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

哮喘是一种可逆性气道阻塞的病理状态，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存在的物质是G蛋白偶联受体（GPCR）的配体，如过敏原蛋白酶、凝血酶和过敏原-IgE激活的肥大细胞颗粒中所含的物质（如组胺、半胱氨酰白三烯（LTD 4）、内皮素1、腺苷和缓激肽）。通常，这些激动剂诱导异源三聚体G蛋白G-α q的活化，其增加平滑肌细胞中细胞内钙的浓度，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，如沙丁胺醇，增加细胞内cAMP（cAMP）水平，促进ASM松弛。虽然嗜酸性粒细胞炎症是过敏性哮喘的典型特征，但它不是气道高反应性（AHR）的先决条件，这表明包括ASM在内的结构细胞的潜在异常有助于哮喘素质。 平滑肌中促收缩信号、GPCR信号的调节异常可介导平滑肌收缩性增强。 10-15%的哮喘患者经历严重的、危及生命的发作，甚至死亡，尽管使用支气管扩张剂和皮质类固醇进行积极治疗。 其中近一半（1000 - 2000万）对丝状真菌（例如烟曲霉，Af）过敏（即具有IgE介导的过敏反应），这已被指定为“真菌致敏的严重哮喘”（SAFS）。目前SAFS的治疗包括抗真菌药或靶向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暴露诱导的小鼠过敏性气道炎症模型，本项目有两个首要目标：1）确定炎症介质下游ASM收缩信号的紊乱; 2）检查过敏原蛋白酶活性在AHR中的功能，特别是与过敏原-ASM相互作用的关系。蛋白酶活性是能够诱导哮喘的过敏原的常见且重要的特征，最显著的是来自普遍存在的真菌如Af。以前我们发现真菌和哮喘之间的因果关系独立于变应原性发生;换句话说，宿主对变应原的炎症反应。 分泌的Af蛋白酶碱性蛋白酶1（Alp 1）在哮喘受试者的气道中被检测到，但在对照组中未检测到。在小鼠和体外研究中，我们发现Alp 1通过降解细胞外基质（ECM）组分直接促进AHR，导致ASM收缩失调。随后，我们证明了Alp 1在缺乏嗜酸性粒细胞或蛋白酶激活受体2（PAR 2）的小鼠中诱导AHR，并且Alp 1在体外直接增加人ASM细胞的收缩力。 在20财年，我们通过R21资助与Krishnan博士合作，启动了一项化合物筛选，以识别Alp 1抑制剂。一个关键的治疗差距是鉴定对真菌Alp 1具有特异性亲和力但对人气道中存在的内源性丝氨酸蛋白酶没有活性的部分。高通量筛选现有库的商业可用的蛋白酶抑制剂将用于确定抑制剂的Alp 1蛋白酶。初级命中可以基于对Alp 1诱导的ASM过度收缩的抑制进行等级排序。可以评估一组优先的有效和无毒的命中物对以下各项的影响：（i）来自患有真菌性哮喘的受试者的ASM细胞;（ii）用Alp 1处理的鼠和人精确切割肺切片（PCLS）的气道;（iii）真菌性哮喘的鼠模型。我们希望这些研究能够确定新的抗SAFS候选药物，并为支气管收缩的ASM内在机制和过敏原蛋白酶依赖机制提供新的见解。 该项目的第二个重点领域是研究肺中G蛋白信号传导（RGS）蛋白的调节因子。RGS通过保守的RGS结构域与G蛋白α亚基Gi和Gq（但不与Gs）结合，并通过催化其固有的GT3活性和通过阻断下游效应物相互作用使其失活。尽管它们通常被认为是GPCR信号通路的负调节剂，但RGS蛋白在肺中的生理功能大多是未知的。 我们鉴定了几种RGS蛋白（RGS 4，RGS 5）在人和小鼠支气管平滑肌中的表达。 在20财年，我们检查了具有全局和平滑肌特异性Rgs 4基因缺失的小鼠的表型。过敏原引起宿主产生作用于G蛋白偶联受体的介质以调节气道张力。其中包括前列腺素E2（PGE 2），它除了作为支气管扩张剂的作用外，还具有抗炎作用。一些哮喘患者在摄入阿司匹林和其他非甾体抗炎药后会发生支气管痉挛，这种疾病称为阿司匹林加重的呼吸道疾病（AERD）。这种情况可能部分是由于对PGE 2的支气管保护作用的异常依赖。在患有严重哮喘的人类受试者中呼吸道上皮中RGS 4表达增加。在Rgs 4-/-小鼠中，过敏原诱导的AHR出乎意料地减少，这一发现与气道PGE 2水平增加有关。RGS 4调节过敏原诱导的人支气管上皮细胞PGE 2分泌和小鼠前列腺素依赖性支气管扩张RGS 4拮抗剂CCG 203769分别减弱野生型或ptges 1-/-小鼠的过敏原或阿司匹林激发诱导的AHR，并与气道PGE 2水平升高相关。这些发现表明，RGS 4通过减少过敏原和阿司匹林诱导的哮喘中气道PGE 2的生物合成而促进AHR的发展。

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 ASM 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 Alkaline protease 1 (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 extracellular matrix (ECM) components, leading to dysregulation of ASM contraction. Subsequently, we demonstrated that Alp1 induces AHR in mice devoid of eosinophils or the protease activated receptor 2 (PAR2) and that Alp1 directly increased contractile force of human ASM cells in vitro. In Fiscal Year (FY) 20, we initiated a compound screen to identify Alp1 inhibitors in collaboration with Dr. Krishnan through an R21 grant. A critical therapeutic gap is to identify moieties with specific affinity for fungal Alp1 but without activity on endogenous serine proteases present in the human airway. High throughput screening of existing libraries of commercially available protease inhibitors will be used to identify inhibitors of Alp1 protease. Primary hits can be rank-ordered based on inhibition of Alp1-induced ASM hypercontraction. A prioritized set of efficacious and non-toxic hits can be evaluated for its effects on (i) ASM cells from subjects with fungal asthma; (ii) airways of murine and human precision-cut lung slices (PCLS) treated with Alp1; (iii) murine models of fungal asthma. We expect these studies to identify novel anti-SAFS drug candidates as well as provide novel insight into both ASM-intrinsic and allergen-protease-dependent mechanisms of bronchoconstriction. 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 20, we examined the phenotype of mice with global and smooth muscle-specific Rgs4 gene deletion. Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease (AERD). This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. RGS4 expression in respiratory epithelium is increased in human subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation in mice. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. These findings suggest that RGS4 contributes to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.