The Role of Caveolins in Lung Inflammation

描述（由申请人提供）：严重的气道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+内流和肌浆（SR） Ca2+释放介导）和Ca2+对气道平滑肌（ASM）产生力的敏感性决定，由促炎细胞因子如TNFa增强。在这方面，小泡和小泡蛋白在肺中的作用是一个令人兴奋的新兴研究领域。小泡是大多数细胞类型的质膜（PM）的小的，无涂层的瓶状内陷，含有称为小泡蛋白的结构蛋白（小泡蛋白-1，-2和/或-3）。最近的研究包括我们自己的研究表明，正常的人类ASM表达小窝和小窝蛋白-1，与特定的蛋白参与[Ca2+]i和力调节与小窝蛋白-1共定位。初步研究表明，气道炎症（tnf fa暴露后的体外或小鼠体内模型）增加了caveolin-1的表达，并增强了腔隙对ASM收缩性的调节，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在正常情况下和气道疾病时，小泡和小泡蛋白调节ASM收缩性的机制。总的假设是，小泡促进PM- SR相互作用，从而影响ASM中的[Ca2+]i和力。此外，我们假设气道炎症增加了小泡和小泡蛋白-1的表达，从而增强了PM-SR相互作用，导致ASM中[Ca2+]i和力的增加。我们的研究的新颖之处在于将先前的解剖和生化证据与ASM中caveolin-1在调节ASM收缩性中的生理作用联系起来。总体方法：Aim 1中人类ASM细胞和组织的体外研究将检查在正常情况下由激动剂（ACh）刺激的caveolin-1可以调节的机制，重点关注激动剂受体，第二信使（PLC/IP3, cADPR），通过（STIM1/Orai1）调节储存操作的Ca2+进入（SOCE），以及RhoA （Ca2+致敏）。随后，我们将研究炎症（由TNFa诱导）时小窝蛋白-1表达改变（Aim 2）和小窝蛋白-1促进ASM收缩性增强（Aim 3）的机制，重点研究具体机制（TNFR1、PTRF、MAPK、NF- B）。最后，在Aim 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）模型在体内水平整合体外数据。本提案的具体目的是：1)确定caveolae/caveolin-1调节人ASM对激动剂的[Ca2+]i和力反应的机制2)确定细胞因子（TNFa）刺激改变人ASM中caveolin-1表达的机制3)确定caveolin-1促进TNFa诱导的人ASM中[Ca2+]i和力的增强的机制；4)确定caveolin-1在气道高反应性中的作用。公共卫生相关性：越来越多的人认识到，在哮喘和慢性支气管炎等临床重要疾病中，气道平滑肌收缩性异常（由炎症加剧）会导致气道过度狭窄并伴有呼吸短促。在这方面，称为小泡的细胞结构及其组成小泡蛋白在气道收缩性调节中的潜在作用是一个令人兴奋的新兴研究领域。通过确定小泡和小泡蛋白在有或无炎症的气道狭窄中的作用，本研究将为更好地了解气道疾病和开发新的治疗靶点奠定基础。

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. PUBLIC HEALTH RELEVANCE: There is increasing recognition that abnormalities in airway smooth muscle contractility (exacerbated by inflammation) contribute to exaggerated airway narrowing and accompanying shortness of breath in clinically- important diseases such as asthma and chronic bronchitis. In this regard, the potential role of cellular structures called caveolae and their constituent caveolin proteins in regulation of airway contractility is an exciting and emerging area of research. By establishing the role of caveolae and caveolins in airway narrowing with or without inflammation, the proposed studies will the foundation for better understanding of airway diseases, and potential development of new therapeutic targets.