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Dartmouth Medical School, Lebanon, New Hampshire
Submitted 29 June 2004 ; accepted in final form 7 November 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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- and 
-ENaC, but not 
-ENaC. Compared with cells expressing FL-SGK1, K127M-SGK1 decreased the expression of - and -subunit mRNA by 
45 and 90%, respectively. Next, to determine whether SGK1 is one of the proteins mediating the induction of -ENaC mRNA by CS, we compared steroid induction of -ENaC in cells expressing K127M-SGK1 vs. FL-SGK1. The maximum level of -ENaC mRNA levels following CS was significantly (45%) higher in FL-SGK1- vs. K127M-SGK1-expressing cells, although the fold-induction by CS was similar in both FL-SGK1- and K127M-SGK1-expressing cells. In summary, we report for the first time that SGK1 regulates transcription of ENaC subunits. We propose that the effect of SGK1 on ENaC transcription is mediated by the activation of unidentified transcription factors. aldosterone; transcriptional regulation; cortical collecting ducts; sodium transport; epithelial sodium channel
, , and , which share 35% identity in amino acid sequence (3, 11, 19). Aldosterone, through binding to the mineralocorticoid receptor, has both an immediate action on ENaC and a delayed effect (>3 h) that involve the synthesis of new ENaC subunits (4, 6, 27, 31, 40). The immediate response has been attributed to increased trafficking or activity of ENaC in the apical membrane. Serum- and glucocorticoid-inducible kinase-1 (SGK1) is thought to play an important role in the immediate response to aldosterone, although the exact mechanism remains unknown. SGK1 is an early aldosterone-induced gene, whose transcription is increased within 15–30 min of exposure to aldosterone (13, 31). Several studies have demonstrated that the coexpression of SGK1 and ENaC increases the sodium current by three- to sevenfold in Xenopus laevis oocytes and A6 cells, whereas the kinase-dead dominant negative form of SGK1 decreases the sodium current (2, 5, 13, 17, 27, 31). Our laboratory recently demonstrated a similar trend in mouse CCD (M1) cells, where overexpression of SGK1 significantly increased the sodium current, whereas a dominant negative kinase-dead SGK1 decreased it (20). Taken together, these studies exemplify the importance of SGK1 in the early aldosterone-mediated induction of ENaC, but they do not address whether SGK1 may also mediate the delayed effects of aldosterone, including ENaC transcription.
In the CCD, exogenous aldosterone or endogenous aldosterone induced by a low-sodium diet has been shown to increase -ENaC gene transcription, without increasing expression of the - or -subunit (7, 16, 27, 32, 38). Therefore, in these cells, the synthesis of the -subunit may be the rate-limiting step in channel formation. The -ENaC promoter contains a glucocorticoid response element (GRE), which could facilitate the transcriptional regulation (35), but because the effect is not rapid, it is not known whether the entire corticosteroid effect is mediated through glucocorticoid receptor/mineralocorticoid receptor binding to the GRE or whether part of the -ENaC gene induction is indirect. We hypothesized that SGK1 plays a role in the aldosterone-mediated ENaC transcription by phosphorylating transcription factors that regulate ENaC transcription.
Our hypothesis is based on the knowledge that SGK1 belongs to the "AGC" family of protein kinases, and these kinases are known to be involved in the regulation of transcription (36). This family includes PKA, PKG, PKC, and PKB. SGK1 has a 45% identity with PKB (43). PKB and SGK1 are known to phosphorylate transcription factors such as FKHR and FKHRL1 (10, 34), whereas PKB has been shown to activate the transcription factor NF-
B (9, 28). Therefore, it is possible that SGK1 phosphorylates transcription factors that, in turn, activate ENaC subunit transcription, which could play a role in the delayed regulation of ENaC by aldosterone. We report here that SGK1 plays an important role in the transcriptional regulation of - and -, but not -ENaC, in renal CCD cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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M1 cell lines stably expressing human full-length (FL)-SGK1 or the dominant negative kinase-dead (K127M)-SGK1 were generated, and their function was characterized as described elsewhere (20). Cells were grown in PC-1 medium until reaching confluence, then lysed in 500 ml of Tri-Reagent for RNA preparation (Molecular Research Center, Cincinnati, OH).
To study the effects of corticosteroids, after reaching confluence, cells were switched to steroid-free/low-serum medium for 24 h. Steroid-free medium was made from DMEM/F-12 (Cellgro, Herndon, VA) containing 5% twice-charcoal stripped FBS (stripping removes >99% of small-molecular-mass materials), 15 mM HEPES, 2 mM glutamine, 7.5% bicarbonate, and antibiotics. After 24 h, one-half of the steroid-starved cells were treated with dexamethasone (Dex; 10–6 M) for 24 h, whereas the other half remained in steroid-free/low-serum medium. After a 24-h incubation, both steroid-starved and Dex-treated cells were lysed for RNA in Tri-Reagent.
Amiloride treatment of cells grown on a permeable membrane. M1 cells expressing K127M-SGK1 and FL-SGK1 were grown in duplicate on Millicell permeable membranes (Millipore, Bedford, MA) in complete growth media for 48 h. After cells reached confluence, one-half of the cells were treated with 10 µM amiloride for 24 h. Cells were then lysed with Tri-Reagent for RNA extraction.
Protein synthesis inhibition with cycloheximide and anisomycin. M1 cells were grown in a 12-well plate in complete growth medium until reaching confluence. Then cells were steroid starved for 24 h and treated in duplicate for 8 h as follows: 1) steroid-free medium, 2) Dex (10–6 M), 3) cycloheximide (10 µM), 4) anisomycin (1 µg/µl), 5) cycloheximide+Dex, and 6) anisomycin+Dex. After treatment, cells were lysed in Tri-Reagent for RNA extraction.
Quantitative RT-PCR. To compare the relative amounts of ENaC subunit mRNA levels among parent-M1, FL-SGK1-, and K127M-SGK1-expressing M1 cells, we used quantitative RT-PCR as described (18). RNA was isolated from cells using Tri-Reagent (Molecular Research Center) according to the manufacturer's protocol. Reverse transcription was performed on 2 µg of total RNA, calculated from the absorbance measured at 260 nm, using MMLV Reverse Transcriptase (GIBCO, Gaithersburg, MD). Sense and antisense primers were designed for all three mouse ENaC subunits using the Oligo 6 computer program.
A 159-bp product of mouse -ENaC was amplified by the following primers: 5'-GGC AGC CCA CCG AGG AGG A-3' (sense) and 5'-GCC ACA GCA CCG CCC AGA A-3' (antisense). Four serial dilutions of cDNA were used as templates. For the cells grown in steroid-free medium, the amounts of cDNA were 20, 6.7, 2.2, and 0.74 ng. For cells grown in complete growth medium or treated with Dex, we used 5, 1.7, 0.57, and 0.19 ng of cDNA as templates. Reactions were performed under standard conditions with AmpliTaq DNA polymerase (Roche, Indianapolis, IN). After an initial 2-min denaturation at 96°C, -ENaC PCR was carried out for 29 cycles (95, 61, and 72°C for 45 s each). The reaction mixture was incubated for a final extension at 72°C for 8 min.
A 349-bp product of mouse -ENaC was amplified by the primers 5'-CCG ATG TTG CCA TAA AGA ACC T-3' (sense) and 5'-CTC TGG TCC CGC TCC TGA GAC-3' (antisense), using 100, 50, 25, and 12.5 ng cDNA. PCR was carried out using touchdown conditions by decreasing the annealing temperature 3°C every 5 cycles, beginning with 95, 61, and 72°C for 45 s each, 5 cycles; 95, 58, and 72°C and 95, 55, and 72°; and ending with 95, 52, and 72°C for 45 s each, 24 cycles.
A 317-bp product of mouse -ENac was amplified by the primers 5'-CGC CCT CCT CGT CTT CTC TTT C-3' (sense) and 5'-TGG CCT TTC CCT TCT CGT TCT C-3' (antisense), using 80, 26.6, 8.88, and 2.96 ng of cDNA as templates. PCR was carried out using touchdown conditions with annealing temperatures of 63, 60, and 57°C for 45 s, 5 cycles each, and ending with 54°C, 45 s each, 17 cycles.
Results were normalized to -actin mRNA levels determined in each sample and amplified by the primers 5'-GCC GGG ACC TGA CAG ACT A-3' (sense) and 5'-GGC CAT CTC CTG CTC GAA-3' (antisense), using serial dilutions (2, 0.66, 0.22, and 0.07 ng) of cDNA, amplified for 25 cycles (96, 57, and 72°C for 1 min each). After amplification of cDNA, 20 µl of each sample were separated on a 5% polyacrylamide gel and stained with ethidium bromide. Gels were scanned on a FluorImager 575, and the amount of PCR product was quantified using ImageQUANT software (Molecular Dynamics, Sunnyvale, CA).
Real-time quantitative RT-PCR.
To verify the accuracy of the results of traditional RT-PCR, we performed real-time RT-PCR on -ENaC mRNA from cells grown in complete growth medium. The -ENaC primers were the same as described above, and the -actin primers were 5'-AGA GGG AAA TCG TGC GTG AC-3' (sense) and 5'-CAA TAG TGA TGA CCT GGC CGT-3' (antisense). The amount of cDNA amplified was 10 ng in triplicate for an initial 8-min denaturation at 95°C. Both -ENaC and -actin PCR were carried out at the same time with the conditions 95, 57, and 72°C for 30 s each and a final annealing at 72°C for 5 min. The melt curve data were then collected at 10-s cycles (55°C for 80 cycles), using an iCycler Thermal Cycler (Bio-Rad, Hercules, CA). Data were analyzed using the MyiQ Single-Color Real-Time PCR detection system (Bio-Rad).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-ENaC gene expression compared with K127M-SGK1, whereas the - and -subunit levels were too low for detection (A. Náray-Fejes-Tóth, unpublished observations). To determine whether the effect of SGK1 on the current is mediated, in part, by differences in ENaC subunit expression, we measured the levels of mRNA for all three ENaC subunits in M1 cells with different levels of SGK1 activity. As shown in Fig. 1A, when cells were maintained in complete growth medium, expression levels of - and -ENaC were significantly lower in cells expressing the dominant negative K127M-SGK1 compared with cells expressing the FL-SGK1. mRNA levels of the -subunit decreased by 45% (P < 0.05) due to expression of K127M-SGK1 compared with cells expressing FL-SGK1. Levels of -subunit mRNA exibited the most significant decrease in response to K127M-SGK1, with a decrease of 80% (P < 0.05). The decrease in -ENaC mRNA due to the expression of K127M-SGK1, although significant, was not as obvious as the decrease in -ENaC, so we verified our -ENaC expression data using real-time quantitative RT-PCR. As seen in Fig. 1B, these experiments confirmed the results obtained by traditional RT-PCR.
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- and -ENaC mRNA, our results revealed that the expression levels of the -subunit were not statistically different in response to changes in SGK1 activity (P = 0.08), although FL-SGK1 showed a trend toward increasing -ENaC transcription compared with K127M-SGK1 (Fig. 1A). Also, we did not observe a statistical difference between ENaC mRNA levels of parent M1 cells vs. FL-SGK1-expressing cells.
Does ENaC transcription occur independently of sodium transport in the presence of varying levels of SGK1 activity?
Since K127M-SGK1 was previously shown to decrease sodium transport in M1 cells (20), the possibility exists that the concurrent decrease in ENaC transcription was a result of decreased ENaC function. Therefore, SGK1 could be directly affecting ENaC function and only inadvertently affecting ENaC transcription. To determine whether SGK1 activity is directly affecting the transcription of -ENaC, we grew M1 cells expressing K127M-SGK1 and FL-SGK1 on permeable membranes and then treated cells with 10 µM amiloride to block ENaC function. The results shown in Fig. 1C suggest that it is not primarily ENaC function that affects ENaC transcription, because when function is inhibited by amiloride, the varying levels of SGK1 activity continued to cause similar differences in -ENaC transcription compared with cells without amiloride treatment.
Does corticosteroid induction of -ENaC gene expression require de novo protein synthesis?
Several groups have previously demonstrated that corticosteroids, such as Dex or aldosterone, and/or a low-sodium diet induce mRNA and protein expression of the -ENaC subunit in CCD cells, whereas they do not affect the expression of the - or -subunit (7, 16, 26, 27, 29, 30, 32, 38). Our results shown in Fig. 2 confirm these previous findings. In parent M1 cells, a 24-h treatment with 10–6 M Dex increased the expression of -ENaC mRNA about fourfold (P < 0.05), whereas there was no significant change in the expression of the (P = 0.5)- or -subunit (P = 0.2) in steroid-free vs. Dex-treated M1 cells.
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-ENaC transcription is a direct action through the GRE in the promoter of the -ENaC gene, or whether de novo protein synthesis is required for this induction. It has been observed that when corticosteroids bind to their receptors, they can trans-activate the GRE and stimulate -ENaC transcription (35). However, this is probably not the only mechanism of ENaC induction by steroids, because -ENaC responds to corticosteroids depending on the cell type. For example, treatment of intestinal epithelium with corticosteroids does not significantly induce -subunit mRNA, while increasing - and -ENaC gene expression (7, 16, 25, 38), suggesting that tissues specifically express transcription factors that are needed for steroid regulation of ENaC subunits. To test whether de novo protein synthesis is required for corticosteroid induction of -ENaC transcription in M1 cells, cells were treated with two different inhibitors, anisomycin and cycloheximide, at concentrations previously shown to inhibit protein synthesis (21). M1 cells were treated with the inhibitors for 8 h, because time course experiments measuring transepithelial resistance suggested that 8 h were sufficient for observing a Dex induction while avoiding the potential cytotoxic effects of protein synthesis inhibitors. In the presence of anisomycin, the levels of -ENaC mRNA were significantly lower in both steroid-free medium and after corticosteroid treatment vs. cultures without anisomycin (80 and 60%, respectively, P < 0.05; Fig. 3), suggesting that protein synthesis is necessary for this action. However, this trend was not seen in cells treated with cycloheximide. The level of -ENaC mRNA in cycloheximide-treated cells grown in either steroid-free medium or Dex-treated medium was not statistically different from control levels (P = 0.4, P = 0.1, respectively). Such an apparent contradiction between effects observed with two different protein synthesis inhibitors was previously reported by Itani et al. (21). These authors observed that in Madin-Darby canine kidney (MDCK)-C7 cells, cycloheximide treatment for 24 h stimulated -ENaC transcription and explained their findings by cycloheximide's activation of p38 MAP kinase, which, in turn, increases -ENaC transcription (21). It is possible that cycloheximide is stimulating the p38 MAP kinase in M1 cells; thus cycloheximide treatment cannot be used to determine whether -ENaC induction is direct or indirect. However, the results obtained with anisomycin suggest that de novo protein synthesis is necessary for -ENaC transcription because the absolute levels of -ENaC mRNA are decreased compared with cells without protein synthesis inhibition. At the same time, the nearly fourfold Dex induction suggests that corticosteroids maintain a direct effect on -ENaC transcription that does not require de novo synthesis of other steroid-induced proteins.
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-ENaC mRNA?
The results from our cells grown in complete growth medium suggest that SGK1 activity is important for - and -ENaC transcription. The next step was to determine whether this trend continued for cells grown in steroid-free/low-serum conditions, which removes from the complete medium the hormones and/or growth factors that may affect ENaC transcription. Data in Fig. 4 show that cells grown in steroid-free medium respond to varying levels of SGK1 activity similarly to cells grown in complete growth medium. In K127M-SGK1-expressing cells, -ENaC was decreased by 45% compared with cells expressing FL-SGK1 (P < 0.05). Next, we determined the effects of corticosteroids on ENaC transcription in the presence of varying levels of SGK1 activity. The proteins that might mediate, at least in part, the corticosteroid induction of the -ENaC gene have not yet been identified. Since aldosterone and glucocorticoids induce both SGK1 and -ENaC, we asked whether SGK1 is one of the proteins involved in this pathway. Our previous work showed that M1 cells stably expressing K127M-SGK1 were unable to respond to corticosteroids with an increase in the ENaC current, whereas the FL-SGK1-expressing cells were able to increase the ENaC current (20). A possible explanation for this observation is that active SGK1 is necessary for the steroid induction of the ENaC current. As shown in Fig. 4, the maximum level of -ENaC expression in K127M-SGK1-expressing cells treated with Dex was significantly lower, by 45%, than in cells expressing FL-SGK1 (P < 0.05). At the same time, Dex still induced -ENaC mRNA levels by about fourfold, even in the presence of the K127M-SGK1 (Fig. 4). From these results and previous results regarding corticosteroid-induced current, we conclude that SGK1 is a partial mediator of -ENaC transcription.
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- and -ENaC subunits in the presence of corticosteroids?
The next question we asked was whether SGK1 also affects the expression of - and -ENaC in steroid-free/low-serum medium and in corticosteroid-treated cells. Our results, along with others, have shown that corticosteroids increase the transcription of -ENaC while having no effect on the - or -subunit (Fig. 2). On the other hand, SGK1 affects both - and -ENaC transcription in cells grown in complete growth medium (Fig. 1A), and corticosteroids can induce - and -ENaC in intestinal epithelia (7, 16, 25, 38). Our results summarized in Fig. 4 demonstrate the combined effects of SGK1 and corticosteroids on - and -ENaC mRNA expression levels in the presence of K127M-SGK1 vs. FL-SGK1. Expression of K127M-SGK1 in steroid-free/low-serum-treated cells resulted in a 95% decrease in the levels of -ENaC mRNA compared with cells expressing FL-SGK1 (P < 0.05). Following Dex treatment, K127M-SGK1-expressing cells still had a 95% decrease in -ENaC levels compared with Dex-treated FL-SGK1-expressing cells. Surprisingly, FL-SGK1-expressing cells treated with Dex expressed significantly higher levels (45%, P < 0.05) of -ENaC mRNA than cells in steroid-free/low-serum medium. -ENaC was detectable in all experiments when cells were maintained in complete growth medium, but in steroid-free/low-serum medium, and following Dex treatment, the levels decreased more dramatically, so that K127M-SGK1-expressing cells had detectable -ENaC mRNA levels in only a minority of experiments. This suggests that there is a factor in the complete growth medium necessary for -ENaC transcription. Similarly, as with the other growth media, the Dex-containing medium had no significant change in -ENaC mRNA levels in K127M-SGK1- vs. FL-SGK1-expressing cells (P = 0.4; Fig. 4). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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During the last few years, SGK1 has emerged as a potentially important protein regulating the early phase of aldosterone-induced sodium transport. Prior research has established a role for SGK1 in ENaC regulation and function in M1 cells, A6 cells, and oocytes (5, 13, 17, 20, 27, 31). In the functional studies with M1 cells, SGK1 levels were regulated through the stable expression of K127M-SGK1 or FL-SGK1. The K127M mutation in the catalytic domain of SGK1 results in a kinase-dead mutant that is not only inactive but acts as a dominant negative by inhibiting endogenous SGK1 activity (27). The overexpression of FL-SGK1 results in an increase in SGK1 expression that is posttranscriptionally activated through the phosphatidylinositol 3-kinase pathway. Our previous work showed that M1 cells expressing K127M-SGK1 had a significant decrease in current (Isc) compared with parent M1 cells, whereas Isc was significantly increased in FL-SGK1-expressing cells (20).
The exact mechanism by which SGK1 increases ENaC function is not known. It was originally suggested that SGK1 directly phosphorylates ENaC, resulting in the increase in activation and translocation. However, several studies have shown that SGK1 does not directly phosphorylate ENaC (5, 23, 42). Further studies suggest that SGK1, by phosphorylating Nedd4–2, prevents Nedd4–2 from binding to the PY motif of ENaC, and thereby preventing it from endocytosis and/or degradation (14, 37). In addition to direct posttranslational effects, both the early and late phases of aldosterone induction could involve changes in the transcription of certain genes that may affect vesicle trafficking. Even more probable is the assumption that regulation of transcription plays an important role in the late effects of aldosterone action. Figure 5 illustrates our proposed model for ENaC regulation by hormones and SGK1. During the early phase, SGK1, induced by corticosteroids, might phosphorylate preexisting transport machinery involved in increasing ENaC function or activate silent ENaC molecules present in the membrane (15, 41). During the delayed phase, corticosteroids, in addition to other hormones and growth factors, may act on ENaC through two mechanisms that facilitate dual regulation of ENaC gene expression. ENaC transcription may occur directly through the GRE and/or indirectly through SGK1. We propose that SGK1 regulates the transcription of the - and -subunit by phosphorylating the consensus sequence RXRXXS/T (22, 33) on specific transcription factors, which then stimulate ENaC transcription.
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B, NF-AT, and activator protein-1 (1, 8). SGK1 and PKB can phosphorylate Forkhead transcription factors (10, 34), and PKB also phosphorylates NF-B through p38 MAP kinase (9, 28). Most recently, Chen et al. (12) have shown that SGK1 stimulates the promoter region of the NPR-A gene by inducing a threefold increase in NPR-A promoter luciferase reporter activity.
In summary, the combined results from the three culture conditions (complete growth medium, steroid-free/low-serum medium, and corticosteroid-containing medium) establish that expression of FL-SGK1 significantly increases the levels of - and -ENaC compared with K127M-SGK1, while having no effect on -ENaC. At the same time, the fold-induction of -ENaC due to corticosteroids remains similar for both FL-SGK1- and K127M-SGK1-expressing cells, suggesting that corticosteroids are affecting ENaC regardless of SGK1 activity. Independent of corticosteroid treatment, SGK1 remains an important regulator of -ENaC transcription because the absolute value of -ENaC transcription is significantly increased in cells expressing FL-SGK1 compared with K127M-SGK1. These results are also supported by the studies using the protein synthesis inhibitor anisomycin. Our anisomycin results, along with those of Itani et al. (21), suggest that de novo synthesis is necessary for the corticosteroid induction of -ENaC mRNA. The inhibition of protein synthesis significantly decreases the absolute amount of -ENaC mRNA for cells grown in both steroid-free medium and corticosteroid-treated medium. We propose that SGK1 may be one of the de novo proteins mediating the transcription of -ENaC.
To our knowledge, this is the first report implicating SGK1 as having a role in the regulation of ENaC transcription. In complete growth medium, we did not observe a significant difference in ENaC gene expression between paired parent M1 cells and cells expressing FL-SGK1. This might be because transcription is occurring at a maximal rate in parent cells stimulated with complete growth medium. The findings were somewhat surprising because we previously showed that expression of FL-SGK1 increased Isc compared with parent WT-M1 cells (20). This increase could be due to the effect of SGK1 on ENaC activation and translocation and not an increase in gene expression.
The mechanism by which SGK1 leads to increased expression of - and -ENaC is not known. We speculate that the mechanism involves SGK1 phosphorylating and activating specific transcription factor(s) that, in turn, bind the promoter region of - and -ENaC and stimulate their transcription. Alternately, SGK1 might phosphorylate kinases upstream from transcription factors that would, in turn, activate those factors responsible for increased ENaC transcription. An additional possibility is that SGK1 inactivates an inhibitor that interferes with ENaC transcripton. Further studies in our laboratory will be aimed at defining those mechanisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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