Dysregulation of RGS2 Protein and Airway Hyperresponsiveness in Asthma

描述（由申请人提供）：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蛋白信号调节因子2（RGS 2），一种支气管收缩GPCR的细胞内选择性调节剂，是否是治疗哮喘的新的有效靶点。由于我们最近的研究表明，气道平滑肌（ASM）RGS 2在调节哮喘的病理生理气道高反应性（AHR）的关键作用，我们的直接目标是确定AHR发展中的ASM RGS 2失调的机制和病理生理重要性。我们的发现：1）哮喘患者气道RGS 2表达减少。2)RGS 2是过度的人哮喘ASM（HASM-A）细胞收缩的最有效的调节剂，并且RGS 2敲低增强HASM细胞收缩。3)杂合和纯合RGS 2敲除（KO）小鼠均表现出自发性AHR。4)RGS 2 KO增强了Ca 2+振荡，导致增强的ASM细胞收缩，这对AHR至关重要。5)增加的miR- 1271有助于HASM-A细胞中的RGS 2抑制。6)白细胞介素-13（IL-13）是过敏性哮喘中AHR的关键介质，上调miR-1271，但下调RGS 2，增强ASM细胞收缩。7)抗miR-1271减弱HASM细胞中RGS 2的IL-13下调。我们的假设：IL-13通过上调miR-1271诱导ASM细胞中的RGS 2抑制在AHR发展中起关键作用。我们将使用分子、细胞、组织和动物模型来检验这一假设。目的1：确定RGS 2调节ASM收缩的机制。我们将使用诱变来精确定位RGS 2区域，该区域对于调节RGS 2 KO小鼠ASM细胞和HASM-A细胞的过度收缩至关重要。我们还将使用抑制剂和诱变剂来确定RGS 2对细胞内Ca 2+振荡的调节和控制ASM收缩性的敏化。我们的研究将确定RGS 2调节的途径，这些途径在哮喘中是有缺陷的。目的2：阐明RGS 2在ASM细胞中的抑制机制。我们将首先研究外源性miR-1271对RGS 2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RGS 2来减少IL-13诱导的ASM过度收缩。我们还将研究RGS 2过表达是否减弱IL-13的作用，并确定抗miR-1271是否降低HASM-A细胞的过度收缩性。目的3：探讨RGS 2抑制在ASM中的病理生理意义。我们将首先使用侵入性气管造口术和精确切割的肺切片来确定RGS 2 KO对屋尘螨（HDM）诱导的小鼠AHR的影响。将通过原位杂交和免疫组织化学检查HDM对小鼠气道中miR-1271和RGS 2表达的影响。我们还将确定IL-13阻断是否能在体内防止HDM效应。将检查RGS 2损失对气道炎症和上皮功能障碍的影响，这是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是否在体内恢复β 2激动剂支气管保护作用并改善IL-13诱导的小鼠AHR。

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.