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、凝血酶，以及过敏原-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信号通路的负调控因子，但RGS蛋白在肺中的生理功能大多尚不清楚。用免疫组织化学和免疫印迹法检测了几种RGS蛋白(RGS4、RGS5、RGS10)在支气管平滑肌中的表达。 虽然嗜酸性炎症是过敏性哮喘的典型表现，但它不是AHR的先决条件，这表明潜在的结构细胞异常，如气道平滑肌(ASM)，参与了哮喘的素质。ASM中促收缩、G蛋白偶联受体(GPCR)信号的失调可介导收缩能力的增强。RGS5的缺失促进了ASM的结构性AHR，这是由于增强了GPCR诱导的ASM内钙动员。来自NAVE RGS5-/-小鼠的精密切割肺切片(PCL)在基线时收缩最大，与过敏原攻击无关。RGS5缺乏对过敏性炎症指标影响不大，包括支气管肺泡灌洗液(BALF)中细胞计数、粘蛋白产生、ASM质量和上皮下胶原沉积。出乎意料的是，与WT相比，RGS5-/-小鼠BALF中的IL-13水平要低得多。这些研究表明，在没有过敏性炎症的情况下，RGS5的缺乏会导致小鼠自发的AHR。 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。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。目前的研究正在检测RGS4-/-小鼠在急性和慢性哮喘模型中的表型。我们将与密歇根大学的纽比格博士合作，在动物模型和细胞培养中研究RGS4特异性抑制剂对哮喘表型和ASM增殖和收缩的影响。这是一种第一代RGS抑制化合物。 环磷酸腺苷-蛋白激酶A(PKA)-CREB途径是呼吸道松弛和ASM增殖的关键调节因子。我们实验室以前的研究表明，一些但不是所有的RGS蛋白调节这一信号途径。RGS10在免疫系统中高度表达，在包括海马体、纹状体、中缝背侧和腹侧中脑在内的广泛脑区中都有表达，在哮喘的ASM中也可以检测到。我们与埃默里大学医学院的Tansey博士合作，发现RGS10的稳定过表达使一个模型多巴胺能神经元细胞株(MN9D)能够通过在PKA水平上调节这一信号通路来抵抗肿瘤坏死因子诱导的细胞毒性。这些结果表明，PKA是RGS10抗炎性应激神经保护作用的关键介质，提示RGS10可能通过这一机制调节气道张力。 最后，我们与Catallamo博士(LIR/NIAID)合作探索了蛋白酶受体信号的调控。创伤和其他组织损伤对血管完整性的破坏会导致炎症和凝血级联反应的激活，凝血级联反应通过丝氨酸蛋白酶凝血酶激活蛋白酶激活受体1(PAR-1)而联系在一起。我们发现，人类免疫缺陷病毒(HIV)感染者外周血记忆效应T细胞中的CD4(+)和CD8(+)T细胞表达PAR-1，CD8(+)T细胞中PAR-1的表达增强。凝血酶促进健康对照和HIV感染者CD8(+)T细胞分泌细胞因子。此外，凝血酶诱导CD8(+)T细胞的趋化运动，而不是趋化，导致结构变化，包括细胞极化和形成富含F-肌动蛋白和磷酸化的Ezrin-radexin-moesin蛋白的结构。

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, 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. 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 mostly unknown. Using immunohistochemistry and immunoblotting with specific antibodies, we identified expression of several RGS proteins (RGS4, RGS5, RGS10) in bronchial smooth muscle. Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for AHR, suggesting that underlying abnormalities in structural cells such as airway smooth muscle (ASM) contribute to the asthmatic diathesis. Dysregulation of procontractile, G protein-coupled receptor (GPCR) signaling in ASM could mediate enhanced contractility. Loss of RGS5 promoted constitutive AHR due to enhanced GPCR-induced Ca2+ mobilization in ASM. Precision-cut lung slices (PCLS) from nave Rgs5-/- mice contracted maximally at baseline, independent of allergen challenge. RGS5 deficiency had little effect on parameters of allergic inflammation including cell counts in bronchoalveolar lavage fluid (BALF), mucin production, ASM mass, and subepithelial collagen deposition. Unexpectedly, IL-13 levels were much lower in BALF from Rgs5-/- mice relative to WT. These studies showed that deficiency of RGS5 confers spontaneous AHR in mice in the absence of allergic inflammation. In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung ASM mass and stiffness. 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. Current studies are examining the phenotype of Rgs4-/- mice in models of acute and chronic asthma. In collaboration with Dr. Neubig at the University of Michigan, 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 a first-generation RGS inhibitory compound. A key regulator of airway relaxation and ASM proliferation is the cyclic AMP-protein kinase A (PKA)-CREB pathway. Previous studies in our laboratory indicated that some but not all RGS proteins regulate this signaling route. RGS10, which is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain, is also detected in asthmatic ASM. In collaboration with Dr. Tansey at Emory University School of Medicine, we found that stable over-expression of RGS10 rendered a model dopaminergic neuronal cell line (MN9D) resistant to TNF-induced cytotoxicity through regulation of this signaling pathway at the level of PKA. These results identified PKA as a key mediator of the neuroprotective effect of RGS10 against inflammatory stress and suggest that RGS10 may regulate airway tone through this mechanism. Finally, we explored the regulation of protease receptor signaling in collaboration with Dr. Catalfamo (LIR/NIAID). Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade, which are linked by activation of protease-activated receptor 1 (PAR-1) by the serine protease thrombin. We found that peripheral blood effector memory CD4(+) and CD8(+) T lymphocytes expressed PAR-1 and that expression was increased in CD8(+) T cells from human immunodeficiency virus (HIV)-infected patients. Thrombin enhanced cytokine secretion in CD8(+) T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8(+) T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins.