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• W2147513612 abstract "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. 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) 1The abbreviations used are: ENaC, epithelial sodium channel; ERK, extracellular signal-regulated kinase; CCD cells, mpkCCDc14 cells; FBS, fetal bovine serum; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; TNF, tumor necrosis factor; ROMK, renal outer medullary potassium channel; BSA, bovine serum albumin; AFC, alveolar fluid clearance; MBS, modified Barth's saline. is located in the apical membrane in a variety of Na+-transporting epithelia including the cortical collecting duct of the kidney, the distal colon, the ducts of secretory glands, and the lung (1Duc C. Farman N. Canessa C.M. Bonvalet J.P. Rossier B.C. J. Cell Biol. 1994; 127: 1907-1921Crossref PubMed Scopus (360) Google Scholar, 2Renard S. Voilley N. Bassilana F. Lazdunski M. Barbry P. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 299-307Crossref PubMed Scopus (224) Google Scholar). ENaC mediates Na+ 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 (3Hansson J.H. Nelson-Williams C. Suzuki H. Schild L. Shimkets R. Lu Y. Canessa C. Iwasaki T. Rossier B. Lifton R.P. Nat. Genet. 1995; 11: 76-82Crossref PubMed Scopus (724) Google Scholar, 4Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (399) Google Scholar) and salt wasting seen in variants of pseudohypoaldosteronism (5Rotin D. Curr. Opin. Nephrol. Hypertens. 2000; 9: 529-534Crossref PubMed Scopus (46) Google Scholar, 6Arai K. Zachman K. Shibasaki T. Chrousos G.P. J. Clin. Endocrinol. Metab. 1999; 84: 2434-2437PubMed Google Scholar). 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, α, β, and γ, which share a 30% sequence homology (7Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar). When expressed alone in Xenopus oocytes, these subunits are capable of generating a Na+ 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-μm range, and slow gating kinetics (8Canessa C.M. Horisberger J.D. Schild L. Rossier B.C. Kidney Int. 1995; 48: 950-955Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 9Fyfe G.K. Canessa C.M. J. Gen. Physiol. 1998; 112: 423-432Crossref PubMed Scopus (95) Google Scholar, 10Palmer L.G. Frindt G. Fed. Proc. 1986; 45: 2708-2712PubMed Google Scholar). 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 (11Gormley K. Dong Y. Sagnella G.A. Biochem. J. 2003; 371: 1-14Crossref PubMed Scopus (66) Google Scholar, 12Snyder P.M. Endocr. Rev. 2002; 23: 258-275Crossref PubMed Scopus (189) Google Scholar). ENaC activity has also been shown to be regulated by channel-activating proteases (CAPs) (13Vuagniaux G. Vallet V. Jaeger N.F. Hummler E. Rossier B.C. J. Gen. Physiol. 2002; 120: 191-201Crossref PubMed Scopus (199) Google Scholar) and furin (14Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279: 18111-18114Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) 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 (15Shi H. Asher C. Chigaev A. Yung Y. Reuveny E. Seger R. Garty H. J. Biol. Chem. 2002; 277: 13539-13547Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Using fractionated cytosol extracted from rat distal colon to phosphorylate glutathione S-transferase (GST) fusion proteins constructed with the carboxyl terminus of γ-ENaC, Shi et al. (15Shi H. Asher C. Chigaev A. Yung Y. Reuveny E. Seger R. Garty H. J. Biol. Chem. 2002; 277: 13539-13547Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 16Shi H. Asher C. Yung Y. Kligman L. Reuveny E. Seger R. Garty H. Eur. J. Biochem. 2002; 269: 4551-4558Crossref PubMed Scopus (40) Google Scholar) 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 β-ENaC. We now report that one of these proteins, IKKβ (the β-subunit of the kinase that cleaves the inhibitor of nuclear factor κB), significantly interacts with and augments ENaC activity. Cell Lines—mpkCCDc14 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. (17Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. Rafestin-Oblin M.E. Rossier B. Vandewalle A. J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar). Cells were grown on semipermeable supports (6-well, 0.4-μm 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 μg/ml insulin, 20 mmd-glucose, 2% v/v fetal bovine serum (FBS), and 20 mm HEPES, pH 7.4) at 37 °C in 5% CO2, 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—β- and γ-ENaC antibodies were generated and affinity-purified as described previously (18Rokaw M.D. Wang J.M. Edinger R.S. Weisz O.A. Hui D. Middleton P. Shlyonsky V. Berdiev B.K. Ismailov I. Eaton D.C. Benos D.J. Johnson J.P. J. Biol. Chem. 1998; 273: 28746-28751Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Antibodies to IKKβ 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 10-6m aldosterone for 3 or 18 h at 37 °C. Yeast Two-hybrid—Using the BD Matchmaker Gal4 Two-hybrid System 3 (Clontech, BD Biosciences), carboxyl termini of α-, β-, and γ-murine ENaC (mENaC) were subcloned into the DNA binding domain vector pGBKT7, and full-length IKKβ kinase was subcloned into the DNA activation domain vector pGADT7 (19Clontech Matchmaker Gal4 Two-hybrid System 3 & Libraries User Manual. Clontech, Palo Alto, CA1999Google Scholar). Competent AH109 yeast cells were made using the Frozen-EZ Yeast Transformation II kit (Zymo Research, Orange, CA). Yeast were transformed with vectors according to the manufacturer's instructions and grown on solid medium with appropriate dropout powder (Clontech) and BD Bacto agar (Difco). Yeast were grown in a room air incubator at 30 °C. Primers for carboxyl termini of murine ENaC were constructed as follows: α-ENaC, 5′-AAGAATTCCACAGGTTCCGGAGCCGG and 3′-TCGGATCCTTAGAGTGCCATGGCCGGAGC; β-ENaC, 5′-AAGAATTCAAAGGCCTGCGCAGGAGG and 3′-TTGGATCCTTAGATGGCCTCCACCTCACT; and γ-ENaC, 5′-AAGAATTCCGCCGCCAGTGGCAGAAA and 3′-TTGGATCCTTAGAACTCATTGGTCAACTG. Yeast Two-hybrid β-Galactosidase/X-Gal Assay—A standard filter lift assay was performed as outlined in the BD Matchmaker Gal-4 Two-hybrid System 3 manual (20Clontech Yeast Protocols Handbook. Clontech, Palo Alto, CA2001Google Scholar). Yeast were permeabilized in liquid nitrogen, and β-galactosidase activity was assayed in a Z buffer (Na2HPO4·7H2O (16.1 g/liter), NaH2PO4·H2O (5.5 g/liter), KCl (0.75 g/liter), MgSO4·7H2O (0.246 g/liter), pH 7)/β-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-κB Luciferase Activity Assay—The NF-κB-responsive gene (pNF-κB-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 μg of pNF-κB-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 (10-6m) or TNFα (120 ng/ml) for 18 h. NF-κB activity was quantified by measuring luciferase activity using standard methods (Promega, Madison, WI) and a Turner TD 20/20 illuminometer 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 (21Lebowitz J. An B. Edinger R.S. Zeidel M.L. Johnson J.P. Am. J. Physiol. Renal Physiol. 2003; 285: F524-F531Crossref PubMed Scopus (10) Google Scholar). 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 calcium-free saline solution. Murine ENaC cRNAs (1-3 ng/subunit in 50 nl of H2O) were microinjected into all oocytes. Oocytes in the experimental group were additionally injected with 5 ng of cRNA of murine IKKβ (IMAGE clone 4482634). As a control, αβγ-ENaC cRNAs were co-injected with 5 ng of kinase-dead human IKKβ(K44A) (22Ten R.M. McKinstry M.J. Trushin S.A. Asin S. Paya C.V. J. Immunol. 1999; 163: 3851-3857PubMed Google Scholar) (a gift from Dr. Carlos Paya) cRNA. All oocytes were incubated at 18 °C in modified Barth's saline (MBS) (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.3 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 15 mm HEPES-NaOH, pH 7.2, supplemented with 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate, and 100 μg/ml gentamycin sulfate). Whole cell currents were measured 24-46 h after cRNA injections. To determine whether the effects of IKKβ on ENaC were not the result of generic effects of IKKβ on transcription, protein trafficking, or cell surface expression, we used ROMK-expressing oocytes (23Bruns J.B. Hu B. Ahn Y.J. Sheng S. Hughey R.P. Kleyman T.R. Am. J. Physiol. Renal Physiol. 2003; 285: F600-F609Crossref PubMed Scopus (22) Google Scholar) with and without IKKβ. Wild-type ROMK cRNA (2 ng) was injected with or without IKKβ cRNA (5 ng). Whole Cell Current Measurements—A two-electrode voltage clamp technique was used as described previously (24Carattino M. Sheng S. Kleyman T.R. J. Biol. Chem. 2004; 279: 4120-4126Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Whole cell inward amiloride-sensitive currents were measured in control oocytes expressing αβγ-ENaC alone or experimental oocytes expressing αβγ-ENaC + IKKβ 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 CaCl2, 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 -140 to +60 mV in 20-mV increments with a duration of 900 ms. Currents were measured at a holding potential of -100 mV 600 ms after initiation of the clamp potential. For ROMK measurements, whole cell potassium currents were measured at -100 mV in the absence or presence of 5 mm BaCl2. Cell Surface ENaC Labeling—The general approach was based on the method of Zerangue et al. (25Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar) as modified by Condliffe et al. (26Condliffe S.B. Carattino M.D. Frizzell R.A. Zhang H. J. Biol. Chem. 2003; 278: 12796-12804Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Control oocytes expressing mouse α-, β-FLAG-, and γ-ENaC subunits and experimental oocytes co-injected with αβFLAGγ-ENaC and IKKβ 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, β-ENaC containing the FLAG epitope (DYDKKKD) at the extracellular loop does not alter INa relative to wild-type ENaC expression as first demonstrated by Firsov et al. (4Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (399) Google Scholar). 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(ab′)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 illuminometer with the signal integrated over a 60-s interval. Yeast Two-hybrid Results—Initial results of the yeast two-hybrid library screen plated on medium-selective media (-His/-Leu/-Trp) using a commercially available mouse kidney library (Clontech, BD Biosciences) against carboxyl termini of ENaC revealed an interaction between the β-ENaC and a number of clones including IKKβ. Full-length IKKβ was subcloned into the pGADT7 vector. We then screened IKKβ against the carboxyl terminus of α-, β-, and γ-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 (-His/-Leu/-Trp), nor did they exhibit spontaneous β-galactosidase activity. As shown in Fig. 1, only co-transformation of IKKβ with the β-carboxyl terminus of ENaC yielded robust growth on medium-selective media (-His/-Leu/-Trp) after 5 days of incubation. α-ENaC exhibited no growth, and γ-ENaC grew only fine colonies on medium-selective media. However, only β-ENaC co-transformants exhibited significant β-galactosidase activity. This β-galactosidase activity was evident in the β-ENaC/IKKβ co-transformants after 2 h but was robust after 4 h as shown in Fig. 1, bottom. γ-ENaC only exhibited minimal β-galactosidase activity after 4 h (Fig. 1, bottom), which was unchanged at 8 h. Only co-transformation of β-ENaC with IKKβ yielded both growth on restrictive media and β-galactosidase activity. Immunoprecipitation with β- and γ-ENaC Antibodies—To determine whether the ENaC-IKKβ 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 (17Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. Rafestin-Oblin M.E. Rossier B. Vandewalle A. J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar) 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 β- and γ-ENaC, we performed Western blots using a commercially available antibody for IKKβ. Fig. 2 shows the result of Western blots with the IKKβ antibody. The first lane of Fig. 2 demonstrates that the IKKβ antibody can detect IKKβ 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-IKKβ after immunoprecipitation of CCD cell lysate with β-ENaC and γ-ENaC antibodies. IKKβ co-immunoprecipitates with β- but not γ-ENaC. The next two lanes of Fig. 2 are controls to demonstrate that the β- and γ-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-IKKβ. Co-expression of IKKβ and αβγ-ENaC in Xenopus Oocytes Significantly Increases Amiloride-sensitive Current—To determine whether the interaction between the carboxyl terminus of β-ENaC and IKKβ 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 αβγ-ENaC with IKKβ cRNA significantly increased amiloride-sensitive current by 26% (n = 42 oocytes, n = 3 separate groups of oocytes, p = 0.0007). To determine whether the kinase activity of IKKβ was required for its effect on ENaC, we examined the effect of a kinase-dead mutant IKKβ (K44A) (22Ten R.M. McKinstry M.J. Trushin S.A. Asin S. Paya C.V. J. Immunol. 1999; 163: 3851-3857PubMed Google Scholar). Fig. 3B shows that co-expression of αβγ-ENaC and kinase-dead IKKβ(K44A) resulted in a 50% decrease in normalized currents (n = 47, n = 3, p < 0.001). We examined the effects of IKKβ and kinase-dead IKKβ on the expression of a distinct ion channel, ROMK (23Bruns J.B. Hu B. Ahn Y.J. Sheng S. Hughey R.P. Kleyman T.R. Am. J. Physiol. Renal Physiol. 2003; 285: F600-F609Crossref PubMed Scopus (22) Google Scholar). Fig. 3, C and D, shows the effect of IKKβ and IKKβ(K44A) co-expression with ROMK. Neither construct appears to have a significant effect on barium-sensitive K+ current, suggesting that IKKβ activation and IKKβ(K44A)-mediated down-regulation of channel activity are ENaC-specific effects. Co-expression of IKKβ 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 IKKβ and αβFLAGγ-ENaC as compared with controls expressing αβFLAGγ-ENaC alone. This 30% increase in surface expression of ENaC parallels the increase in amiloride-sensitive current induced by IKKβ, implying that the mechanism of IKKβ-mediated ENaC up-regulation is secondary to an increase in the number of channels at the surface rather than an increase in open probability. IKKβ Is An Aldosterone-regulated Protein—Given that ENaC is responsive to aldosterone, we sought to determine whether IKKβ is regulated by aldosterone. CCD cell monolayers were exposed to 10-6m 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 IKKβ that can be seen after 18 h of exposure to aldosterone. Western blots were quantified by densitometry (n = 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 IKKβ as compared with controls. However, exposure to 18 h of aldosterone increased the amount of whole cell IKKβ significantly (p < 0.02). NF-κB Is Activated by Aldosterone—IKKβ is a serine-threonine kinase that is integral to the activation of NF-κB (27Senftleben U. Karin M. Crit. Care Med. 2002; 30: S18-S26Crossref Scopus (266) Google Scholar, 28Wallach D. Arumugam T.U. Boldin M.P. Cantarella G. Ganesh K.A. Goltsev Y. Goncharov T.M. Kovalenko A.V. Rajput A. Varfolomeev E.E. Zhang S.Q. Arthritis Res. 2002; 4: S189-S196Crossref PubMed Scopus (38) Google Scholar, 29Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). To determine whether aldosterone has a direct effect on the transcription of NF-κB-sensitive genes in this cell line, we transiently transfected CCD cells with an NF-κB-sensitive luciferase reporter gene. As noted in Fig. 6, cells exposed to 18 h of aldosterone or TNFα exhibited a significant increase in luciferase activity as compared with transfected controls. There was no statistical difference between the aldosterone response when compared with the response of TNFα. Our present studies show that there is a physiologically meaningful interaction between the carboxyl terminus of β-ENaC and IKKβ kinase. The true two-hybrid demonstrated a robust interaction between β-ENaC and IKKβ. This interaction was confirmed using immunoprecipitation with specific antibodies to ENaC subunits. The immunoprecipitation results confirmed that the interaction is exclusive to β-ENaC. The physiological relevance of this interaction was demonstrated by augmentation of ENaC activity as measured by amiloride-sensitive current when IKKβ and αβγ-ENaC are co-expressed in the Xenopus oocyte system. Kinase-dead IKKβ 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 IKKβ are specific to ENaC because co-expression of IKKβ or IKKβ(K44A) and ROMK did not affect whole cell K+ currents. It appears that the kinase activity of IKKβ is required for its effects on ENaC. Kinase-dead IKKβ not only blocked the stimulation by IKKβ 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 IKKβ, 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-κB system through IKKβ may augment both NF-κB-mediated transcription and ENaC activity. Although a direct interaction between ENaC and IKKβ has never been proposed, the groundwork establishing a link between ENaC activity and inflammation/apoptosis has been established. Fukuda et al. (30Fukuda N. Jayr C. Lazrak A. Wang Y. Lucas R. Matalon S. Matthay M.A. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 280: L1258-L1265Crossref PubMed Google Scholar) conducted whole animal studies which established that alveolar fluid clearance (AFC) increased in rats administered TNFα, a potent agonist of NF-κB transcription. Amiloride was shown to block basal AFC and also blocked TNFα-induced up-regulation of AFC, implying that the TNFα-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 TNFα and were noted to have an 85% increase in amiloride-sensitive current as measured by whole cell patch clamping. TNFα has also been shown to increase AFC during acute bacterial pneumonia (31Rezaiguia S. Garat C. Delclaux C. Meignan M. Fleury J. Legrand P. Matthay M.A. Jayr C. J. Clin. Investig. 1997; 99: 325-335Crossref PubMed Scopus (157) Google Scholar) and intestinal reperfusion (32Borjesson A. Norlin A. Wang X. Andersson R. Folkesson H.G. Am. J. Physiol. Lung Cell Mol. Physiol. 2000; 278: L3-L12Crossref PubMed Google Scholar). Acute TNFα stimulation also induces sodium retention in diabetic rats (33DiPetrillo K. Coutermarsh B. Soucy N. Hwa J. Gesek F. Kidney Int. 2004; 65: 1676-1683Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This sodium retention is blocked by amiloride, indicating that this sodium retention is mediated by ENaC. These results confirm the relationship of TNFα and ENaC activation and imply a connection between the activation of ENaC and stimulation of NF-κB. A growing number of studies have examined the relationship between the NF-κB system and activation of" @default.
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