Abstract
Oxidative stress plays a major role in hyperoxia-induced acute lung injury. We have shown previously that mice lacking the Nrf2 are more susceptible to hyperoxia than are wild-type mice. Nrf2 activates antioxidant response element (ARE)-mediated gene expression involved in cellular protection against toxic insults. The present study was designed to investigate the mechanisms that control the activation of Nrf2 by hyperoxia using a non-malignant murine alveolar epithelial cell line, C10. No significant alteration in the levels of Nrf2 mRNA and protein was found following exposure to hyperoxia. In contrast, hyperoxia caused the translocation of Nrf2 from the cytoplasm to the nucleus within 30-60 min of exposure. Consistent with these observations, gel shift and reporter analyses demonstrated a correlation between the hyperoxia-enhanced ARE DNA-binding activity of Nrf2 and an up-regulation of ARE-driven transcription. Inhibition of NADPH oxidase with diphenyleneiodonium (DPI) blocked both Nrf2 translocation and ARE-mediated transcription. Inhibition of the MEK/ERK pathway caused a similar effect. Consistent with this finding, hyperoxia stimulated ERK-1 and ERK-2 phosphorylation, whereas DPI or N-acetyl-l-cysteine blocked such activation. Hyperoxia stimulated the phosphorylation of endogenous Nrf2, but not in the presence of U0126, suggesting a critical role for ERK signaling in the activation of Nrf2. Consistent with this notion, hyperoxia did not stimulate the phosphorylation of Nrf2 in fibroblasts lacking the ERK-1. Collectively, our findings suggest that hyperoxia-induced, ARE-driven, Nrf2-dependent transcription is controlled by NADPH oxidase and ERK-1 signaling.
Highlights
Oxidative stress plays a major role in hyperoxia-induced acute lung injury
To understand the mechanisms controlling the activation of NF-E2-related transcription factor 2 (Nrf2), we initially studied the effects of hyperoxia on Nrf2 expression in A549 and C10 cell lines, which exhibit alveolar epithelial-like characteristics [27]
These results suggest that hyperoxia does not affect the levels of Nrf2 mRNA or protein expression in C10 or A549 cells, and they are consistent with the reported regulation of Nrf2 in a majority of cell types in response to toxic stimuli [16, 26]
Summary
Vol 279, No 40, Issue of October 1, pp. 42302–42312, 2004 Printed in U.S.A. NADPH Oxidase and ERK Signaling Regulates Hyperoxia-induced Nrf2-ARE Transcriptional Response in Pulmonary Epithelial Cells*. In contrast to wild-type, the Nrf2-deficient mice are more susceptible to inflammatory and hyper-permeability responses induced by hyperoxia [20] Consistent with this finding, studies from other laboratories have shown an important role for Nrf in the regulation of AOE expression in response to various oxidative and cytotoxic insults in many cells and tissues [14, 18, 22,23,24,25]. To delineate the molecular basis of Nrf2-mediated protection against hyperoxia-induced toxicity in the lung, we investigated the mechanisms controlling Nrf activation and subsequent AREmediated gene transcription in response to hyperoxia in an alveolar epithelial cell culture system. Our results indicate that ROS-induced ERK MAPK signaling plays a critical role in regulating ARE-mediated, hyperoxia-induced gene expression in pulmonary epithelial cells
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