后的体外还是小鼠模型体内的炎症）都会增加 Caveolin-1 的表达并增强 ASM 收缩性的小凹调节，这表明总体而言，Caveolin-1 与 ASM 收缩性相关或促进 ASM 收缩性。拟议研究的长期目标是了解小凹和小凹蛋白在正常条件下和气道疾病下调节 ASM 收缩性的机制。 Ca2+ 总体假设是小凹促进 PM-SR 相互作用，从而影响 ASM 中的 [Ca2+]i 和力。此外，我们假设气道炎症会增加小窝和小窝蛋白-1 的表达，从而增强 PM-SR 相互作用，导致 ASM 中 [Ca2+]i 和力增加。我们研究的新颖性在于将先前 ASM 中的 Caveolin-1 的解剖学和生化证据与 ASM 收缩性调节中的生理作用联系起来。总体方法：目标 1 中对人类 ASM 细胞和组织的体外研究将检查在正常条件下，caveolin-1 在激动剂 (ACh) 刺激下可调节的机制，重点关注激动剂受体、第二信使（PLC/IP3、cADPR）、（STIM1/Orai1）对钙池操纵的 Ca2+ 内流（SOCE）的调节和 RhoA（Ca2+ 敏化）。然后将检查 Caveolin-1 表达改变（目标 2）和 Caveolin-1 通过炎症（TNFa 诱导）增强 ASM 收缩性（目标 3）的作用机制，重点关注特定机制（TNFR1、PTRF、MAPK、NF-κB）。最后，在目标 4 中，将使用野生型 (WT) 和 Caveolin-1 敲除 (KO) 小鼠气道高反应性的卵清蛋白 (OVA) 模型将体外数据整合到体内水平。该提案的具体目标是： 1) 确定 Caveolae/caveolin-1 调节 [Ca2+]i 并迫使人 ASM 对激动剂做出反应的机制 2) 确定细胞因子 (TNFa) 刺激改变人 ASM 中的 Caveolin-1 表达的机制 3) 确定 Caveolin-1 促进 TNFa 诱导的 [Ca2+]i 增强和增强的机制 人类 ASM 中的力量； 4)确定caveolin-1在体内气道高反应性中的作用。