IκB Kinase-β (IKKβ) Modulation of Epithelial Sodium Channel Activity*

Using the yeast two-hybrid system, we identified a number of proteins that interacted with the carboxyl termini of murine epithelial sodium channel (ENaC) subunits. Initial screens indicated an interaction between the carboxyl terminus of β-ENaC and IκB kinase-β (IKKβ), the kinase that phosphorylates Iκβ and results in nuclear targeting of NF-κB. A true two-hybrid reaction employing full-length IKKβ and the carboxyl termini of all three subunits confirmed a strong interaction with β-ENaC, a weak interaction with γ-ENaC, and no interaction with α-ENaC. Co-immunoprecipitation studies for IKKβ were performed in a murine cortical collecting duct cell line that endogenously expresses ENaC. Immunoprecipitation with β-ENaC, but not γ-ENaC, resulted in co-immunoprecipitation of IKKβ. To examine the direct effects of IKKβ on ENaC activity, co-expression studies were performed using the two-electrode voltage clamp technique in Xenopus oocytes. Oocytes were injected with cRNAs for αβγ-ENaC with or without cRNA for IKKβ. Co-injection of IKKβ significantly increased the amiloride-sensitive current above controls. Using cell surface ENaC labeling, we determined that an increase of ENaC in the plasma membrane accounted for the increase in current. The injection of kinase-dead IKKβ (K44A) in ENaC-expressing oocytes resulted in a significant decrease in current. Treatment of mpkCCDc14 cells with aldosterone increased whole cell amounts of IKKβ. Because this result suggested that aldosterone might activate NF-κB, mpkCCDc14 cells were transiently transfected with a luciferase reporter gene responsive to NF-κB activation. Both aldosterone and tumor necrosis factor-α (TNFα) stimulation caused a similar and significant increase in luciferase activity as compared with controls. We conclude that IKKβ interacts with ENaC by up-regulating ENaC at the plasma membrane and that the presence of IKKβ is at very least permissive to ENaC function. These studies also suggest a previously unexpected interaction between the NF-κB transcription pathway and steroid regulatory pathways in epithelial cells.

The epithelial sodium channel (ENaC) 1 is located in the apical membrane in a variety of Na ϩ -transporting epithelia including the cortical collecting duct of the kidney, the distal colon, the ducts of secretory glands, and the lung (1,2). ENaC mediates Na ϩ absorption in most epithelia with high resistance and is critical to the regulation of fluid homeostasis, blood pressure, and airway fluid volume. Structural abnormalities of ENaC are linked to human diseases including hypertension seen with Liddle's syndrome (3,4) and salt wasting seen in variants of pseudohypoaldosteronism (5,6).
ENaC is a member of the degenerin (DEG)/ENaC family, a group of structurally related and phylogenetically conserved ion channels with diverse functions. Structurally, ENaC is composed of three subunits, ␣, ␤, and ␥, which share a 30% sequence homology (7). When expressed alone in Xenopus oocytes, these subunits are capable of generating a Na ϩ current. Only fully reconstituted channels have the same characteristics of the wild-type channel, exhibiting voltage independence, relatively low conductance, distinctive cation selectivity, sensitivity to amiloride in the sub-M range, and slow gating kinetics (8 -10).
ENaC is subject to regulation by a number of hormones, including aldosterone. Additionally, ENaC activity is modulated by a number of different proteins including serum/glucocorticoid-inducible kinase (SGK), Nedd4, syntaxin 1A, the cystic fibrosis transmembrane conductance regulator (CFTR), and K-Ras2A (11,12). ENaC activity has also been shown to be regulated by channel-activating proteases (CAPs) (13) and furin (14) presumably by cleavage of ENaC subunits. Although there is strong evidence that direct phosphorylation may play an important role in ENaC regulation, only two kinases have been positively identified that directly phosphorylate ENaC subunits (15). Using fractionated cytosol extracted from rat distal colon to phosphorylate glutathione S-transferase (GST) fusion proteins constructed with the carboxyl terminus of ␥-ENaC, Shi et al. (15,16) were able to identify ERK2 and casein kinase 2. Both kinases were able to phosphorylate ENaC subunits, and ERK was noted to modulate channel activity in an oocyte expression system. Of note, a third fraction of cytosol was also found to phosphorylate ENaC, but the identity of the kinase responsible for this phosphorylation is not yet known.
Using the yeast two-hybrid system we were able to identify a number of proteins that interacted with the carboxyl terminus of ␤-ENaC. We now report that one of these proteins, IKK␤ (the ␤-subunit of the kinase that cleaves the inhibitor of nuclear factor B), significantly interacts with and augments ENaC activity.

EXPERIMENTAL PROCEDURES
Cell Lines-mpkCCD c14 cells (hereafter referred to as CCD cells), an immortal cell line derived from transgenic mice containing a 2.7-kDa fragment of the SV40 early region, were maintained in culture as described by Bens et al. (17). Cells were grown on semipermeable supports (6-well, 0.4-m pore size Transwell polycarbonate membranes, Costar, Cambridge, MA) to confluency. Only monolayers that exhibited high transepithelial resistance were used in experiments. CCD cells grown on plastic were allowed to grow to confluence prior to use. All cells were maintained in defined medium (Dulbecco's modified Eagle's medium:Ham's F-12 medium (1:1, v/v), 60 nM sodium selenate, 5 mg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothryonine, 10 ng/ml epidermal growth factor, 5 g/ml insulin, 20 mM D-glucose, 2% v/v fetal bovine serum (FBS), and 20 mM HEPES, pH 7.4) at 37°C in 5% CO 2 , 95% air atmosphere. Medium was changed three times/week. Prior to experiments, cells were maintained in steroid-free and FBS-free medium for 24 h.
Antibodies and Reagents-␤-and ␥-ENaC antibodies were generated and affinity-purified as described previously (18). Antibodies to IKK␤ were obtained from Upstate Cell Signaling Solutions (Lake Placid, NY). All other reagents were purchased from Sigma unless otherwise noted.
Aldosterone Treatment-Prior to aldosterone treatment, cells were placed in steroid-free medium for 24 h. CCD cells were treated with 10 Ϫ6 M aldosterone for 3 or 18 h at 37°C.
Yeast Two-hybrid ␤-Galactosidase/X-Gal Assay-A standard filter lift assay was performed as outlined in the BD Matchmaker Gal-4 Two-hybrid System 3 manual (20). Yeast were permeabilized in liquid nitrogen, and ␤-galactosidase activity was assayed in a Z buffer (Na 2 HPO 4 ⅐7H 2 O (16.1 g/liter), NaH 2 PO 4 ⅐H 2 O (5.5 g/liter), KCl (0.75 g/liter), MgSO 4 ⅐7H 2 O (0.246 g/liter), pH 7)/␤-mercaptoethanol (0.27 ml/ 100 ml of Z buffer)/X-gal solution (1.67 ml of stock solution/100 ml of Z buffer). X-gal stock solution was made immediately prior to each experiment by dissolving X-gal in N,N-dimethylformamide at a concentration of 20 mg/ml. Filters were placed in a room air incubator at 30°C and checked periodically until they turned a blue color.
NF-B Luciferase Activity Assay-The NF-B-responsive gene (pNF-B-Luc, Clontech) was transiently transfected into CCD cells using LipofectAMINE 2000 (Invitrogen) per the manufacturer's instructions. CCD cells were grown directly on plastic in 12-well clusters, transfected with 1.6 g of pNF-B-Luc/well, and grown to confluence. Cells were placed in steroid-free and FBS-free medium for 24 h prior to experiments. Cells were then kept in control conditions or incubated with either aldosterone (10 Ϫ6 M) or TNF␣ (120 ng/ml) for 18 h. NF-B activity was quantified by measuring luciferase activity using standard methods (Promega, Madison, WI) and a Turner TD 20/20 luminometer with the signal integrated over a 15-s interval. Luciferase activity was measured in arbitrary luminometry units.
Immunoprecipitation and Western Blot Analysis-CCD cells were grown on 6-well filter inserts and were maintained in steroid-and FBS-free medium for 24 h. Monolayers were then subjected to control, 3-h aldosterone, and 18-h aldosterone conditions. The immunoprecipitation and the Western blotting protocol were performed as described previously (21). Equivalency of loading for Western blot analysis and immunoprecipitation was ensured by performing protein assays (BCA, Pierce) and using equal amounts of protein for each experimental condition.
Channel Expression in Xenopus Oocytes-Xenopus oocytes (stage V-VI) were pretreated with 2 mg/ml collagenase (type IV) in calciumfree saline solution. Murine ENaC cRNAs (1-3 ng/subunit in 50 nl of H 2 O) were microinjected into all oocytes. Oocytes in the experimental group were additionally injected with 5 ng of cRNA of murine IKK␤ (IMAGE clone 4482634). As a control, ␣␤␥-ENaC cRNAs were co-injected with 5 ng of kinase-dead human IKK␤ (K44A) ( Whole Cell Current Measurements-A two-electrode voltage clamp technique was used as described previously (24). Whole cell inward amiloride-sensitive currents were measured in control oocytes expressing ␣␤␥-ENaC alone or experimental oocytes expressing ␣␤␥-ENaC ϩ IKK␤ using a DigiData 1200 interface (Axon Instruments, Foster City, CA) and a TEV 200A voltage clamp amplifier (Dagan Corp., Minneapolis, MN). Data acquisition and analysis were performed using pClamp 7.0. Amiloride-sensitive currents were defined as the difference of the current in the absence and the presence of 0.1 mM amiloride. Oocytes were bathed in a solution containing 110 mM NaCl, 2 mM CaCl 2 , 2 mM KCl, 10 mM HEPES-NaOH, pH 7.40. All measurements were made at room temperature (22-25°C), and the bath solution was continuously perfused at 5 ml/min by gravity. Oocytes were typically incubated in the bath solution for at least 10 min before the current was recorded to allow currents to stabilize. Membrane potentials were clamped from Ϫ140 to ϩ60 mV in 20-mV increments with a duration of 900 ms. Currents were measured at a holding potential of Ϫ100 mV 600 ms after initiation of the clamp potential. For ROMK measurements, whole cell potassium currents were measured at Ϫ100 mV in the absence or presence of 5 mM BaCl 2 .
Cell Surface ENaC Labeling-The general approach was based on the method of Zerangue et al. (25) as modified by Condliffe et al. (26). Control oocytes expressing mouse ␣-, ␤-FLAG-, and ␥-ENaC subunits and experimental oocytes co-injected with ␣␤ FLAG ␥-ENaC and IKK␤ were blocked with MBS supplemented with 1 mg/ml of bovine serum albumin (MBS-BSA) after 2 days of incubation. Oocytes were then exposed to MBS-BSA with 1 mg/ml mouse monoclonal anti-FLAG antibody (M2, Sigma) at 4°C for 1 h. Of note, ␤-ENaC containing the FLAG epitope (DYDKKKD) at the extracellular loop does not alter I Na relative to wild-type ENaC expression as first demonstrated by Firsov et al. (4). After the oocytes were labeled with primary antibody, they were washed six times in MBS-BSA at 4°C and incubated in MBS-BSA supplemented with 1 mg/ml horseradish peroxidase-conjugated secondary antibody (peroxidase-conjugated AffiniPure F(abЈ) 2 fragment goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA)) for 1 h at 4°C. After 12 additional washes, individual oocytes were placed in 100 ml of SuperSignal enzyme-linked immunosorbent assay Femto solution (Pierce) and incubated at room temperature for 1 min. Chemiluminescence was quantified in arbitrary light units using TD 20/20 luminometer with the signal integrated over a 60-s interval.

RESULTS
Yeast Two-hybrid Results-Initial results of the yeast twohybrid library screen plated on medium-selective media (ϪHis/ ϪLeu/ϪTrp) using a commercially available mouse kidney library (Clontech, BD Biosciences) against carboxyl termini of ENaC revealed an interaction between the ␤-ENaC and a number of clones including IKK␤. Full-length IKK␤ was subcloned into the pGADT7 vector. We then screened IKK␤ against the carboxyl terminus of ␣-, ␤-, and ␥-ENaC placed in the pGBKT7 vector. All constructs were transformed into yeast individually and selected for with appropriate medium. Single-plasmid transformants did not grow on medium-selective media (ϪHis/ ϪLeu/ϪTrp), nor did they exhibit spontaneous ␤-galactosidase activity. As shown in Fig. 1, only co-transformation of IKK␤ with the ␤-carboxyl terminus of ENaC yielded robust growth on medium-selective media (-His/-Leu/-Trp) after 5 days of incubation. ␣-ENaC exhibited no growth, and ␥-ENaC grew only fine colonies on medium-selective media. However, only ␤-ENaC co-transformants exhibited significant ␤-galactosidase activity. This ␤-galactosidase activity was evident in the ␤-ENaC/IKK␤ co-transformants after 2 h but was robust after 4 h as shown in Fig. 1, bottom. ␥-ENaC only exhibited minimal ␤-galactosidase activity after 4 h (Fig. 1, bottom), which was unchanged at 8 h. Only co-transformation of ␤-ENaC with IKK␤ yielded both growth on restrictive media and ␤-galactosidase activity.
Immunoprecipitation with ␤and ␥-ENaC Antibodies-To determine whether the ENaC-IKK␤ interaction was detectable in a physiologically relevant system, we employed in continuous culture a line of mouse CCD cells that was developed by Bens, Vandewalle, and co-workers (17) and that expresses endogenous ENaC activity, which is regulated by hormones in a manner similar to the intact cortical collecting duct. After immunoprecipitation of cell lysate using specific antibodies for ␤and ␥-ENaC, we performed Western blots using a commercially available antibody for IKK␤. Fig. 2 shows the result of Western blots with the IKK␤ antibody. The first lane of Fig. 2 demonstrates that the IKK␤ antibody can detect IKK␤ in CCD cell lysate with a characteristic band at 87-90 kDa, identical to its appearance in Jurkat cell lysate supplied by the manufacturer of the antibody (not shown). The next two lanes of Fig. 2 show the Western blots using anti-IKK␤ after immunoprecipitation of CCD cell lysate with ␤-ENaC and ␥-ENaC antibodies. IKK␤ co-immunoprecipitates with ␤but not ␥-ENaC. The next two lanes of Fig. 2 are controls to demonstrate that the ␤and ␥-ENaC antibodies bring down the appropriate subunits, and the final lane is a control showing that incubation of lysate and immunoprecipitation beads without antibody does not result in detection of any protein using the anti-IKK␤.
Co-expression of IKK␤ and ␣␤␥-ENaC in Xenopus Oocytes Significantly Increases Amiloride-sensitive Current-To determine whether the interaction between the carboxyl terminus of ␤-ENaC and IKK␤ modulated ENaC activity, we used the Xenopus oocyte expression system. Currents shown in Fig. 3 are all normalized to control. As shown in Fig. 3A, co-expression of ␣␤␥-ENaC with IKK␤ cRNA significantly increased amiloride-sensitive current by 26% (n ϭ 42 oocytes, n ϭ 3 separate groups of oocytes, p ϭ 0.0007). To determine whether the kinase activity of IKK␤ was required for its effect on ENaC, we examined the effect of a kinase-dead mutant IKK␤ (K44A) (22). Fig. 3B shows that co-expression of ␣␤␥-ENaC and kinasedead IKK␤ (K44A) resulted in a 50% decrease in normalized currents (n ϭ 47, n ϭ 3, p Ͻ 0.001). We examined the effects of IKK␤ and kinase-dead IKK␤ on the expression of a distinct ion channel, ROMK (23). Fig. 3, C and D, shows the effect of IKK␤ and IKK␤ (K44A) co-expression with ROMK. Neither construct appears to have a significant effect on barium-sensitive K ϩ current, suggesting that IKK␤ activation and IKK␤ (K44A) -mediated down-regulation of channel activity are ENaC-specific effects.
Co-expression of IKK␤ Increases Surface Expression of ENaC-Surface expression of ENaC is shown in Fig. 4. As measured by chemiluminescence, there is a 30% increase in signal with co-expression of IKK␤ and ␣␤ FLAG ␥-ENaC as compared with controls expressing ␣␤ FLAG ␥-ENaC alone. This 30% increase in surface expression of ENaC parallels the increase in amiloride-sensitive current induced by IKK␤, implying that the mechanism of IKK␤-mediated ENaC up-regulation is secondary to an increase in the number of channels at the surface rather than an increase in open probability.
IKK␤ Is An Aldosterone-regulated Protein-Given that ENaC is responsive to aldosterone, we sought to determine whether IKK␤ is regulated by aldosterone. CCD cell monolayers were exposed to 10 Ϫ6 M aldosterone or diluent for 3 or 18 h. Fig. 5 shows typical Western blot results of CCD cell lysate. Fig. 5A indicates a clear increase in the whole cell amount of IKK␤ that can be seen after 18 h of exposure to aldosterone. Western blots were quantified by densitometry (n ϭ 5). Fig. 5B shows these densitometry results as a percent of control. After 3 h of exposure to aldosterone, there is no significant increase in whole cell IKK␤ as compared with controls. However, exposure to 18 h of aldosterone increased the amount of whole cell IKK␤ significantly (p Ͻ 0.02).
NF-B Is Activated by Aldosterone-IKK␤ is a serine-threonine kinase that is integral to the activation of NF-B (27)(28)(29). To determine whether aldosterone has a direct effect on the transcription of NF-B-sensitive genes in this cell line, we transiently transfected CCD cells with an NF-B-sensitive luciferase reporter gene. As noted in Fig. 6, cells exposed to 18 h of aldosterone or TNF␣ exhibited a significant increase in lu-  1. The yeast two-hybrid indicates an interaction between IKK␤ and the carboxyl terminus of ␤-ENaC. Carboxyl termini of mouse ␣-, ␤-, and ␥-ENaC were subcloned into the DNA binding domain "bait" vector. Individual constructs were co-transformed into competent yeast with full-length murine IKK␤ subcloned into the DNA activation domain "prey" vector. Top, growth characteristics on medium-selective media (ϪHis/ϪLeu/ϪTrp). Colonies co-transformed with ␤-ENaC exhibited more robust growth than those co-transformed with ␥-ENaC. ␣-ENaC co-transformation did not yield colonies. Bottom, the results of the ␤-galactosidase/X-gal filter assay. Of note, the assay for the IKK␤/ ␤-ENaC co-transformation was positive after 2 h. This became markedly positive at 4 h as shown in the image of the filter above. There was only a weakly positive blue color noted in the ␥-ENaC co-transformants as seen above. This did not change appreciably after 8 h (not shown).
ciferase activity as compared with transfected controls. There was no statistical difference between the aldosterone response when compared with the response of TNF␣. DISCUSSION Our present studies show that there is a physiologically meaningful interaction between the carboxyl terminus of ␤-ENaC and IKK␤ kinase. The true two-hybrid demonstrated a robust interaction between ␤-ENaC and IKK␤. This interaction was confirmed using immunoprecipitation with specific antibodies to ENaC subunits. The immunoprecipitation results confirmed that the interaction is exclusive to ␤-ENaC. The physiological relevance of this interaction was demonstrated by augmentation of ENaC activity as measured by amiloride-sensitive current when IKK␤ and ␣␤␥-ENaC are co-expressed in the Xenopus oocyte system. Kinase-dead IKK␤ had the opposite effect with a decrease in amiloride-sensitive current. The change in ENaC activity appears to be because of an increase in surface expression, as opposed to changes in open probability, as measured by the chemiluminescence assay in oocytes. The effects of IKK␤ are specific to ENaC because co-expression of IKK␤ or IKK␤ (K44A) and ROMK did not affect whole cell K ϩ currents. It appears that the kinase activity of IKK␤ is required FIG. 3. Xenopus oocyte expression studies. ␣␤␥-ENaC cRNAs were microinjected into all oocytes. Oocytes in the experimental group were injected with 5 ng of cRNA of IKK␤. Whole cell currents were measured 24 -46 h after cRNA injections. All currents shown were normalized to control. A shows a 26% increase in amiloride-sensitive current (p ϭ 0.0007) noted in the ␣␤␥-ENaC/IKK␤-co-expressing oocytes (n ϭ 42 oocytes, n ϭ 3 separate groups of oocytes) as compared with ␣␤␥-ENaC-injected controls (n ϭ 31, n ϭ 4) as measured by a two-electrode voltage clamp at a holding potential of Ϫ100 mV. B shows a 50% decrease in amiloride-sensitive current (p Ͻ 0.001) in the ␣␤␥-ENaC/IKK␤(K44A)-co-expressing oocytes (n ϭ 47, n ϭ 3) as compared with ␣␤␥-ENaC-expressing controls (n ϭ 49, n ϭ 3). C and D show oocyte controls for ROMK expression with and without IKK␤ or IKK␤ (K44A) co-expression, respectively. In either case, co-expression does not alter barium-sensitive potassium currents (p ϭ not significant for both).

FIG. 4. Surface expression of ENaC is altered with co-expression of IKK␤.
Surface expression is measured using FLAG-tagged ␤-ENaC subunits and wild-type ␣and ␤-ENaC as described under "Experimental Procedures." The arbitrary luminometric units are normalized to ␣␤ FLAG ␥-ENaC controls. There is a 30% increase (p ϭ 0.019) in oocytes that co-expressed ␣␤ FLAG ␥-ENaC and IKK␤ (n ϭ 5) as compared with ␣␤ FLAG ␥-ENaC controls (n ϭ 4). This parallels the change in amiloride-sensitive current noted with co-expression of ␣␤␥-ENaC and IKK␤. These data imply that the increase in current is secondary to an increase in the number of channels expressed at the surface, rather than an increase in ENaC open probability. Of note, non-epitopetagged ␣␤␥-ENaC controls did not exhibit significant autonomous chemiluminescence. for its effects on ENaC. Kinase-dead IKK␤ not only blocked the stimulation by IKK␤ but also markedly reduced ENaC activity. The dramatic decrease in ENaC activity when co-expressed with the kinase-dead mutant implies that constitutive activation of IKK␤, or a similar kinase, is necessary for a significant proportion of basal ENaC surface expression and that this interaction occurs in oocytes.
The present study demonstrates that activation of the NF-B system through IKK␤ may augment both NF-B-mediated transcription and ENaC activity. Although a direct interaction between ENaC and IKK␤ has never been proposed, the groundwork establishing a link between ENaC activity and inflammation/apoptosis has been established. Fukuda et al. (30) conducted whole animal studies which established that alveolar fluid clearance (AFC) increased in rats administered TNF␣, a potent agonist of NF-B transcription. Amiloride was shown to block basal AFC and also blocked TNF␣-induced up-regulation of AFC, implying that the TNF␣-mediated increase in fluid clearance was because of up-regulation of ENaC. In separate experiments, A549 cells, an immortal cell line that possesses characteristics of type II alveolar cells, were exposed to TNF␣ and were noted to have an 85% increase in amiloride-sensitive current as measured by whole cell patch clamping. TNF␣ has also been shown to increase AFC during acute bacterial pneumonia (31) and intestinal reperfusion (32). Acute TNF␣ stimulation also induces sodium retention in diabetic rats (33). This sodium retention is blocked by amiloride, indicating that this sodium retention is mediated by ENaC. These results confirm the relationship of TNF␣ and ENaC activation and imply a connection between the activation of ENaC and stimulation of NF-B.
A growing number of studies have examined the relationship between the NF-B system and activation of ENaC in pulmonary epithelial cells. NF-B can be activated by numerous stimuli including activation of TNF receptor 1, chemokine receptors, and lymphotoxin-␤ receptors, lipopolysaccharide activation of Toll-like receptors, and activation of interleukin receptors. NF-B can also be activated by changes in oxygen tension, cell injury, and radiation exposure (27)(28)(29). Initial interest in this interaction came from the observation that the activation of sodium transport is integral to alveolar fluid clearance at birth (34). Rat fetal distal lung epithelial cells exposed to a shift from low P O2 to high P O2 exhibit a parallel increase in both amiloride-sensitive current and NF-B activity as measured by an electrophoretic mobility shift assay (35) or luciferase reporter gene activity (36). The NF-B response is bimodal with an initial peak of activity at 15 min and a second peak occurring at 60 h. ENaC activity as measured by amiloride-sensitive current increased between 6 and 24 h. Increases in amiloride-sensitive current and NF-B activity by changes in oxygen tension were blocked by the cell-permeable superoxide scavenger tetramethylpiperidine-N-oxyl (TEMPO), indicating that superoxide activation of the NF-B system was the modality that could account for the increase in ENaC activity. Although amiloride-sensitive current and NF-B activity clearly increased in parallel and blockade of NF-B activation ablated augmentation of amiloride-sensitive current, the mechanism by which these changes occurred in parallel was not elucidated. Increases in ENaC activity by activation of NF-B may also help explain the phenomenon of sodium retention in nephrotic syndrome (37)(38)(39), a state where NF-B also appears to be activated.
Other proteins that have been shown to modulate ENaC activity also have been shown to activate NF-B. Murr1, a chaperone protein that helps direct copper to ceruloplasm and has been shown to decrease NF-B activity (40), also decreases amiloride-sensitive current when co-expressed with ␣␤␥-ENaC and ␦␤␥-ENaC in Xenopus oocytes (41). Murr1 appears to directly interact with the carboxyl termini of ␤-, ␥-, and ␦-ENaC. Additionally, Murr1 has other effects that are not related to sodium channel activity. In concordance with the results of our present study, Murr1 has been shown to down-regulate NF-B signaling, and knockdown of Murr1 has been shown to increase NF-B activity (40). In contrast to Murr1, ERK is a kinase that has been shown to phosphorylate ENaC subunits and downregulate amiloride-sensitive transport. ERK and NF-B have been demonstrated to have a reciprocal role in osteoclasts (42), and a constitutively active mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase (MEK) 3 ERK pathway has been shown to negatively regulate NF-B-driven transcription (43). These two examples support the hypothesis that some activators of ENaC appear to up-regulate NF-Bmediated transcription and that down-regulators of ENaC activity may inhibit the NF-B system.
Steroid activation appears to affect transcription of NF-B (44,45). Glucocorticoids appear to inhibit NF-B transcription by activating an NF-B inhibitory protein ␣ (IB␣). The antagonism appears to be reciprocal with NF-B activation repressing glucocorticoid transactivation (46,47). Nevertheless, glucocorticoids do not appear to repress NF-B activation in all renal cell epithelial lines (48,49). The response of NF-B to mineralocorticoid stimulation is less well described. In vivo studies in rats have shown that salt loading and the infusion of aldosterone increase NF-B activation, a phenomenon that is thought to worsen cardiac fibrosis (50 -52). Activation of NF-B has also been shown to repress the transcriptional activity of the mineralocorticoid receptor (MR) (53).
It should be noted that other studies have shown that TNF␣ has a minimal effect on Na ϩ transport (54) or have shown that long term administration of TNF␣ down-regulates ENaC expression and reduces amiloride-sensitive current (55). This apparent disparity could be caused by differences in cell lines used or experimental methodology. However, these results could also be explained by the length of exposure to TNF␣. With chronic up-regulation of NF-B transcription, a negative feedback loop could predominate that decreases ENaC activity through inhibition of the mineralocorticoid receptor or through activation of as yet unidentified inhibitory pathways.
In our present study, we have demonstrated that IKK␤ appears to interact with the carboxyl terminus of ␤-ENaC and increases ENaC activity. The interactions between IKK␤ and ENaC and between aldosterone and NF-B are surprising. Whether activation of ENaC is the result of a phosphorylation event remains a subject for future study. One hypothesis con-FIG. 6. NF-B reporter gene activity with 18 h of exposure to aldosterone or TNF␣. The NF-B-responsive reporter gene was transiently transfected into CCD cells. As noted, cells exposed to 18 h of aldosterone or TNF␣ exhibited a significant increase in luciferase activity as compared with transfected and non-transfected controls (p Ͻ 0.05). There was no statistical difference between the aldosterone response when compared with the response of TNF␣. sistent with these observations is that IKK␤ activation by aldosterone leads to activation of ENaC and also subsequent activation of NF-B by the classic pathway (29). Stimulation of NF-B-mediated transcription could then lead to down-regulation of mineralocorticoid receptor activation, representing a novel negative feedback loop modulating long term ENaC activation by steroid hormones.