Oxidative stress disrupts glucocorticoid hormone-dependent transcription of the amiloride-sensitive epithelial sodium channel alpha-subunit in lung epithelial cells through ERK-dependent and thioredoxin-sensitive pathways.
The amiloride-sensitive epithelial Na(+) channel (ENaC) plays a critical role in the maintenance of alveolar fluid balance. It is generally accepted that reactive oxygen and nitrogen species can inhibit ENaC activity and aggravate acute lung injury; however, the molecular mechanism for free radical-mediated ENaC inhibition is unclear. Previously, we showed that the expression of the alpha-subunit of ENaC, alpha-ENaC, which is indispensable for ENaC activity, is repressed by Ras activation in salivary epithelial cells. Here, we investigated whether exogenous H(2)O(2) modulates alpha-ENaC gene expression in lung epithelial cells through a similar molecular mechanism. Utilizing transient transfection reporter assays and site-directed mutagenesis analyses, we found that the glucocorticoid response element (GRE), located at -1334 to -1306 base pairs of the alpha-ENaC 5'-flanking region, is the major enhancer for the stimulated alpha-ENaC expression in A549 lung epithelial cells. We further demonstrate that the presence of an intact GRE is necessary and sufficient for oxidants to repress alpha-ENaC expression. Consistent with our hypothesis, exogenous H(2)O(2)-mediated repression of alpha-ENaC GRE activity is partially blocked by either a specific inhibitor for extracellular signal-regulated kinase (ERK) pathway activation, U0126, or dominant negative ERK, suggesting that, in part, activated ERK may mediate the repressive effects of H(2)O(2) on alpha-ENaC expression. In addition, overexpression of thioredoxin restored glucocorticoid receptor action on the alpha-ENaC GRE in the presence of exogenous H(2)O(2). Taken together, we hypothesize that oxidative stress impairs Na(+) transport activity by inhibiting dexamethasone-dependent alpha-ENaC GRE activation via both ERK-dependent and thioredoxin-sensitive pathways. These results suggest a putative mechanism whereby cellular redox potentials modulate the glucocorticoid receptor/dexamethasone effect on alpha-ENaC expression in lung and other tight epithelia.
Wang
H C
HC
Department of Molecular Pharmacology and Toxicology, Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California 90033, USA.
Zentner
M D
MD
Deng
H T
HT
Kim
K J
KJ
Wu
R
R
Yang
P C
PC
Ann
D K
DK
D002070:Butadienes / Q000494:pharmacology; D003907:Dexamethasone / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D053503:Epithelial Sodium Channels; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D048728:MAP Kinase Kinase Kinase 1; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008954:Models, Biological; D009570:Nitriles / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020239:Receptor Cross-Talk; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D020218:Response Elements; D015398:Signal Transduction; D015222:Sodium Channels / Q000235:genetics*; D013879:Thioredoxins / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D018631:ras Proteins / Q000378:metabolism
D002070:Butadienes; D053503:Epithelial Sodium Channels; D005938:Glucocorticoids; D009570:Nitriles; D011965:Receptors, Glucocorticoid; D015222:Sodium Channels; C113580:U 0126; D013879:Thioredoxins; D003907:Dexamethasone; D006861:Hydrogen Peroxide; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D018631:ras Proteins