Relative contribution of Nedd4 and Nedd4-2 to ENaC regulation in epithelia determined by RNA interference.
Epithelial Na+ transport is regulated in large part by mechanisms that control expression of the epithelial Na+ channel (ENaC) at the cell surface. Nedd4 and Nedd4-2 are candidates to control ENaC surface expression, but it is not known which of these proteins contributes to ENaC regulation in epithelia. To address this question, we used RNA interference to selectively reduce expression of Nedd4 or Nedd4-2. We found that endogenous Nedd4-2, but not Nedd4, negatively regulates ENaC in two epithelial cell lines (Fischer rat thyroid and H441); small interfering RNA (siRNA) against Nedd4-2 increased amiloride-sensitive Na+ current (compared with control siRNA), but Nedd4 siRNA did not. A mutation associated with Liddle's syndrome (betaR566X) abolished the effect of Nedd4-2 siRNA, suggesting that a defect in ENaC regulation by Nedd4-2 contributes to the pathogenesis of this inherited form of hypertension. Previous work found that Nedd4-2 is phosphorylated by serum and glucocorticoid-regulated kinase, a Ser/Thr kinase induced by steroid hormones. Here we found that Nedd4-2 phosphorylation contributes to ENaC regulation by steroid hormones. Consistent with this model, ENaC stimulation by dexamethasone was reduced by Nedd4-2 siRNA and by overexpression of a mutant Nedd4-2 lacking serum and glucocorticoid-regulated kinase phosphorylation sites. Thus, endogenous Nedd4-2 negatively regulates ENaC in epithelia and is a component of a signaling pathway by which steroid hormones regulate ENaC. Defects in this regulation may contribute to the pathogenesis of hypertension.
Snyder
Peter M
PM
Department of Internal Medicine and Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Steines
Jennifer C
JC
Olson
Diane R
DR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D056827:Endosomal Sorting Complexes Required for Transport; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels; D006801:Humans; D006973:Hypertension / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D015398:Signal Transduction; D015222:Sodium Channels / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D007527:Isoenzymes; D034741:RNA, Small Interfering; D015222:Sodium Channels; D013256:Steroids; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases