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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 induces nuclear NRF2 accumulation in a time-dependent manner.A549 cells were exposed to 5.0 µg/ml (A–D) and 12.5 µg/ml (E–H) of PCN, respectively, for indicated time intervals. Total nuclear and cytoplasmic proteins were harvested for Western blot using an anti-NRF2 monoclonal antibody. Histone H3 and GAPDH were used as loading controls. The experiments were repeated three times with similar results. The western blots from one typical experiment are shown. Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when PCN-exposed cells were compared against the control cells exposed to same volume of sterile water. (A–B) Western blots and densitometry analyses of nuclear NRF2 after 24 hr of exposure to 5.0 µg/ml PCN. (C–D) Western blots and densitometry analyses of cytoplasmic NRF2 after 24 hr of exposure to 5.0 µg/ml PCN. (E–F) Western blots and densitometry analyses of nuclear NRF2 after 24 hr of exposure to 12.5 µg/ml PCN. (G–H) Western blots and densitometry analyses of cytoplasmic NRF2 after 24 hr of exposure to 12.5 µg/ml PCN.
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pone-0072528-g002: PCN induces nuclear NRF2 accumulation in a time-dependent manner.A549 cells were exposed to 5.0 µg/ml (A–D) and 12.5 µg/ml (E–H) of PCN, respectively, for indicated time intervals. Total nuclear and cytoplasmic proteins were harvested for Western blot using an anti-NRF2 monoclonal antibody. Histone H3 and GAPDH were used as loading controls. The experiments were repeated three times with similar results. The western blots from one typical experiment are shown. Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when PCN-exposed cells were compared against the control cells exposed to same volume of sterile water. (A–B) Western blots and densitometry analyses of nuclear NRF2 after 24 hr of exposure to 5.0 µg/ml PCN. (C–D) Western blots and densitometry analyses of cytoplasmic NRF2 after 24 hr of exposure to 5.0 µg/ml PCN. (E–F) Western blots and densitometry analyses of nuclear NRF2 after 24 hr of exposure to 12.5 µg/ml PCN. (G–H) Western blots and densitometry analyses of cytoplasmic NRF2 after 24 hr of exposure to 12.5 µg/ml PCN.

Mentions: To further verify this result, we examined the dynamics of NRF2 in a time-dependent manner following exposure to PCN. Cells were exposed to PCN in the concentrations of 5.0 and 12.5 µg/ml, respectively, which have been shown to induce higher nuclear accumulation of NRF2 (Figure 1). At 5 µg/ml concentration, PCN markedly stimulated NRF2 translocation from the cytoplasm to the nucleus as early as 3 hr after exposure (1.5 to 2-fold), reached its peak at 12 hours (2 to 3-fold) (Figure 2A–D). Again, the accumulation of NRF2 in nuclei was correlated with a decrease (1 to 1.5-fold) in the cytoplasm. At 12.5 µg/ml concentration, PCN induced a more robust nuclear localization and corresponding cytoplasmic depletion of NRF2 (Figure 2E–H). Collectively, these results suggest that PCN-mediated ROS trigger the translocation of NRF2 from the cytoplasm to the nucleus in concentration and time-dependent manners.


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 induces nuclear NRF2 accumulation in a time-dependent manner.A549 cells were exposed to 5.0 µg/ml (A–D) and 12.5 µg/ml (E–H) of PCN, respectively, for indicated time intervals. Total nuclear and cytoplasmic proteins were harvested for Western blot using an anti-NRF2 monoclonal antibody. Histone H3 and GAPDH were used as loading controls. The experiments were repeated three times with similar results. The western blots from one typical experiment are shown. Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when PCN-exposed cells were compared against the control cells exposed to same volume of sterile water. (A–B) Western blots and densitometry analyses of nuclear NRF2 after 24 hr of exposure to 5.0 µg/ml PCN. (C–D) Western blots and densitometry analyses of cytoplasmic NRF2 after 24 hr of exposure to 5.0 µg/ml PCN. (E–F) Western blots and densitometry analyses of nuclear NRF2 after 24 hr of exposure to 12.5 µg/ml PCN. (G–H) Western blots and densitometry analyses of cytoplasmic NRF2 after 24 hr of exposure to 12.5 µg/ml PCN.
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getmorefigures.php?uid=PMC3755003&req=5

pone-0072528-g002: PCN induces nuclear NRF2 accumulation in a time-dependent manner.A549 cells were exposed to 5.0 µg/ml (A–D) and 12.5 µg/ml (E–H) of PCN, respectively, for indicated time intervals. Total nuclear and cytoplasmic proteins were harvested for Western blot using an anti-NRF2 monoclonal antibody. Histone H3 and GAPDH were used as loading controls. The experiments were repeated three times with similar results. The western blots from one typical experiment are shown. Densitometry data represent the mean ± SD from all three experiments. *p<0.05 when PCN-exposed cells were compared against the control cells exposed to same volume of sterile water. (A–B) Western blots and densitometry analyses of nuclear NRF2 after 24 hr of exposure to 5.0 µg/ml PCN. (C–D) Western blots and densitometry analyses of cytoplasmic NRF2 after 24 hr of exposure to 5.0 µg/ml PCN. (E–F) Western blots and densitometry analyses of nuclear NRF2 after 24 hr of exposure to 12.5 µg/ml PCN. (G–H) Western blots and densitometry analyses of cytoplasmic NRF2 after 24 hr of exposure to 12.5 µg/ml PCN.
Mentions: To further verify this result, we examined the dynamics of NRF2 in a time-dependent manner following exposure to PCN. Cells were exposed to PCN in the concentrations of 5.0 and 12.5 µg/ml, respectively, which have been shown to induce higher nuclear accumulation of NRF2 (Figure 1). At 5 µg/ml concentration, PCN markedly stimulated NRF2 translocation from the cytoplasm to the nucleus as early as 3 hr after exposure (1.5 to 2-fold), reached its peak at 12 hours (2 to 3-fold) (Figure 2A–D). Again, the accumulation of NRF2 in nuclei was correlated with a decrease (1 to 1.5-fold) in the cytoplasm. At 12.5 µg/ml concentration, PCN induced a more robust nuclear localization and corresponding cytoplasmic depletion of NRF2 (Figure 2E–H). Collectively, these results suggest that PCN-mediated ROS trigger the translocation of NRF2 from the cytoplasm to the nucleus in concentration and time-dependent manners.

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