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Pseudomonas aeruginosa pyocyanin activates NRF2-ARE-mediated transcriptional response via the ROS-EGFR-PI3K-AKT/MEK-ERK MAP kinase signaling in pulmonary epithelial cells.

Xu Y, Duan C, Kuang Z, Hao Y, Jeffries JL, Lau GW - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America ; Laboratory of Clinical Immunology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, People's Republic of China.

ABSTRACT
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.

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PCN activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway that positively regulates NRF2 during oxidative stress.Cytoplasmic or crude plasma membrane proteins of A549 cells previously exposed to PCN or sterile H2O were probed with anti-phospho–specific antibodies. (A–D) PCN induces the phosphorylation of EGFR (A), AKT (B), MEK1/2 (C) and ERK1/2 (D). The experiments were repeated three times with similar results. Right panels represent densitometry analysis of western blots in (A–D). Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when PCN-exposed cells were compared with the control cells exposed to same volume of sterile H2O.
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pone-0072528-g006: PCN activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway that positively regulates NRF2 during oxidative stress.Cytoplasmic or crude plasma membrane proteins of A549 cells previously exposed to PCN or sterile H2O were probed with anti-phospho–specific antibodies. (A–D) PCN induces the phosphorylation of EGFR (A), AKT (B), MEK1/2 (C) and ERK1/2 (D). The experiments were repeated three times with similar results. Right panels represent densitometry analysis of western blots in (A–D). Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when PCN-exposed cells were compared with the control cells exposed to same volume of sterile H2O.

Mentions: Previous studies have shown that hyperoxic conditions activate the EGFR signaling pathway, leading to nuclear translocation of NRF2, and the transcription of ARE-containing antioxidant genes [33], [34]. Most recently, we and others have shown that PCN induces GCHM and the biosynthesis of major airway mucins MUC5AC and MUC5B in mouse airways, in part, by inducing the ROS-dependent EGFR signaling [11], [15]. We compared the levels of phosphorylated EGFR in A549 cells after 12 h of exposure to sterile H2O or various concentrations of PCN. Western blot analysis indicated that PCN at between 1.3 to 25 µg/ml increased the levels of phosphorylated EGFR between 1.4 - to 2-fold (Figure 6A). Furthermore, PCN exposure induced the phosphorylation of downstream components of the EGFR signaling cascade (Figure 6B, 6C, 6D). For example, after 12 hr of exposure, PCN (12.5 µg/ml) induced 4.0, 4.1 and 3.5-fold increases in p-AKT, p-MEK1/2 and p-ERK1/2, respectively (Figure 6B, 6C, 6D). Taken together, these results suggest that PCN activate the signaling of EGFR and its downstream PI3K-AKT/MEK1/2-ERK1/2 MAP kinase cascade, triggering the nuclear translocation of NRF2.


Pseudomonas aeruginosa pyocyanin activates NRF2-ARE-mediated transcriptional response via the ROS-EGFR-PI3K-AKT/MEK-ERK MAP kinase signaling in pulmonary epithelial cells.

Xu Y, Duan C, Kuang Z, Hao Y, Jeffries JL, Lau GW - PLoS ONE (2013)

PCN activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway that positively regulates NRF2 during oxidative stress.Cytoplasmic or crude plasma membrane proteins of A549 cells previously exposed to PCN or sterile H2O were probed with anti-phospho–specific antibodies. (A–D) PCN induces the phosphorylation of EGFR (A), AKT (B), MEK1/2 (C) and ERK1/2 (D). The experiments were repeated three times with similar results. Right panels represent densitometry analysis of western blots in (A–D). Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when PCN-exposed cells were compared with the control cells exposed to same volume of sterile H2O.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3755003&req=5

pone-0072528-g006: PCN activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway that positively regulates NRF2 during oxidative stress.Cytoplasmic or crude plasma membrane proteins of A549 cells previously exposed to PCN or sterile H2O were probed with anti-phospho–specific antibodies. (A–D) PCN induces the phosphorylation of EGFR (A), AKT (B), MEK1/2 (C) and ERK1/2 (D). The experiments were repeated three times with similar results. Right panels represent densitometry analysis of western blots in (A–D). Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when PCN-exposed cells were compared with the control cells exposed to same volume of sterile H2O.
Mentions: Previous studies have shown that hyperoxic conditions activate the EGFR signaling pathway, leading to nuclear translocation of NRF2, and the transcription of ARE-containing antioxidant genes [33], [34]. Most recently, we and others have shown that PCN induces GCHM and the biosynthesis of major airway mucins MUC5AC and MUC5B in mouse airways, in part, by inducing the ROS-dependent EGFR signaling [11], [15]. We compared the levels of phosphorylated EGFR in A549 cells after 12 h of exposure to sterile H2O or various concentrations of PCN. Western blot analysis indicated that PCN at between 1.3 to 25 µg/ml increased the levels of phosphorylated EGFR between 1.4 - to 2-fold (Figure 6A). Furthermore, PCN exposure induced the phosphorylation of downstream components of the EGFR signaling cascade (Figure 6B, 6C, 6D). For example, after 12 hr of exposure, PCN (12.5 µg/ml) induced 4.0, 4.1 and 3.5-fold increases in p-AKT, p-MEK1/2 and p-ERK1/2, respectively (Figure 6B, 6C, 6D). Taken together, these results suggest that PCN activate the signaling of EGFR and its downstream PI3K-AKT/MEK1/2-ERK1/2 MAP kinase cascade, triggering the nuclear translocation of NRF2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America ; Laboratory of Clinical Immunology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, People's Republic of China.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus