The amiloride-sensitive epithelial sodium channel alpha-subunit is transcriptionally down-regulated in rat parotid cells by the extracellular signal-regulated protein kinase pathway.

Previous studies have shown that an inducible Raf-1 kinase protein, DeltaRaf-1:ER, activates the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK)-signaling pathway, which is required for the transformation of the rat salivary epithelial cell line, Pa-4. Differential display polymerase chain reaction was employed to search for mRNAs repressed by DeltaRaf-1:ER activation. Through this approach, the gene encoding the alpha-subunit of the amiloride-sensitive epithelial sodium channel (alpha-ENaC) was identified as a target of activated Raf-1 kinases. alpha-ENaC down-regulation could also be seen in cells treated with 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA), indicating that the repression of steady-state alpha-ENaC mRNA level was dependent upon the activity of protein kinase C, the target of TPA, as well. Pretreatment of cells with a specific inhibitor of the ERK kinase pathway, PD 98059, markedly abolished the down-regulation of alpha-ENaC expression, consistent with the hypothesis that the ERK kinase-signaling pathway is involved in TPA-mediated repression. Moreover, through the use of transient transfection assays with alpha-ENaC-reporter and activated Raf expression construct(s), we provide the first evidence that activation of the ERK pathway down-regulates alpha-ENaC expression at the transcriptional level. Elucidating the molecular programming that modulates the expression of the alpha-subunit may provide new insights into the modulation of sodium reabsorption across epithelia.