No abstract

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 ␣-subunit of ENaC, ␣-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 ␣-ENaC gene expression in lung epithelial cells through a similar molecular mechanism. Utilizing transient transfection reporter assays and sitedirected mutagenesis analyses, we found that the glucocorticoid response element (GRE), located at ؊1334 to ؊1306 base pairs of the ␣-ENaC 5-flanking region, is the major enhancer for the stimulated ␣-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 ␣-ENaC expression. Consistent with our hypothesis, exogenous H 2 O 2 -mediated repression of ␣-ENaC GRE activity is partially blocked by either a specific inhibitor for extracellular signalregulated 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 ␣-ENaC expression. In addition, overexpression of thioredoxin restored glucocorticoid receptor action on the ␣-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 ␣-ENaC GRE activation via both ERKdependent and thioredoxin-sensitive pathways. These results suggest a putative mechanism whereby cellular redox potentials modulate the glucocorticoid receptor/ dexamethasone effect on ␣-ENaC expression in lung and other tight epithelia.