Limits...
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.

Show MeSH

Related in: MedlinePlus

EGFR inhibitor and antioxidant GSH prevent the inhibition of PCN-mediated activation of AKT and ERK1/2 signaling.A549 cells were exposed to the EGFR inhibitor AG1478 (A–B) or GSH (C–D) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. Control cells were exposed to same volume of sterile H2O. Cytoplasmic proteins were probed with anti-phospho–specific antibodies. (A–B) The phosphorylation of AKT and ERK1/2 in A549 cells treated with AG1478 and PCN. (C–D) The phosphorylation of AKT and ERK1/2 in A549 cells treated with GSH and PCN. Right panels represent densitometry analysis of western blots. The experiments were repeated three times with similar results. Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when cells that were exposed to PCN were compared to control cells exposed to the same volume of sterile water. **p<0.05 when cells that were exposed to AG1478 or GSH were compared to cells exposed to PCN alone.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3755003&req=5

pone-0072528-g008: EGFR inhibitor and antioxidant GSH prevent the inhibition of PCN-mediated activation of AKT and ERK1/2 signaling.A549 cells were exposed to the EGFR inhibitor AG1478 (A–B) or GSH (C–D) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. Control cells were exposed to same volume of sterile H2O. Cytoplasmic proteins were probed with anti-phospho–specific antibodies. (A–B) The phosphorylation of AKT and ERK1/2 in A549 cells treated with AG1478 and PCN. (C–D) The phosphorylation of AKT and ERK1/2 in A549 cells treated with GSH and PCN. Right panels represent densitometry analysis of western blots. The experiments were repeated three times with similar results. Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when cells that were exposed to PCN were compared to control cells exposed to the same volume of sterile water. **p<0.05 when cells that were exposed to AG1478 or GSH were compared to cells exposed to PCN alone.

Mentions: To provide a clearer demonstration AKT and ERK1/2 activation is downstream of PCN-induced EGFR activation, we examined whether inhibition of EGFR would block the activation of AKT and ERK. The induction of AKT (Figure 8A) and ERK1/2 (Figure 8B) phosphorylation by PCN were reduced in the presence of the EGFR inhibitor AG1478. These results confirm that PCN activates both AKT and ERK1/2 through the induction of EGFR signaling. Furthermore, provision of antioxidant GSH also blocked the phosphorylation of AKT and ERK1/2 by PCN (Figure 8C, 8D), suggesting that PCN-generated ROS were the indirect activators of EGFR. Collectively, our results indicate that PCN-generated ROS induce the EGFR-PI3K-AKT/MEK1/2-ERK1/2 signaling pathway, resulting in the nuclear translocation of NRF2 to activate the transcription of the antioxidative genes γGCSh and NQO1.


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)

EGFR inhibitor and antioxidant GSH prevent the inhibition of PCN-mediated activation of AKT and ERK1/2 signaling.A549 cells were exposed to the EGFR inhibitor AG1478 (A–B) or GSH (C–D) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. Control cells were exposed to same volume of sterile H2O. Cytoplasmic proteins were probed with anti-phospho–specific antibodies. (A–B) The phosphorylation of AKT and ERK1/2 in A549 cells treated with AG1478 and PCN. (C–D) The phosphorylation of AKT and ERK1/2 in A549 cells treated with GSH and PCN. Right panels represent densitometry analysis of western blots. The experiments were repeated three times with similar results. Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when cells that were exposed to PCN were compared to control cells exposed to the same volume of sterile water. **p<0.05 when cells that were exposed to AG1478 or GSH were compared to cells exposed to PCN alone.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0072528-g008: EGFR inhibitor and antioxidant GSH prevent the inhibition of PCN-mediated activation of AKT and ERK1/2 signaling.A549 cells were exposed to the EGFR inhibitor AG1478 (A–B) or GSH (C–D) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. Control cells were exposed to same volume of sterile H2O. Cytoplasmic proteins were probed with anti-phospho–specific antibodies. (A–B) The phosphorylation of AKT and ERK1/2 in A549 cells treated with AG1478 and PCN. (C–D) The phosphorylation of AKT and ERK1/2 in A549 cells treated with GSH and PCN. Right panels represent densitometry analysis of western blots. The experiments were repeated three times with similar results. Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when cells that were exposed to PCN were compared to control cells exposed to the same volume of sterile water. **p<0.05 when cells that were exposed to AG1478 or GSH were compared to cells exposed to PCN alone.
Mentions: To provide a clearer demonstration AKT and ERK1/2 activation is downstream of PCN-induced EGFR activation, we examined whether inhibition of EGFR would block the activation of AKT and ERK. The induction of AKT (Figure 8A) and ERK1/2 (Figure 8B) phosphorylation by PCN were reduced in the presence of the EGFR inhibitor AG1478. These results confirm that PCN activates both AKT and ERK1/2 through the induction of EGFR signaling. Furthermore, provision of antioxidant GSH also blocked the phosphorylation of AKT and ERK1/2 by PCN (Figure 8C, 8D), suggesting that PCN-generated ROS were the indirect activators of EGFR. Collectively, our results indicate that PCN-generated ROS induce the EGFR-PI3K-AKT/MEK1/2-ERK1/2 signaling pathway, resulting in the nuclear translocation of NRF2 to activate the transcription of the antioxidative genes γGCSh and NQO1.

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