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）是哮喘相关细胞的重要调节因子，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调节剂（RGS2）是否是治疗哮喘的一个新的有效靶点，RGS2是一种支气管收缩gpcr细胞内选择性调节剂。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）中起关键作用，我们的直接目标是确定ASM RGS2失调在AHR发展中的机制和病理生理重要性。我们发现：1)哮喘患者气道RGS2表达降低。2) RGS2是人类哮喘ASM （HASM- a）细胞过度收缩的最有效调节剂，RGS2的敲除增强了HASM细胞的收缩。3)异型和纯合子RGS2敲除（KO）小鼠均表现出自发性AHR。4) RGS2 KO增强Ca2+振荡，导致ASM细胞收缩增强，这对AHR至关重要。5) miR- 1271的增加参与了HASM-A细胞中RGS2的抑制。6)变应性哮喘AHR的关键介质白介素-13 （IL-13）上调miR-1271，下调RGS2， ASM细胞收缩增强。7) Anti-miR-1271可减弱HASM细胞中IL-13对RGS2的下调。我们的假设是：il -13通过上调miR-1271在ASM细胞中诱导RGS2抑制在AHR的发展中起着至关重要的作用。我们将使用分子、细胞、组织和动物模型来验证这一假设。目的1：确定RGS2调控ASM收缩的机制。我们将使用诱变技术来确定RGS2的关键区域，该区域调节RGS2 KO小鼠ASM细胞和hsm - a细胞的过度收缩。我们还将使用抑制剂和诱变来确定RGS2对细胞内Ca2+振荡和敏化的调节，这两者都控制着ASM的收缩性。我们的研究将确定在哮喘中被认为有缺陷的rgs2调控通路。目的2：阐明ASM细胞中RGS2抑制的机制。我们将首先研究外源性miR-1271对RGS2表达和HASM细胞收缩的影响。我们将确定anti-miR-1271是否可以通过恢复RGS2来降低il -13诱导的ASM超收缩性。我们还将研究RGS2过表达是否会减弱IL-13的作用，并确定anti-miR-1271是否会降低HASM-A细胞的超收缩性。目的3：探讨RGS2抑制在ASM体内的病理生理意义。我们将首先通过有创气管切开术和精确肺切片来确定RGS2 KO对屋尘螨（HDM）诱导的小鼠AHR的影响。通过原位杂交和免疫组织化学检测HDM对小鼠气道中miR-1271和RGS2表达的影响。我们还将确定IL-13阻断是否能阻止体内hdm效应。RGS2缺失对气道炎症和上皮功能障碍（hdm治疗小鼠的其他关键哮喘特征）的影响将被检测。最后，我们将在体内研究anti-miR-1271是否能恢复2-受体激动剂的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.