Dysregulation of RGS2 Protein and Airway Hyperresponsiveness in Asthma

描述(申请人提供)：G蛋白偶联受体(GPCRs)是参与哮喘的细胞的重要调节因子，GPCRs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蛋白信号转导调节因子2(RGS2)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)RGS2在调节哮喘的病理生理学气道高反应性(AHR)中起着关键作用，我们的近期目标是明确ASM RGS2异常调节在AHR发生发展中的机制和病理生理学意义。我们的发现：1)哮喘患者的呼吸道RGS2表达减少。2)RGS2是人类哮喘ASM(HASM-A)细胞过度收缩的最强调节剂，RGS2基因敲除可增强HASM细胞的收缩。3)RGS2基因敲除(KO)杂合子和纯合RGS2基因敲除小鼠均表现出自发性AHR。4)RGS2 KO增强了钙振荡，导致ASM细胞收缩增强，这对AHR至关重要。5)miR-1271表达增加参与了HASM-A细胞中RGS2的抑制。6)IL-13是过敏性哮喘AHR的关键介质，上调miR-1271，下调RGS2，使ASM细胞收缩增强。7)抗miR-1271可减弱IL-13对HASM细胞RGS2的下调作用。我们的假设是：IL-13通过上调miR-1271诱导ASM细胞中RGS2的抑制在AHR的发展中起关键作用。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：探讨RGS2调节ASM收缩的机制。我们将使用突变来精确定位RGS2区域，该区域对其调节RGS2 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突变来确定RGS2对细胞内钙振荡的调节和敏化，这两者都控制着ASM的收缩能力。我们的研究将确定RGS2调节的通路在哮喘中被认为是有缺陷的。目的：阐明RGS2在ASM细胞中的抑制机制。我们将首先检测外源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过度收缩。我们还将研究RGS2过表达是否减弱IL-13的影响，并确定抗miR-1271是否降低HASM-A细胞的过度收缩能力。目的：探讨活体ASM中RGS2抑制的病理生理学意义。我们将首先使用有创气管切开术和精密肺切片来确定RGS2 KO对屋尘螨(HDM)诱导的小鼠AHR的影响。用原位杂交和免疫组织化学方法检测HDM对小鼠呼吸道miR-1271和RGS2表达的影响。我们还将确定阻断IL-13是否能在体内阻止HDM效应。将研究RGS2缺失对呼吸道炎症和上皮功能障碍的影响，以及HDM治疗的小鼠哮喘的其他关键特征。最后，我们将在体内研究抗miR-1271是否能恢复2-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.