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

G 蛋白偶联受体对正常和哮喘肺功能的调节

• 批准号: 8336178
• 负责人: Kirk m Druey
• 金额: $ 33.18万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8336178
• 关键词: Actins; Adenosine; Adrenal Cortex Hormones; Adrenergic Agonists; Adrenergic beta-Agonists; Affect; Affinity; Agonist; Allergens; Allergic Reaction; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Asthma; Binding; Bradykinin; Breathing; Bronchoconstriction; Bronchodilation; Bronchodilator Agents; Calcium; Calcium-Sensing Receptors; Carbachol; Cell Culture Techniques; Cells; Collaborations; Contracts; Cyclic AMP; Cytoplasmic Granules; Depressed mood; Development; Disease; Drug Delivery Systems; Endothelin-1; Family; G(q) Alpha; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gene Deletion; Goals; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric GTP-Binding Proteins; Histamine; Human; Hyperplasia; IgE; IgE Receptors; Immunoblotting; Immunohistochemistry; Inflammatory Infiltrate; Isoproterenol; Lead; Leukotriene Antagonists; Leukotriene D4; Ligands; Lung; Lung Inflammation; Mediating; Mitogens; Modeling; Mus; Muscle Contraction; Muscle Fibers; Muscle relaxation phase; Myosin ATPase; Myosin Light Chains; Normal tissue morphology; Obstruction; Organ; Parathyroid Adenoma; Parathyroid gland; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Physiological; Plasma; Platelet-Derived Growth Factor; Production; Protein Binding; Proteins; RGS Domain; RGS Proteins; Regulation; Relaxation; Respiratory physiology; Role; Serotonin azidobenzamidine; Serum; Signal Pathway; Signal Transduction; Slice; Smooth Muscle; Smooth Muscle Myocytes; Steroids; Surface; Symptoms; Thrombin; Tissues; Up-Regulation; Work; airway hyperresponsiveness; airway obstruction; allergic airway inflammation; beta-Myosin; cell type; crosslink; cysteinyl-leukotriene; disorder risk; inhibitor/antagonist; interest; mast cell; muscle form; overexpression; phospholipase C beta; pulmonary function; receptor; receptor coupling; release of sequestered calcium ion into cytoplasm; respiratory smooth muscle; response

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.

哮喘是一种可逆性气道阻塞的病理状态，由肺部炎症和细支气管平滑肌过度收缩组成。 这种气道高反应性（AHR）可以在没有明显炎症浸润的情况下存在，然而，这表明在这种疾病中可能存在气道平滑肌（ASM）数量或收缩的原发性异常。 引起支气管平滑肌收缩的主要物质是G蛋白偶联受体（GPCRs）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发的，这被认为是哮喘中最常见的病理生理机制。 肥大细胞颗粒中含有的或由肥大细胞合成的许多化合物作用于促收缩GPCR以诱导支气管收缩。 实例包括组胺、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，如异丙肾上腺素，增加细胞内cAMP（cAMP）水平，促进ASM松弛。 G蛋白信号传导调节因子（Regulators of G protein signaling，RGS）蛋白的大家族通过保守的RGS结构域结合G蛋白α亚基Gi和Gq（但不结合Gs），并通过增强其内在的GT3活性和通过阻断下游效应物相互作用使其失活。RGS蛋白在肺中的生理功能是未知的。 该项目的主要目的是确定哪些RGS蛋白在肺中的特定细胞类型中表达，并列举它们在该器官中的功能。 第一个目的主要是通过免疫组织化学和免疫印迹使用特异性抗体。 通过PCR和免疫印迹显示RGS 5在人和小鼠支气管平滑肌细胞中表达。 尽管短效和长效吸入性β 2-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，单独使用任何一种药物而不合并使用抗炎药（皮质类固醇）可能会增加疾病加重的风险。我们以前发现，预处理的人精密切割肺切片（PCLS）与SABA损害随后β 2-激动剂诱导的支气管扩张，发生独立的受体数量的变化。在目前的工作中，我们提供的证据表明，培养的人气道平滑肌（HuASM）细胞长期暴露于β 2受体激动剂直接增强促收缩信号通路引起的几种化合物，包括凝血酶，缓激肽，组胺。 这种处理不影响表面受体、G蛋白或下游效应子（磷脂酶C-β和肌球蛋白轻链）途径组分的表达，而是诱导RGS 4和RGS 5蛋白的显著减少，这两种蛋白是G蛋白偶联受体（GPCR）的抑制剂。HuASM中RGS 5的敲低增加了细胞内钙通量和肌球蛋白轻链（MLC）磷酸化，以响应这些配体，这是收缩的先决条件，而RGS 5的过表达抑制了这些反应。 来自RGS 5缺陷小鼠的精确切割的肺切片比WT切片对卡巴胆碱收缩更多。这些结果表明，RGS 5控制ASM中GPCR诱发的G-α-q依赖性信号通路。重复使用β 2-激动剂不仅可能导致支气管保护作用降低，还可能由于RGS 5表达降低而导致兴奋-收缩途径致敏。 为了确定RGS 5是否也调节其他细胞类型中的Ca 2+信号传导，我们与Olson博士合作研究了RGS 5在甲状旁腺中的功能。我们发现：（1）RGS 5在甲状旁腺细胞中高度表达;（2）与配对正常组织相比，甲状旁腺腺瘤表达升高水平的RGS 5;（3）RGS 5可抑制钙敏感受体（CaSR）刺激引起的钙诱导的IP 3产生;（4）RGS 5缺失的小鼠血浆PTH水平异常降低，但血清钙正常。 与Panettieri博士合作的第二个项目确定了RGS 4在人类和小鼠支气管平滑肌中的表达。 在重度哮喘中，支气管扩张剂和类固醇不敏感的气流阻塞通过一种未知的机制发展，其特征在于肺气道平滑肌（ASM）质量和硬度增加。 有丝分裂原如血小板衍生生长因子（PDGF）部分地通过激活磷酸肌醇-3-OH激酶（PI 3 K）信号通路诱导ASM的这种可塑性。 我们发现，有丝分裂原特异性上调RGS 4可诱导一种过度增殖和低收缩的ASM表型，与在哮喘中观察到的表型相似。 RGS 4在重度哮喘患者支气管平滑肌束中的表达显著增加，并且表达与肺功能降低显著相关。 尽管RGS 4抑制GPCR介导的支气管收缩，但出乎意料的是，在培养的人ASM细胞中，PDGF诱导的增殖和持续的PI 3 K活化需要RGS 4。 这些研究支持了一个模型，其中增加RGS 4表达促进ASM的表型转换，引起严重哮喘患者的不可逆气道阻塞。与Neubig博士合作，我们将在动物模型和细胞培养中研究RGS 4特异性抑制剂对哮喘表型和ASM增生和收缩发展的影响。 这是第一个出现的RGS抑制化合物。