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.

PUBLIC HEALTH RELEVANCE: Health relatedness: This project will identify specific aberrant signaling pathways and molecules involved in the enhanced ASM contractility and AHR in patients with asthma, leading to increased understanding of asthma pathogenesis. The aberrant signaling molecules identified may serve as targets for asthma treatment, which could eventually help in development of more specific therapies for controlling the AHR of asthma. A major strength of our project is that the original data driving this project came from human patients and animal models, thus the likelihood for this project to be directly relevant to human asthma and to lead to new therapies is strong.