Cycloheximide increases glucocorticoid-stimulated α-ENaC mRNA in collecting duct cells by p38 MAPK-dependent pathway

Aldosterone and glucocorticoids (GCs) stimulate Na+ reabsorption in the collecting ducts by increasing the activity of the epithelial Na+ channel (ENaC). Our laboratory has used Madin-Darby canine kidney-C7 cells to demonstrate that this effect is associated with an increase in alpha-ENaC gene transcription (Mick VE, Itani OA, Loftus RW, Husted RF, Schmidt TJ, and Thomas CP, Mol Endocrinol 15: 575-588, 2001). Cycloheximide (CHX) superinduced the GC-stimulated alpha-ENaC expression in a dose-dependent manner, but had no effect on basal or aldosterone-stimulated alpha-ENaC expression, whereas anisomycin inhibited basal and corticosteroid-stimulated alpha-ENaC expression. The superinduction of alpha-ENaC expression was also seen with hypotonicity, was blocked by RU-38486, and was independent of protein synthesis. CHX had no effect on alpha-ENaC mRNA half-life, confirming that its effect was via an increase in alpha-ENaC transcription. The effect of CHX and hypotonicity on alpha-ENaC expression was abolished by SB-202190, indicating an effect mediated via p38 MAPK. Consistent with this scheme, CHX increased pp38 and MKK6, an upstream activator of p38, stimulated alpha-ENaC promoter activity. These data confirm a model in which CHX activates p38 in Madin-Darby canine kidney-C7 cells to increase alpha-ENaC gene transcription in a GC-dependent manner.