Abstract
The ubiquitin ligase Nedd4-like (Nedd4L, or Nedd4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out Nedd4L in mice intestinal epithelium (Nedd4Lf/f ;Vil-CreERT2 ) we show here that Nedd4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl-, suggesting that Na+/K+/2Cl- (NKCC1/SLC12A2) co-transporter and ENaC are targets of Nedd4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the Nedd4L knock-out animals was increased, indicating that Nedd4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between Nedd4L and NKCC1, suggesting that Nedd4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in β and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for Nedd4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.
Highlights
The ubiquitin ligase neuronal precursor cell expressed developmentally downregulated 4 (Nedd4)-like (Nedd4L, or Nedd4-2) binds to and regulates stability of the epithelial Na؉ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues
Nedd4L Regulates Function of NKCC1 and ENaC in the Colon—Nedd4L is expressed in the distal colon (Fig. 1A), a tissue known to express ENaC (Refs. 7 and 22 and see below)
Because we could not detect NKCC1 phosphorylation in the colons of our mice, we tested for such phosphorylation in the NKCC1-expressing Hek293T cells depleted of Nedd4L and treated with low ClϪ/hypotonic solution plus calyculin A; our results revealed no effect of Nedd4L depletion on NKCC1 phosphorylation (Fig. 8A) or SGK1 phosphorylation or abundance of WNK3 (Fig. 8B), which is known to regulate NKCC1 in some epithelial cells [27]
Summary
Nedd4L Regulates Function of NKCC1 and ENaC in the Colon—Nedd4L is expressed in the distal colon (Fig. 1A), a tissue known to express ENaC (Refs. 7 and 22 and see below). In the case of ␥ENaC, Nedd4L depletion greatly increased the abundance of its cleaved, active form upon salt restriction (Fig. 4G, boxed) Together, these results suggest that a low salt diet elevates ␥ENaC protein abundance and ENaC activity but does not enhance NKCC1 activity despite increased mRNA and protein expression. Regulation of NKCC1 Abundance by Nedd4L in the Colon Does Not Involve NKCC1 or Sgk Phosphorylation or the WNK1/3-SPAK Pathway—Nedd4L was recently found to degrade the (aldosterone-inducible) PY motif-containing isoform of the kinase WNK1 in the kidney and to regulate NCC activity [25]; this raises the possibility that Nedd4L deficiency in the colon may stabilize WNK1 and enhance the NKCC1 activation we observed here, similar to its effect on NCC in the kidney To test this possibility, we analyzed the abundance of WNK1 as well as activation of the WNK-target kinase SPAK (or its relative OSR1) in controls and Nedd4L KO colons after (or not) a low salt diet to elevate plasma aldosterone. Our results suggest that Nedd4L normally suppresses expression and function of NKCC1 in the colon, a suppression that likely does not involve the WNK/SPAK pathway
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