Dysregulation of RGS2 Protein and Airway Hyperresponsiveness in Asthma

哮喘中 RGS2 蛋白的失调和气道高反应性

• 批准号: 8838246
• 负责人: YAPING TU
• 金额: $ 35.83万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8838246
• 关键词: Agonist; Allergic inflammation; Animal Model; Asthma; Attenuated; Automobile Driving; Bronchoconstrictor Agents; Cell model; Cells; Coupled; Data; Development; Down-Regulation; Drug Design; Epithelial; Epithelial Cells; Exhibits; Extrinsic asthma; Family; Functional disorder; G alpha q Protein; G-Protein-Coupled Receptors; GTP-Binding Protein Regulators; GTPase-Activating Proteins; Goals; Health; Human; Immune; Immunohistochemistry; In Situ Hybridization; Inflammation; Inflammatory; Interleukin-13; Knock-out; Knockout Mice; Lead; Link; Lung; Measures; Mediator of activation protein; Messenger RNA; Modeling; Molecular Models; Mus; Muscle Contraction; Mutagenesis; Myosin Light Chains; Pathogenesis; Pathway interactions; Patients; Phosphorylation; Play; Protein Dephosphorylation; Protein Family; Proteins; Pyroglyphidae; RGS Proteins; Regulation; Repression; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Slice; Smooth Muscle Myocytes; Testing; Tissue Model; Tracheostomy procedure; Translations; Wild Type Mouse; airway hyperresponsiveness; airway inflammation; antagonist G; asthmatic; asthmatic airway; base; cell type; constriction; cytokine; human RGS2 protein; in vivo; inhibitor/antagonist; knock-down; member; molecular modeling; mutant; overexpression; prevent; protein expression; receptor; respiratory smooth muscle; response

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are important regulators of cells involved in asthma, and agonists or antagonists of GPCRs are used to treat asthma. Our long-term goal is to determine if Regulator of G-protein Signaling 2 (RGS2), an intracellular selective modulator of bronchoconstrictor GPCRs, is a new and effective target for treatment of asthma. Since our recent studies suggest a critical role of airway smooth muscle (ASM) RGS2 in regulating pathophysiologic airway hyperresponsiveness (AHR) of asthma, our immediate objective is to define mechanisms and pathophysiologic importance of ASM RGS2 dysregulation in AHR development. Our findings: 1) Asthmatics have reduced airway RGS2 expression. 2) RGS2 is the most potent modulator of excessive human asthmatic ASM (HASM-A) cell contraction and RGS2 knock-down enhances HASM cell contraction. 3) Both hetero- and homozygous RGS2 knockout (KO) mice exhibit spontaneous AHR. 4) RGS2 KO augments Ca2+ oscillations, causing enhanced ASM cell contraction critical for AHR. 5) Increased miR- 1271 contributes to RGS2 repression in HASM-A cells. 6) Interleukin-13 (IL-13), a key mediator of AHR in allergic asthma, up-regulates miR-1271 but down-regulates RGS2 with enhanced ASM cell contraction. 7) Anti-miR-1271 attenuates IL-13 down-regulation of RGS2 in HASM cells. Our hypothesis: IL-13-induced RGS2 repression in ASM cells via upregulated miR-1271 plays a crucial role in AHR development. We will test this hypothesis using molecular, cellular, tissue, and animal models. Aim 1: To determine the mechanisms by which RGS2 regulates ASM contraction. We will use mutagenesis to pinpoint the region of RGS2 critical for its regulation of excessive contraction of RGS2 KO mouse ASM cells and HASM-A cells. We will also use inhibitors and mutagenesis to determine RGS2 regulation of intracellular Ca2+ oscillations and sensitization that both control ASM contractility. Our studies will identify the RGS2-regulated pathways that are putatively defective in asthma. Aim 2: To elucidate the mechanism of RGS2 repression in ASM cells. We will first examine the effects of exogenous miR-1271 on RGS2 expression and HASM cell contraction. We will determine if anti-miR-1271 can reduce IL-13-induced ASM hypercontractility by restoring RGS2. We will also investigate if RGS2 overexpression attenuates IL-13 effects and determine if anti-miR-1271 reduces hypercontractility of HASM-A cells. Aim 3: To investigate the pathophysiologic importance of RGS2 repression in ASM in vivo. We will first determine effects of RGS2 KO on house dust mite (HDM)-induced mouse AHR using invasive tracheostomy and precision-cut lung slices. Effects of HDM on miR-1271 and RGS2 expression in mouse airways will be examined by in situ hybridization and immunohistochemistry. We will also determine if IL-13 blockade prevents HDM-effects in vivo. The impact of RGS2 loss on airway inflammation and epithelial dysfunction, other key asthmatic features of HDM-treated mice, will be examined. Finally, we will investigate if anti-miR-1271 restores ¿2-agonist bronchoprotective effect and ameliorates IL-13-induced mouse AHR in vivo.

描述（由适用提供）：G蛋白偶联受体（GPCR）是参与哮喘的细胞的重要调节剂，GPCR的激动剂或拮抗剂用于治疗哮喘。我们的长期目标是确定G蛋白信号传导2（RGS2）的调节剂是支气管收缩GPCR的细胞内选择性调节剂，是治疗哮喘的新目标。由于我们最近的研究表明气道平滑肌（ASM）RGS2在调节哮喘的病理生理气道高反应性（AHR）中的关键作用，我们的直接目标是定义ASM RGS2非正调在AHR发展中的机制和病理生理重要性。我们的发现：1）哮喘患者降低了气道RGS2表达。 2）RGS2是过量人类哮喘ASM（HASM-A）细胞收缩和RGS2敲低增强的最潜在的调节剂HASM细胞收缩。 3）异质和纯合RGS2敲除（KO）小鼠暴露了赞助商AHR。 4）RGS2 KO增强Ca2+振荡，导致ASM细胞收缩的增强对AHR至关重要。 5）增加的miR-1271在HASM-A细胞中有助于RGS2表达。 6）白介素-13（IL-13），是过敏性哮喘中AHR的关键介体，上调miR-1271，但通过增强的ASM细胞收缩来下调RGS2。 7）抗MIR-1271减弱HASM细胞中RGS2的IL-13下调。我们的假设是：IL-13诱导的ASM细胞中通过更新的miR-1271在AHR发育中起着至关重要的作用。我们将使用分子，细胞，组织和动物模型检验这一假设。目标1：确定RGS2调节ASM收缩的机制。我们将使用诱变来指出RGS2的区域至关重要，以调节其过度收缩RGS2 KO小鼠ASM细胞和HASM-A细胞。我们还将使用抑制剂和诱变来确定均控制ASM收缩力的细胞内Ca2+振荡和灵敏度的RGS2调节。我们的研究将识别RGS2调节的途径，这些途径在哮喘中具有预期的缺陷。目标2：阐明ASM细胞中RGS2表达的机理。我们将首先检查外源性miR-1271对RGS2表达和HASM细胞收缩的影响。我们将确定抗MIR-1271是否可以通过恢复RGS2来降低IL-13诱导的ASM超收入。我们还将调查RGS2过表达是否会减弱IL-13的影响，并确定抗MIR-1271是否会降低HASM-A细胞的超合同性。目标3：研究RGS2表达在体内的病理生理重要性。我们首先使用侵入性气管切开术和精确切割肺切片来确定RGS2 KO对房屋粉尘螨（HDM）诱导的小鼠AHR的影响。 HDM对小鼠气道中miR-1271和RGS2表达的影响将通过原位杂交和免疫组织化学检查。我们还将确定IL-13封锁是否阻止体内HDM效应。将检查RGS2损失对气道炎症和上皮功能障碍的影响，即HDM处理的小鼠的其他关键哮喘特征。最后，我们将研究抗MIR-1271是否恢复了2个激动剂支气管保护作用，并改善了IL-13诱导的小鼠AHR体内。