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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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EGFR and PI3K inhibitors diminish PCN-enhanced NRF2 accumulation in the nuclei of A549 cells.A549 cells were exposed to the EGFR inhibitor AG1478 (A), the PI3K inhibitor LY294002 (B), or a combination of both AG1478 and LY294002 (C), or AKT or ERK inhibitors (D) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. The expression of nuclear NRF2 was examined by monoclonal anti-NRF2 antibody and quantified by densitometry. 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 LY294002 or AG1478 plus LY294002, or AKT and ERK inhibitors were compared to cells exposed to PCN alone.
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pone-0072528-g007: EGFR and PI3K inhibitors diminish PCN-enhanced NRF2 accumulation in the nuclei of A549 cells.A549 cells were exposed to the EGFR inhibitor AG1478 (A), the PI3K inhibitor LY294002 (B), or a combination of both AG1478 and LY294002 (C), or AKT or ERK inhibitors (D) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. The expression of nuclear NRF2 was examined by monoclonal anti-NRF2 antibody and quantified by densitometry. 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 LY294002 or AG1478 plus LY294002, or AKT and ERK inhibitors were compared to cells exposed to PCN alone.

Mentions: To further address whether the EGFR-PI3K-AKT/MEK1/2-ERK1/2 signaling pathway act as the upstream activator that induces NRF2-ARE-mediated transcriptional response to PCN, we examined the nuclear translocation of NRF2 in A549 cells in the presence or absence of the EGFR specific inhibitor AG1478, PI3K-specific inhibitor LY294002, ERK1/2 inhibitor 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, and AKT Inhibitor VIII trifluoroacetate salt hydrate. The EGFR inhibitor AG1478 at concentrations of 2.5, 5.0, 10 and 25 µM significantly reduced the PCN-mediated translocation of NRF2 from the cytoplasm to the nucleus. In the absence of AG1478, the nuclear-localized NRF2 increased by 2-fold after 12 hr of exposure to PCN (12.5 µg/ml) (Figure 7A). In contrast, cells treated with 2.5, 5.0, 10 and 25 µM EGFR inhibitor AG1478 diminished PCN-enhanced NRF2 accumulation in the nuclei by 8%, 15%, 30% and 47.5%, respectively (Figure 7A). Similarly in the absence of PI3K inhibitor LY294002, nuclear-localized NRF2 was increased by 2.5-fold after 12 hr of exposure to PCN (12.5 µg/ml) (Figure 7B). In contrast, in the presence of 2.5, 5.0, 10 and 25 µM LY294002, the PCN-mediated NRF2 accumulation in the nuclei decreased by 27%, 32%, 60.6% and 80%, respectively. Next, we examined whether a combination of both AG1478 and LY294002 would allow a more robust inhibition of PCN-mediated NRF2 accumulation in the nuclei. The western blotting results showed that a combination of both inhibitors at the concentration of 2.5, 5.0, 10 and 25 µM, attenuated PCN-mediated NRF2 accumulation in the nuclei by 25%, 53%, 85.7% and 90%, respectively (Figure 7C). Finally, addition of both AKT and ERK1/2 inhibitors also significantly reduced the NRF2 nuclear translocation during PCN exposure (Figure 7D). Collectively, these results suggest that EGFR-PI3K-dependent signaling mediates the nuclear translocation of NRF2 in lung epithelial cells during PCN-mediated oxidative stress.


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 and PI3K inhibitors diminish PCN-enhanced NRF2 accumulation in the nuclei of A549 cells.A549 cells were exposed to the EGFR inhibitor AG1478 (A), the PI3K inhibitor LY294002 (B), or a combination of both AG1478 and LY294002 (C), or AKT or ERK inhibitors (D) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. The expression of nuclear NRF2 was examined by monoclonal anti-NRF2 antibody and quantified by densitometry. 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 LY294002 or AG1478 plus LY294002, or AKT and ERK inhibitors were compared to cells exposed to PCN alone.
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Related In: Results  -  Collection

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pone-0072528-g007: EGFR and PI3K inhibitors diminish PCN-enhanced NRF2 accumulation in the nuclei of A549 cells.A549 cells were exposed to the EGFR inhibitor AG1478 (A), the PI3K inhibitor LY294002 (B), or a combination of both AG1478 and LY294002 (C), or AKT or ERK inhibitors (D) for 90 min before the addition of PCN (12.5 µg/ml) for 12 hr. The expression of nuclear NRF2 was examined by monoclonal anti-NRF2 antibody and quantified by densitometry. 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 LY294002 or AG1478 plus LY294002, or AKT and ERK inhibitors were compared to cells exposed to PCN alone.
Mentions: To further address whether the EGFR-PI3K-AKT/MEK1/2-ERK1/2 signaling pathway act as the upstream activator that induces NRF2-ARE-mediated transcriptional response to PCN, we examined the nuclear translocation of NRF2 in A549 cells in the presence or absence of the EGFR specific inhibitor AG1478, PI3K-specific inhibitor LY294002, ERK1/2 inhibitor 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, and AKT Inhibitor VIII trifluoroacetate salt hydrate. The EGFR inhibitor AG1478 at concentrations of 2.5, 5.0, 10 and 25 µM significantly reduced the PCN-mediated translocation of NRF2 from the cytoplasm to the nucleus. In the absence of AG1478, the nuclear-localized NRF2 increased by 2-fold after 12 hr of exposure to PCN (12.5 µg/ml) (Figure 7A). In contrast, cells treated with 2.5, 5.0, 10 and 25 µM EGFR inhibitor AG1478 diminished PCN-enhanced NRF2 accumulation in the nuclei by 8%, 15%, 30% and 47.5%, respectively (Figure 7A). Similarly in the absence of PI3K inhibitor LY294002, nuclear-localized NRF2 was increased by 2.5-fold after 12 hr of exposure to PCN (12.5 µg/ml) (Figure 7B). In contrast, in the presence of 2.5, 5.0, 10 and 25 µM LY294002, the PCN-mediated NRF2 accumulation in the nuclei decreased by 27%, 32%, 60.6% and 80%, respectively. Next, we examined whether a combination of both AG1478 and LY294002 would allow a more robust inhibition of PCN-mediated NRF2 accumulation in the nuclei. The western blotting results showed that a combination of both inhibitors at the concentration of 2.5, 5.0, 10 and 25 µM, attenuated PCN-mediated NRF2 accumulation in the nuclei by 25%, 53%, 85.7% and 90%, respectively (Figure 7C). Finally, addition of both AKT and ERK1/2 inhibitors also significantly reduced the NRF2 nuclear translocation during PCN exposure (Figure 7D). Collectively, these results suggest that EGFR-PI3K-dependent signaling mediates the nuclear translocation of NRF2 in lung epithelial cells during PCN-mediated oxidative stress.

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