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G蛋白偶联受体的天然配体。过敏反应是由IgE致敏的肺肥大细胞上的高亲和力IgE受体的过敏原交联引起的，这被认为是哮喘最常见的病理生理机制。肥大细胞颗粒中的许多化合物或由肥大细胞合成的许多化合物作用于促收缩的GPCRs，从而诱导支气管收缩。例子包括组胺、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，如异丙肾上腺素，可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。RGS蛋白在肺中的生理功能尚不清楚。这个项目的主要目标是确定哪些RGS蛋白在肺中特定的细胞类型中表达，并列举它们在这个器官中的功能。第一个目标主要是通过使用特定抗体的免疫组织化学和免疫印迹来实现的。聚合酶链式反应和免疫印迹实验表明，RGS5在人和小鼠的支气管平滑肌细胞中均有表达。 虽然短效和长效吸入的β2-肾上腺素能受体激动剂(分别为SABA和LABA)可缓解哮喘症状，但在某些患者中，单独使用这两种药物与伴随的抗炎药(皮质类固醇)可能会增加疾病恶化的风险。我们先前发现，用SABA对人精密切割肺切片(PCL)进行预处理会削弱β2激动剂诱导的随后的支气管扩张，这种作用不依赖于受体数量的变化。在目前的工作中，我们提供了证据表明，培养的人呼吸道平滑肌(HuASM)细胞长期暴露于β2激动剂可以直接增强几种化合物(包括凝血酶、缓激肽和组胺)引发的促收缩信号通路。这种处理不影响表面受体、G蛋白或下游效应分子(磷脂酶C-β和肌球蛋白轻链)的表达，而是诱导G蛋白偶联受体(GPCR)的抑制物RGS4和RGS5蛋白的显著减少。RGS5在HuASM中的敲除增加了细胞内钙通量和肌球蛋白轻链(MLC)的磷酸化，这是收缩的先决条件，而RGS5的过表达则抑制了这些反应。RGS5缺陷小鼠的精切肺切片比WT切片收缩更多的氨基甲胆碱。这些结果表明，RGS5在ASM中控制着GPCR诱导的G-α-Q依赖的信号通路。反复使用β2-激动剂不仅会降低对支气管的保护作用，还会由于RGS5表达减少而导致兴奋-收缩通路的敏化。 为了确定RGS5是否也调节其他类型细胞中的钙信号，我们与Olson博士合作研究了RGS5在甲状旁腺中的功能。我们发现：(1)RGS5在甲状旁腺细胞中高表达；(2)甲状旁腺腺瘤与正常组织相比，RGS5表达水平升高；(3)RGS5可以抑制钙诱导的IP3的产生，以响应钙感应受体(CaSR)的刺激；以及(4)RGS5基因缺失的小鼠血浆PTH水平异常降低，但血清钙水平正常。 与Panettieri博士合作的第二个项目确定了RGS4在人和小鼠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是通过一种未知的机制发展起来的，其特征是肺通道平滑肌(ASM)质量和僵硬增加。有丝分裂原如血小板衍生生长因子(PDGF)通过激活磷脂酰肌醇-3-羟基激酶(PI3K)信号通路部分地诱导ASM的可塑性。我们发现，有丝分裂原特异性上调RGS4可诱导ASM过度增殖和收缩减慢，与顽固性哮喘相似。重症哮喘患者支气管平滑肌中RGS4的表达显著增加，且与肺功能下降显著相关。虽然RGS4抑制了GPCR介导的支气管收缩，但意外的是，RGS4是PDGF诱导的人ASM细胞增殖和持续激活PI3K所必需的。这些研究支持一种模型，在该模型中，RGS4表达增加促进ASM的表型转换，在严重哮喘患者中引发不可逆转的气道阻塞。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 G protein coupled receptors (GPCRs). Allergic reactions are initiated by allergen crosslinking of high affinity IgE receptors on lung mast cells sensitized by IgE, and this is considered the most common pathophysiological mechanism in asthma. Many of the compounds contained in mast cell granules or synthesized by mast cells act on procontractile GPCRs to induce bronchoconstriction. Examples include 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 isoproterenol, 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. The physiological function of RGS proteins in the lung is unknown. The principal objective of this project is to determine which RGS proteins are expressed in specific cell types in the lung and to enumerate their functions in this organ. The first objective is accomplished primarily by immunohistochemistry and immunoblotting using specific antibodies. RGS5 was shown to be expressed by PCR and immunoblotting in human and mouse bronchial smooth muscle cells. Although short-acting and long-acting inhaled beta2-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent beta2-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. In current work, we provided evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to beta2-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not affect expression of surface receptor, G protein, or downstream effector (phospholipase C-beta and myosin light chain) pathway components, but rather induced a dramatic reduction in RGS4 and RGS5 proteins, which are inhibitors of G-protein-coupled receptors (GPCR). Knockdown of RGS5 in HuASM increased intracellular calcium flux and myosin light chain (MLC) phosphorylation in response to these ligands, which are prerequisites for contraction, while overexpression of RGS5 inhibited these responses. Precision-cut lung slices from RGS5-deficient mice contracted more to carbachol than WT slices. These results indicate that RGS5 controls GPCR-evoked G-alpha-q-dependent signaling pathways in ASM. Repetitive beta2-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction pathways as a result of reduced RGS5 expression. To determine whether RGS5 also regulates Ca2+ signaling in other cell types, we examined the function of RGS5 in parathyroid in collaboration with Dr. Olson. We found that (1) RGS5 is highly expressed in parathyroid cells; (2) parathyroid adenomas express elevated levels of RGS5 compared to matched pair normal tissue; (3) RGS5 can inhibit calcium-induced IP3 production in response to Calcium sensing receptor (CaSR) stimulation; and (4) mice nullizygous for RGS5 have abnormally depressed plasma PTH levels but normal serum calcium. A second project in collaboration with Dr. Panettieri identified the expression of RGS4 in human and mouse bronchial smooth muscle. In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through an unknown mechanism characterized by increased lung airway smooth muscle (ASM) mass and stiffness. Mitogens such as platelet-derived growth factor (PDGF) induce such plasticity of ASM in part by activating the phosphoinositide-3-OH kinase (PI3K) signaling pathway. We showed that specific upregulation of RGS4 by mitogens induces a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle bundles of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited GPCR-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained PI3K activation in cultured human ASM cells. These studies support a model in which increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in severe asthmatics. 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.