Cycloheximide increases glucocorticoid-stimulated alpha -ENaC mRNA in collecting duct cells by p38 MAPK-dependent pathway

Omar A. Itani1,2, Kristyn L. Cornish1, Kang Z. Liu1, and Christie P. Thomas1,2,3

1 Department of Internal Medicine, and 2 Graduate Program in Molecular Biology, University of Iowa College of Medicine, and 3 Veterans Affairs Medical Center, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Aldosterone and glucocorticoids (GCs) stimulate Na+ reabsorption in the collecting ducts by increasing the activity of the epithelial Na+ channel (ENaC). Our laboratory has used Madin-Darby canine kidney-C7 cells to demonstrate that this effect is associated with an increase in alpha -ENaC gene transcription (Mick VE, Itani OA, Loftus RW, Husted RF, Schmidt TJ, and Thomas CP, Mol Endocrinol 15: 575-588, 2001). Cycloheximide (CHX) superinduced the GC-stimulated alpha -ENaC expression in a dose-dependent manner, but had no effect on basal or aldosterone-stimulated alpha -ENaC expression, whereas anisomycin inhibited basal and corticosteroid-stimulated alpha -ENaC expression. The superinduction of alpha -ENaC expression was also seen with hypotonicity, was blocked by RU-38486, and was independent of protein synthesis. CHX had no effect on alpha -ENaC mRNA half-life, confirming that its effect was via an increase in alpha -ENaC transcription. The effect of CHX and hypotonicity on alpha -ENaC expression was abolished by SB-202190, indicating an effect mediated via p38 MAPK. Consistent with this scheme, CHX increased pp38 and MKK6, an upstream activator of p38, stimulated alpha -ENaC promoter activity. These data confirm a model in which CHX activates p38 in Madin-Darby canine kidney-C7 cells to increase alpha -ENaC gene transcription in a GC-dependent manner.

epithelial sodium channel; aldosterone; glucocorticoid; gene regulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SODIUM REABSORPTION IN THE cortical and medullary collecting duct is tightly regulated in response to the perceived extracellular fluid volume and to dietary Na+ intake. Sodium transport in this segment of the nephron occurs via the epithelial Na+ channel (ENaC), and an important class of physiological regulators of this transport pathway is the corticosteroids, aldosterone, and glucocorticoids (GCs). GCs are also important regulators of ENaC-dependent Na+ transport in the lung, whereas aldosterone influences Na+ transport in other mineralocorticoid-responsive tissues, such as the distal colon, sweat ducts, and salivary glands (21, 48). One important molecular target for aldosterone and GC action in the collecting duct is the alpha -subunit of ENaC itself, which is transcriptionally regulated via a GC response element (GRE) in the 5'-flanking region of the gene (27, 32, 38, 44).

In addition to corticosteroids, many other signaling pathways appear to regulate Na+ transport in the collecting duct, including those activated by AVP, prostaglandins, and EGF. The effects of AVP are synergistic to the effects of corticosteroids and, in some cases, can be mimicked by activators of adenylate cyclase and by membrane-permeant analogs of cAMP (21, 45). Short- and long-term infusion of 1-desamino-8-D-AVP to Brattleboro rats increases the abundance of each of the three ENaC subunit proteins, an effect that may be mediated by an increase in ENaC mRNA abundance (15). In contrast to corticosteroids and AVP, EGF and PGE2 inhibit collecting-duct Na+ transport (53). Although the mechanism of inhibition of Na+ transport in the collecting duct is unknown, growth factors can modulate gene expression by activation of protein kinase C and mitogen-activated protein (MAP) kinases. In parotid salivary epithelial cells, protein kinase C activation leads to transcriptional downregulation of the alpha -subunit of ENaC, an effect mediated by the ERK (57). The ERK pathway antagonizes the GC-dependent trans-activation of the alpha -ENaC subunit gene and provided the first evidence for a direct cross talk between a nuclear hormone receptor and a MAP kinase signaling pathway in the regulation of this Na+ channel (27).

In the course of our investigation of the mechanism of GC stimulation of ENaC gene expression, we found evidence that a second MAP kinase, p38 MAP kinase, stimulates alpha -ENaC gene transcription in a GC-dependent manner in Madin-Darby canine kidney (MDCK)-C7 cells, a collecting duct cell line with regulated Na+ transport.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Dexamethasone, aldosterone, cycloheximide, anisomycin, emetine hydrochloride, puromycin, and puromycin aminonucleoside were purchased from Sigma (St. Louis, MO), and RU-28362, and RU-38486 were generous gifts from Roussel Uclaf (Romainville, France). SB-202190, SB-203580, and U-0126 were obtained from Calbiochem-Novabiochem (San Diego, CA), and actinomycin D was from Roche Molecular Biochemicals (Indianapolis, IN). Culture materials were from Life Technologies (Gaithersburg, MD), and [alpha -32P]UTP and [35S]methionine were from NEN Life Science Products (Boston, MA). Stock solutions of actinomycin D, cycloheximide, emetine, puromycin, U-0126, SB-202190 and SB-203580 were made in Me2SO, whereas dexamethasone, aldosterone, RU-28362, RU-38486, and anisomycin were made in ethanol.

Tissue culture and RNA preparation. MDCK-C7 cells (gift from B. Blazer-Yost and H. Oberleithner) were maintained in MEM with 10% fetal bovine serum (7). For RNA experiments, cells were grown in 100 mM petri dishes or six-well plates, switched to serum-free media, and incubated with 100 nM dexamethasone, aldosterone, or vehicle (ethanol) in the presence or absence of other reagents for 24 h, except where indicated. To determine mRNA turnover, MDCK-C7 cells were stimulated for 12 h with 100 nM dexamethasone alone or with cycloheximide, and then treatment continued with these reagents for various times in the presence of 1 µM actinomycin D. For tonicity experiments, MDCK-C7 cells were exposed to an external osmolality of 250 or 200 mosmol/kgH2O by the addition of 0.2 or 0.5 vol of H2O to the culture media for 4 h. As a control for dilution of media contents, isotonic conditions were maintained in other samples by the addition of 0.2 or 0.5 vol of 150 mM NaCl for 4 h. RNA from cultured cells was prepared with the RNeasy minikit (Qiagen, Valencia, CA), following the manufacturer's recommendations.

RNAse protection assay. A template containing the cloned canine alpha -ENaC and sgk1 cDNA and an 18S rRNA cDNA fragment (pTR1 RNA 18S; Ambion, Austin, Tx) were used to generate radiolabeled antisense cRNA probes, as previously described (32). RNA samples were cohybridized overnight with alpha -ENaC or sgk1 and 18S cRNAs and then digested with RNase A and T1 and nuclease-protected fragments resolved by polyacrylamide gel electrophoresis. To quantitate mRNA expression, autoradiograms were scanned, and the density of individual bands was measured by using Kodak Digital Science Image Analysis software (Rochester, NY). The alpha -ENaC band was normalized for the density of the 18S rRNA band, and the data from multiple experiments were pooled and analyzed by one-way analysis of variance and/or by Student's t-test. For calculation of mRNA half-life, data points were plotted on a semilogarithmic scale, and exponential regression lines were derived for both experimental conditions.

Radiolabeling of MDCK-C7 proteins. For metabolic labeling of MDCK-C7 proteins, cells were seeded into 24-well plates, grown until subconfluent, and switched to serum-free media for an additional 18-24 h. After they were washed with PBS, cells were incubated for 10 min with methionine-free RPMI 1640, and then for 2 h with the same medium containing 15 µCi/ml of [35S]methionine in the presence or absence of cycloheximide or serum. The cells were then washed twice with ice-cold PBS, twice with 5% TCA, and solubilized in 0.5 ml of 0.25 N NaOH, and an aliquot was counted in a liquid scintillation counter.

Western blotting. MDCK-C7 cells were grown in six-well plates until subconfluent, switched to serum-free media for 24 h, and then treated with various reagents for the indicated times. Samples were then lysed in a solution containing 50 mM Tris, pH 7.4, 76 mM NaCl, 2 mM EGTA, 1% Nonidet P-40, 0.5% SDS, 10% glycerol, 0.4 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 2 µg/ml aprotinin for 1 h at 4°C and then homogenized by passing through an 18-gauge needle several times. Protein concentrations were determined by the Bradford method, and 100 µg of each lysate were run on a 10% polyacrylamide gel at 30 V for 16 h. Resolved proteins were transferred onto nitrocellulose (Transblot, Bio-Rad, Hercules, CA) by using the Owl Separation System (Midwest Scientific, Valley Park, MO) at 400 mA for 45 min and blocked in 5% nonfat dry milk/0.05% TBS-Tween (TTBS) for 1 h. Blots were incubated with 1:250 to 1:1,000 dilution of p38 MAP kinase antibody, stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) (p54/46) antibody, phospho-SAPK/JNK (p54/46) antibody (all from Cell Signaling Technologies, Beverly, MA), or phospho-p38 MAP kinase antibody (Sigma) in 3% nonfat dry milk/TTBS for 1 h. Blots were then washed once in TTBS and incubated with 1:1,000 to 1:3,000 horseradish peroxidase-conjugated secondary IgG antibody for 1 h. After three 10-min washes in TTBS, horseradish peroxidase detection was performed with the SuperSignal chemiluminescence substrate (Pierce, Rockford, IL).

Transient transfection assays. An ~1,400-nt fragment of the halpha -ENaC 5'-flanking region (-1,388+55/alpha -ENaC-luc) cloned upstream of the firefly luciferase coding region of pGL3basic (Promega, Madison, WI) was used for transfection experiments (32). This construct was transiently transfected into MDCK-C7 by using LipofectAMINE Plus (Life Technologies), along with a control plasmid, pRLSV40, which expresses the sea pansy luciferase, to correct for differences in transfection efficiency and in recovery of cytosolic extracts. The day after transfection, cells were treated for 24 h with 100 nM dexamethasone or aldosterone and 20 µM SB-202190 or its vehicle. Some wells were also treated with 1 µM cycloheximide for the last 6 h. The transfected cells were maintained in serum-free media from the time of transcription until cells were lysed. Cell lysates were prepared ~48 h after transfection, and reporter gene activity was measured as previously described (44).

In another set of experiments, the plasmid, -1,388+55/alpha -ENaC-luc or TAT3-luc, a GC-responsive reporter plasmid (gift from David Pearce and Keith Yamamoto), was cotransfected into MDCK-C7 cells along with pRLSV40 and MKK6b, a constitutively active form of the upstream kinase for p38 MAP kinase (39). Six hours after transfection, 100 nM dexamethasone or its vehicle was added, and 24 h later, lysates were prepared and analyzed as described.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our laboratory has previously reported that dexamethasone and aldosterone increase amiloride-sensitive Na+ transport in MDCK-C7 cells, an effect temporally associated with increased expression of alpha -ENaC mRNA (32). The stimulation of alpha -ENaC expression is mediated in both cases by GC receptor (GR)-mediated activation of a GRE in the 5'-flanking region of the alpha -ENaC gene. To determine whether protein synthesis was required for the corticosteroid effect on alpha -ENaC gene transcription, we used cycloheximide, a general protein synthesis inhibitor, simultaneously with dexamethasone, aldosterone, or vehicle for 24 h and measured alpha -ENaC mRNA levels. Although cycloheximide had no effect on vehicle or aldosterone-stimulated alpha -ENaC, a more than twofold increase in alpha -ENaC mRNA levels was seen in the presence of dexamethasone (Fig. 1, A and B). The initial interpretation of this finding was that cycloheximide reduced the synthesis of an intermediary protein that functioned to inhibit the GC-stimulated but not basal alpha -ENaC gene transcription. It is important to note that MDCK-C7 cells lack the mineralocorticoid receptor (MR) and that aldosterone-stimulated alpha -ENaC expression occurs through GR (32). The inability of cycloheximide to superinduce alpha -ENaC mRNA levels in the presence of aldosterone was, therefore, surprising, because the effect of aldosterone and GC on alpha -ENaC mRNA is mediated by GR. To determine whether the cycloheximide effect was dependent on activation of GR, we used a specific GR agonist, RU-28362, and a specific GR antagonist, RU-38486, and examined alpha -ENaC mRNA levels. As expected, RU-28362 stimulated alpha -ENaC mRNA levels, and this was abolished by simultaneous treatment with RU-38486 (Fig. 1, C and D). Cycloheximide had additional stimulatory effects on RU-28362-treated MDCK-C7 cells, an effect that was completely abrogated by the GR antagonist RU-38486 (Fig. 1, C and D).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of cycloheximide (chx) on alpha -epithelial Na+ channel (ENaC) expression. Madin-Darby canine kidney (MDCK)-C7 cells were exposed to dexamethasone (dex) or aldosterone (aldo) in the presence or absence of 10 µM chx for 24 h, and alpha -ENaC mRNA was measured by RNase protection assay (RPA) and compared with vehicle (ctrl). A: representative RPA is shown, and the last lane indicates yeast (Y) control. B: RPA data are quantitated and pooled (n = 3; means ± SE). The data are significantly different by one-way analysis of variance: dex and aldo increase alpha -ENaC expression (* P < 0.02 compared with control); chx superinduces dex-stimulated alpha -ENaC expression (# P < 0.05 compared with dex alone) but has no effect on basal or aldo-stimulated gene expression. C: the effect of chx on RU-28362-stimulated alpha -ENaC expression. D: representative RPA and pooled data are shown (n = 3; means ± SE). The data are significantly different by one-way analysis of variance: RU-28362 increases alpha -ENaC gene expression (* P < 0.005 compared with ctrl alone), an effect that is blocked by the glucocorticoid receptor (GR) antagonist RU-38486 and further stimulated by chx (# P < 0.01 compared with RU-28362 alone). The effect of chx and RU-38486 on alpha -ENaC gene expression is completely inhibited by RU-38486.

To confirm the cycloheximide effect, we used another protein synthesis inhibitor, emetine, with vehicle and dexamethasone treatment. Both emetine and cycloheximide dose dependently increased dexamethasone-stimulated alpha -ENaC expression, with negligible effects in vehicle-treated cells (Fig. 2A). We also tested the effect of hypotonicity on dexamethasone-stimulated alpha -ENaC expression, because, in an amphibian model of the collecting duct, hypotonicity stimulates Na+ transport and increases the expression of another GC-regulated gene, sgk1 (42, 55). When MDCK-C7 cells were switched to hypotonic media in the presence of dexamethasone, an increase in alpha -ENaC expression was seen that was more pronounced the greater the degree of hypotonicity (Fig. 2B). We then asked whether cycloheximide could have a similar effect on other dexamethasone-regulated genes in MDCK-C7 cells. Sgk1 is a serine-threonine kinase that appears, at least in part, to mediate the corticosteroid effects on Na+ transport in the collecting duct (5, 10, 18, 33). As our laboratory has previously reported, dexamethasone increases sgk1 expression in MDCK-C7 cells (32). Simultaneous incubation with cycloheximide substantially increased dexamethasone-stimulated sgk1 expression (Fig. 2C).


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of chx, emetine, and hypotonicity on gene expression. A: MDCK-C7 cells were exposed to increasing concentrations of emetine or chx in the presence of dex or its vehicle for 24 h, and alpha -ENaC mRNA levels were measured. RPA is shown, and the last lane indicates yeast (Y) control. Emetine and chx increase glucocorticoid-stimulated alpha -ENaC expression in a dose-dependent manner. B: MDCK-C7 exposed to dex under isotonic (300 mosmol/kgH2O) or hypotonic (250 and 200 mosmol/kgH2O) conditions for 4 h, and alpha -ENaC mRNA levels were measured. Hypotonicity increases alpha -ENaC expression. C: MDCK-C7 cells treated with 100 nM dex for 1 or 2 h in the presence or absence of 10 mM chx, and sgk1 mRNA levels were measured. Chx increases glucocorticoid-stimulated sgk1 expression.

The results with cycloheximide and emetine raised the possibility that their effects on alpha -ENaC expression may be mediated by inhibition of protein synthesis. To determine whether the effect of cycloheximide correlated with its effects on protein synthesis inhibition, we measured protein synthesis rates in MDCK-C7 cells labeled with [35S]methionine. As expected, cycloheximide inhibited protein synthesis but profoundly only at 10 µM, while there was a modest but significant effect at 1 µM, and no effect at 0.1 µM, concentrations that were sufficient to superinduce alpha -ENaC gene transcription (Fig. 3). These results suggested that the effects of cycloheximide were unlikely to be secondary to inhibition of protein synthesis.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   MDCK cell proteins were metabolically labeled with [35S]methionine in the presence of complete medium (serum), various concentrations of chx (in µM), or its vehicle; and methionine incorporation into cell lysates was measured. Values are means ± SE; n = 3. * P < 0.05 and # P < 0.001 compared with ctrl. cpm, Counts per minute.

We then tested two other protein synthesis inhibitors and saw dramatically different effects on alpha -ENaC mRNA expression. Puromycin and its inactive analog puromycin aminonucleoside had no effect on alpha -ENaC mRNA levels, whereas anisomycin inhibited vehicle- and dexamethasone-treated alpha -ENaC mRNA levels in a dose-dependent manner (Fig. 4, A and B). These contrasting results clearly indicated that the effects of these agents on alpha -ENaC gene expression could not be explained by inhibition of protein synthesis. We then evaluated the effect of anisomycin on aldosterone-stimulated alpha -ENaC expression and confirmed that anisomycin inhibited aldosterone-stimulated alpha -ENAC expression in a dose-dependent manner (Fig. 4C). These results demonstrate that, in contrast to the cycloheximide effect to superinduce GC-stimulated but not aldosterone-stimulated alpha -ENaC expression, anisomycin was able to inhibit constitutive alpha -ENAC expression, as well as that stimulated by GC and aldosterone.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of puromycin (Puro) and anisomycin on alpha -ENaC expression. A: MDCK-C7 cells were exposed to increasing concentrations of puromycin or puromycin aminonucleoside in the presence of dex or its vehicle for 24 h, and alpha -ENaC mRNA levels were measured. RPA is shown, and the last lane indicates yeast (Y) control. Neither puromycin nor its inactive analog alters glucocorticoid-stimulated alpha -ENaC expression. B: MDCK-C7 cells were exposed to increasing concentrations of anisomycin in the presence of dex or its vehicle for 24 h, and alpha -ENaC mRNA levels were measured. RPA is shown, with the position of alpha -ENaC mRNA and 18S rRNA indicated. The last lane indicates Y control. While chx superinduces dex-stimulated alpha -ENaC expression, anisomycin inhibits ctrl and dex-stimulated alpha -ENaC expression in a dose-dependent manner. C: MDCK-C7 cells were exposed to increasing concentrations of anisomycin in the presence of aldo or its vehicle for 24 h, and alpha -ENaC mRNA levels were measured. Similar to dex, the ctrl and aldo-stimulated alpha -ENaC expression is inhibited by anisomycin.

To begin to understand the basis for the effect of cycloheximide, we examined the kinetics of the cycloheximide response on dexamethasone-stimulated alpha -ENaC expression. Dexamethasone stimulated alpha -ENaC expression, which was barely evident by 1 h, but in the presence of cycloheximide the transcript was clearly evident at 1 h, and with cycloheximide was more abundant at every time point tested (Fig. 5A). Because alpha -ENaC mRNA appeared earlier, these results suggested that cycloheximide increased the rate of transcription of alpha -ENaC. To evaluate this further, we measured alpha -ENaC mRNA decay characteristics in the presence of dexamethasone alone and in the presence of dexamethasone and cycloheximide (Fig. 5B). The mRNA half-life under both conditions was remarkably similar at ~9 h, confirming that cycloheximide did not alter alpha -ENaC mRNA stability. This finding further argues for an effect of cycloheximide on GC-stimulated alpha -ENaC gene transcription.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 5.   Transcription and turnover of calpha -ENaC mRNA. A: effect of chx on the kinetics of alpha -ENaC expression after stimulation for 24 h with dex. RPA is shown, with the position of alpha -ENaC mRNA and 18S rRNA indicated. Chx increases the rate of appearance of alpha -ENaC mRNA. B: decay of alpha -ENaC mRNA with dex (D) or dex + chx (D+C) in the presence of actinomycin D (A). The means ± SE of duplicate samples were used to derive an exponential regression line for both sets. The chx-treated samples have more alpha -ENaC mRNA and hence begin their decay at a higher level. The half-time for both sets is 9 h.

A mechanism by which protein synthesis inhibitors may superinduce gene expression is by activation of MAP kinases (3, 58). We used SB-202190 or SB-203580 p38 MAP kinase inhibitors along with cycloheximide and dexamethasone and measured alpha -ENaC mRNA levels. The superinduction of dexamethasone-stimulated alpha -ENaC expression by cycloheximide was abolished by simultaneous treatment with SB-202190 and inhibited by SB-203580 (Fig. 5A). U-0126, an inhibitor of MEK, the upstream activator of ERK, had no effect on cycloheximide-stimulated alpha -ENaC expression (Fig. 6A). These data suggested that cycloheximide increased alpha -ENaC expression by activation of p38 MAP kinase. The hypotonicity-mediated increase in GC-regulated alpha -ENaC expression was also blocked by SB-202190 and SB-203580, but not by U-0126 or PD-98059 (Fig. 6B). These results indicated that the effect of hypotonicity was also mediated by p38 MAP kinase.


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of mitogen-activated protein (MAP) kinase inhibitors on alpha -ENaC expression. A: effect of the specific p38 MAP kinase inhibitor SB-202190 on the chx stimulation of alpha -ENaC expression. MDCK-C7 cells were placed in serum-free media for 24 h and then treated with 100 nM dex for 4 h; 10 µM chx for 24 h; and/or 20 µM SB-202190, SB-203580, or U-0126 for 24 h. SB-202190 and SB-203580 inhibit the chx effect on alpha -ENaC expression. A is representative of at least 2 experiments. B: effect of MAP kinase inhibitors on the stimulation of alpha -ENaC expression by hypotonicity. MDCK-C7 cells were placed in serum-free media for 24 h and then treated with 100 nM dex under hypotonic conditions (200 mosmol/kgH2O) for 4 h, in the presence of 20 µM SB-202190, SB-203580, PD-98059, or 10 µM U-0126. SB-202190 and SB-203580 inhibit the tonicity effect on alpha -ENaC expression. B is representative of at least 2 experiments.

To examine the effect of cycloheximide on MAP kinase activation, we measured total and phosphorylated p38 and JNK in MDCK-C7 lysates after treatment with either agent. Whereas total p38 did not vary between conditions, short-term treatment with cycloheximide or anisomycin increased phospho-p38 abundance (Fig. 7). In addition to the principal band, a lower band was also seen, especially in cycloheximide-treated lanes, which may represent another isoform of p38 or a breakdown product. By contrast, anisomycin led to a dramatic increase in phosphorylated JNKs, principally p46 (pJNK), whereas the corresponding treatment with cycloheximide showed a barely detectable increase in pJNK, even when higher concentrations of cycloheximide (25 µM) were used (Fig. 7). There was, however, no change in the total JNK between conditions. The data suggest that cycloheximide principally activates p38 MAP kinase, whereas anisomycin is a potent activator of JNK and p38. Based on the data in Fig. 6A, the activation of p38 MAP kinase by cycloheximide appears to increase transcription of the alpha -ENaC gene. Despite activation of p38, anisomycin reduces alpha -ENaC gene expression, presumably because activation of other pathways results in inhibition of alpha -ENAC gene transcription.


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 7.   Immunoblotting for inactive and active forms of p38 and c-Jun NH2-terminal kinase (JNK). MDCK-C7 cells were placed in serum-free media for 24 h and then treated with various reagents for the indicated times. Lysates were resolved by SDS-PAGE and then immunoblotted with the indicated antibodies. Anisomycin (Aniso) and chx-activated p38 (pp38) were compared with vehicle-treated (ctrl) lane. Anisomycin increases phospho-JNK (pJNK), whereas vehicle and chx treatments do not lead to a detectable change in pJNK.

To confirm the effect of cycloheximide and SB-202190 on alpha -ENaC gene transcription, we used the 1,388-bp human alpha -ENaC promoter coupled to luciferase in transfection assays. This promoter contains the alpha -ENaC GRE, and, as our laboratory has shown before (32), is robustly stimulated by treatment with 100 nM dexamethasone for 24 h (Fig. 8A). To minimize the effects of cycloheximide on translation of luciferase, this agent was used at a lower dose (1 µM) and for the last 6 h only. The results demonstrate that cycloheximide significantly increased alpha -ENaC promoter-driven luciferase activity in the presence of dexamethasone, indicating that the cycloheximide effect was at the level of gene transcription and that cis-elements within the included 5'-flanking sequence were sufficient to confer this effect. SB-202190 abolished the effect of cycloheximide (Fig. 8A), correlating with the inhibition of alpha -ENaC gene expression seen in Fig. 6A. As in Fig. 6A, SB-202190 appeared to partially inhibit the dexamethasone effect, suggesting that p38 MAP kinase activation may also support basal or dexamethasone-stimulated alpha -ENaC gene expression. We next asked whether cycloheximide would have an effect on aldosterone-stimulated gene transcription. Consistent with the data seen in Fig. 1, the effect of cycloheximide to increase alpha -ENaC gene transcription is seen only with dexamethasone and not with aldosterone (Fig. 8B).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 8.   Transient transfection assays. A: MDCK-C7 cells were transfected with -1,388+55/alpha -ENaC-luc and pRLSV40 and treated with dex in the presence or absence of 20 µM SB-202190 (SB) for 24 h, with 1 µM chx added for the last 6 h. Values are means ± SE; n = 4. # P < 0.005 compared with dex + chx + vehicle. B: MDCK-C7 cells were transfected with -1,388+55/alpha -ENaC-luc and pRLSV40 and treated with 100 nM dex or aldo for 24 h, with 1 µM chx added for the last 6 h. Values are means ± SE; n = 4. * P < 0.005 compared with dex alone; # P < 0.01 compared with ctrl. C: MDCK-C7 cells were transfected with -1,388+55/alpha -ENaC-luc (ENaC-luc) or TAT3-luc with pRLSV40 and MKK6b and then treated for 24 h with 100 nM dex or vehicle. Values are means ± SE; n = 3. Results significantly different by one-way ANOVA: * P < 0.05 compared with dex-treated ENaC-luc without cotransfected MKK6b; # P < 0.05 compared with dex-treated TAT3-luc without cotransfected MKK6b. The respective control samples are not significantly different. In all panels, luciferase assays were performed and results are expressed as a ratio of firefly luciferase (luciferase I) to sea pansy luciferase (luciferase II).

Finally, we asked whether activation of p38 MAP kinase was sufficient to stimulate alpha -ENaC gene transcription. We used a plasmid that overexpressed a MAP kinase kinase, MKK6b, which constitutively activates p38 in transient transfection assays (1). After transfection with the alpha -ENaC promoter-luciferase construct, MDCK-C7 cells were treated with 100 nM dexamethasone for 24 h, and our results demonstrate that cotransfection of MKK6b increases alpha -ENaC gene transcription (Fig. 8C). Similar results were seen with TAT3-luc, a GC-responsive reporter plasmid in which three tandem copies of the GRE in the rat tyrosine amino transferase gene are placed upstream of a TATA-driven firefly luciferase construct (29). These results suggest that the effects of p38 MAP kinase on GC-dependent gene transcription may be seen with other genes that are regulated by GREs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GC and mineralocorticoids are important physiological regulators of ENaC, and most, if not all, of their effects are mediated by increases in transcription of target genes. In vivo, in kidney cortex and medulla and in the lung, alpha -ENaC is itself a target of hormone action (2, 17, 49, 50). The increase in transcription of the alpha -ENaC subunit is mediated by trans-activation of a GRE in the 5'-flanking region of the alpha -ENaC gene by hormone-bound GR or MR (27, 32, 38, 44). However, the presence of hormone-bound receptor is not sufficient for alpha -ENaC gene activation, because, in colonic epithelia, treatment with GC or mineralocorticoids increases steady-state levels of beta - and gamma -ENaC mRNA without any effect on alpha -ENaC mRNA (28, 40, 49). One explanation for these results is that cell-specific coactivators present in the collecting duct or lung epithelia are required for the corticosteroid effect or that cell-specific repressors in colonic epithelia prevent the corticosteroid response.

To begin to explore the mechanism of GC-stimulated alpha -ENaC mRNA transcription in the collecting duct, we used cycloheximide, a widely used inhibitor of protein synthesis, with dexamethasone and aldosterone and found that it specifically increased dexamethasone-stimulated, but not aldosterone-stimulated or basal, alpha -ENaC mRNA levels. The phenomenon of a further increase in mRNA levels with a protein synthesis inhibitor over that seen with an inducing agent such as dexamethasone is well known and is termed superinduction (12, 26). This phenomenon was first observed for labile immediate early-response genes such as c-fos, c-jun, and egr-1, which function as transcription factors, but more recently has been seen with enzymes such as cyclooxygenase and protein phosphatases and signaling molecules such as cytokines (14, 16, 31). Classically, these gene products are expressed rapidly and transiently, and protein synthesis inhibitors typically induce basal and superinduce hormone or growth factor-stimulated gene expression. Our observations with cycloheximide and the alpha -ENaC transcript were intriguing in many ways. First, the alpha -ENaC transcript is not an immediate early gene product, because, after stimulation by aldosterone or dexamethasone, mRNA levels begin to increase by ~2 h and continue to increase for the next 24-48 h (24, 32). Second, this transcript encodes a channel protein rather than a transcription factor, enzyme, or signaling molecule. Third, the superinduction is seen with dexamethasone and not with aldosterone, although both agonists bind to the same transcription factor, GR, and activate alpha -ENaC gene transcription via the same cis element in these cells (32).

Several mechanisms have been proposed to explain superinduction of immediate early genes by inhibitors of protein synthesis. These include 1) an increase in mRNA stability (37, 56); 2) increase in transcription, perhaps by inhibition of transcriptional downregulation (23, 41); and 3) stimulation of intracellular signaling pathways (30, 58). Increasingly, differential effects of various protein synthesis inhibitors have been reported, and it appears that inhibition of protein synthesis may not be required for some of these effects. In our own studies, we show that, whereas emetine and cycloheximide superinduce alpha -ENaC expression, puromycin has no effect and anisomycin inhibits gene expression, suggesting that protein synthesis inhibition is not sufficient for superinduction (Figs. 2A and 4, A and B). Our metabolic labeling studies also confirmed that inhibition of protein synthesis is not required for the cycloheximide effect (Fig. 3).

Given the disparate effects between cycloheximide and anisomycin, we began to wonder whether our results with alpha -ENaC expression could be explained by differential stimulation of intracellular signaling pathways. Recently, anisomycin has been shown to be a potent stimulant of JNK and is increasingly being used as a tool to examine the role of JNK in subcellular biological phenomena (8, 9). It has become clear, however, that, in some cell systems, anisomycin may activate each of the three known MAP kinase-signaling pathways, including JNK, p38, and ERK. In HeLa cells, for example, anisomycin activates JNK and ERK and p38 (43, 58), whereas, in other cells such as NIH3T3 cells, ERK activation is not seen (11). Cycloheximide is a considerably weaker activator of JNK (3, 58), and differing effects on MAP kinases could explain the contrasting effects of cycloheximide and anisomycin. When we used SB-202190, a specific p38 MAP kinase inhibitor, the effect of cycloheximide was abolished in MDCK-C7 cells, confirming that cycloheximide stimulated alpha -ENaC mRNA expression in a p38-dependent manner. Indeed, by immunoblot analysis, we demonstrated that cycloheximide activated p38 alone, whereas anisomycin increased phosphorylation of JNK and p38. Cycloheximide also enhanced GC- but not aldosterone-stimulated alpha -ENaC promoter activity in luciferase assays, an activity that was blocked by SB-202190, confirming that its effect was mediated by p38 MAP kinase. Finally, direct activation of p38 MAP kinase by overexpression of its upstream kinase MKK6 was also able to increase alpha -ENaC gene transcription in a reporter gene assay. Taken together, the data strongly suggest that cycloheximide activates p38 MAP kinase and that this activation increases alpha -ENaC transcription in a GC-dependent manner in MDCK-C7 cells.

What is the relevance of GC-stimulated transcription of genes that are involved in Na+ transport in the collecting duct? In vivo, under physiological conditions, aldosterone rather than cortisol engages the MR to increase gene transcription and stimulate Na+ transport (19, 20, 34). The selectivity of MR for aldosterone comes from the actions of the enzyme 11beta -hydroxysteroid dehydrogenase type 2 (11beta -OHSD2), which inactivates cortisol to cortisone, a metabolite that no longer has affinity for MR or GR (19). Under pathophysiological conditions in which the activity of 11beta -OHSD2 is diminished or when circulating levels of cortisol are high enough to overwhelm the capacity of this enzyme, endogenous GC can bind either the GR or MR to stimulate Na+ transport. The syndrome of apparent mineralocorticoid excess, which is due to mutations in 11beta -OHSD2, exemplifies a situation in which cortisol functions as a mineralocorticoid (13, 54). Additionally, several synthetic GCs in clinical use, such as dexamethasone, have very low affinity for MR and are not substrates for metabolism by 11beta -OHSD2 (35). Thus the effects of these agents on alpha -ENaC mRNA expression and on amiloride-sensitive Na+ transport pathways in vivo and in cultured cells derived from the collecting duct must be mediated virtually exclusively via activation of GR (2, 36, 46, 49, 51).

In contrast to the MR, GR binds with high affinity to cortisol and dexamethasone but with much lower affinity to aldosterone (19). With supraphysiological doses, aldosterone increases alpha -ENaC mRNA expression and Na+ transport in some collecting duct cell lines that lack functional MR (6, 32, 46, 52), suggesting that aldosterone may be able to exert mineralocorticoid effects through GR under certain circumstances. Under physiological conditions, it is unlikely that aldosterone binds to GR in mineralocorticoid-responsive tissues, but, under conditions in which circulating levels of aldosterone are very high or where aldosterone is competitively displaced from MR (e.g., with the use of spironolactone), crossover binding to GR can occur. The identification of loss of function mutations of MR in human disease and the creation of transgenic mice with targeted deletion of MR have begun to provide us with information on MR-independent pathways that contribute to regulation of ENaC and the reabsorption of Na+ in the collecting duct (4, 22, 47). Understanding the downstream effects of GR occupation when aldosterone, rather than a classic GC, is the activating ligand has arguably become important. Our studies demonstrate that the p38 MAP kinase pathway has stimulatory effects on GC-mediated alpha -ENaC gene expression. This effect is GR dependent and is only evident in the presence of its high-affinity ligand, dexamethasone or RU-28362, and not aldosterone. These results suggest one of two possibilities: a direct physical interaction between a p38 MAP kinase-dependent coactivator and the GR-ligand complex, or cooperative interactions between p38 and GR-activated cis elements in the regulatory regions of the alpha -ENaC gene. The data obtained with TAT3-luc, in which the regulatory elements consist only of a minimal promoter and multiple GREs, would suggest that the former is more likely. The data presented here thus fit a model in which cycloheximide or hypotonicity activates p38 MAP kinase, which in turns recruits a transcriptional coactivator that recognizes GR bound to GCs such as dexamethasone, but not aldosterone (Fig. 9). In fact, a nuclear receptor coactivator, PGC-1, has recently been described that, on activation by p38 MAP kinase, enhances GR-dependent gene transcription, at least when reconstituted in HeLa cells (25). Our studies point to mechanisms whereby ENaC gene transcription can be enhanced by activation of p38 MAP kinase following the engagement of a variety of membrane receptors or in response to adverse conditions such as ischemia.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 9.   Model for stimulation of dex-regulated gene expression by chx and hypotonicity. p38 MAP kinase activation via its upstream kinase MKK6 leads to the recruitment of a transcriptional coactivator that enhances transcription from glucocorticoid response elements (GREs) in the presence of GR bound to dex but not GR bound to aldo.


    ACKNOWLEDGEMENTS

The authors thank H. Oberleithner and B. Blazer-Yost for the gift of the MDCK-C7 cell line, Jiahuai Han for the gift of plasmid MKK6b, David Pearce and Keith Yamamoto for the gift of the plasmid TAT3-luc, and acknowledge the DNA synthesis and sequencing services provided by the University of Iowa DNA core facility.


    FOOTNOTES

This work was supported in part by United States Public Health Service Grant DK54348 and March of Dimes Birth Defects Foundation Grant-in-Aid 6-FY99-444. C. P. Thomas is an Established Investigator of the American Heart Association.

Portions of this work were presented in abstract form at the Experimental Biology 2002, New Orleans, LA.

Address for reprint requests and other correspondence: C. P. Thomas, Dept. of Internal Medicine, E300H GH, Univ. of Iowa, 200 Hawkins Drive, Iowa City, IA 52242 (E-mail: christie-thomas{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.

First published December 27, 2002;10.1152/ajprenal.00088.2002

Received 6 March 2002; accepted in final form 15 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alpert, D, Schwenger P, Han J, and Vilcek J. Cell stress and MKK6b-mediated p38 MAP kinase activation inhibit tumor necrosis factor-induced Ikappa B phosphorylation and NF-kappa B activation. J Biol Chem 274: 22176-22183, 1999[Abstract/Free Full Text].

2.   Asher, C, Wald H, Rossier BC, and Garty H. Aldosterone-induced increase in abundance of Na+ channel subunits. Am J Physiol Cell Physiol 271: C605-C611, 1996[Abstract/Free Full Text].

3.   Barros, LF, Young M, Saklatvala J, and Baldwin SA. Evidence of two mechanisms for the activation of the glucose transporter GLUT1 by anisomycin: p38(MAP kinase) activation and protein synthesis inhibition in mammalian cells. J Physiol 504: 517-525, 1997[Abstract].

4.   Berger, S, Bleich M, Schmid W, Cole TJ, Peters J, Watanabe H, Kriz W, Warth W, Greger R, and Schutz G. Mineralocorticoid receptor knockout mice: pathophysiology of Na+ metabolism. Proc Natl Acad Sci USA 95: 9424-9429, 1998[Abstract/Free Full Text].

5.   Bhargava, A, Fullerton MJ, Myles K, Purdy TM, Funder JW, Pearce D, and Cole TJ. The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action. Endocrinology 142: 1587-1594, 2001[Abstract/Free Full Text].

6.   Blazer-Yost, BL, Paunescu TG, Helman SI, Lee KD, and Vlahos CJ. Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption. Am J Physiol Cell Physiol 277: C531-C536, 1999[Abstract/Free Full Text].

7.   Blazer-Yost, BL, Record RD, and Oberleithner H. Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line. Pflügers Arch 432: 685-691, 1996[ISI][Medline].

8.   Bogoyevitch, MA, Ketterman AJ, and Sugden PH. Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes. J Biol Chem 270: 29710-29717, 1995[Abstract/Free Full Text].

9.   Cano, E, Doza YN, Ben-Levy R, Cohen P, and Mahadevan LC. Identification of anisomycin-activated kinases p45 and p55 in murine cells as MAPKAP kinase-2. Oncogene 12: 805-812, 1996[ISI][Medline].

10.   Chen, SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96: 2514-2519, 1999[Abstract/Free Full Text].

11.   Chung, KC, Kim SM, Rhang S, Lau LF, Gomes I, and Ahn YS. Expression of immediate early gene pip92 during anisomycin-induced cell death is mediated by the JNK- and p38-dependent activation of Elk1. Eur J Biochem 267: 4676-4684, 2000[Abstract/Free Full Text].

12.   Cochran, BH, Reffel AC, and Stiles CD. Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell 33: 939-947, 1983[ISI][Medline].

13.   Cooper, M, and Stewart P. The syndrome of apparent mineralocorticoid excess. QJM 91: 453-455, 1998[Free Full Text].

14.   Deans, JP, Serra HM, Shaw J, Shen YJ, Torres RM, and Pilarski LM. Transient accumulation and subsequent rapid loss of messenger RNA encoding high molecular mass CD45 isoforms after T cell activation. J Immunol 148: 1898-1905, 1992[Abstract/Free Full Text].

15.   Ecelbarger, CA, Kim GH, Terris J, Masilamani S, Mitchell C, Reyes I, Verbalis JG, and Knepper MA. Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney. Am J Physiol Renal Physiol 279: F46-F53, 2000[Abstract/Free Full Text].

16.   Efrat, S, Zelig S, Yagen B, and Kaempfer R. Superinduction of human interleukin-2 messenger RNA by inhibitors of translation. Biochem Biophys Res Commun 123: 842-848, 1984[ISI][Medline].

17.   Escoubet, B, Coureau C, Bonvalet JP, and Farman N. Noncoordinate regulation of epithelial Na+ channel and Na+ pump subunit mRNAs in kidney and colon by aldosterone. Am J Physiol Cell Physiol 272: C1482-C1491, 1997[Abstract/Free Full Text].

18.   Faletti, CJ, Perrotti N, Taylor SI, and Blazer-Yost BL. Sgk: an essential convergence point for peptide and steroid hormone regulation of ENaC-mediated Na+ transport. Am J Physiol Cell Physiol 282: C494-C500, 2002[Abstract/Free Full Text].

19.   Farman, N, and Rafestin-Oblin ME. Multiple aspects of mineralocorticoid selectivity. Am J Physiol Renal Physiol 280: F181-F192, 2001[Abstract/Free Full Text].

20.   Funder, JW. Glucocorticoid and mineralocorticoid receptors: biology and clinical relevance. Annu Rev Med 48: 231-240, 1997[ISI][Medline].

21.   Garty, H, and Palmer LG. Epithelial sodium channels: function and regulation. Physiol Rev 77: 359-396, 1997[Abstract/Free Full Text].

22.   Geller, DS, Rodriguez-Soriano J, Vallo Boado A, Schifter S, Bayer M, Chang SS, and Lifton RP. Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I. Nat Genet 19: 279-281, 1998[ISI][Medline].

23.   Greenberg, ME, Hermanowski AL, and Ziff EB. Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription. Mol Cell Biol 6: 1050-1057, 1986[ISI][Medline].

24.   Itani, OA, Auerbach SD, Husted RF, Volk KA, Ageloff S, Knepper MA, Stokes JB, and Thomas CP. Glucocorticoid-stimulated Na+ transport in human lung epithelia is associated with regulated ENaC and sgk1 expression. Am J Physiol Lung Cell Mol Physiol 282: L631-L641, 2002[Abstract/Free Full Text].

25.   Knutti, D, Kressler D, and Kralli A. Regulation of the transcriptional coactivator PGC-1 via MAPK-sensitive interaction with a repressor. Proc Natl Acad Sci USA 98: 9713-9718, 2001[Abstract/Free Full Text].

26.   Lau, LF, and Nathans D. Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc. Proc Natl Acad Sci USA 84: 1182-1186, 1987[Abstract].

27.   Lin, HH, Zentner MD, Ho HLL, Kim KJ, and Ann DK. The gene expression of the amiloride-sensitive epithelial sodium channel alpha -subunit is regulated by antagonistic effects between glucocorticoid hormone and ras pathways in salivary epithelial cells. J Biol Chem 274: 21544-21554, 1999[Abstract/Free Full Text].

28.   Lingueglia, E, Renard S, Waldmann R, Voilley N, Champigny G, Plass H, Lazdunski M, and Barbry P. Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone. J Biol Chem 269: 13736-13739, 1994[Abstract/Free Full Text].

29.   Liu, W, Wang J, Sauter N, and Pearce D. Steroid receptor heterodimerization demonstrated in vitro and in vivo. Proc Natl Acad Sci USA 92: 12480-12484, 1995[Abstract].

30.   Mahadevan, LC, and Edwards DR. Signalling and superinduction. Nature 349: 747-748, 1991[ISI][Medline].

31.   Maier, JA, Hla T, and Maciag T. Cyclooxygenase is an immediate-early gene induced by interleukin-1 in human endothelial cells. J Biol Chem 265: 10805-10808, 1990[Abstract/Free Full Text].

32.   Mick, VE, Itani OA, Loftus RW, Husted RF, Schmidt TJ, and Thomas CP. The alpha  subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts, and this transcriptional response is mediated via distinct cis-elements in the 5' flanking region of the gene. Mol Endocrinol 15: 575-588, 2001[Abstract/Free Full Text].

33.   Naray-Fejes-Toth, A, Canessa C, Cleaveland ES, Aldrich G, and Fejes-Toth G. Sgk is an aldosterone-induced kinase in the renal collecting duct. J Biol Chem 274: 16973-16978, 1999[Abstract/Free Full Text].

34.   Naray-Fejes-Toth, A, Colombowala I, and Fejes-Toth G. The role of 11beta -hydroxysteroid dehydrogenase in steroid hormone specificity. J Steroid Biochem Mol Biol 65: 311-316, 1998[ISI][Medline].

35.   Naray-Fejes-Toth, A, and Fejes-Toth G. 11beta -Hydroxysteroid dehydrogenase-2 is a high affinity corticosterone-binding protein. Mol Cell Endocrinol 134: 157-161, 1997[ISI][Medline].

36.   Naray-Fejes-Toth, A, and Fejes-Toth G. Glucocorticoid receptors mediate mineralocorticoid-like effects in cultured collecting duct cells. Am J Physiol Renal Fluid Electrolyte Physiol 259: F672-F678, 1990[Abstract/Free Full Text].

37.   Ohh, M, and Takei F. Regulation of ICAM-1 mRNA stability by cycloheximide: role of serine/threonine phosphorylation and protein synthesis. J Cell Biochem 59: 202-213, 1995[ISI][Medline].

38.   Otulakowski, G, Rafii B, Bremner HR, and O'Brodovich H. Structure and hormone responsiveness of the gene encoding the alpha-subunit of the rat amiloride-sensitive epithelial sodium channel. Am J Respir Cell Mol Biol 20: 1028-1040, 1999[Abstract/Free Full Text].

39.   Raingeaud, J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, and Davis RJ. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270: 7420-7426, 1995[Abstract/Free Full Text].

40.   Renard, S, Voilley N, Bassilana F, Lazdunski M, and Barbry P. Localization and regulation by steroids of the alpha , beta  and gamma  subunits of the amiloride-sensitive Na+ channel in colon, lung and kidney. Pflügers Arch 430: 299-307, 1995[ISI][Medline].

41.   Roger, T, Out TA, Jansen HM, and Lutter R. Superinduction of interleukin-6 mRNA in lung epithelial H292 cells depends on transiently increased C/EBP activity and durable increased mRNA stability. Biochim Biophys Acta 1398: 275-284, 1998[ISI][Medline].

42.   Rozansky, DJ, Wang J, Doan N, Purdy T, Faulk T, Bhargava A, Dawson K, and Pearce D. Hypotonic induction of SGK1 and Na+ transport in A6 cells. Am J Physiol Renal Physiol 283: F105-F113, 2002[Abstract/Free Full Text].

43.   Sayed, M, Kim SO, Salh BS, Issinger OG, and Pelech SL. Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase. J Biol Chem 275: 16569-16573, 2000[Abstract/Free Full Text].

44.   Sayegh, R, Auerbach SD, Li X, Loftus R, Husted R, Stokes JB, and Thomas CP. Glucocorticoid induction of epithelial sodium channel expression in lung and renal epithelia occurs via trans-activation of a hormone response element in the 5' flanking region of the human epithelial sodium channel alpha subunit gene. J Biol Chem 274: 12431-12437, 1999[Abstract/Free Full Text].

45.   Schafer, J, and Hawk C. Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids. Kidney Int 41: 255-268, 1992[ISI][Medline].

46.   Schmidt, TJ, Husted RF, and Stokes JB. Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors. Am J Physiol Cell Physiol 264: C875-C884, 1993[Abstract/Free Full Text].

47.   Schulz-Baldes, A, Berger S, Grahammer F, Warth R, Goldschmidt I, Peters J, Schutz G, Greger R, and Bleich M. Induction of the epithelial Na+ channel via glucocorticoids in mineralocorticoid receptor knockout mice. Pflügers Arch 443: 297-305, 2001[ISI][Medline].

48.   Stokes, JB. Disorders of the epithelial sodium channel: insights into the regulation of extracellular volume and blood pressure. Kidney Int 56: 2318-2333, 1999[ISI][Medline].

49.   Stokes, JB, and Sigmund RD. Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue and steroid heterogeneity. Am J Physiol Cell Physiol 274: C1699-C1707, 1998[Abstract/Free Full Text].

50.   Tchepichev, S, Ueda J, Canessa CM, Rossier BC, and O'Brodovich HM. The lung epithelial Na+ channel subunits are differentially regulated during development and by steroids. Am J Physiol Cell Physiol 269: C805-C812, 1995[Abstract].

51.   Volk, KA, Sigmund RD, Snyder PM, McDonald FJ, Welsh MJ, and Stokes JB. rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct. J Clin Invest 96: 2748-2757, 1995[ISI][Medline].

52.   Wang, J, Barbry P, Maiyar AC, Rozansky DJ, Bhargava A, Leong M, Firestone GL, and Pearce D. SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. Am J Physiol Renal Physiol 280: F303-F313, 2001[Abstract/Free Full Text].

53.   Warden, DH, and Stokes JB. EGF and PGE2 inhibit rabbit CCD Na+ transport by different mechanisms: PGE2 inhibits Na+-K+ pump. Am J Physiol Renal Fluid Electrolyte Physiol 264: F670-F677, 1993[Abstract/Free Full Text].

54.   White, PC. 11beta -Hydroxysteroid dehydrogenase and its role in the syndrome of apparent mineralocorticoid excess. Am J Med Sci 322: 308-315, 2001[ISI][Medline].

55.   Wills, NK, Millinoff LP, and Crowe WE. Na+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity. J Membr Biol 121: 79-90, 1991[ISI][Medline].

56.   Wilson, T, and Treisman R. Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3' AU-rich sequences. Nature 336: 396-399, 1988[ISI][Medline].

57.   Zentner, MD, Lin HH, Wen X, Kim KJ, and Ann DK. The amiloride-sensitive epithelial sodium channel alpha-subunit is transcriptionally down-regulated in rat parotid cells by the extracellular signal-regulated protein kinase pathway. J Biol Chem 273: 30770-30776, 1998[Abstract/Free Full Text].

58.   Zinck, R, Cahill MA, Kracht M, Sachsenmaier C, Hipskind RA, and Nordheim A. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1. Mol Cell Biol 15: 4930-4938, 1995[Abstract].


Am J Physiol Renal Fluid Electrolyte Physiol 284(4):F778-F787