Shi Weifeng, Hou Xueling, Peng Hongjun, Zhang Li, Li Yuanyuan, Gu Zhiwen, Jiang Qingbo, Shi Mei, Ji Yun, Jiang Jingting
Department of Clinical Laboratory, The Third Affiliated Hospital of Soochow University, 185 Juqian Street, Changzhou, Jiangsu, 213003, PR China.
Department of Tumor Biological Treatment, The Third Affiliated Hospital of Soochow University, 185 Juqian Street, Changzhou, 213003, China.
Virol J. 2014 Dec 30;11:227. doi: 10.1186/s12985-014-0227-7.
The mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/ERK) signaling pathway is involved in viral life cycle. However, the effect of MEK/ERK pathway in enterovirus 71(EV71)-infected immature dendritic cells (iDCs) is still unclear.
Human peripheral blood mononuclear cells (PBMCs) were isolated and induced to generate iDCs. Unifected iDCs and EV71-infected iDCs with a multiplicity of infection (MOI = 5) were analyzed by flow cytometry. Differential gene expressions of MEK/ERK signaling pathway molecules in EV71-infected iDCs were performed by PCR arrays. The phosphorylation of MEK/ERK pathway molecules in EV71-infected iDCs preincubated without or with U0126 (20 μM) at indicated times was detected by Western blot. The concentrations of IL-1α, IL-2, IL-6, IL-12, TNF-α, IFN-α1, IFN-β and IFN-γ in culture supernatant were analyzed by the luminex fluorescent technique.
When iDCs were infected with EV71 for 24 h, the percentage of CD80, CD83, CD86 and HLA-DR expressed on iDCs significantly increased. PCR arrays showed that gene expressions of molecules in MEK/ERK signaling pathway were remarkably upregulated in EV71-infected iDCs. EV71 infection activated both MEK1/2 and ERK1/2, which phosphorylated their downstream transcription factor c-Fos, c-Jun, c-myc and Elk1. Importantly, the treatment of U0126 significantly inhibited MEK/ERK signaling pathway molecules and severely impaired virus replication., Additionally, EV71 infection promoted the expression of son of sevenless (SOS1) and increased the secretion of IL-1α, IL-2, IL-6, IL-12, TNF-α,IFN-β and IFN-γ. Furthermore,the release of IL-1α, IL-2,IL-6 and TNF-α could be effectively suppressed by inhibitor U0126.
Our data suggest that the MEK/ERK signaling pathway plays an important role in EV71-infected iDCs and these molecules may be potential targets for the development of new anti-EV71 drugs.
丝裂原活化蛋白激酶激酶/细胞外信号调节激酶（MEK/ERK）信号通路参与病毒生命周期。然而，MEK/ERK通路在肠道病毒71型（EV71）感染的未成熟树突状细胞（iDCs）中的作用仍不清楚。
分离人外周血单核细胞（PBMCs）并诱导生成iDCs。通过流式细胞术分析未感染的iDCs和感染复数（MOI = 5）的EV71感染的iDCs。通过PCR阵列检测EV71感染的iDCs中MEK/ERK信号通路分子的差异基因表达。通过蛋白质印迹法检测在指定时间预先孵育无或有U0126（20 μM）的EV71感染的iDCs中MEK/ERK通路分子的磷酸化。通过Luminex荧光技术分析培养上清液中IL-1α、IL-2、IL-6、IL-12、TNF-α、IFN-α1、IFN-β和IFN-γ的浓度。
当iDCs感染EV71 24小时时，iDCs上表达的CD80、CD83、CD86和HLA-DR的百分比显著增加。PCR阵列显示，MEK/ERK信号通路中分子的基因表达在EV71感染的iDCs中显著上调。EV71感染激活了MEK1/2和ERK1/2，它们使下游转录因子c-Fos、c-Jun、c-myc和Elk1磷酸化。重要的是，U0126处理显著抑制MEK/ERK信号通路分子并严重损害病毒复制。此外，EV71感染促进了七号染色体失活蛋白（SOS1）的表达并增加了IL-1α、IL-2、IL-6、IL-12、TNF-α、IFN-β和IFN-γ的分泌。此外，抑制剂U0126可有效抑制IL-1α、IL-2、IL-6和TNF-α的释放。
我们的数据表明，MEK/ERK信号通路在EV71感染的iDCs中起重要作用，这些分子可能是开发新型抗EV71药物的潜在靶点。
