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，包括肺部炎症和细支气管平滑肌的过度收缩。然而，这种气道高反应性(AHR)可以在没有明显炎性浸润物的情况下存在，这表明该病可能存在原发的气道平滑肌(ASM)数量或收缩异常。引起支气管平滑肌收缩的主要物质是GPCRs的天然配体，如过敏原蛋白水解酶和过敏原-IgE激活的肥大细胞颗粒中的那些物质(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。 G蛋白信号转导蛋白(RGS)的一个大家族通过保守的RGS结构域与G蛋白α亚单位Gi和Gq(而不是Gs)结合，并通过增强其固有的GTPase活性和阻断下游效应器的相互作用来使其失活。虽然它们通常被认为是GPCR信号通路的负调节因子，但RGS蛋白在肺中的生理功能尚不清楚。用免疫组织化学和免疫印迹法检测RGS4和RGS5在支气管平滑肌中的表达。 在严重哮喘中，对支气管扩张剂和类固醇不敏感的气流阻塞通过未知的机制发展，其特征是肺ASM质量和僵硬增加。我们探讨了RGS4和RGS5在晚期哮喘ASM增殖和收缩能力降低中的作用。通过免疫细胞化学染色，我们研究了RGS4在健康受试者和轻、中、重度哮喘患者的支气管内活检组织中的表达。在培养的人ASM细胞和人精切肺切片上对细胞增殖、激动剂诱导的钙动员和支气管收缩进行了评估。利用获得和丧失功能的方法，我们研究了RGS蛋白在刺激人类ASM增殖和抑制支气管收缩中的确切作用。RGS4的表达仅限于ASM的一个亚群，并被有丝分裂原特异性上调，这导致了与顽固性哮喘相似的ASM过度增殖和收缩减慢的表型。重症哮喘患者肺组织中RGS4的表达显著增加，且与肺功能下降显著相关。RGS4抑制GPCR介导的支气管收缩，而RGS4意外地是PDGF诱导的增殖和PI3K的持续激活所必需的，PI3K是调节ASM增殖的有丝分裂信号分子。这些研究表明，RGS4的表达增加促进了ASM的表型转换，在严重哮喘患者中引发了不可逆转的气道阻塞。 我们还研究了RGS5在固定性气道阻塞发生中的作用。我们比较了死于非呼吸系统原因的受试者和死于哮喘的受试者死后ASM中GPCR诱导的钙动员和与促收缩信号相关的GPCRs和信号蛋白的表达。尽管与健康供者相比，哮喘ASM细胞中促收缩GPCRs(缓激肽B2、组胺H1和PAR1)的表达增加或相似，但哮喘ASM细胞显示组胺诱导的钙动员减少，对缓激肽和凝血酶的反应类似，提示存在受体后信号缺陷。相应地，与健康的人类供体或幼小鼠相比，哮喘患者和过敏原攻击小鼠的ASM细胞和支气管平滑肌束中ASM收缩抑制因子RGS5的表达都增加。RGS5的过度表达损害了钙向凝血酶、组胺和卡巴胆碱的释放，并减少了精密切割肺切片对卡巴胆碱的收缩。这些结果表明，在严重和致命的哮喘中，RGS5表达增加也是导致心肌细胞缩短减少的原因之一。 与纽比格博士合作，我们将在动物模型和细胞培养中检查RGS4特异性抑制剂对哮喘表型和ASM增殖和收缩的影响。这是第一个出现的RGS抑制化合物。

Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and hyper-contractility of the bronchiolar smooth muscle. Such airway hyperresponsiveness (AHR) can exist in the absence of frank inflammatory infiltrates, however, suggesting that primary abnormalities in airway smooth muscle (ASM) number or contraction may exist in this disease. The major substances that induce bronchial smooth muscle contraction are natural ligands of GPCRs, such as allergen proteases 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. A large family of Regulators of G protein signaling (RGS) proteins binds to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivates them by accentuating 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 unknown. Using immunohistochemistry and immunoblotting with specific antibodies, we identified expression of RGS4 and RGS5 in bronchial smooth muscle. In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung ASM mass and stiffness. We explored the role of RGS4 and RGS5 in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthmatic ASM. Using immunocytochemical staining, we investigated RGS4 expression in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were evaluated in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, we studied the precise role of RGS proteins in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited GPCR-mediated bronchoconstriction, RGS4 was unexpectedly required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma. We also investigated the role of RGS5 in the development of fixed airway obstruction. We compared the GPCR-induced calcium mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2, histamine H1, and PAR1) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced calcium mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of RGS5, an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both asthmatic human subjects and allergen-challenged mice, relative to those of healthy human donors or naive mice. The overexpression of RGS5 impaired the release of calcium to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression also contributes to decreased myocyte shortening in severe and fatal asthma. In collaboration with Dr. Neubig, we will examine the effect of an RGS4-specific inhibitor on the development of the asthma phenotype and ASM hyperplasia and contraction in animal models and cell culture. This is the first RGS inhibitory compound that has emerged.