活性氧依赖的表皮生长因子受体磷酸化参与醛固酮诱导的系膜细胞胞外信号调节激酶活化

Oxidative Stress-Dependent Epidermal Growth Factor Receptor Trans-Activation Phosphorylation on Aldosterone-Induced Extracellular Signal-Regulated Kinase Signaling Activation

目的探讨活性氧(ROS)介导的表皮生长因子受体(EGFR)磷酸化在醛固酮(ALDO)诱导的肾小球系膜细胞胞外信号调节激酶(ERK1/2)信号通路活化中的作用。方法体外培养人肾小球系膜细胞,应用ALDO刺激或应用不同阻断剂预处理后再加入ALDO刺激,应用荧光探针2,7-二氯二氢荧光素乙酰乙酸(DCFDA)检测细胞内ROS的产生;Western blot检测EGFR、Ki-RasA、c-Raf、丝裂原活化蛋白激酶(MEK1/2)和ERK1/2活化。结果 ALDO可呈时间依赖性和剂量依赖性的方式促进肾小球系膜细胞ROS产生,100 nmol.L-1ALDO刺激60 min,系膜细胞ROS的产生是对照组的2.54倍。ALDO可显著活化系膜细胞EGFR,ALDO刺激60 min,EGFR活性是对照组的5.50倍,盐皮质激素受体(MR)抑制剂依普利酮和抗氧化剂乙酰半胱氨酸(NAC)几乎完全阻断ALDO诱导的EGFR磷酸化。ALDO刺激系膜细胞3 h,活化的Ki-RasA、c-Raf、MEK1/2和ERK1/2表达显著增强,分别是对照组的4.05倍、3.62倍、4.52倍和3.40倍。抗氧化剂NAC和EGFR特异性抑制剂AG1478几乎完全阻断ALDO诱导的Ki-RasA、c-Raf、MEK1/2和ERK1/2活化。结论 ALDO可通过ROS依赖的EGFR磷酸化活化Ki-RasA/c-Raf/MEK/ERK信号通路。

Objective To investigate the role of oxidative stress-dependent epidermal growth factor receptor（EGFR） phosphorylation on aldosterone（ALDO）-induced extracellular signal-regulated kinase（ERK1/2） signaling activation.Methods Human mesangial cells（MCs） were isolated and cultured,and the cells were treated with ALDO in the presence or absence of different antagonist.Reactive oxygen species（ROS） was determined by 2,7-dichlorofluorescein diacetate（DCFDA fluorescence）.Western blot analysis was used to detect the activation of EGFR,Ki-RasA,c-Raf,mitogen activated protein kinase（MEK1/2） and ERK1/2.Results ALDO induced ROS production in time-and dose-dependent manner in cultured human MCs,incubation with 100 nmol·L-1 ALDO for 60 minutes,ROS generation was increased by 2.54-fold.ALDO induced EGFR trans-activation,incubation with 100 nmol·L-1 ALDO for 60 minutes,EGFR phosphorylation was increased by 5.50-fold,which was completely inhibited by eplerenone,the antagonist of mineracorticoid receptor,and antioxidant N-acetylcysteine（NAC）.Stimulation with ALDO for 3 hours,Ki-RasA,c-Raf,MEK1/2,and ERK1/2 activity increased by 4.05,3.62,4.52 and 3.40-fold,respectively.NAC and EGFR specific inhibitor AG1478 almost completely blocked ALDO-induced Ki-RasA,c-Raf,MEK1/2,and ERK1/2 activation.Conclusion ALDO-induced Ki-RasA/c-Raf/MEK/ERK activation was dependent on ROS-mediated EGFR trans-activation.