Glia maturation factor-gamma modulation of signaling pathways in macrophages

巨噬细胞介导的炎症反应在肥胖相关组织炎症和胰岛素抵抗的发生发展中起关键作用。游离脂肪酸(FFAs)是导致炎症因子产生和胰岛素抵抗的典型因素。 胶质成熟因子γ(GMFG)是ADF/cofilin家族中的一员，调节肌动蛋白的细胞骨架重组，主要表达于炎症细胞，但其在巨噬细胞免疫反应中的作用尚不清楚。在这项研究中，我们研究了GMFG是否可能影响FFAs诱导的巨噬细胞炎症反应。我们通过实时定量聚合酶链式反应和酶联免疫吸附试验证实，在人外周血单核细胞来源的巨噬细胞中，通过转染GMFG siRNA(抑制内源性表达水平的80%)显著增强了FFAs诱导的促炎细胞因子和趋化因子的产生，包括肿瘤坏死因子、IL-6和IL-8。这些炎性细胞因子的增加是由于FFAs刺激后NF-B和ERK1/2 MAPK信号通路的激活增加(基于NF-954；B p65磷酸化增加，蛋白质印迹分析中ERK1/2缺失和ERK1/2磷酸化增强)和PI3K/Akt/GSK3-/946；通路的磷酸化减少所致。在GMFG沉默的细胞中过表达GFP-GMFG抑制了NF-954；B p65，ERK1/2 MAPK的磷酸化增强，Akt和GSK3的磷酸化降低。此外，在GMFG被敲除的细胞中，FFAs诱导SHIP1的表达增加，SHIP1是一种负调控PI3K信号通路的磷酸酶，提示SHIP1的表达增加可能与FFAs刺激后炎症细胞因子的增加有关。我们还发现，沉默GMFG可增强氧化低密度脂蛋白(OxLDL)诱导的MDM中肿瘤坏死因子和白介素6的表达。鉴于GMFG在巨噬细胞中有结构性表达，并受FFAs刺激后表达下调，GMFG可能通过参与PI3K/Akt信号通路而发挥新的负性调节作用，提示巨噬细胞特异性调节GMFG在FFAs诱导的炎症反应中可能是有益的。

The macrophage-mediated inflammatory response plays a critical role in the development of obesity-related tissue inflammation and insulin resistance. Free-fatty acids (FFAs) are well-characterized factors causing production of inflammatory factors and insulin resistance. Glia maturation factor gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in the macrophages immune response remains unclear. In this study, we investigated whether GMFG may affect FFAs-induced inflammatory reaction in macrophages. We show here that silencing of GMFG (80% inhibition of the endogenous expression levels) by transfected with GMFG siRNA significantly enhanced FFAs-induced production of proinflammatory cytokines and chemokines, including TNF-α, IL-6 and IL-8 compared to transfected non-targeting silencing siRNA in human peripheral blood monocytes-derived macrophage as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay. These increased inflammatory cytokines resulted from an increased activation of NF-κB and the ERK1/2 MAPK signaling pathway (based on increased NF-κB p65 phosphorylation, IκBα loss and enhanced phosphorylation of ERK1/2 in Western blot analysis) and a reduced phosphorylation of the PI3K/Akt/GSK3-β pathway following FFAs stimulation. Overexpression of GFP-GMFG in GMFG-silenced cells abrogated enhanced phosphorylation of NF-κB p65, ERK1/2 MAPK and reduced phosphorylation of Akt and GSK3−β. Furthermore, in cells in which GMFG was knockdown, FFAs induced an increase in the expression of SHIP1, a phosphatase that negatively regulates the PI3K signaling pathway, suggesting increased SHIP1 expression may be responsible for the augmentation of inflammatory cytokines following FFAs stimulation. We also show that silencing of GMFG enhances the oxidized low density lipoprotein (oxLDL) induced expression of TNF-α and IL-6 in MDM. Taking together with the data that GMFG is constitutively expressed in macrophages and its expression is down-regulated by FFAs stimulation, GMFG might function as a novel negative regulator through participating in the PI3K/Akt signaling pathway, suggesting that macrophage-specific modulation of GMFG may be beneficial in the treatment of FFAs induced inflammation.