Pseudomonas aeruginosa Pyocyanin Activates NRF2-ARE-Mediated Transcriptional Response via the ROS-EGFR-PI3K-AKT/MEK-ERK MAP Kinase Signaling in Pulmonary Epithelial Cells

The redox-active pyocyanin (PCN) secreted by the respiratory pathogen Pseudomonas aeruginosa generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.由呼吸道致病菌铜绿假单胞菌（Pseudomonas aeruginosa）分泌的氧化还原活性绿脓菌素（PCN）可产生活性氧（ROS），并对肺上皮细胞诱发氧化应激。核因子（红细胞衍生2样2，NRF2）可通过结合顺式作用抗氧化反应元件（ARE），诱导解毒酶与蛋白的表达，从而对ROS介导的细胞死亡起到防护作用。然而，NRF2与PCN介导的氧化应激之间的明确关联尚未通过实验得到证实。本研究针对肺上皮细胞中PCN诱导的NRF2-ARE应答展开探究。我们分析了PCN对培养的人气道上皮细胞以及慢性PCN暴露小鼠模型中NRF2表达与核转位的影响，并评估了NRF2依赖的抗氧化酶转录情况。此外，我们利用抑制剂考察了介导PCN应答下NRF2-ARE激活的表皮生长因子受体（EGFR）及其下游信号组分的参与作用。PCN可促进核内NRF2的积累，并激活ARE介导的抗氧化基因转录。进一步而言，PCN可通过诱导表皮生长因子受体（EGFR）-磷脂酰肌醇3-激酶（PI3K）信号通路及其主要下游效应分子AKT与MEK1/2-ERK1/2丝裂原活化蛋白激酶（MAP kinases）来激活NRF2。抑制EGFR-PI3K信号通路可显著减弱PCN刺激诱导的核内NRF2积累。本研究首次证实，PCN介导的氧化应激可激活EGFR-PI3K-AKT/MEK1/2-ERK1/2丝裂原活化蛋白激酶信号通路，进而促使肺上皮细胞发生核NRF2转位并产生ARE应答。