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 α-subunit of ENaC, α-ENaC, which is indispensable for ENaC activity, is repressed by Ras activation in salivary epithelial cells. Here, we investigated whether exogenous H2O2 modulates α-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 α-ENaC 5'-flanking region, is the major enhancer for the stimulated α-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 α-ENaC expression. Consistent with our hypothesis, exogenous H2O2-mediated repression of α- 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 H2O2 on α-ENaC expression. In addition, overexpression of thioredoxin restored glucocorticoid receptor action on the α-ENaC GRE in the presence of exogenous H2O2. Taken together, we hypothesize that oxidative stress impairs Na+ transport activity by inhibiting dexamethasone-dependent α-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 α-ENaC expression in lung and other tight epithelia.
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