Regulation of Normal and Asthmatic Lung Function by G-Protein-Coupled Receptors

G 蛋白偶联受体对正常和哮喘肺功能的调节

基本信息

批准号：
10014104
负责人：
Kirk m Druey
金额：
$ 51.11万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10014104
关键词：
Actins Adenosine Adrenal Cortex Hormones Adrenergic beta-Agonists Affect Agonist Albuterol Allergens Antifungal Agents Arachidonic Acids Area Aspergillus fumigatus Aspirin Asthma Autopsy Binding Bradykinin Bronchial Spasm Bronchodilator Agents Calcium Cellular Structures Cessation of life Collaborations Coupled Cyclic AMP Dinoprostone Disease susceptibility Drug Targeting Endothelin-1 Epithelial Cells Epithelium Extracellular Matrix Extrinsic asthma G Protein-Coupled Receptor Signaling G alpha q Protein G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gene Deletion Goals Guanosine Triphosphate Phosphohydrolases Heterotrimeric GTP-Binding Proteins Histamine Human Hypersensitivity IgE Immune response Impairment In Vitro Inflammation Mediators Inflammatory Response Ingestion Inhibitory G-Protein Gi Injury Intervention Leukotriene Antagonists Leukotriene D4 Life Ligands Link Lung Lung Inflammation Mammalian Cell Mediating Metabolic Diseases Modeling Molds Monoclonal Antibodies Mus Muscle Contraction Muscle Fibers Muscle function Muscle relaxation phase Myosin ATPase Non-Steroidal Anti-Inflammatory Agents PAR-2 Receptor Pathologic Patients Peptide Hydrolases Phenotype Physiological Predisposition Production RGS Domain RGS Proteins Recombinants Regulation Relaxation Respiratory physiology Role Signal Pathway Signal Transduction Signaling Protein Slice Smooth Muscle Smooth Muscle Myocytes Specimen Thrombin Tissues Trypsin airway hyperresponsiveness airway obstruction allergic airway inflammation allergic response aspirin-exacerbated respiratory disease asthmatic asthmatic airway cysteinyl-leukotriene eosinophilic inflammation experience functional disability fungus granule cell high throughput screening inhibitor/antagonist interest mast cell omalizumab potential biomarker receptor repository respiratory respiratory smooth muscle screening small molecule success

项目摘要

Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and hyper-contractility of the bronchial smooth muscle. The major naturally occurring substances that induce bronchial smooth muscle contraction are ligands of G-protein-coupled receptors (GPCRs), such as allergen proteases, thrombin, and those contained in allergen-IgE activated mast cell granules (e.g. histamine, cysteinyl leukotrienes (LTD4), endothelin 1, adenosine, and bradykinin). In general, these agonists induce activation of the heterotrimeric G protein G-alpha q, which increases the concentration of intracellular calcium in smooth muscle cells, promoting actin-myosin interactions and muscle fiber shortening. In contrast, ligands acting on G-alpha-s-coupled receptors, such as albuterol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for airway hyper-responsiveness (AHR), suggesting that underlying abnormalities in structural cells including airway smooth muscle (ASM) cells contribute to the asthmatic diathesis. Dysregulation of procontractile, GPCR signaling in ASM could mediate enhanced contractility. 10-15% of people with asthma experience severe, life threatening attacks and even death despite aggressive treatment with bronchodilators and corticosteroids. Nearly half of these (10-20 million) are sensitized (i.e. have IgE-mediated allergy) to filamentous fungi (e.g. Aspergillus fumigatus, Af), which has been designated "severe asthma with fungal sensitization" (SAFS). Current therapies for SAFS including antifungals or omalizumab monoclonal antibody (mAb) targeting IgE have not achieved uniform success. Utilizing a model of allergic airway inflammation in mice induced by respiratory Af exposure, we have 2 overarching aims for this project: 1) identify derangements in ASM contraction signaling downstream of inflammatory mediators; 2) examine the functions of allergen protease activity in AHR, particularly in relation to allergen-ASM interactions. Protease activity is a common and important feature of allergens capable of inducing asthma, most notably from ubiquitous fungi such as Af. Whether any allergens affect ASM contraction directly has never been explored. Previously we found a causal link between fungi and asthma occurring independently of allergenicity; in other words, host inflammatory response to the allergen. The secreted Af protease Alp1 was detected in the airways of asthmatic subjects but not controls. In mouse and in vitro studies, we found that Alp1 directly promoted AHR by degrading ECM components, leading to dysregulation of ASM contraction. In fiscal year 19, we demonstrated that Alp1 from Aspergillus fumigatus can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease-activated receptor 2/F2 trypsin-like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices and human airway smooth muscle cells, we demonstrated that Alp1 directly increased contractile force. The second area of emphasis for this project is the study of Regulators of G protein signaling (RGS) proteins in the lung. RGSs bind to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivates them by catalyzing their intrinsic GTPase activity and by blocking downstream effector interactions. Although they are generally considered to act as negative regulators of GPCR signaling pathways, the physiological function of RGS proteins in the lung is mostly unknown. We identified expression of several RGS proteins (RGS4, RGS5) in bronchial smooth muscle of humans and mice. In fiscal year 19, we examined the phenotype of mice with global and smooth muscle-specific Rgs4 gene deletion. Nearly 15% of patients with severe asthma develop bronchospasm following ingestion of aspirin and other non-steroidal anti-inflammatory drugs, a condition known as aspirin-exacerbated respiratory disease (AERD), which may result in part from disordered metabolism of arachidonic acid metabolites such as prostaglandin E2 (PGE2). We also examined the role of RGS4 in AERD by challenging AERD-like (ptges1-/-) mice with aspirin. We found that PGE2 secretion is a negative homeostatic signal within the lung epithelium to allergen-induced injury, and we identify RGS4 as a critical NSAID-inducible factor in respiratory epithelial cells that impairs this host response and increases susceptibility to bronchospasm. RGS4 expression in lung epithelium is increased in subjects with severe asthma, with expression that correlates strongly with the degree of functional impairment. Allergen-induced AHR was significantly diminished in Rgs4-/- mice, a finding that was dependent on increased airway PGE2 levels. RGS4 inhibited allergen-induced PGE2 secretion in human bronchial epithelial cells, and treatment with the RGS4 inhibitor CCG203769 in WT or (ptges1-/-) mice alleviated AHR induced by allergen and/or aspirin challenge, respectively, in association with increased PGE2 production. We conclude that RGS4 is a potential biomarker and interventional target in severe and aspirin-associated asthma. In collaboration with Dr. Krishnan and colleagues, we propose high throughput screening to identify inhibitors of Alp1 protease. Such compounds will be used to study the functions of protease allergens in AHR and elucidate ASM contraction mechanisms in fungal-associated asthma. Eventually, we hope to identify small molecules targeting Alp1 protease for the treatment of patients with SAFS. Toward this end, we have generated recombinant Alp1 protease in mammalian cells to use for compound screening.

哮喘是一种可逆性气道阻塞的病理状况，由肺部炎症和支气管平滑肌过度收缩组成。诱导支气管平滑肌收缩的主要天然物质是G蛋白偶联受体（GPCR）的配体，例如过敏原蛋白酶、凝血酶以及过敏原IgE激活的肥大细胞颗粒中所含的物质（例如组胺、半胱氨酰白三烯（LTD4） )、内皮素 1、腺苷和缓激肽)。一般来说，这些激动剂会诱导异源三聚体 G 蛋白 G-α q 的激活，从而增加平滑肌细胞内钙的浓度，促进肌动蛋白-肌球蛋白相互作用和肌纤维缩短。相反，作用于 G-α-s 偶联受体的配体（例如沙丁胺醇）会增加细胞内环 AMP (cAMP) 的水平，从而促进 ASM 松弛。尽管嗜酸性粒细胞炎症是过敏性哮喘的典型表现，但它并不是气道高反应性（AHR）的先决条件，这表明包括气道平滑肌（ASM）细胞在内的结构细胞的潜在异常导致了哮喘素质。 ASM 中的前收缩 GPCR 信号传导失调可能会介导收缩性增强。尽管使用支气管扩张剂和皮质类固醇进行积极治疗，但 10-15% 的哮喘患者仍会出现严重的、危及生命的发作，甚至死亡。其中近一半（10-2000 万）对丝状真菌（例如烟曲霉，Af）过敏（即 IgE 介导的过敏），这被称为“真菌致敏性严重哮喘”(SAFS)。目前的 SAFS 疗法，包括抗真菌药物或针对 IgE 的奥马珠单抗单克隆抗体 (mAb)，尚未取得一致的成功。利用呼吸道AF暴露引起的小鼠过敏性气道炎症模型，我们这个项目有两个总体目标：1）识别炎症介质下游ASM收缩信号传导的紊乱； 2）检查AHR中过敏原蛋白酶活性的功能，特别是与过敏原-ASM相互作用相关的功能。蛋白酶活性是能够诱发哮喘的过敏原的一个常见且重要的特征，尤其是来自无处不在的真菌，例如 Af。是否有任何过敏原直接影响 ASM 收缩尚未被探索过。此前我们发现真菌与哮喘之间存在独立于过敏性的因果关系。换句话说，宿主对过敏原的炎症反应。在哮喘受试者的气道中检测到分泌的 Af 蛋白酶 Alp1，但在对照组中未检测到。在小鼠和体外研究中，我们发现Alp1通过降解ECM成分直接促进AHR，导致ASM收缩失调。在第 19 财年，我们证明烟曲霉中的 Alp1 可以在无法产生嗜酸性粒细胞炎症的小鼠中诱导 AHR。引人注目的是，Alp1 在缺乏蛋白酶激活受体 2/F2 胰蛋白酶样受体 1 (PAR2/F2RL1) 的小鼠中诱导 AHR，这是一种在肺上皮中表达的受体，对含蛋白酶过敏原的过敏反应至关重要。相反，我们使用精确切割的肺切片和人气道平滑肌细胞，证明 Alp1 直接增加收缩力。该项目的第二个重点领域是肺部 G 蛋白信号传导 (RGS) 蛋白调节因子的研究。 RGS 通过保守的 RGS 结构域与 G 蛋白 α 亚基 Gi 和 Gq（但不是 Gs）结合，并通过催化其内在 GTP 酶活性和阻断下游效应子相互作用来灭活它们。尽管它们通常被认为是 GPCR 信号通路的负调节因子，但 RGS 蛋白在肺部的生理功能大多未知。我们鉴定了人和小鼠支气管平滑肌中几种 RGS 蛋白（RGS4、RGS5）的表达。在第 19 财年，我们检查了具有整体和平滑肌特异性 Rgs4 基因缺失的小鼠的表型。近 15% 的严重哮喘患者在摄入阿司匹林和其他非甾体抗炎药后出现支气管痉挛，这种情况被称为阿司匹林加重性呼吸系统疾病 (AERD)，部分原因可能是花生四烯酸代谢物代谢紊乱所致，例如花生四烯酸代谢物代谢紊乱。如前列腺素 E2 (PGE2)。我们还通过用阿司匹林攻击 AERD 样 (ptges1-/-) 小鼠来检查 RGS4 在 AERD 中的作用。我们发现 PGE2 分泌是肺上皮内对过敏原诱导损伤的负稳态信号，并且我们将 RGS4 确定为呼吸道上皮细胞中关键的 NSAID 诱导因子，会损害宿主反应并增加对支气管痉挛的易感性。在患有严重哮喘的受试者中，肺上皮中的 RGS4 表达增加，其表达与功能损伤的程度密切相关。 Rgs4-/- 小鼠中过敏原诱导的 AHR 显着减少，这一发现依赖于气道 PGE2 水平的增加。 RGS4 抑制人支气管上皮细胞中过敏原诱导的 PGE2 分泌，并且在 WT 或 (ptges1-/-) 小鼠中使用 RGS4 抑制剂 CCG203769 治疗分别减轻了过敏原和/或阿司匹林激发诱导的 AHR，并与 PGE2 产量增加相关。我们得出的结论是，RGS4 是严重哮喘和阿司匹林相关哮喘的潜在生物标志物和干预靶点。我们与 Krishnan 博士及其同事合作，提出高通量筛选来鉴定 Alp1 蛋白酶的抑制剂。此类化合物将用于研究 AHR 中蛋白酶过敏原的功能，并阐明真菌相关哮喘中的 ASM 收缩机制。最终，我们希望找到针对 Alp1 蛋白酶的小分子来治疗 SAFS 患者。为此，我们在哺乳动物细胞中生成了重组 Alp1 蛋白酶，用于化合物筛选。