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

哮喘是一种可逆性呼吸道阻塞的病理状态，包括肺部炎症和支气管平滑肌的过度收缩。引起支气管平滑肌收缩的主要天然物质是G蛋白偶联受体(GPCRs)的配体，如变应原蛋白水解酶、凝血酶，以及那些包含在过敏原-IgE激活的肥大细胞颗粒中的物质(如组胺、半胱氨基白三烯(LTD4)、内皮素1、腺苷和缓激肽)。一般来说，这些激动剂诱导异源三聚体G蛋白G-αQ的激活，从而增加平滑肌细胞内钙的浓度，促进肌动蛋白-肌球蛋白的相互作用和肌肉纤维的缩短。相反，作用于G-α-S偶联受体的配体，如沙丁胺醇，可以增加细胞内环磷酸腺苷(CAMP)的水平，从而促进ASM的松弛。虽然嗜酸性炎症是过敏性哮喘的典型表现，但它不是呼吸道高反应性(AHR)的先决条件，这表明包括ASM在内的潜在结构细胞异常参与了哮喘的素质。ASM中前收缩、GPCR信号的失调可能介导了收缩能力的增强。 10-15%的哮喘患者经历了严重的危及生命的发作，甚至死亡，尽管使用了积极的支气管扩张剂和皮质类固醇治疗。其中近一半(1,000-2,000万人)对丝状真菌(如烟曲霉，Af)致敏(即有IgE介导的过敏)，这种真菌已被指定为“严重哮喘伴真菌致敏”(SAFS)。目前治疗SAFS的方法包括抗真菌药物或针对IgE的奥马珠单抗(MAb)，但并未取得一致的成功。利用呼吸道Af暴露诱导的小鼠过敏性呼吸道炎症模型，我们有两个主要目标：1)确定炎症介质下游ASM收缩信号的紊乱；2)检测AHR中变应原蛋白酶活性的功能，特别是与变应原-ASM相互作用有关的功能。蛋白酶活性是能够诱发哮喘的过敏原的一个共同和重要的特征，最明显的是来自普遍存在的真菌，如Af。是否有任何过敏原直接影响ASM收缩还从未被研究过。此前，我们发现真菌和哮喘之间的因果联系独立于过敏性发生；换句话说，宿主对过敏原的炎症反应。在哮喘患者的呼吸道中检测到分泌型Af蛋白水解酶碱性蛋白水解酶1(Alp1)，而在对照组中未检测到。在小鼠和体外研究中，我们发现Alp1通过降解细胞外基质(ECM)成分直接促进AHR，导致ASM收缩的失调。随后，我们证明了Alp1在没有嗜酸性粒细胞或蛋白酶激活受体2(PAR2)的小鼠中诱导AHR，并且Alp1在体外直接增加了人ASM细胞的收缩能力。 在20财年，我们与Krishnan博士合作，通过R21拨款启动了一项复合筛查，以确定Alp1抑制剂。一个关键的治疗缺口是确定对真菌Alp1具有特定亲和力但对人类呼吸道中存在的内源性丝氨酸蛋白酶没有活性的部分。高通量筛选现有的商业可获得的蛋白酶抑制剂文库将用于鉴定Alp1酶的抑制剂。根据对Alp1诱导的ASM过度收缩的抑制程度，一次击打可以排序。一组优先的有效和无毒的打击可以从以下方面进行评估：(I)真菌性哮喘受试者的ASM细胞；(Ii)经Alp1处理的小鼠和人类精密切割肺片(PCL)的呼吸道；(Iii)真菌性哮喘的小鼠模型。我们期望这些研究能够确定新的抗SAFS药物候选药物，并为ASM固有的和变应原蛋白水解酶依赖的支气管收缩机制提供新的见解。 该项目的第二个重点领域是研究肺中G蛋白信号(RGS)蛋白的调节。RGSS通过保守的RGS结构域与G蛋白α亚基Gi和Gq(而不是Gs)结合，并通过催化其固有的GTPase活性和阻断下游效应器的相互作用来灭活它们。虽然它们通常被认为是GPCR信号通路的负调控因子，但RGS蛋白在肺中的生理功能大多尚不清楚。我们鉴定了几种RGS蛋白(RGS4、RGS5)在人和小鼠的支气管平滑肌中的表达。 在20财年，我们检测了具有全局和平滑肌特异性RGS4基因缺失的小鼠的表型。过敏原诱导宿主产生作用于G蛋白偶联受体的介质，以调节呼吸道张力。其中包括前列腺素E2(PGE2)，它除了具有支气管扩张剂的作用外，还具有抗炎作用。一些哮喘患者在服用阿司匹林和其他非类固醇抗炎药后出现支气管痉挛，这种疾病被称为阿司匹林加重的呼吸系统疾病(AERD)。这种情况的部分原因可能是对PGE2的支气管保护作用的异常依赖。严重哮喘患者呼吸道上皮细胞RGS4表达增加。在RGS4-/-小鼠中，过敏原诱导的AHR意外减少，这一发现与呼吸道PGE2水平升高有关。RGS4调节变应原诱导的人支气管上皮细胞PGE2分泌和小鼠前列腺素依赖的支气管扩张。RGS4拮抗剂CCG203769分别减弱了过敏原或阿司匹林攻击野生型或ptges1/-小鼠引起的AHR，并与气道PGE2水平升高有关。这些发现表明，在过敏原和阿司匹林诱导的哮喘中，RGS4通过减少呼吸道PGE2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.