Aldosterone and vasopressin affect α- and γ-ENaC mRNA translation

Vasopressin and aldosterone play key roles in the fine adjustment of sodium and water re-absorption in the nephron. The molecular target of this regulation is the epithelial sodium channel (ENaC) consisting of alpha-, beta- and gamma-subunits. We investigated mRNA-specific post-transcriptional mechanisms in hormone-dependent expression of ENaC subunits in mouse kidney cortical collecting duct cells. Transcription experiments and polysome gradient analysis demonstrate that both hormones act on transcription and translation. RNA-binding proteins (RBPs) and mRNA sequence motifs involved in translational control of gamma-ENaC synthesis were studied. gamma-ENaC-mRNA 3'-UTR contains an AU-rich element (ARE), which was shown by RNA affinity chromatography to interact with AU-rich element binding proteins (ARE-BP) like HuR, AUF1 and TTP. Some RBPs co-localized with gamma-ENaC mRNA in polysomes in a hormone-dependent manner. Reporter gene co-expression experiments with luciferase gamma-ENaC 3'-UTR constructs and ARE-BP expression plasmids demonstrate the importance of RNA-protein interaction for the up-regulation of gamma-ENaC synthesis. We document that aldosterone and the V-2 receptor agonist dDAVP act on synthesis of alpha- and gamma-ENaC subunits mediated by RBPs as effectors of translation but not by mRNA stabilization. Immunoprecipitation and UV-crosslinking analysis of gamma-ENaC-mRNA/HuR complexes document the significance of gamma-ENaC-mRNA-3'-UTR/HuR interaction for hormonal control of ENaC synthesis.