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）：过度的气道狭窄和炎症是临床重要疾病（如哮喘）的标志。气道张力主要由细胞内Ca 2+（[Ca 2 +]i）（由Ca 2+内流和肌浆（SR）Ca 2+释放介导）和气道平滑肌（ASM）的力产生的Ca 2+敏感性决定，通过促炎细胞因子如TNF α增强。在这方面，小窝和小窝蛋白在肺中的作用是一个令人兴奋的新兴研究领域。小窝是大多数细胞类型的质膜（PM）的小的、未涂覆的瓶状内陷，并且含有称为小窝蛋白（小窝蛋白-1、-2和/或-3）的结构蛋白。最近的研究表明，包括我们自己的研究表明，正常人ASM表达小窝和小窝蛋白-1，与特定的蛋白参与[Ca 2 +]i和力调节共定位与小窝蛋白-1。初步研究显示，气道炎症（在TNF α暴露后的体外或在小鼠模型中的体内）增加小窝蛋白-1表达并增强ASM收缩性的小窝调节，表明总体上，小窝蛋白-1与ASM收缩性相关或促进ASM收缩性。这些研究的长期目标是了解在正常情况下和气道疾病时，小窝和小窝蛋白调节ASM收缩性的机制。Ca ~（2+）总体假设是小窝促进PM-SR相互作用，从而影响ASM中的[Ca ~（2+）]i和力。此外，我们假设气道炎症增加了小窝和小窝蛋白-1的表达，从而增强了PM-SR相互作用，导致ASM中[Ca 2 +]i和力增加。我们的研究的新奇在于连接以前的解剖学和生物化学的证据，小窝蛋白-1在ASM的生理作用，调节ASM收缩。总体方法：目标1中的人ASM细胞和组织的体外研究将检查在正常条件下可通过小窝蛋白-1与激动剂（ACh）刺激进行调节的机制，重点是激动剂受体、第二信使（PLC/IP 3、cADPR）、（STIM 1/Orai 1）对钙库操纵的钙内流（SOCE）的调节和RhoA（钙致敏）。然后将研究改变小窝蛋白-1表达（Aim 2）和小窝蛋白-1对增强ASM收缩性（Aim 3）与炎症（由TNF α诱导）的贡献的潜在机制，重点关注特定机制（TNFR 1、PTRF、MAPK、NF-？B）。最后，在目的4中，将在野生型（WT）和小窝蛋白-1敲除（KO）小鼠中使用气道高反应性的卵清蛋白（OVA）模型在体内水平整合体外数据。本研究的具体目的是：1）确定caveolae/caveolin-1调节人ASM对激动剂的[Ca ~（2+）]i和力反应的机制2）确定细胞因子（TNF α）刺激改变人ASM中caveolin-1表达的机制3）确定caveolin-1促进TNF α诱导的人ASM中[Ca ~（2+）]i和力的增强的机制; 4）研究caveolin-1在气道高反应性中的作用。