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: 8745411
• 负责人: Kirk m Druey
• 金额: $ 26.2万
• 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/8745411
• 关键词: Actins; Acute; Adenosine; Adrenergic beta-Agonists; Agonist; Albuterol; Allergens; Allergic inflammation; Animal Model; Antibodies; Asthma; Binding; Blood Vessels; Bradykinin; Brain region; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Bronchodilator Agents; CD8-Positive T-Lymphocytes; CD8B1 gene; CREB1 gene; Calcium; Cell Count; Cell Culture Techniques; Cell Line; Cells; Chemotaxis; Chronic; Coagulation Process; Collaborations; Collagen; Contracts; Corpus striatum structure; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Granules; Deposition; Development; Disease; Disease susceptibility; Dorsal; Drug Targeting; Endothelin-1; Extrinsic asthma; F-Actin; Family; G Protein-Coupled Receptor Signaling; 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; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; HIV; Heterotrimeric GTP-Binding Proteins; Hippocampus (Brain); Histamine; Human; Hyperplasia; IgE; Immune system; Immunoblotting; Immunohistochemistry; Inflammation; Inflammatory; Inflammatory Infiltrate; Interleukin-13; Laboratories; Leukotriene Antagonists; Leukotriene D4; Ligands; Link; Lung; Lung Inflammation; Mediating; Mediator of activation protein; Memory; Michigan; Midbrain structure; Mitogens; Modeling; Mucins; Mus; Muscle Contraction; Muscle Fibers; Muscle function; Muscle relaxation phase; Myosin ATPase; National Institute of Allergy and Infectious Disease; Neurons; Obstruction; PAR-1 Receptor; Pathway interactions; Patients; Peptide Hydrolases; Phenotype; Physiological; Platelet-Derived Growth Factor; Production; Protein Binding; Proteins; RGS Domain; RGS Proteins; Receptor Signaling; Regulation; Relative (related person); Relaxation; Resistance; Respiratory physiology; Role; Route; Serine Protease; Signal Pathway; Signal Transduction; Signaling Molecule; Slice; Smooth Muscle; Smooth Muscle Myocytes; Specimen; Steroids; Stress; Structure; T-Lymphocyte; TNF gene; Thrombin; Tissues; Trauma; Universities; airway hyperresponsiveness; airway obstruction; allergic airway inflammation; asthmatic airway; cysteinyl-leukotriene; cytokine; cytotoxicity; eosinophilic inflammation; ezrin; inhibitor/antagonist; interest; mast cell; medical schools; moesin; muscle form; peripheral blood; pulmonary function; receptor coupling; repository; respiratory smooth muscle

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.

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。 诱导支气管平滑肌收缩的主要物质是GPCR的天然配体，如过敏原蛋白酶、凝血酶和过敏原-IgE激活的肥大细胞颗粒中所含的物质（如组胺、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，如沙丁胺醇，增加细胞内cAMP（cAMP）水平，促进ASM松弛。 G蛋白信号调节因子（RGS）蛋白的一个大家族通过保守的RGS结构域与G蛋白α亚基GI和Gq（但不与Gs）结合，并通过增强其内在的GTPase活性和阻断下游效应物相互作用来使它们失活。尽管它们通常被认为是GPCR信号通路的负调节剂，但RGS蛋白在肺中的生理功能大多是未知的。 利用免疫组化和免疫印迹技术，我们确定了几个RGS蛋白（RGS 4，RGS 5，RGS 10）在支气管平滑肌的表达。 虽然嗜酸性粒细胞炎症是过敏性哮喘的典型特征，但它不是AHR的先决条件，这表明结构细胞（如气道平滑肌（ASM））的潜在异常有助于哮喘素质。 ASM中促收缩的G蛋白偶联受体（GPCR）信号转导失调可介导增强的收缩力。RGS 5的缺失促进了ASM中GPCR诱导的Ca 2+动员，从而促进了组成型AHR。来自Rgs 5-/-小鼠的精确切割肺切片（PCLS）在基线时收缩最大，与过敏原激发无关。RGS 5缺乏对变应性炎症的参数影响不大，包括支气管肺泡灌洗液（BALF）中的细胞计数、粘蛋白产生、ASM质量和上皮下胶原沉积。出乎意料的是，来自Rgs 5-/-小鼠的BALF中的IL-13水平相对于WT低得多。这些研究表明，RGS 5的缺乏在不存在过敏性炎症的情况下在小鼠中赋予自发性AHR。 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未知的机制发展，其特征在于肺ASM质量和硬度增加。RGS 4的表达仅限于ASM的一个亚群，并且被有丝分裂原特异性上调，这诱导了与在哮喘中观察到的类似的过度增殖和收缩减退的ASM表型。RGS 4在重度哮喘患者支气管平滑肌中的表达显著增加，并且表达与肺功能降低显著相关。而RGS 4抑制GPCR介导的支气管收缩，RGS 4出乎意料地需要PDGF诱导的增殖和持续激活PI 3 K，一种有丝分裂信号分子，调节ASM增殖。这些研究表明，RGS 4表达的增加促进了ASM的表型转换，在严重哮喘受试者中引起不可逆的气道阻塞。目前的研究正在检查急性和慢性哮喘模型中Rgs 4-/-小鼠的表型。与密歇根大学的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。 这是第一代RGS抑制化合物。 气道松弛和ASM增殖的关键调节因子是环AMP-蛋白激酶A（PKA）-CREB途径。我们实验室以前的研究表明，一些但不是所有的RGS蛋白调节这一信号通路。RGS 10在免疫系统和广泛的脑区域（包括海马、纹状体、中缝背侧和中脑腹侧）中高度表达，也在哮喘ASM中检测到。与埃默里大学医学院的Tansey博士合作，我们发现RGS 10的稳定过表达使模型多巴胺能神经元细胞系（MN 9D）通过在PKA水平调节该信号通路而对TNF诱导的细胞毒性具有抗性。这些结果表明PKA是RGS 10对抗炎症应激的神经保护作用的关键介质，并表明RGS 10可能通过该机制调节气道张力。 最后，我们与Catalfamo博士（LIR/NIAID）合作探索了蛋白酶受体信号传导的调节。创伤和其他组织损伤对血管完整性的破坏导致炎症和凝血级联反应的激活，这与丝氨酸蛋白酶凝血酶激活蛋白酶激活受体1（PAR-1）有关。 我们发现，外周血效应记忆CD 4（+）和CD 8（+）T淋巴细胞表达PAR-1，并且在人类免疫缺陷病毒（HIV）感染患者的CD 8（+）T细胞中表达增加。凝血酶增强健康对照组和HIV感染患者CD 8（+）T细胞的细胞因子分泌此外，凝血酶诱导了CD 8（+）T细胞的趋化作用，但没有趋化性，这导致了结构变化，包括细胞极化和富含F-肌动蛋白和磷酸化ezrin-radexin-moesin蛋白的结构的形成。