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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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Translocation of NRF2 into nuclei correlates with the induction of γ-GCSh and NQO1 gene expression.The expression of γ-GCSh and NQO1 genes were quantified by qRT-PCR using total RNA extracted from A549 cells after exposure to 12.5 µg/ml PCN in the presence or absence of trigonelline or GSH for the indicated time intervals and concentrations. (A) qRT-PCR analyses of γ-GCSh gene expression. (B) qRT-PCR analyses of NQO1 gene expression. (C) Trigonelline blocked the PCN-induced nuclear translocation of NRF2. (D–E) qRT-PCR of γ-GCSh and NQO1 expression in the presence of trigonelline. (F) GSH blocked PCN-induced nuclear translocation of NRF2. (G–H) qRT-PCR of γ-GCSh and NQO1 expression in the presence of 1 or 5 mM GSH. qRT-PCR was performed in triplicates in three independent experiments. Results from one typical experiment are shown. *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 control cells exposed to PCN alone. Western blots were performed in three independent experiments. Results from one typical experiment are shown.
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pone-0072528-g005: Translocation of NRF2 into nuclei correlates with the induction of γ-GCSh and NQO1 gene expression.The expression of γ-GCSh and NQO1 genes were quantified by qRT-PCR using total RNA extracted from A549 cells after exposure to 12.5 µg/ml PCN in the presence or absence of trigonelline or GSH for the indicated time intervals and concentrations. (A) qRT-PCR analyses of γ-GCSh gene expression. (B) qRT-PCR analyses of NQO1 gene expression. (C) Trigonelline blocked the PCN-induced nuclear translocation of NRF2. (D–E) qRT-PCR of γ-GCSh and NQO1 expression in the presence of trigonelline. (F) GSH blocked PCN-induced nuclear translocation of NRF2. (G–H) qRT-PCR of γ-GCSh and NQO1 expression in the presence of 1 or 5 mM GSH. qRT-PCR was performed in triplicates in three independent experiments. Results from one typical experiment are shown. *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 control cells exposed to PCN alone. Western blots were performed in three independent experiments. Results from one typical experiment are shown.

Mentions: It is well established that activated NRF2 in the nucleus can bind to ARE by forming a heterodimer with other transcription factors, such as MAF, and regulate the transcription of antioxidant genes harboring the ARE cis-acting element [22]. To evaluate the effects of the nuclear accumulation of NRF2 induced by PCN, we monitored the expression of the ARE-containing antioxidant genes γ-GCSh and NQO1. The γ-GCS catalyzes the rate limiting de novo biosynthesis of glutathione (GSH) whereas NQO1 is a cytosolic protein that reduces and detoxifies quinones and their derivatives, thus protecting cells against redox cycling and oxidative stress [20]. The transcription level of both γ-GCSh (Figure 5A) and NQO1 (Figure 5B) genes increased 2.4 and 4.3-fold, respectively, at 12 hr post-PCN exposure. The peak expression of both γ-GCSh and NQO1 coincides with peak nuclear localization of NRF2 induced by PCN (Figure 2). In contrast, the expression of γ-GCSh and NQO1 decreased at 18 and 24 hr, corresponding to decreasing levels of nuclear NRF2 (Figure 2).


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)

Translocation of NRF2 into nuclei correlates with the induction of γ-GCSh and NQO1 gene expression.The expression of γ-GCSh and NQO1 genes were quantified by qRT-PCR using total RNA extracted from A549 cells after exposure to 12.5 µg/ml PCN in the presence or absence of trigonelline or GSH for the indicated time intervals and concentrations. (A) qRT-PCR analyses of γ-GCSh gene expression. (B) qRT-PCR analyses of NQO1 gene expression. (C) Trigonelline blocked the PCN-induced nuclear translocation of NRF2. (D–E) qRT-PCR of γ-GCSh and NQO1 expression in the presence of trigonelline. (F) GSH blocked PCN-induced nuclear translocation of NRF2. (G–H) qRT-PCR of γ-GCSh and NQO1 expression in the presence of 1 or 5 mM GSH. qRT-PCR was performed in triplicates in three independent experiments. Results from one typical experiment are shown. *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 control cells exposed to PCN alone. Western blots were performed in three independent experiments. Results from one typical experiment are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3755003&req=5

pone-0072528-g005: Translocation of NRF2 into nuclei correlates with the induction of γ-GCSh and NQO1 gene expression.The expression of γ-GCSh and NQO1 genes were quantified by qRT-PCR using total RNA extracted from A549 cells after exposure to 12.5 µg/ml PCN in the presence or absence of trigonelline or GSH for the indicated time intervals and concentrations. (A) qRT-PCR analyses of γ-GCSh gene expression. (B) qRT-PCR analyses of NQO1 gene expression. (C) Trigonelline blocked the PCN-induced nuclear translocation of NRF2. (D–E) qRT-PCR of γ-GCSh and NQO1 expression in the presence of trigonelline. (F) GSH blocked PCN-induced nuclear translocation of NRF2. (G–H) qRT-PCR of γ-GCSh and NQO1 expression in the presence of 1 or 5 mM GSH. qRT-PCR was performed in triplicates in three independent experiments. Results from one typical experiment are shown. *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 control cells exposed to PCN alone. Western blots were performed in three independent experiments. Results from one typical experiment are shown.
Mentions: It is well established that activated NRF2 in the nucleus can bind to ARE by forming a heterodimer with other transcription factors, such as MAF, and regulate the transcription of antioxidant genes harboring the ARE cis-acting element [22]. To evaluate the effects of the nuclear accumulation of NRF2 induced by PCN, we monitored the expression of the ARE-containing antioxidant genes γ-GCSh and NQO1. The γ-GCS catalyzes the rate limiting de novo biosynthesis of glutathione (GSH) whereas NQO1 is a cytosolic protein that reduces and detoxifies quinones and their derivatives, thus protecting cells against redox cycling and oxidative stress [20]. The transcription level of both γ-GCSh (Figure 5A) and NQO1 (Figure 5B) genes increased 2.4 and 4.3-fold, respectively, at 12 hr post-PCN exposure. The peak expression of both γ-GCSh and NQO1 coincides with peak nuclear localization of NRF2 induced by PCN (Figure 2). In contrast, the expression of γ-GCSh and NQO1 decreased at 18 and 24 hr, corresponding to decreasing levels of nuclear NRF2 (Figure 2).

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