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Am J Physiol Renal Physiol 293: F813-F820, 2007. First published July 3, 2007; doi:10.1152/ajprenal.00225.2007
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Discordant effects of corticosteroids and expression of subunits on ENaC activity

Russell F. Husted,1 Kenneth A. Volk,1 Rita D. Sigmund,2 and John B. Stokes1,2

1Department of Internal Medicine, University of Iowa, and 2Veterans Affairs Medical Center, Iowa City, Iowa

Submitted 14 May 2007 ; accepted in final form 28 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In renal distal nephron and airway epithelial cells, adrenocortical steroids increase epithelial Na+ channel (ENaC) activity and also markedly increase the expression of the {alpha}-subunit. The present experiments were designed to reconstitute this steroid effect in ENaC-expressing cells by overexpressing the subunits whose expression is enhanced by corticosteroids. In renal collecting duct monolayers, corticosteroids increased ENaC activity 5- to 8-fold, endogenous {alpha}-ENaC mRNA and protein ~10-fold, and beta-ENaC protein and mRNA 1.2- to 2-fold. {gamma}-ENaC expression was unchanged. To determine whether this increase in expression was sufficient to increase ENaC activity, we used a regulated adenovirus system to increase expression of each subunit alone and in combination. Unexpectedly, increased expression of the {alpha}- and/or beta-subunit had no effect on ENaC activity in collecting duct cells or lung epithelial cells. In contrast, a small increase in {gamma}-ENaC expression increased ENaC activity about threefold. This increase in activity was additive to the effect of steroids. Thus, even though corticosteroids strongly increase {alpha}-ENaC expression and moderately increase beta-ENaC expression, these effects are not, by themselves, sufficient to increase ENaC activity. Knockdown experiments are consistent with the idea that the increased expression of {alpha}-ENaC is necessary for the full steroid effect on ENaC. Increased expression of {gamma}-ENaC and corticosteroid treatment enhances ENaC activity by parallel, noninteracting pathways. These results underscore the importance of other actions of steroid hormones for long-term enhancement of ENaC activity and raise new possibilities for regulation of ENaC activity by {gamma}-ENaC expression.

adenovirus; Na+ transport; kidney; lung; aldosterone

THE LONG-TERM REGULATION of the epithelial Na+ channel (ENaC) activity is of central importance for Na+ homeostasis, blood pressure regulation, lung fluid clearance, and other vital homeostatic processes. ENaC is comprised of three subunits with similar topologies; each is encoded by a separate gene. It is well-recognized that the full constitutive activity of ENaC is dependent on adequate amounts of each of these subunits (6, 26, 35). ENaC activity is highly regulated by a variety of agents and pathways. Among the most potent agents for increasing ENaC activity are corticosteroids. The precise molecular actions underlying the enhanced ENaC activity are not well-understood, but regulation of transcription is required for this steroid effect (15, 40).

In many cell models, the enhancement of ENaC activity by corticosteroids appears to have two phases. The early phase is sometimes evident within an hour while the later phase is usually evident after 4 h (40). One of the key mediators of the early response is serum and glucocorticoid-regulated kinase 1 (SGK1), the activity of which is regulated both by transcription and by phosphorylation (21). The observations that corticosteroids rapidly induce SGK1 mRNA (7, 29) and that its induction is critical for the full effect of corticosteroids on ENaC activity (12, 14) have led to the widely accepted idea that SGK1 plays a critical role in the early increase in ENaC activity following exposure to corticosteroids.

There is a general acceptance of the idea that the late and sustained effect of corticosteroids on ENaC activity is due to enhanced expression of one or more of the individual subunits (1, 32). In the renal distal nephron, corticosteroids generally increase the expression of the {alpha}-ENaC subunit, with smaller or no effects on beta- and {gamma}-ENaC (3, 22, 24, 38, 41). These results have led to the idea that the {alpha}-ENaC subunit is rate-limiting for the assembly of a functional ENaC complex (32). This idea is supported by the fact that expression of the {alpha}-ENaC subunit in oocytes or in lipid bilayers can induce a small Na+ current (6, 17, 25). Further support for this idea comes from the observation that increased expression of SGK1 increases the expression of {alpha}-ENaC (5).

The idea that enhanced expression of the {alpha}-ENaC subunit could explain the majority of the long-term effect of corticosteroids on ENaC function is not completely consistent with the effects on all ENaC-expressing tissues. Although corticosteroids increase {alpha}-ENaC expression in the lung and kidney (38), they do not do so in colon. In colon, corticosteroids increase beta- and {gamma}-ENaC expression without an effect on {alpha}-ENaC expression (10, 20, 23, 38).

The present experiments were designed to test the idea that reproducing the corticosteroid effects on the expression of the individual ENaC subunits would be sufficient to reconstitute the long-term corticosteroid effect on ENaC function. We report that increased expression of the subunits enhanced by corticosteroids is not sufficient to enhance ENaC activity. In contrast, enhanced expression of the {gamma}-ENaC subunit produces increased ENaC activity that appears mechanistically parallel to the effects of corticosteroids.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Fisher rat thyroid (FRT) cells were a gift from Dr. P. Snyder. Rabbit anti-{alpha}-, beta-, and {gamma}-ENaC antibodies were gifts from Drs. M. Knepper and Z. Ergonul and have been fully characterized (11, 22). Mouse monoclonal anti-myc antibody was from Invitrogen; mouse monoclonal anti-HA antibody was from Sigma; mouse monoclonal anti-V5 antibody was from Invitrogen; goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were from Chemicon. Cell culture media were from the University of Iowa Cancer Center. Unless otherwise indicated, all reagents were purchased from Sigma.

Cell culture and electrical measurements. M1 cells, a model of the renal collecting duct (39), were grown as described (42). FRT cells, a generic epithelial cell line, were propagated in plastic flasks and seeded on filters as described (35). H441 cells, a line derived from a human lung tumor, were grown as described (18, 34). In general, cells were seeded at confluent density on Millicell PCF filters for 3 days at which time they were infected with the appropriate virus at 10 multiplicity of infection (MOI). Monolayers were treated with corticosteroids (100 nM dexamethasone and 100 nM aldosterone) for 24 h starting on day 4 after seeding on filters.

Measurements of transepithelial resistance (RT) and short-circuit current (Isc) were made under sterile conditions as described (16) by placing the 12-mm Millicell filters into modified Ussing chambers and voltage-clamped (University of Iowa Bioengineering) to record electrical values in culture medium at 37°C. In these experiments, Isc reflects Na+ transport by ENaC as extensive testing showed that all but ~1 µA was sensitive to amiloride or the analog benzamil.

Adenovirus constructs. The {alpha}-, beta-, and {gamma}-human ENaC sequences used for these experiments have been described (43) and the {gamma}-hENaC adenovirus construct has been described (42). The {alpha}-hENaC and beta-hENaC constructs were cloned into the same Ad5 shuttle vector used to make the {gamma}-hENaC-expressing adenovirus. The COOH-terminal epitope tags were changed to V5 for {alpha}-hENaC, and HA for beta-hENaC. The {gamma}-hENaC COOH-terminal epitope tag was myc. The {alpha}-hENaC construct expressing an HA epitope in the extracellular domain (19) was obtained from Dr. P. Snyder and subcloned into the Ad5 shuttle vector. The mutation AAA594 was made using the QuickChange method (Stratagene) as previously described (43). Mutated constructs were fully sequenced to ensure that there were no errors introduced in the process of mutation. Replication-deficient adenovirus particles were grown in 293 cells in the University of Iowa Vector Core Facility according to published methods (2). The Ad5 virus encoding the transactivator was also obtained at the University of Iowa Vector Core facility.

M1 cells were infected at a MOI of 10 (both the transactivator and the hENaC vectors) based on preliminary experimentation to determine optimal conditions. As reported previously, this MOI produced infection of >90% of M1 cells on filters when applied to confluent monolayers from the apical surface (42). Virus particles were allowed to incubate in the apical solution for 4 h after which the virus-containing medium was removed and new medium was added. Measurements were made 48 h after infection with the adenovirus particles unless otherwise indicated.

Immunoblot analysis. Monolayers on 12-mm filters were washed with ~2 ml of PBS, and three to four filters were cut out and placed in a 1.5-ml Eppendorf tube. For each filter in the tube, 100 µl of Laemmli buffer (2% SDS, 8 mM Tris buffer, 40 mM dithiothreitol, and 6% glycerol) were added. Cells on plastic (35-mm dish) were solubilized in 0.5 ml of Laemmli buffer. All samples were incubated for 15 min at 60°C and stored at 4°C until used. Protein analysis was conducted by fluorescence assay (4) with bovine albumin as the standard. The indicated amount of total protein was separated by SDS-PAGE using 8% acrylamide and transferred to Immobilon-NC (Millipore) using a FB-SDB-2020 semidry blotting unit (Fisher). The analysis of epitope-tagged proteins was carried out at room temperature. The nitrocellulose membrane was blocked in phosphate-buffered saline with 0.05% Tween 20 (PBST) plus 5% milk for 20 min followed by 1 h of a 1:50,000 dilution of the primary antibody. The membrane was washed twice in PBST and incubated for 1 h in a 1:50,000 dilution of the secondary horseradish peroxidase-conjugated anti-mouse antibody. After three PBST washes and two PBS washes, the horseradish peroxidase was detected by exposing the membrane to SuperSignal femto chemiluminescent substrate (Pierce).

Analysis of native mENaC protein was conducted using the antisera and protocols similar to those reported by Masilamani et al. (22). Because we were unable to detect {gamma}-mENaC in M1 cell lysate, we prepared a microsomal fraction from M1 cells grown on filters. Cells grown on 10- to 30-mm Millicell PCF were placed in a test tube with 3 ml of an isolation solution containing triethanolamine hydrochloride (1.86 g/l), sucrose (86 g/l), and protease inhibitor cocktail (Roche Complete Mini, EDTA-free; 1 tablet/10 ml) at pH 7.6. The solution and filters were frozen at –80°C and then thawed and sonicated on ice for 2 min at 80% amplitude and 10-s pulser with a Cole Parmer Ultrasonic Processor. The solution was then transferred to Eppendorf tubes and centrifuged at 4,000 g for 20 min at 4°C in a Hermle Z233MK centrifuge to remove unbroken cells and nuclei. The supernatant was transferred to 34-mm tubes and centrifuged at 200,000 g for 1 h at 4°C in a Beckman Optima TLX centrifuge. The pellet (membrane preparation) was resuspended in Laemmli buffer.

Immunoblot results were acquired digitally and quantitated using the Optichemi Bioimaging system (UVP).

Surface biotinylation of apical membrane proteins. Cells grown on filters were rinsed three times with PBS containing Ca2+ and Mg2+. EZ-Link Sulfo-NHS-LC-Biotin (Pierce) was added to the apical compartment (to 0.5 mg/ml) and incubated for 30 min at 4°C. Both the apical and basolateral solutions were rinsed three times and incubated for 30 min at 4°C with PBS containing 10 mM glycine as a quenching solution. After three cold rinses with PBS, cells from 12- to 30-mm filters were scraped into one microfuge tube and spun for 2 min at top speed. The resulting pellet was homogenized and solubilized by drawing through a 20-gauge needle 20 times in 1.5 ml of PBS containing 2% SDS and supplemented with the recommended amount of the Complete protease inhibitor cocktail (Roche). The resulting mixture was diluted with 13.5 ml of PBS containing 1% Triton X-100 and Complete and spun at 18,000 g to remove the insoluble material. One hundred fifty microliters of streptavidin (Pierce) beads were added and the mixture was rotated overnight at 4°C. The streptavidin beads were harvested by spinning at 2,500 g for 2 min and then washed five times with cold PBS supplemented with Complete. Two hundred microliters of Laemmli sample buffer were added and boiled for 5 min before loading on an 8% SDS-PAGE gel for subsequent Western blot analysis.

Quantitative assessment of mRNA. Total RNA was prepared from M1 cells grown on filters following the method of Chomczynski and Sacchi (8). Purified RNA was treated with DNase-I and further processed with the RNeasy kit (Ambion). Reverse transcription was performed using standard protocols. In brief, an aliquot of purified DNA-free RNA was combined with M-MLV reverse transcriptase, random hexamers, dNTPs, MgCl2, and RNasin in an appropriate buffer system. The mixture was incubated for 10 min at 20°C followed by 1 h at 42°C and the reaction was terminated by incubating for 5 min at 99°C.

For real-time PCR, {alpha}-mENaC mRNA was quantitated using primers and a probe purchased from Applied Biosystems (catalog no. Mm00803386_m1). We cloned and sequenced the amplicon and confirmed that it was a fragment of {alpha}-mENaC that spanned an intron. The GAPDH primers and probe were also purchased from Applied Biosystems. The fluorescent probes for beta- and {gamma}-mENaC were designed using the Primer Express software (Applied Biosystems) and also spanned an intron. The primers and probes were manufactured by IDT (Coralville, IA) and had the following sequences: beta-mENaC forward primer -GGGCATGACAGAGGAGACACTT, reverse primer -CGATGTCCAGGATCAACTTGAG, and probe -CTTCTGCCAACCCTGGGACTGAATTTG; {gamma}-mENaC forward primer -GGATTTCCCCGCTGTCACTA, reverse primer -GTCTAGAACATCTTTGACCCCATACA, and probe -TGCAATATCAACCCCTACAAGTACAGTGCTGTG. The TaqMan-based quantitative PCR was run on a PRISM 7700 Sequence Detection System (Applied Biosystems).

RNase protection assay was performed and analyzed by scanning densitometry as described (28).

siRNA directed against {alpha}-mENaC. The HPLC-purified {alpha}-mENaC siRNA construct was purchased from Ambion (siRNA ID no. 151572). M1 cells (106 cells) were suspended in 500 µl cytomix solution (in mM: 120 KCl, 0.15 CaCl2, 10 K2HPO4, 10 KH2PO4, 25 HEPES, 2 EGTA, 5 MgCl2, 2 Na-ATP, and 5 glutathione) and then mixed with 7 µg siRNA (scrambled or {alpha}-mENaC specific). The cells were electroporated with Gene Pulser II (Bio-Rad, Hercules, CA) at 400 V and 500 µF, seeded on a 12-mm filter, and grown for 4 days in standard M1 media containing 100 nM dexamethasone. Electrical measurements were made in sterile Ussing chambers after which total RNA was isolated as described above. Only those experiments where monolayers electroporated with control and siRNA developed an average RT of >50 {Omega}·cm2 were included in this analysis; ~50% of the experiments failed to grow to confluency.

Statistics. Values are reported as means ± SE. Unless otherwise indicated, statistical analysis was by ANOVA with subsequent paired or unpaired analysis with corrections for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of corticosteroids on ENaC subunit expression in M1 cells. Exposure to corticosteroids (100 nM dexamethasone and 100 nM aldosterone) for 24 h produced a 10-fold increase in {alpha}-mENaC mRNA and about a 2-fold increase in beta-mENaC mRNA. There was no significant effect on {gamma}-mENaC mRNA (Fig. 1). The effects of corticosteroids on ENaC protein paralleled these effects (Fig. 2); {alpha}-mENaC protein increased about ninefold and beta-mENaC protein increased only slightly. With the antibody we had available, we were not able to detect {gamma}-mENaC from whole cell lysates. However, we were able to detect {gamma}-mENaC protein in a microsomal fraction. Steroids had no effect on {gamma}-mENaC as assessed in this fashion. These results demonstrate that the major effect of steroids on mENaC expression in M1 cells is to increase the abundance of {alpha}-mENaC. The response is similar to that reported in kidney collecting ducts (9, 22, 27, 38, 41).


Figure 1
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  		Fig. 1. Effect of corticosteroids (Ster) on mRNA abundance of {alpha}-, beta-, and {gamma}-ENaC in M1 cells. All measurements were made using RNase protection assay after 24-h exposure to corticosteroids (or vehicle) in confluent monolayers grown on filters. Values referenced to control [no steroids (NS)] and reported as means ± SE. Note log scale; n = 5 experiments. **P < 0.01; *P < 0.05 compared with NS values.

 

Figure 2
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  		Fig. 2. Effect of 24-h corticosteroids (Ster) on the protein abundance of {alpha}-, beta-, and {gamma}-mENaC in M1 cells. Examples of an individual experiment on right; graphs represent quantitation of 3–5 experiments. Cells were grown on filters and treated with steroids for 24 h. Protein was separated using PAGE and transferred to a nitrocellulose membrane for immunoblotting. Determination of {alpha}- and beta-mENaC protein was made using whole cell lysate. The {gamma}-mENaC subunit was not detectable using this preparation, so we used microsomal preparation as described in METHODS. Protein loading was 100 µg/lane for {alpha}-mENaC, 425 µg/lane for beta-mENaC, and 275 µg/lane for {gamma}-mENaC. *P < 0.01, compared with NS.

 
Expression of {alpha}-, beta-, and {gamma}-hENaC in M1 cells by adenovirus. Figure 3 shows the expression of each of the hENaC subunits in M1 monolayers produced by the respective Ad5 virus. Increasing concentrations of doxycycline added at the time of infection caused a reduction in protein expression for each construct, as expected. The range of protein expression quantitated by densitometry was >1,000. We also conducted immunocytochemistry for each of the three subunits in monolayers infected with all three of the ENaC subunits and found that >90% of the cells expressed some protein of a single subunit and that most cells demonstrated expression of multiple subunits (data not shown). These results are similar to those we previously reported for {gamma}-hENaC expression in M1 cells (42) and confirm the regulated expression with doxycycline.


Figure 3
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  		Fig. 3. Immunoblot demonstrating expression of {alpha}-, beta-, and {gamma}-hENaC subunits via a doxycycline (DOX)-sensitive adenovirus system. Each monolayer of M1 cells was grown on filters and exposed to 10 MOI of each viral construct for 48 h as detailed in METHODS. Immunoblot analysis was conducted on 100 µg protein in each lane. Densitometry of the major bands for each construct showed expression ranging more than 1,000-fold as a function of the DOX concentration. Arrows indicate full-length protein.

 
Effect of expressing {alpha}-hENaC in M1 monolayers. We first asked whether increasing expression of {alpha}-hENaC in M1 cells would increase ENaC-mediated Na+ transport. As shown in Fig. 4A, we were unable to detect an increase in Na+ transport above baseline with any level of {alpha}-hENaC expression. Because this result was unexpected, we asked whether the M1 cells could properly produce {alpha}-hENaC and process it to the apical membrane. As shown in Fig. 4B, biotinylation of proteins on the apical membrane clearly demonstrated the expressed {alpha}-hENaC on the surface. Thus the lack of a functional effect of overexpressing {alpha}-hENaC is not because the expressed protein cannot reach the apical membrane.


Figure 4
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  		Fig. 4. Increased expression of {alpha}-hENaC in M1 cells does not increase Na+ transport. A: effect of varying expression of {alpha}-hENaC on Na+ transport by M1 cells. Cells were infected with the Ad5 construct expressing {alpha}-hENaC and incubated for 48 h with the indicated concentrations of DOX. Na+ transport values (bullet), expressed as short-circuit current (Isc), from control (bottom solid lines showing means ± SE) and steroid-treated monolayers (top lines, means ± SE) conducted in parallel are shown as means ± SE; n = 13–34 monolayers from 5–10 experiments in each group. Values of SE are generally smaller than the size of bullet. B: {alpha}-hENaC delivered by the adenovirus system is expressed on the apical membrane. Surface expression was detected by biotinylation, precipitation with streptavidin beads, and immunoblotted as in Fig 3. Lanes showing "cell lysate" represent all soluble proteins separated and immunoblotted at 30 (1x) and 3 (0.1x) µg total protein per lane. C: Ad5 hENaC constructs are functional in Fisher rat thyroid (FRT) cells. Infecting FRT cells with the virus expressing beta- and {gamma}-hENaC for 24 h does not induce Na+ transport, but expression of all three subunits does produce amiloride-sensitive current (representative experiment, n = 3 monolayers in each group). FRT cells were incubated with 10 MOI of each subunit (plus the AdtTA) in the apical solution for 24 h. AdtTA, adenovirus expressing the transactivator protein.

 
To test whether the Ad5 construct we used to express this {alpha}-hENaC was functional, we compared the effect of expressing beta- and {gamma}-hENaC or {alpha}-, beta-, and {gamma}-hENaC subunits in FRT cells. Because FRT cells do not have endogenous ENaC (35), we expected that expression of all three subunits would be required to produce ENaC activity. As shown in Fig. 4C, addition of the {alpha}-hENaC subunit to monolayers expressing beta- and {gamma}-hENaC substantially increased the amiloride-sensitive current. These results demonstrate that the Ad5 hENaC constructs can function when expressed together and thus the Ad5 {alpha}-hENaC construct as well as the beta- and {gamma}-ENaC constructs are functional.

Even though the Ad5 {alpha}-hENaC was functional in FRT cells, we considered the possibility that its expression in M1 cells might somehow be impaired. We therefore made two other Ad5 {alpha}-hENaC constructs, each with an HA epitope tag in the extracellular domain (19). One construct contained only the HA epitope in the extracellular domain, and the second contained, in addition, mutations in the COOH terminus where residues 594–596 were changed to alanines (AAA594). This mutation reduces ENaC activity in oocytes when it is expressed with wild-type beta- and {gamma}-hENaC (43). As shown in Fig. 5A, both of these constructs expressed protein in M1 cells. M1 cells infected with Ad5 {alpha}-hENaC-HA showed the same ENaC current as uninfected M1 cells (Fig. 5B). These results are the same as those obtained using the Ad5 {alpha}-hENaC-V5 construct (Fig. 4A). Figure 5B also shows that expressing the {alpha}-hENaC-HA construct did not alter the corticosteroid-induced enhancement of Na+ transport.


Figure 5
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  		Fig. 5. Expression of HA epitope-tagged {alpha}-hENaC subunits in M1 cells. The HA epitope was inserted in the extracellular domain and constructs expressed in M1 cells using the Ad5 viral system identical to that used for {alpha}-, beta-, and {gamma}-hENaC. AAA594 contained mutations in positions 594–596 to alanine residues that reduce ENaC activity when expressed in oocytes. A: protein expression as detected by immunoblot using an anti-HA antibody. B: effect of expressing the wild-type {alpha}-hENaC construct on Na+ transport (Isc). Expression of this construct (hatched) had no effect on basal or steroid enhanced ENaC currents. C: expression of a construct with an AAA594 mutation (hatched) decreased ENaC function. The reduction of steroid-stimulated ENaC function is consistent with the idea that the {alpha}-ENaC protein expressed by adenovirus can be processed and incorporated into a functioning ENaC complex in M1 cells. *P < 0.01, n = 8 monolayers in each group.

 
We tested the effect of expressing the {alpha}-hENaC-HA construct containing the AAA594 mutation. The effect of this construct in monolayers not exposed to steroids was modest; however, in monolayers exposed to corticosteroids the inhibitory effect of the mutation was clear (Fig. 5C). Thus this construct can integrate into the ENaC complex and reduce its function.

Expressing combinations of hENaC subunits on Na+ transport. Since corticosteroids increased beta-mENaC expression to a modest degree (Figs. 1 and 2), we tested the idea that increased expression of the beta-ENaC subunit, either alone or in combination with {alpha}-hENaC, would enhance Na+ transport. However, expression of beta-hENaC over the range possible with the Ad5 system did not increase Na+ transport (Fig. 6A). We found the same results when we coexpressed the combination of {alpha}- and beta-hENaC in M1 cells over a wide range (Fig. 6B). These results suggest that the increased expression of {alpha}- and beta-ENaC produced by corticosteroids in M1 cells is not sufficient to enhance Na+ transport. In contrast to the lack of effect of {alpha}- and beta-hENaC, expression of {gamma}-hENaC increased Na+ at all levels of expression (Fig. 6C), a result similar to what we previously reported (42).


Figure 6
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  		Fig. 6. Expression of beta- and {gamma}-hENaC on Na+ transport (Isc) by M1 cells. A: effect of expressing varying amounts of beta-hENaC. B: effect of expressing varying amounts of {alpha}- plus beta-hENaC. C: effect of overexpressing varying amounts of {gamma}-hENaC. Bottom lines are means ± SE of control monolayers; top (dashed) lines are means ± SE of monolayers treated with steroids from the same experiments; n = 5–7 experiments with 3 monolayers in each experiment at each [doxycycline]. There was no effect of overexpressing beta-hENaC alone or {alpha}- plus beta-hENaC on Na transport (ANOVA). In contrast, expression of {gamma}-hENaC increased Na+ transport at any [doxycycline]. There was no effect of [doxycycline] on the magnitude of Na+ transport (ANOVA).

 
Because increased expression of {gamma}-hENaC increased Na+ transport, we investigated the effect of combining its expression with other subunits. As shown in Fig. 7, coexpression of {alpha}- and {gamma}-hENaC produced no greater Na+ transport than {gamma}-hENaC alone. However, increased expression of all three subunits produced more Na+ transport than increased {gamma}-hENaC alone while expression of beta- and {gamma}-hENaC produced a marginal increase in Na+ transport.


Figure 7
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  		Fig. 7. Effect of overexpressing {gamma}-hENaC in combination with other subunits on Na+ transport by M1 cells. Isc measured 48 h after infection, no steroid exposure, and [doxycycline] was 1 ng/ml in all monolayers; n = 18–37 monolayers in each group from 6 experiments. *P < 0.01 compared with {gamma}-hENaC expressed alone (hatched bar) by ANOVA.

 
We considered the possibility that the pattern of responsiveness to the hENaC subunits in M1 cells might be owing to species differences between mouse and human ENaC. We tested this idea in H441 cells, a line from human lung adenocarcinoma that expresses functional ENaC complexes and responds to corticosteroids by increasing the abundance of {alpha}-ENaC as well as beta- and {gamma}-ENaC (18). As shown in Fig. 8, increased expression of {gamma}-hENaC, but not {alpha}- and/or beta-hENaC, increased Na+ transport in these monolayers. Interestingly, the increase in Na+ transport produced by {gamma}-hENaC overexpression was about the same as that produced by corticosteroid treatment, and there was no synergistic effect of expressing all three ENaC subunits. The basis for these differences is unknown at present. However, the major point is that only increased expression of {gamma}-hENaC produced an increase in Na+ transport in both M1 cells and in H441 cells.


Figure 8
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  		Fig. 8. Effect of increased expression {alpha}-, beta-, or {gamma}-hENaC alone or in combination on Na+ transport by H441 cells, a line of epithelial cells derived from a human lung tumor. The pattern of their effect on ENaC activity is similar to that of expressing these constructs in M1 cells. Overexpression of {alpha}- or beta-hENaC alone produced no change in Na+ transport. Corticosteroid treatment (hatched) and overexpression of {gamma}-hENaC alone or in combination with other subunits give similar effects; n = 6–12 monolayers, 2–4 experiments each group. Values from monolayers expressing {gamma}-hENaC alone and in combination with other subunits are not different from steroids, but greater than other groups (P < 0.001 by ANOVA).

 
Additive effect of corticosteroids and {gamma}-hENaC in M1 cells. We tested the idea that increased expression of {gamma}-ENaC might contribute to the increased Na+ transport produced by corticosteroids. Our hypothesis was that even though we could not detect an increase in {gamma}-ENaC in response to corticosteroids in M1 cells, there might be a small but undetectable increase in {gamma}-ENaC abundance. Even an undetectable increase might be sufficient to increase Na+ in response to steroids. The results of experiments testing all combinations of hENaC expression with and without corticosteroids are shown in Fig. 9. Expression of hENaC subunits alone and in combination in the absence of corticosteroids produced Na+ transport rates similar to those shown in Figs. 4–7. However, in monolayers treated with steroids, the magnitude of Na+ transport showed only two populations of responses. Monolayers expressing {gamma}-hENaC treated with corticosteroids had consistently higher magnitude of Na+ transport than steroid-treated monolayers not expressing {gamma}-hENaC. Statistical analysis showed independent effects of {gamma}-hENaC and corticosteroids.


Figure 9
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  		Fig. 9. Effect of corticosteroid treatment (hatched bars) on Na+ transport by M1 cells expressing one or more of the hENaC subunits. Expression of {gamma}-hENaC (and not {alpha}- or beta-hENaC) increased Na+ transport in the absence of steroid treatment and produced an additive effect to steroids. Two-way ANOVA revealed independent effects of {gamma}-hENaC and steroids; n = 9 monolayers from 3 experiments in each group. Measurements made 48 h after infection and 24 h after steroid treatment. DOX (1 ng/ml) present in all experiments.

 
Requirement for {alpha}-ENaC in steroid-induced effects on Na+ transport. Having concluded that the increase in {alpha}-ENaC was not sufficient to account for the increase in Na+ transport induced by corticosteroids, we asked whether the increase in expression was necessary for the full corticosteroid effect. We electroporated M1 cells with siRNA directed against {alpha}-mENaC and compared them to monolayers electroporated with a scrambled siRNA. As shown in Fig. 10, there was no difference in RT between these two groups, but siRNA against {alpha}-mENaC reduced Na+ transport by 30%. The monolayers so treated showed a 70% reduction in {alpha}-mENaC mRNA. These results are consistent with the idea that the increase in {alpha}-mENaC abundance is necessary for the full effect of corticosteroids.


Figure 10
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  		Fig. 10. Effect of siRNA directed against {alpha}-mENaC on Na+ transport (Isc), transepithelial electrical resistance, and {alpha}-mENaC mRNA in M1 cells. siRNA against {alpha}-mENaC (hatched bars) or scrambled siRNA (open bars) were electroporated into M1 cells which were then seeded at confluent density on filters. After 4 days, electrical values were measured followed by real-time RT-PCR measurement of mRNA for {alpha}-mENaC and mGAPDH. All monolayers were treated with steroids. mRNA represents the a-mENaC/GAPDH ratio; n = 9 monolayers from 3 experiments. *P < 0.05, **P < 0.01 by unpaired analysis.

 

   DISCUSSION
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The present results bring us to the somewhat counterintuitive conclusion that the increased expression of {alpha}-ENaC, the subunit most strongly upregulated by steroids in kidney and lung, is not sufficient to increase ENaC activity. In addition, regulation of the {gamma}-ENaC subunit expression, which is not affected by corticosteroids in collecting duct, produces an independent action to regulate ENaC activity.

We provide six lines of evidence that the lack of an effect of increased {alpha}-ENaC expression is not the result of technical inadequacy. 1) The infected monolayers expressed the indicated proteins (Fig. 3) and did so in the vast majority of cells as determined by immunocytochemistry (data not shown and Ref. 42). Furthermore, the abundance of the expressed {alpha}-hENaC (and beta-hENaC) protein could be regulated by doxycycline over a range exceeding 1,000 (Fig. 3) with no effect on function (Figs. 4A, 6, A and B). 2) The {alpha}-hENaC construct was functional in FRT cells when expressed with beta- and {gamma}-hENaC (Fig. 4C). 3) Heterologous expression of {alpha}-hENaC in human lung epithelial cells also had no effect on Na+ transport (Figs. 4A and 8). Thus species incompatibility does not explain the lack of effect of increased {alpha}-ENaC expression. 4) The overexpressed {alpha}-hENaC protein was detectable on the apical membrane of M1 cells (Fig. 4B), suggesting that it could be sorted correctly. 5) Expression of two different Ad5 {alpha}-hENaC constructs produced a similar lack of an effect (Figs. 4A and 5B), despite the fact that an inhibitory mutation of one construct was able to reduce ENaC activity (Fig. 5C). 6) Expression of {alpha}-hENaC with beta-hENaC and {gamma}-hENaC produced higher ENaC activity in M1 cells than {gamma}-hENaC alone (Fig. 7) further documenting the functionality of the {alpha}-hENaC construct. We also provide evidence that increased expression of either {alpha}-hENaC and/or beta-hENaC does not increase ENaC activity in either the collecting duct cells or lung epithelial cells. Thus increased expression of either {alpha}-ENaC or beta-ENaC (or both) does not reconstitute the steroid effect (Figs. 6, A and B).

While increased expression of {alpha}-ENaC may not be sufficient for enhancing ENaC function, its increased expression may be necessary for the full steroid effect. Knockdown of endogenous {alpha}-mENaC with siRNA reduced the ENaC activity (and mRNA) in the presence of steroids without affecting electrical resistance (Fig. 10). However, based on these results, this conclusion carries some caveats. It is well-established that all three subunits are necessary for full ENaC activity (6, 25, 35) and Fig. 4C. In the siRNA experiments, the reduction of the abundance of {alpha}-mENaC mRNA in a population of cells may have lowered its expression to below that needed for functional expression. Thus while these siRNA results are consistent with the idea that full expression of {alpha}-ENaC is necessary for the steroid-induced enhancement of ENaC activity, we cannot exclude heterogeneous effects on a population of cells.

The second unexpected result is that of the three subunits, {gamma}-hENaC alone is sufficient to increase ENaC activity (Figs. 6C, 8, and 9). Consistent with our previously reported results (42), the amount of {gamma}-hENaC protein needed to produce a maximal increase in ENaC activity was below the limits of our ability to detect it by immunoblot. It is clear that even the highest concentration of doxycycline does not fully suppress all protein production by this Ad5 system (42), but the lack of a further enhancement of ENaC function with higher amounts of {gamma}-hENaC protein is also unexpected.

The enhancement of ENaC activity resulting from increased expression of {gamma}-ENaC appears to be separate from the steroid-enhancing effect. This conclusion is supported by the fact that even with high concentrations of steroids, overexpression of a small amount of {gamma}-hENaC produces an additive enhancement of ENaC activity (Fig. 9 and Ref. 42). The mechanism for this effect of overexpressing {gamma}-hENaC is not clear, but our previous analysis suggests that the increased {gamma}-hENaC expression produces a complex with a longer functional half-life than ENaC treated with steroids but no additional {gamma}-ENaC (42).

Increased expression of all three ENaC subunits produces greater ENaC activity than increased expression of {gamma}-hENaC alone in the absence of steroid treatment (Fig. 9). Thus increased expression of the three subunits produces synergy. However, this synergistic effect disappears in the presence of corticosteroids (Fig. 9). These results further support the conclusion that an increase in the expression of {gamma}-hENaC and treatment with corticosteroids act by parallel and noninteracting mechanisms to enhance ENaC activity.

The parallel effects of increased {gamma}-ENaC expression and steroid-enhanced ENaC raise the possibility that regulation of ENaC might occur by agents that alter the expression of {gamma}-ENaC primarily. Examples of agents that downregulate {gamma}-ENaC include activation of protein kinase C (37) and Li+ exposure (31). Agents that can upregulate {gamma}-ENaC expression include chronic administration of vasopressin (30, 33, 36) and activation of pPAR-{gamma} (13). These results, taken together, suggest that regulation of ENaC activity can be achieved by altering expression of {gamma}-ENaC (with or without altered expression of beta-ENaC). Such regulatory pathways may be parallel to and independent of steroid regulation of ENaC activity.

The failure of increased expression of the {alpha}-ENaC (with or without the beta-ENaC) subunit to enhance ENaC activity implies that the mechanisms whereby steroids enhance ENaC activity are far more complex than "simply" overexpressing the channel subunits. It seems likely that steroids produce a variety of effects that ultimately increase ENaC activity. Such effects may include enhanced expression of Na/K pump subunits, metabolic proteins, trafficking proteins, kinases and phosphatases to regulate these pathways, pathways to alter posttranscriptional ENaC processing, and alteration of transcription factors that regulate expression of other gene products.


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 ABSTRACT
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This work was supported in part by The O'Brien Kidney Research Center at the University of Iowa (DK-52617) and by a grant from the Department of Veterans Affairs. The University of Iowa Center for Gene Therapy (supported by DK-54759) provided assistance with vector production. University of Iowa Cancer Center and the University of Iowa DNA core facility also provided services.


   ACKNOWLEDGMENTS
 
We appreciate the gift of FRT cells from Dr. P. Snyder.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. B. Stokes, E300 GH, Dept. of Internal Medicine, 200 Hawkins Drive, Univ. of Iowa, Iowa City, IA 52246 (e-mail: john-stokes{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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