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
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ABSTRACT |
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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 -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
-ENaC expression in a dose-dependent
manner, but had no effect on basal or aldosterone-stimulated
-ENaC
expression, whereas anisomycin inhibited basal and
corticosteroid-stimulated
-ENaC expression. The superinduction of
-ENaC expression was also seen with hypotonicity, was blocked by
RU-38486, and was independent of protein synthesis. CHX had no effect
on
-ENaC mRNA half-life, confirming that its effect was via an
increase in
-ENaC transcription. The effect of CHX and hypotonicity
on
-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
-ENaC promoter
activity. These data confirm a model in which CHX activates p38 in
Madin-Darby canine kidney-C7 cells to increase
-ENaC gene transcription in a GC-dependent manner.
epithelial sodium channel; aldosterone; glucocorticoid; gene regulation
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INTRODUCTION |
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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 -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 -subunit of ENaC, an effect mediated by the
ERK (57). The ERK pathway antagonizes the GC-dependent
trans-activation of the
-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 -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.
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METHODS |
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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 [-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 -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
-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
-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 h-ENaC 5'-flanking region
(
1,388+55/
-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).
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RESULTS |
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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 -ENaC mRNA (32). The stimulation of
-ENaC expression is mediated in both cases by GC receptor
(GR)-mediated activation of a GRE in the 5'-flanking region of the
-ENaC gene. To determine whether protein synthesis was required for
the corticosteroid effect on
-ENaC gene transcription, we used
cycloheximide, a general protein synthesis inhibitor, simultaneously
with dexamethasone, aldosterone, or vehicle for 24 h and measured
-ENaC mRNA levels. Although cycloheximide had no effect on vehicle
or aldosterone-stimulated
-ENaC, a more than twofold increase in
-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
-ENaC gene transcription. It is
important to note that MDCK-C7 cells lack the mineralocorticoid receptor (MR) and that aldosterone-stimulated
-ENaC expression occurs through GR (32). The inability of cycloheximide to
superinduce
-ENaC mRNA levels in the presence of aldosterone was,
therefore, surprising, because the effect of aldosterone and GC on
-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
-ENaC mRNA levels. As expected, RU-28362 stimulated
-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).
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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 -ENaC expression, with negligible effects in vehicle-treated cells (Fig.
2A). We also tested the effect of hypotonicity on dexamethasone-stimulated
-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
-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).
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The results with cycloheximide and emetine raised the possibility that
their effects on -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
-ENaC gene
transcription (Fig. 3). These results
suggested that the effects of cycloheximide were unlikely to be
secondary to inhibition of protein synthesis.
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We then tested two other protein synthesis inhibitors and saw
dramatically different effects on -ENaC mRNA expression. Puromycin and its inactive analog puromycin aminonucleoside had no effect on
-ENaC mRNA levels, whereas anisomycin inhibited vehicle- and dexamethasone-treated
-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
-ENaC gene expression could not be explained by inhibition
of protein synthesis. We then evaluated the effect of anisomycin on
aldosterone-stimulated
-ENaC expression and confirmed that
anisomycin inhibited aldosterone-stimulated
-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
-ENaC expression,
anisomycin was able to inhibit constitutive
-ENAC expression, as
well as that stimulated by GC and aldosterone.
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To begin to understand the basis for the effect of cycloheximide,
we examined the kinetics of the cycloheximide response on dexamethasone-stimulated -ENaC expression. Dexamethasone stimulated
-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
-ENaC
mRNA appeared earlier, these results suggested that cycloheximide
increased the rate of transcription of
-ENaC. To evaluate this
further, we measured
-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
-ENaC mRNA stability. This finding
further argues for an effect of cycloheximide on GC-stimulated
-ENaC
gene transcription.
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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 -ENaC mRNA levels. The
superinduction of dexamethasone-stimulated
-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
-ENaC expression (Fig.
6A). These data suggested that
cycloheximide increased
-ENaC expression by activation of p38 MAP
kinase. The hypotonicity-mediated increase in GC-regulated
-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.
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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 -ENaC gene.
Despite activation of p38, anisomycin reduces
-ENaC gene expression, presumably because activation of other pathways results in inhibition of
-ENAC gene transcription.
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To confirm the effect of cycloheximide and SB-202190 on -ENaC
gene transcription, we used the 1,388-bp human
-ENaC promoter coupled to luciferase in transfection assays. This promoter contains the
-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
-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
-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
-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
-ENaC gene transcription is seen only with dexamethasone and not
with aldosterone (Fig. 8B).
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Finally, we asked whether activation of p38 MAP kinase was sufficient
to stimulate -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
-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
-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.
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DISCUSSION |
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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, -ENaC is itself a target of hormone
action (2, 17, 49, 50). The increase in transcription of
the
-ENaC subunit is mediated by trans-activation of a
GRE in the 5'-flanking region of the
-ENaC gene by hormone-bound GR
or MR (27, 32, 38, 44). However, the presence of
hormone-bound receptor is not sufficient for
-ENaC gene activation,
because, in colonic epithelia, treatment with GC or mineralocorticoids increases steady-state levels of
- and
-ENaC mRNA without any effect on
-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 -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,
-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
-ENaC transcript were
intriguing in many ways. First, the
-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
-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 -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 -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
-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
-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
-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
-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
11-hydroxysteroid dehydrogenase type 2 (11
-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 11
-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 11
-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 11
-OHSD2
(35). Thus the effects of these agents on
-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
-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
-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
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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