Functional Regulation of the Epithelial Na+ Channel by IκB Kinase-β Occurs via Phosphorylation of the Ubiquitin Ligase Nedd4-2*

We have previously shown that IκB kinase-β (IKKβ) interacts with the epithelial Na+ channel (ENaC) β-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKβ-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKβ in these cells by ∼50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKβ binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKβ in vitro. Because IKKβ stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKKβ-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. 32P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKKβ phosphorylation target. ENaC stimulation by IKKβ was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKKβ are required for IKKβ regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKβ-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.

The epithelial Na ϩ channel (ENaC) 2 is the rate-limiting step in electrogenic Na ϩ reabsorption in a variety of epithelia that develop steep transepithelial Na ϩ concentration gradients, including the aldosterone-sensitive distal nephron, colon, and alveoli (1). ENaC is therefore critical to the regulation of extracellular and airway fluid volume (1,2). Given its importance to normal homeostasis, channel activity is highly regulated by a number of hormonal influences, including aldosterone and vasopressin, as well as numerous intra-and extracellular stimuli such as flow rate, Na ϩ concentration, and proteolytic activation (1).
Several kinases have been shown to be important in mediating these regulatory influences, including SGK1, protein kinase A (PKA), protein kinase C, ERK, the G-protein-coupled receptor kinase (Grk2), and AMP-activated protein kinase (AMPK) (1,(3)(4)(5)(6)(7)(8). In some cases, as with ERK and Grk2, these kinases phosphorylate ENaC directly (5,6); but in many cases, kinase activity alters the activity of key intracellular regulatory proteins that control the apical membrane insertion or retrieval of ENaC (1,9). We have previously described the interaction of IB kinase (IKK)-␤ with the ␤-subunit of ENaC and have provided evidence that IKK␤ increases ENaC activity in an oocyte expression system, primarily by increasing cell surface expression of the channel (10). Moreover, expression of a kinase-dead IKK␤ had the opposite effect of decreasing ENaC activity, implying that tonic activation of IKK␤ may be required for a significant proportion of basal ENaC activity (10).
IKK␤ is a part of the IKK complex formed by IKK␣, IKK␤, and IKK␥ (or NEMO, the nuclear factor-B (NF-B) essential modulator), which mediates the activation of the NF-B pathway by proinflammatory cytokines, bacterial products such as lipopolysaccharide (LPS), and other stimuli such as heat shock or oxidative stress (11,12). Interaction between this canonical proinflammatory and stress signaling pathway and ENaC function has been suggested by a number of observations and appears to be biphasic. Acute exposure to the cytokine tumor necrosis factor-␣ has been shown to increase amiloride-sensitive Na ϩ transport in alveolar epithelium (13) and distal tubule cells from diabetic rats (14), whereas LPS acutely increases Na ϩ transport in mouse cortical collecting duct cells (15). Amiloride-sensitive alveolar fluid clearance, a function of ENaC activ-ity, increases in a rat model of pneumonia by a tumor necrosis factor-␣-dependent mechanism (16). In contrast, prolonged (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) h) exposure of alveolar epithelial cells to tumor necrosis factor-␣ (17) or of mouse collecting duct cells to LPS (18) results in down-regulation of ENaC activity and expression. We have found that aldosterone, the major hormonal regulator of ENaC, increases IKK␤ levels in renal epithelia and activates NF-B and have suggested that long term stimulation of NF-B might represent a negative feedback loop modulating long term ENaC activation in conditions of oxidative or metabolic stress (10).
Despite evidence of cross-talk between the IKK complex and ENaC-mediated Na ϩ transport, the significance of the interaction between IKK␤ and ENaC remains unclear, as does the potential mechanism of IKK␤-dependent up-regulation of ENaC activity. The current studies were performed to examine whether ENaC is a substrate for IKK␤ phosphorylation, the significance of the IKK␤-ENaC interaction in natively expressing cells, and the mechanism by which IKK␤ regulates ENaC expression. We have found that IKK-dependent regulation of ENaC is physiologically relevant in mouse kidney collecting duct cells and that the regulatory mechanism involves phosphorylation of the E3 ubiquitin-protein ligase Nedd4-2 by IKK␤ at a previously identified SGK1 and PKA phosphorylation site.

EXPERIMENTAL PROCEDURES
Reagents, Chemicals, and Cell Culture-All reagents and chemicals used were purchased from Sigma unless otherwise noted. Plasmids expressing NH 2 -terminal hemagglutinin-tagged and COOH-terminal V5-tagged ENaC subunits were kindly provided by Dr. Thomas Kleyman. HEK-293 and mpkCCD c14 cells were cultured in high glucose, Dulbecco's modified Eagle's medium and defined medium supplemented with hormones and nutrients, respectively, as described previously (7).
Equivalent Short-circuit Current Measurements-A portable epithelial volt ohmmeter was used to measure I sc across polarized mpkCCD c14 cell monolayers as described (19). Amiloride-sensitive equivalent I sc was calculated as the difference in equivalent I sc measured before versus after addition of 10 M amiloride to the apical medium.
In Vitro Phosphorylation Assays-For in vitro phosphorylation assays of ENaC, plasmids expressing NH 2 -terminal hemagglutinin-tagged and COOH-terminal V5-tagged ENaC subunits were translated in vitro using the TNT coupled reticulocyte lysate system (Promega) in the presence or absence of [ 35 S]Met/Cys following the manufacturer's instructions. ENaC subunits were immobilized using 1 l of anti-V5 monoclonal antibody (Invitrogen) per reaction coupled to protein A/G beads (15-l bead volume; Pierce). In vitro phosphorylation was performed by adding 1 l of purified active IKK␤ enzyme (Upstate) versus control buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, and 0.1% 2-mercaptoethanol) with [␥-32 P]ATP (20 Ci; MP Biomedicals, Irvine, CA) at room temperature for 1 h. As a positive control for ENaC phosphorylation, parallel in vitro phosphorylation of ␤-ENaC was performed using 1 l of purified casein kinase-2 (Promega) following the manufacturer's recommendations. As a positive control for IKK␤ activity, another parallel in vitro phosphorylation was performed using glutathione-Sepharose beads (Pierce) to pull down purified glutathione S-transferase (GST)tagged IB␣ (Upstate) for use as a reaction substrate. After SDS-PAGE and transfer to nitrocellulose membranes, phosphorylated bands on the membrane were imaged by exposure to a phospho-screen (Molecular Dynamics), and the bands were quantitated using a Bio-Rad phosphorimaging device.
Assays of Nedd4-2 in vitro phosphorylation were performed as described previously (20). HEK-293T cells transiently transfected with 3 g of plasmid DNA/60-mm dish using Lipofectamine 2000 (Invitrogen) to express wild-type or mutant FLAG-xNedd4-2 were lysed 1 day after transfection. Nedd4-2 was immunoprecipitated from cell lysates using 1 l of anti-FLAG monoclonal antibody M2 coupled to protein A/G beads (20-l bead volume). In vitro phosphorylation reactions were performed as above using either purified active IKK␤ enzyme or control buffer. As a positive control for FLAG-xNedd4-2 phosphorylation, parallel in vitro phosphorylations were performed using 1 l of purified SGK1 (Upstate). As a positive control to check IKK␤ enzymatic activity, GST-tagged IB␣ was again used as a reaction substrate. After SDS-PAGE and transfer to nitrocellulose membranes, immunoblotting for expression of FLAG-xNedd4-2 was first performed (anti-Nedd4 WW2 domain antibody (Upstate) at 1:10,000 and secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (Amersham Biosciences) at 1:5000) and quantitated using a Versa-Doc imager with Quantity One software (Bio-Rad). After the chemiluminescent signal had decayed, phosphorylated bands on the membrane were imaged by exposure to a phospho-screen, and the bands were quantitated using a Bio-Rad phosphorimaging device. The intensity of each band was corrected by subtracting out the local background in the same lane.
In Vivo Phosphorylation Assays-In vivo phosphorylation assays were performed based on our previously described protocol (20). Either Mam-X (stably transfected with a short hair-pin RNA that does not target any known mammalian gene), HEK-293 cells, or HEK-293T cells were transiently co-transfected using Lipofectamine 2000 with the pMO-FLAG-xNedd4-2 plasmid and either SMARTpool IKK␤ siRNA or siGENOME non-targeting siRNA according to the manufacturer's instructions 2 days prior to experimentation. For labeling, cells were washed twice in phosphate-free efflux buffer containing 140 mM NaCl, 3 mM KCl, 1 mM MgSO 4 , 1 mM CaCl 2 , 10 mM glucose, and 10 mM HEPES, pH 7.4 (with 1 M Tris base). Each dish of cells was then incubated at 37°C for 3 h in 1.5 ml of this buffer containing 0.3 mCi of [ 32 P]orthophosphate (MP Biomedicals). After incubation, cells were washed once in ice-cold phosphate-free buffer before lysis in ice-cold radioimmune precipitation assay buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 0.1 mg/ml aprotinin, 1ϫ complete protease inhibitor mixture (Roche Applied Science), 0.1% SDS, 1% sodium deoxycholate, and 1% Triton X-100 (phosphatase and protease inhibitors added just prior to use). After clearing the lysates by high speed centrifugation, FLAG-xNedd4-2 was immunoprecipitated using the anti-FLAG monoclonal antibody M2 coupled to protein A/G beads, washed, eluted in Laemmli sample buffer, subjected to SDS-PAGE, and then transferred to a nitrocellulose membrane. Nedd4-2 Western blotting was performed first, followed by a phosphorimaging device analysis of the same membrane. The ratio of the phosphorylation signal to the immunoblot signal in each lane was compared across conditions to derive relative phosphorylation levels.
Statistics-Statistical analyses were performed using either StatView (SAS) or SigmaPlot software (Jandel Scientific). Analysis of variance was used to compare data obtained from different batches of oocytes for two-electrode voltage clamp experiments. For other biochemical experiments, statistics were performed using Student's t tests, as described in each figure legend. In all cases, p values Ͻ0.05 were considered significant.

Endogenous Regulation of ENaC by IKK␤ in mpkCCD c14
Cells-We previously discovered that IKK␤ interacts with the COOH-terminal cytoplasmic tail of ␤-ENaC through a yeast two-hybrid screen, and this interaction was confirmed biochemically through co-immunoprecipitation studies. Functionally, IKK␤ co-expression was found to enhance ENaC activity by increasing its surface expression in Xenopus oocytes (10). To test whether the regulation of ENaC by IKK␤ is physiologically relevant in an endogenously expressing cell system, we used polarized mpkCCD c14 cells and knocked down IKK␤ expression through RNA interference (Fig. 1). Transfection of siRNAs directed specifically against IKK␤ knocked down its protein expression by a mean of ϳ40 -50% relative to control siRNA-transfected cells (Fig. 1, A and B). Concomitant with this knockdown of IKK␤ expression, amiloride-sensitive, ENaCdependent equivalent I sc was similarly reduced by ϳ50% (Fig.  1C). These findings suggest that IKK␤ expression in these cells is required to sustain ENaC currents under basal conditions; when IKK␤ levels are reduced, ENaC activity is also inhibited.
IKK␤ Does Not Directly Phosphorylate ENaC in Vitro-As IKK␤ is a kinase that binds to ␤-ENaC, we next tested whether any of the ENaC subunits may serve as direct substrates for IKK␤ phosphorylation in vitro. In vitro transcribed and translated epitope-tagged mouse ␣-, ␤-, or ␥-ENaC subunits were immunoprecipitated and then incubated with [␥-32 P]ATP in the presence or absence of purified IKK␤ as described under "Experimental Procedures." The samples were then subjected to SDS-PAGE and transferred to nitrocellulose membranes (Fig. 2). [ 35 S]Met/Cys labeling of ENaC was performed for some samples so that the ENaC subunit mobilities could be assessed on the membrane. No appreciable 32 P incorporation into ENaC was apparent in the samples exposed to IKK␤, yet IKK␤ autophosphorylation bands were observed at ϳ90 kDa, as indicated in Fig. 2A. The presence of co-precipitated IKK␤ confirms that this kinase binds to both ␣and ␤-ENaC and, to a much lesser extent, ␥-ENaC, findings consistent with our earlier work (10). GST-tagged IB␣ was used as an additional positive control to demonstrate IKK␤ activity, and casein kinase-2 was used as a positive control to demonstrate phosphorylation of ENaC by another kinase (Fig. 2B). Similar negative ENaC in vitro phosphorylation data were derived from experiments where ENaC subunits were transiently expressed in HEK-293 cells and then immunoprecipitated from cell lysates prior to performing in vitro phosphorylation assays (data not shown). The failure of IKK␤ to phosphorylate ENaC in vitro suggests that its regulation of ENaC is indirect, not occurring through direct phosphorylation of the channel.
IKK␤-dependent Phosphorylation of Nedd4-2 in Vitro and in Vivo-We have previously reported that IKK␤ stimulation of ENaC activity occurs through enhanced channel plasma membrane expression in Xenopus oocytes (10). As the E3 ubiquitinprotein ligase Nedd4-2 has emerged as a central locus for the regulation of ENaC expression at the apical plasma membrane (21), we considered that ENaC regulation by IKK␤ may be mediated indirectly through Nedd4-2. Of note, Nedd4-2 has been recently reported to be a substrate for various kinases, including SGK1, PKA, AMPK, and Grk2, which may modulate Nedd4-2 function (3,4,20,22). To test whether IKK␤ phosphorylates Nedd4-2 in vitro, FLAG-xNedd4-2 was expressed in and immunoprecipitated from HEK-293 cell lysates, and in vitro phosphorylation assays were performed (Fig. 3). As a positive control for Nedd4-2 phosphorylation, we confirmed that purified SGK1 robustly phosphorylated xNedd4-2 (Fig. 3, upper  panel, left). IKK␤ similarly phosphorylated xNedd4-2 (Fig. 3,  upper panel, middle) and its positive control, GST-tagged IB␣ (upper panel, right). As observed in Fig. 2, autophosphorylation of IKK␤ was also detected in these assays.
To determine whether IKK␤-dependent phosphorylation of Nedd4-2 occurs in intact cells, in vivo labeling with [ 32 P]orthophosphate of HEK-293 cells expressing xNedd4-2 was performed under control conditions, after IKK␤ protein expression was knocked down by RNA interference (Fig. 4A), or after overnight stimulation by 100 ng/ml LPS, an upstream activator of the IKK/NF-B pathway. After immunoprecipitation of FLAG-xNedd4-2 from cell lysates, immunoblotting was performed first, followed by a phosphorimaging device analysis of the same membrane (Fig. 4B). The ratio of the phosphorylation signal to the immunoblot signal in each lane was compared across conditions to derive relative phosphorylation levels (Fig.  4C). Knockdown of IKK␤ reduced in vivo [ 32 P]orthophosphate incorporation into xNedd4-2 by 33 Ϯ 8% relative to control siRNA-transfected cells (Fig. 4C, left). Conversely, LPS stimulation of these cells enhanced in vivo [ 32 P]orthophosphate labeling of xNedd4-2 by 57 Ϯ 19% (Fig. 4C, right). Taken together, these data indicate that IKK␤-dependent phosphorylation of Nedd4-2 occurs both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity by IKK␤.
Identification of Ser-444 in xNedd4-2 as the Target for IKK␤ Phosphorylation in Vitro and in Vivo-Although several IKK␤ target phosphorylation sites have been identified within various components of the NF-B⅐IB protein complex (11,(23)(24)(25), no single consensus IKK␤ target peptide sequence motif has emerged that would allow prediction of IKK␤ phosphorylation sites in new protein substrates. It has been shown previously that both SGK1 and PKA stimulate ENaC activity via phosphorylation of Nedd4-2 at two key residues, Ser-338 and Ser-444 (Xenopus numbering), which has an inhibitory effect on Nedd4-2 function (3,4). As IKK␤ also stimulates ENaC activity, we tested whether IKK␤ phosphorylates xNedd4-2 at the same SGK1 and PKA phosphorylation sites. In vitro phosphorylation assays were performed in the presence or absence of purified IKK␤ using immunoprecipitated WT Nedd4-2 or Nedd4-2  with either Ser-338 or Ser-444 mutated to Ala (S338A or S444A) (Fig. 5). Nedd4-2 phosphorylation signal normalized to Nedd4-2 protein expression was quantitated and compared in the two mutant Nedd4-2 constructs relative to that of WT Nedd4-2 (Fig. 5B). Although the relative in vitro phosphorylation of the S338A mutant was not different from WT Nedd4-2, the 32 P incorporation of the S444A mutant was dramatically reduced by 80 -90% in vitro.
To test whether differential IKK␤-dependent phosphorylation at Ser-444 also occurs in vivo, we performed in vivo [ 32 P]orthophosphate labeling studies using the same three Nedd4-2 constructs in control green fluorescent protein-transfected cells and in cells transfected to overexpress IKK␤ (Fig. 6). Of note, IKK␤ overexpression appeared to enhance the steadystate expression of transfected Nedd4-2 in these cells, whereas expression of the S444A mutant was decreased compared with the other Nedd4-2 constructs under both control and IKK␤ co-expression conditions (Fig. 6A). Nevertheless, when normalized for Nedd4-2 protein expression, overexpression of IKK␤ dramatically increased in vivo phosphate labeling of WT and S338A Nedd4-2 by ϳ5-8-fold. However, there was only a smaller, albeit significant, ϳ2-fold increase in phosphate label-ing with the S444A Nedd4-2 mutant (Fig. 6B). These results suggest that Ser-444 is also the dominant target for IKK␤-dependent phosphorylation in vivo, although additional IKK␤dependent Nedd4-2 phosphorylation site(s) could exist.
IKK␤-dependent Regulation of ENaC Requires Functional Nedd4-2 and Phosphorylation at Ser-444-To explore the potential functional role of Nedd4-2 in the regulation of ENaC by IKK␤, we measured amiloride-sensitive whole cell currents in oocytes co-expressing mouse ENaC, either WT or mutant xNedd4-2 constructs, and/or IKK␤ (Fig. 7). Confirming our previous findings (10), co-expression of IKK␤ with ENaC increased ENaC current by ϳ30% over that of oocytes expressing ENaC alone. Wild-type xNedd4-2 co-expression dramatically reduced ENaC currents under control conditions, but this reduction was substantially reversed by IKK␤ co-expression. Indeed, ENaC current was increased ϳ150% by IKK␤ co-expression relative to ENaC alone in the presence of Nedd4-2, consistent with the idea that the presence of additional Nedd4-2 magnified the regulatory effect of IKK␤ on ENaC. Coexpression of a ubiquitin ligase-deficient, dominant-negative xNedd4-2 mutant (C938S) (26) greatly increased ENaC currents, presumably by preventing Nedd4-2-dependent ENaC ubiquitination and degradation. Under this condition, IKK␤ failed to activate ENaC, suggesting that inhibition of Nedd4-2mediated ENaC ubiquitination is involved in the IKK␤dependent activation of ENaC. Finally, co-expression of the IKK␤ phosphorylation-deficient S444A xNedd4-2 mutant sub-  stantially inhibited ENaC current. Again, IKK␤ failed to activate ENaC current with co-expression of this mutant, suggesting that IKK␤-mediated phosphorylation of Ser-444 in xNedd4-2 is required for the IKK␤-dependent regulation of ENaC.

DISCUSSION
In this study, we have further characterized the mechanism and relevance of ENaC regulation by IKK␤, a novel interaction partner and activator of ENaC that we recently identified (10). Knockdown of endogenous IKK␤ in polarized mpkCCD c14 cells inhibited ENaC-dependent transepithelial currents, suggesting that under baseline conditions in these cultured cells, without exogenously induced stimulation of the IKK/NF-B pathway, there is tonic IKK␤ activity supporting basal ENaC activity. This result is consistent with our previous finding that overexpression of a dominant-negative mutant of IKK␤ downregulated ENaC currents expressed in oocytes under basal conditions (10). Although IKK␤ binds to ENaC, we have found that Nedd4-2, rather than ENaC, is the phosphorylation target for this kinase. We have further shown that phosphorylation of Ser-444 in xNedd4-2, a dominant SGK1/PKA target site, is the relevant target of IKK␤ in vitro and in vivo. Nedd4-2 mediates the IKK␤-dependent regulation of ENaC, and phosphorylation at Ser-444 appears to be required for this regulation to occur. Although our data demonstrate that IKK␤ phosphorylates xNedd4-2, we feel it is likely that IKK␤ will also phosphorylate mammalian Nedd4-2 based on the highly conserved sequence near Ser-444 across species (3), but this prediction remains to be tested.
Additional molecular details regarding the mechanism for IKK␤-dependent regulation of ENaC are not yet clear, but a few intriguing possibilities are suggested by our findings. It has been shown previously that SGK1-dependent phosphorylation of Ser-444 in xNedd4-2 enhances the binding of Nedd4-2 to 14-3-3 scaffolding proteins, which sequesters Nedd4-2 and thereby prevents it from interacting with ENaC (27)(28)(29). Therefore, as the effect of IKK␤ on ENaC, like that of SGK1 and PKA, is stimulatory and requires phosphorylation at Ser-444, it is reasonable to propose that these three kinases share this common mechanism for ENaC regulation. However, the SGK1/PKA regulation of ENaC appears to require the additional phosphorylation at one of two other minor SGK1/PKA phosphorylation sites, Ser-338 or Thr-363. Such phosphorylation may enable the binding of another 14-3-3 molecule within a dimeric 14-3-3 complex to the same Nedd4-2 molecule, and this additional interaction appears to be necessary for the efficient sequestration of Nedd4-2 (3,4). However, Ser-338 was not found to be a site for IKK␤ phosphorylation of Nedd4-2. Moreover, at least based on our in vitro analysis of the IKK␤-dependent phosphorylation of Nedd4-2 (cf. Fig. 5), there are no other apparent significant IKK␤ target phosphorylation sites in Nedd4-2 besides Ser-444. Therefore, an interesting question for future study is to determine whether SGK1 or PKA phosphorylation at another site is required to confer the IKK␤-mediated regulation of ENaC.
Normally, IKK␣ and IKK␤ exist together in a complex, along with IKK␥/NEMO and other adaptor proteins such as  The mean Ϯ S.E. of amiloride-sensitive ENaC currents measured by two-electrode voltage clamp at Ϫ100 mV in oocytes that were co-injected with 2 ng each of mouse ␣-, ␤-, and ␥-ENaC cRNAs Ϯ 5 ng of IKK␤ Ϯ 0.7 ng of WT or S444A Nedd4-2 or 5 ng of C938S Nedd4-2, as indicated, is shown. For each condition, ENaC currents were measured and compared with or without IKK␤ co-expression (*, p Ͻ 0.001 compared with corresponding control in the absence of IKK␤ co-expression; two-tailed unpaired t tests; n ϭ 18-68 oocytes, three batches for each of the Nedd4-2 co-expression conditions, and nine batches for ENaC alone condition).
Hsp90 and Cdc37 (12). Of note, as observed for IKK␤, we have also found that siRNA-mediated knockdown of IKK␣ produces a similar inhibition of ENaC-dependent current in mpkCCD c14 cells (data not shown). This finding indicates that disruption of the IKK complex in vivo may be sufficient to modulate ENaC currents. Furthermore, based on the finding that IKK␤ co-precipitates when added to immunoprecipitates of either ENaC subunits (especially ␣ and ␤) (Fig. 2) or Nedd4-2 (Figs. 3 and 5) in vitro, it is likely that at least the IKK complex, ENaC, and Nedd4-2 can exist together as a larger signaling complex in cells. We propose that this complex may afford the efficient coupling and integration of signals from various potential upstream pathways that impinge on IKK to regulate ENaC activity via its phosphorylation and functional regulation of Nedd4-2. Moreover, as Nedd4-2 appears to be an important regulator of a growing list of other ion transport proteins in cells (e.g. voltage-gated Na ϩ and K ϩ channels, KCNQ channels, ClC Cl Ϫ channels, the SGLT1 Na ϩ /glucose co-transporter, and the NaP i IIb Na ϩ / phosphate co-transporter) (30 -35), IKK␤-dependent regulation of Nedd4-2 function may serve as a general transduction mechanism that couples changes in inflammation and potentially other signaling pathway inputs to transport protein regulation.
There are differing reports in the literature as to the functional consequences of IKK/NF-B pathway stimulation on ENaC activity. Certain studies suggest that stimulation of the IKK/NF-B pathway by either tumor necrosis factor-␣ or LPS inhibits ENaC activity, at least over relatively long time intervals (hours to days) (17,18). This inhibition may occur via down-regulation of SGK1 in renal collecting duct epithelial cells and is earliest seen at 6 h (18). However, shorter term, acute effects may occur within 30 min and appear to be stimulatory (13)(14)(15), indicating a probable biphasic response of ENaC to this pathway. Our findings in this study show a stimulatory effect, at least under baseline conditions. There is good evidence that interstitial inflammation enhances renal Na ϩ reabsorption in association with proteinuric glomerular damage (36). We speculate that inflammation-associated activation of IKK/NF-B and ENaC may contribute to this response. Acute up-regulation of renal salt absorption via this mechanism could also help the body defend against hypotension that occurs with systemic vasodilatation in the setting of sepsis and generalized inflammation.
As there is apparent tonic activity of IKK with respect to ENaC function in our system in the absence of induced inflammatory stimuli, it seems likely that other noninflammatory signaling pathways regulate the IKK complex under the conditions of our experiments. However, it is not yet clear what the potential upstream regulators of the IKK pathway are in this case. Interestingly, the metabolic sensor AMPK has been reported to be an upstream inhibitor of IKK in several cell systems (37)(38)(39), and we have shown that AMPK inhibits ENaC, also via effects on Nedd4-2 in an AMPK phosphorylation-dependent manner (7,20). Further studies to test for the potential involvement of AMPK and other cellular signaling pathways as upstream regulators of the IKK␤-dependent regulation of ENaC are thus warranted.
In summary, we have identified a previously characterized node of Nedd4-2 regulation by SGK1 and PKA as the target for IKK␤-dependent regulation of ENaC. Via effects on Nedd4-2 function, the IKK/NF-B pathway may play an important role in integrating the response of ENaC and other epithelial ion transport proteins and thus transepithelial salt transport to various pathways (e.g. inflammation, hormonal (aldosterone versus vasopressin), and metabolic).