The Role of Caveolins in Lung Inflammation

描述(由申请人提供)：过度的呼吸道狭窄和炎症是哮喘等临床重要疾病的特征。气道张力主要由细胞内钙离子([Ca2+]i)(由钙离子内流和肌浆(SR)钙离子释放介导)和对气道平滑肌(ASM)作用力的钙敏感性决定，而促炎症细胞因子如TNFa可增强其敏感性。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)上的小的、未包被的烧瓶状凹陷，包含称为小窝蛋白(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，与小窝蛋白-1共同定位的特定蛋白涉及[Ca~(2+)]i和力量调节。初步研究表明，呼吸道炎症(无论是暴露于TNFa后的体外还是在小鼠模型中的体内)增加了小凹-1的表达，并增强了对ASM收缩的小窝调节，这表明总的来说，小窝-1与ASM的收缩有关或促进了ASM的收缩。拟议研究的长期目标是了解小窝和小窝蛋白在正常情况下和呼吸道疾病时调节ASM收缩能力的机制。总的假设是小窝促进PM-SR的相互作用，从而影响ASM中的[Ca+]i和FORCE。此外，我们假设，气道炎症增加了小窝和小窝-1的表达，从而增强了PM-SR的相互作用，导致ASM内[Ca~(2+)]i和力量的增加。我们研究的新颖性在于将ASM中小窝蛋白-1的解剖学和生化证据与ASM收缩调节中的生理作用联系起来。总体方法：Aim 1中的人ASM细胞和组织的体外研究将检验正常条件下激动剂(ACh)刺激下小窝蛋白-1可以调节的机制，重点放在激动剂受体、第二信使(PLC/IP3，cADPR)、(STIM1/Orai1)对商店操作的钙内流(SOCE)的调节以及RhoA(钙敏化)。然后，我们将研究小窝蛋白-1表达改变(目标2)和小窝蛋白-1对炎症(由TNFa引起)的ASM收缩能力增强(AIM 3)的作用机制，重点是特定的机制(TNFR1、PTRF、MAPK、NF-？B)。最后，在目标4中，将使用野生型(WT)和小凹-1基因敲除(KO)小鼠的呼吸道高反应性卵清蛋白(OVA)模型在体内水平整合体外数据。本建议的具体目的是：1)确定小凹/小窝蛋白-1调节[Ca~(2+)]i和迫使人ASM对激动剂的反应的机制；2)确定细胞因子(TNFa)刺激改变人ASM中小窝蛋白-1表达的机制；3)确定小窝蛋白-1促进TNFa诱导的人ASM[Ca~(2+)]i和FORCE的机制；4)确定小窝蛋白-1在体内气道高反应性中的作用。与公共卫生相关：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，呼吸道平滑肌收缩异常(因炎症而加剧)会导致气道过度狭窄和伴随呼吸短促。在这方面，称为小窝的细胞结构及其组成的小窝蛋白在调节呼吸道收缩能力中的潜在作用是一个令人兴奋的新兴研究领域。通过确定小窝和小窝蛋白在伴有或不伴有炎症的气道狭窄中的作用，拟议的研究将为更好地了解呼吸道疾病和潜在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.