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 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.
Highlights
The amiloride-sensitive epithelial Na؉ channel (ENaC) plays a critical role in the maintenance of alveolar fluid balance
To determine whether or not glucocorticoids increase ␣-ENaC expression in A549 cells and could be used as a model to explore the molecular mechanism by which glucocorticoids modulate ENaC activity in lung epithelial cells, Northern blot analyses were performed on total RNA taken from A549 cells treated with 100 nM Dex for either 4 or 24 h (Fig. 1)
glucocorticoid receptor (GR)/Dex-mediated Stimulation of ␣-ENaC glucocorticoid response element (GRE) Is Attenuated by Either Ras Pathway Activation or Exogenous H2O2 Treatment—To ascertain that the cross-talk between Ras- and GR/Dex-mediated signaling pathways that we recently reported for salivary epithelial Pa-4 cells [21, 22] dictate overall ␣-ENaC expression levels in nonsalivary cells, we assessed the effect of Ras on ␣-ENaC promoter/enhancer activity using transient transfection and reporter assays in lung A549 cells
Summary
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; the molecular mechanism for free radical-mediated ENaC inhibition is unclear. 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. Reactive oxygen species have been demonstrated to participate in physiological or cellular responses in both signaling pathway activation and gene regulation [1, 3]. The specific effect(s) elicited by reactive oxygen and/or nitrogen species on lung epithelial cells during injury is currently under intense investigation
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