Warren Kristi J, Fang Xi, Gowda Nagaraj M, Thompson Joshua J, Heller Nicola M
From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205.
From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
J Biol Chem. 2016 Nov 25;291(48):24922-24930. doi: 10.1074/jbc.M116.756791. Epub 2016 Oct 14.
Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα, and γC after IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation.
肺M2巨噬细胞是气道炎症的调节因子，与过敏性哮喘中肺功能不佳相关。此前，我们证明白细胞介素-4（IL-4）诱导的M2基因表达与巨噬细胞中胰岛素受体底物-2（IRS-2）的酪氨酸磷酸化相关。我们推测IL-4刺激后IRS-2活性的负调控依赖于IRS-2的丝氨酸磷酸化。在此，我们描述了IL-4刺激后IRS-2的酪氨酸磷酸化（Tyr(P)）和丝氨酸磷酸化（Ser(P)）之间的反向关系。抑制丝氨酸磷酸酶活性会增加Ser(P)-IRS-2并降低Tyr(P)-IRS-2，导致M2基因表达（CD200R、CCL22、MMP12和TGM2）减少。我们发现抑制雷帕霉素靶蛋白复合体1（TORC1）下游的p70核糖体蛋白S6激酶（p70S6K）会导致Ser(P)-IRS-2减少，同时Tyr(P)-IRS-2的持续时间延长。抑制p70S6K会增加CD200R和CCL22的表达，表明p70S6K对部分而非全部人类M2基因起负调控作用。敲低TORC1下游的另一种负调控蛋白生长因子受体结合蛋白10（GRB10），会增强Tyr(P)-IRS-2并增加所有四种M2基因的表达。此外，IL-4刺激后GRB10与IRS-2、NEDD4.2（一种E3泛素连接酶）、IL-4Rα和γC相关联。IL-4处理30分钟后，IL-4Rα和γC均被泛素化，这表明GRB10可能通过与NEDD4.2相互作用来调节IL-4受体信号复合体的降解。综上所述，这些数据突出了两种新的调节蛋白，可通过治疗手段对其进行调控，以限制IL-4诱导的IRS-2信号传导以及过敏性炎症中M2巨噬细胞的极化。
