RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms. RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms

Regulators of G protein signaling (RGS proteins) constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase activating (GAP) activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle (ASM) contractility. Rgs2 or Rgs5 gene deletion in mice enhances AHR and ASM contraction whereas RGS4 knockout (KO) mice unexpectedly have decreased AHR due to increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GAP activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchial contraction compared to either RGS4 KO mice or WT controls. RGS4 interacted with the p85a subunit of PI3 kinase (PI3K) and inhibited PI3K-dependent PGE2 secretion elicited by TGFb in airway epithelial cells. Together these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner. Overall design: RGS4 knockout mice were generated to examine the role of RGS4 in asthma. These RGS4 KO mice were compared to WT, under exposure to Aspergillus fumigatus and control exposure to PBS. Poly A mRNAseq was performed.

G蛋白信号调节蛋白（Regulators of G protein signaling, RGS蛋白）主要通过GTP酶激活（GTPase activating, GAP）活性，在全身范围内约束G蛋白偶联受体（G protein-coupled receptor, GPCR）介导的及其他生理应答，但其调控作用并非完全依赖此活性。
哮喘是一种高发性疾病，其特征为气道对环境刺激产生气道高反应性（airway hyper-responsiveness, AHR），该病理过程部分源于GPCR介导的气道平滑肌（airway smooth muscle, ASM）收缩功能异常增强。
小鼠体内Rgs2或Rgs5基因敲除会加剧AHR与ASM收缩，而令人意外的是，RGS4基因敲除（knockout, KO）小鼠的AHR却有所降低，这是因为肺上皮细胞合成的支气管扩张剂前列腺素E2（prostaglandin E2, PGE2）增多所致。
本研究发现，携带编码点突变（N128A）的Rgs4等位基因、可显著削弱RGS4 GAP活性的敲入小鼠，相较于RGS4 KO小鼠与野生型（wild type, WT）对照小鼠，表现出更严重的AHR、更低的气道PGE2水平，以及GPCR诱导的支气管收缩增强。
RGS4可与磷脂酰肌醇3-激酶（PI3 kinase, PI3K）的p85α亚基相互作用，并抑制气道上皮细胞中转化生长因子β（TGF-β）诱导的PI3K依赖性PGE2分泌。
综上，本研究结果提示RGS4可通过不依赖G蛋白的方式调控气道炎症微环境，从而部分影响哮喘的严重程度。
总体实验设计：构建RGS4基因敲除小鼠以探究RGS4在哮喘中的作用。将RGS4 KO小鼠与野生型小鼠分别暴露于烟曲霉（Aspergillus fumigatus）与磷酸盐缓冲液（PBS），随后开展polyA mRNA测序。