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
Subunit Gene*
Raouf
Sayegh
§,
Scott D.
Auerbach
§,
Xiang
Li
,
Randy W.
Loftus
,
Russell F.
Husted
,
John B.
Stokes
¶, and
Christie
P.
Thomas
¶
From the
Department of Internal Medicine, University
of Iowa College of Medicine and the ¶ Veterans Affairs Medical
Center, Iowa City, Iowa 52246
 |
ABSTRACT |
In airway and renal epithelia, the
glucocorticoid-mediated stimulation of amiloride-sensitive
Na+ transport is associated with increased expression
of the epithelial Na+ channel
subunit (
ENaC). In
H441 lung cells, 100 nM dexamethasone increases
amiloride-sensitive short-circuit current (3.3 µA/cm2 to
7.5 µA/cm2), correlating with a 5-fold increase in
ENaC mRNA expression that could be blocked by actinomycin D. To
explore transcriptional regulation of
ENaC, the human
ENaC
5'-flanking region was cloned and tested in H441 cells. By deletion
analysis, a ~150-base pair region 5' to the upstream promoter was
identified that, when stimulated with 100 nM dexamethasone,
increased luciferase expression 15-fold. This region, which contains
two imperfect GREs, also functioned when coupled to a heterologous
promoter. When individually tested, only the downstream GRE functioned
in cis and bound GR in a gel mobility shift assay. In the
M-1 collecting duct line Na+ transport, m
ENaC expression
and luciferase expression from
ENaC genomic fragments were also
increased by 100 nM dexamethasone. In a colonic cell line,
HT29, trans-activation via a heterologously expressed
glucocorticoid receptor restored glucocorticoid-stimulated
ENaC gene
transcription. We conclude that glucocorticoids stimulate
ENaC
expression in kidney and lung via activation of a hormone response
element in the 5'-flanking region of h
ENaC and this response, in
part, is the likely basis for the up-regulation of Na+
transport in these sites.
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INTRODUCTION |
Transepithelial sodium transport in the collecting ducts of the
kidney, in airway epithelia, and in sweat and salivary glands occurs
principally via amiloride-sensitive epithelial sodium channels (ENaC)1 (1). This channel is
a hetero-multimeric complex, composed of three distinct but
homologous subunits termed
,
, and
ENaC, that was first
cloned from rat colon (2, 3). Two of the major physiologic functions of
this channel are to absorb lung liquid at birth and to regulate
Na+ and K+ homeostasis. Targeted disruption of
the
ENaC subunit causes fatal neonatal respiratory failure, whereas
disruption of the
or
ENaC subunit produces death from
hyperkalemia (4-6). Overactivity of the channel causes Na+
retention, hypokalemia, and hypertension, a phenotype that can be
produced by activating mutations in one of the channel subunits, by
excessive production of mineralocorticoids (MC) or by excessive stimulation by endogenous or exogenous glucocorticoids (GC) (7).
GC and MC are two important physiological regulators of
amiloride-sensitive epithelial Na+ transport in target
epithelia where their cognate receptors are expressed. Dexamethasone or
aldosterone increases
ENaC mRNA expression in rat kidney cortex
and in primary cultures of inner medullary collecting duct cells (IMCD)
without any effect on
and
ENaC expression (8-11). This effect
on
ENaC correlates temporally with an increase in
amiloride-sensitive Na+ transport in primary IMCD cultures
(12). The effect of adrenal steroids on the abundance of
ENaC
mRNA is not confined to the kidney. Dexamethasone, but not
aldosterone, increases
ENaC expression in fetal and adult rat lungs
in vivo, and in fetal lung explants and cultured airway
epithelial cells in vitro (8, 13-15). The mechanism of the
increase in steady state levels of ENaC mRNAs following adrenal
steroid treatment is unknown but has been presumed to be at the level
of ENaC gene transcription.
In this study, we identify the promoter and enhancer elements in the
5'-flanking region of the human
ENaC gene that regulate basal and
GC-mediated induction of
ENaC gene expression. In addition we
provide evidence that enhanced Na+ transport by two
different cell lines (lung and kidney) is tightly linked to enhanced
expression of
ENaC.
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EXPERIMENTAL PROCEDURES |
Cycloheximide, dexamethasone, spironolactone, amiloride, and
human placental collagen were purchased from Sigma. Actinomycin D was
obtained from Roche Molecular Biochemicals, poly dI-dC from Amersham
Pharmacia Biotech, and RU38486 was a generous gift from Roussel Uclaf
(Romainville, France). Culture materials were from Life Technologies,
Inc., and all radionucleotides were from NEN Life Science Products.
Stock solutions of cycloheximide and actinomycin D were made in
Me2SO and stocks of dexamethasone, spironolactone, and
RU38486 made in ethanol.
Tissue Culture and RNA Isolation--
The human lung epithelial
cell line, H441, and the human colon carcinoma cell line, HT29, were
cultured as described previously (16). The mouse renal cortical
collecting duct (CCD) cell line, M-1, was grown in Dulbecco's modified
Eagle's medium:F12 with 10% fetal calf serum (17). To examine the
effects of dexamethasone on gene expression, cell cultures were
switched to serum-free media and then exposed to various concentrations
of dexamethasone or its vehicle for 24 h. Cycloheximide (10 µM), actinomycin D (1 µM), and the steroid
receptor blockers, spironolactone (10 µM) and RU38486 (10 µM), were used in some experiments and compared with
control cultures in the presence of vehicle alone. RNA was isolated
from these cells as described previously (16).
Na+ Transport Measurements--
H441 and M-1 cells
were seeded on 12-mm Millicell PCF filters (Millipore, Bedford, MA),
which had been pretreated with human placental collagen. H441 cells
were grown for 6 days in RPMI medium with 6% serum and 100 nM dexamethasone and the medium changed daily. A day prior
to electrical measurements, the cells were placed in steroid-free media
and the following day placed in serum-free RPMI with or without 100 nM dexamethasone. The Millicell PCF filters were then
transferred to a specially designed chamber (Jim's Instruments, Iowa
City, IA) to measure transepithelial voltage, resistance (RT) and short circuit current (Isc) at 37 °C
(18). M-1 cells were grown for 3 days in Dulbecco's modified Eagle's
medium:F12 supplemented with 5 µg/ml insulin, 5 µg/ml transferrin,
5 nM triiodothyronine, 50 nM hydrocortisone, 10 nM sodium selenite, 50 µg/ml gentamicin, 10 mg/ml bovine
serum albumin, and 5 nM dexamethasone. The filters were
then grown for 1 day without albumin and steroids and then with or
without 100 nM dexamethasone and 10 µM
spironolactone for another 24 h.
Ribonuclease Protection Assay (RPA)--
To measure steady state
levels of h
ENaC gene expression, a previously described cDNA
template that would distinguish exon 1A-initiated (
ENaC-1) from exon
1B-initiated (
ENaC-2, 3, 4) transcripts was used to synthesize
antisense [
-32P]cRNAs (16). To control for RNA
extraction, quantitation and gel loading, an 18 S rRNA template
(pTR1-18SRNA, Ambion, Austin, TX) was used. Sample RNAs were
co-hybridized overnight with ENaC and 18 S riboprobes, digested with
RNase A and T1, and analyzed by PAGE as described previously (16).
To measure steady state levels of mouse
ENaC mRNA, a 451-bp
fragment corresponding to sequence from the 3' portions of the coding
region was amplified and cloned into pCRII and linearized at an
internal BsmFI site to protect a 132-nt product. As a
control a mouse
-actin template (pTR1-
-actin, Ambion) was used to
protect a 99-nt fragment. Antisense biotinylated cRNA probes were
synthesized using the Brightstar Psoralen-Biotin kit (Ambion). Sample
RNAs were co-hybridized with both riboprobes, digested with RNase A and
T1, resolved by PAGE, transferred to nylon membranes, and developed by
the Brightstar Biodetect system (Ambion) (8).
To quantitate mRNA expression, the autoradiograms were scanned with
a PDI scanning densitometer and the density of individual bands
measured using Quantity One software (Huntington Station, NY). Each
ENaC band was normalized for the density of 18 S or
-actin band, and
the data from three to four experiments pooled and analyzed by
Student's t test and by analysis of variance.
Cloning of the 5'-Flanking Region of h
ENaC--
Sequence
information from the 5' end of the SCNN1A locus was provided to us by
the Chromosome 12 Mapping and Sequencing Center at Albert Einstein
University. The transcription start sites for h
ENaC have been mapped
and previously reported (16). To clone the ~1500 bp of
5'-flanking region upstream of exon 1A, primer
25
(5'-ACCCAGCACCCAGAGAGCAGACGAA) and primer
23
(5'-TCAGGCCCTGCAGAGAAGAGAGAAGAGGTC) were used to amplify a DNA fragment
from human genomic DNA. This fragment, which includes 55 nt of the 5'
UTR of
ENaC-1, was directionally subcloned into pGL3basic (Promega,
Madison, WI) upstream of the firefly (Photinus pyralis)
luciferase coding region. The primers
18 (5'-GAGGGGGTGGCGAGGAATCA)
and
21 (5'-CTCGAGCTGTGTCCTGATTC) were used to amplify ~700
bp of sequence upstream of
ENaC-2, digested with Bsu36I
and then subcloned into pGL3basic. This genomic DNA fragment begins
downstream of the transcription start site of
ENaC-1 and includes
682 nt 5' to the transcription start site of
ENaC-2 and 100 nt of
its 5' UTR. Deletion variants of these constructs were created by using
internal restriction enzyme sites or by re-amplification using internal primers.
To test the glucocorticoid-responsive enhancer region in reverse
orientation and with a heterologous promoter, an
AvrII site was introduced at
289 using primer
27
(5'-CCTAGGCTCCGGGTCTGTGTCCA). A native Avr
II site at
142 allowed the recovery of a 146-nt fragment, which was
cloned in reverse orientation upstream of a P1 construct at
142 and a
P2 construct at +322. The glucocorticoid-responsive DNA fragment was
also cloned upstream of a 459-nt h
ENaC promoter (19) coupled to
luciferase. To test individual glucocorticoid response elements (GRE)
in transfection assays, single-stranded oligonucleotides that
correspond to the putative GREs were synthesized and then annealed
together. The flanking SacI and AvrII overhangs were used to clone these fragments into the native AvrII
site upstream of
ENaC-1 (see sequence below).
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Transfection and Functional Analysis of the 5'-Flanking DNA
Clones--
Subconfluent H441 cells grown in 12-well plates were used
for transfection. 1 µg of the firefly luciferase construct or the parent plasmid pGL3basic and 1-2 µg of a control plasmid pSV
-gal, where the Escherichia coli lacZ gene is cloned downstream of
the SV40 promoter (Promega) was combined with Lipofectin (Life
Technologies, Inc.) and added to each well. For transfection of M-1
cells, monolayers from two T75 flasks were trypsinized, and then
resuspended in 2 ml of a solution containing 120 mM KCl,
150 µM CaCl2, 10 mM K2HPO4, 10 mM
KH2PO4, 2 mM EGTA 5 mM
MgCl2, 100 mM ATP, 250 mM glutathione, and 25 mM Hepes, pH 7.6. A 500-µl aliquot of
cells was combined with 20 µg of a luciferase construct and 40 µg
of pSV
-gal in a 0.4-cm cuvette and then electroporated
(Electroporator II, Invitrogen, Carlsbad, CA) at 330 V, 1000 microfarads. and 500 ohms. Each aliquot of cells was then diluted with
complete medium and plated into four to six wells of a 12-well plate.
24-48 h following transfection, cells were placed in serum-free medium and dexamethasone or vehicle were added where appropriate; another 24 h later, cell lysates were prepared for measurement of reporter gene activity. For HT29 cells, LipofectAMINE Plus (Life Technologies, Inc.) was used as the transfecting reagent. As HT29 cells contained a
high level of endogenous
-galactosidase activity, we used pRL-SV40 (Promega), where the sea pansy (Renilla reniformis)
luciferase gene is cloned downstream of the SV40 promoter, as the
internal control plasmid. In some experiments, 1 µg of the plasmid
p6RGR, where the rat glucocorticoid receptor cDNA is under the
control of the Rous sarcoma virus promoter (gift from D. Pierce and K. Yamamoto) was co-transfected with the luciferase vectors.
For preparation of cell lysates, the cells were washed in
phosphate-buffered saline and then scraped into Lysis buffer
(Luciferase assay kit, Promega). An aliquot of cell lysate was added to
Luciferase Assay reagent and activity measured in a Monolight 2010 luminometer (Analytical Luminescence Laboratories, Ann Arbor, MI) for
5 s. For measurement of
-galactosidase activity, an equal
aliquot of cell lysate was incubated with the substrate Galacton-plus (Tropix, Bedford, MA) for 30 to 60 min and then activity measured in a
luminometer for 5 s. Lysates from HT29 cells were made with Passive Lysis Buffer (Promega) and firefly luciferase activity, reflecting transcriptional strength of cloned ENaC gene fragments, and
sea pansy luciferase activity was sequentially measured in the same
sample using the Dual Luciferase Reporter assay kit (Promega).
Gel Mobility Shift Assay--
Oligonucleotides that correspond
to the functional
ENaC GRE or to a nonspecific sequence were
synthesized and annealed together (see sequences below).
For gel mobility shift experiments, 50,000 cpm of end-labeled
double-stranded oligonucleotides were incubated with recombinant human
glucocorticoid receptor (Affinity Bioreagents Inc., Golden, CO) in a
20-µl reaction mixture that contained 20 mM Hepes, pH 7.9, 60 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 10 ng/ml poly(dI-dC), 5 µg/µl bovine
serum albumin, and 10% glycerol. For competition experiments, a
50-fold excess of cold oligonucleotides were used. All constituents
except the labeled probe were preincubated at 4 °C for 30 min and
then incubated with the labeled probe at 22 °C for 30 min. Samples
were then resolved on a 3.5% nondenaturing polyacrylamide gel
(acrylamide:bisacrylamide 20:1) in 0.5× TBE buffer run at 150 V.
 |
RESULTS |
GC increases
ENaC mRNA expression in lung and kidney cortex
and in cultured epithelial cells derived from these tissues (8-11, 15). To address the mechanisms whereby GC increase
ENaC mRNA expression, we used a human lung cell line H441 and mouse CCD cell line
M-1. We found that H441 cells, when grown on permeable supports in the
presence of dexamethasone, develop as a tight epithelium and have
electrogenic ion transport measured as a positive Isc from the apical
to the basolateral compartment (Fig.
1A). This current was almost
completely blocked by 10 µM amiloride applied to the
apical side (Fig. 1B), indicating that the bulk of the Isc
was accounted for by amiloride-sensitive electrogenic Na+
transport pathways. To examine the effect of dexamethasone on Na+ transport, H441 cells were placed in steroid-free media
for 24 h and then exposed to 100 nM dexamethasone or
vehicle for another 24 h. Dexamethasone increased Isc from 3.3 µA/cm2 to 7.5 µA/cm2 (Fig. 1C),
similar to results obtained from primary cultures of airway epithelial
cells (15).

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Fig. 1.
Short circuit current (Isc) measurements in
H441 cells. A, an increasing current is seen starting
at day 2 after seeding. Cells were grown continuously in 100 nM dexamethasone. B, the current is almost
entirely blocked by 10 µM amiloride (Amil+)
applied to the apical side of the membrane. C, cells grown
for 24 h in steroid-free complete media and then placed in
serum-free steroid-free media and treated with 100 nM
dexamethasone (dex) or vehicle (ctrl). A robust
dexamethasone-inducible Isc is evident at 24 h. *,
p < 0.001 compared with control; #, p < 0.001 compared with 0 h.
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We have previously reported that heterogeneity in h
ENaC transcripts
arise from alternate transcription start sites and from splicing at the
5' end of h
ENaC (16). Exon 1A begins at the upstream transcription
start site and gives rise to
ENaC-1 while a second transcription
start site 724 bp downstream in an alternate first exon (exon 1B) gives
rise to
ENaC-2,3,4 (Fig. 2). 24 h of treatment with dexamethasone increased expression of both
ENaC transcripts in a dose-dependent manner with the earliest
effect seen at 10 nM (Fig.
3A). The basal expression of
exon 1A-intiated transcripts was greater than exon 1B-initiated
transcripts, and this difference persisted through the dose-response
curve. The effect of GC on
ENaC expression was blocked by RU38486, a
type II (glucocorticoid receptor; GR) antagonist, but not by
spironolactone, a type I (mineralocorticoid receptor; MR) antagonist,
confirming that these effects required GR binding (Fig. 3B).
The effect of GC on
ENaC expression was blocked by co-administration
of actinomycin D, an inhibitor of transcription, suggesting that GC
stimulates
ENaC expression by increasing gene transcription rather
than by affecting mRNA stability (Fig. 3C).
Cycloheximide, a general inhibitor of translation, did not prevent
GC-induction of h
ENaC mRNA expression and may have augmented
expression of the h
ENaC-1 transcript (Fig. 3D).

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Fig. 2.
Schematic of the 5' end of human
ENaC gene including 1400 nt 5' to
ENaC-1 transcription start site. Four
principal transcripts ENaC-1, 2, 3, and 4 and two principal
N-terminal protein variants are created from this arrangement. The
primers used for amplification of genomic sequence are shown as
numbered arrows.
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Fig. 3.
A, dexamethasone increases ENaC
mRNA in a dose-dependent manner. ENaC mRNA
measured by RPA in H441 cells and corrected for 18 S rRNA expression.
Pooled data for three separate dose-response experiments. *,
p < 0.02 compared with control; #, p < 0.05 compared with control. Repeated measures analysis of
variance < 0.02 between both dose-response curves. B,
dexamethasone induction of ENaC mRNA occurs via binding to the
glucocorticoid receptor. Steady state levels of ENaC exon 1A and
1B-initiated transcripts following 24 h of stimulation with 1 µM dexamethasone (Dex) in the presence or
absence of the GR blocker RU38486 (10 µM) or the MR
blocker spironolactone (10 µM) measured by RPA. *,
p < 0.005; #, p < 0.03 compared with
dexamethasone alone (n = 3). C, 1 µM actinomycin D (ACT) blocks dexamethasone
stimulation of ENaC mRNA expression. #, p < 0.01 compared with control; *, p < 0.01 compared with
dexamethasone alone; @, p < 0.01 compared with
control. D, 10 µM cycloheximide
(CHX) enhances dexamethasone-stimulated ENaC mRNA
expression. *, p < 0.01 compared with control; #,
p < 0.05 compared with dexamethasone alone.
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To examine the mechanism of transcriptional regulation of the
ENaC
gene, we cloned 5'-flanking sequences upstream of both transcription
start sites and evaluated these by transient transfection assays. We
focused our attention on identification of the putative promoters as
well as the GC-regulated elements of the
ENaC gene. Constructs that
included either transcription start site and portions of their proximal
5'-flanking sequence were able to stimulate luciferase expression in
H441 cells (Fig. 4A). The
ability of constructs containing sequence 5' to each transcription
start site (e.g.
142 + 44 and +322 +814) to increase
luciferase gene transcription suggest that separate promoters direct
expression of
ENaC-1 and
ENaC-2. Two of the five tested
constructs robustly stimulated luciferase activity when treated with
100 nM dexamethasone (Fig. 4A). Analysis of the
sequence common to these constructs (
487 and
142) revealed that it
contained one or more GREs. The magnitude of the dexamethasone-induced
luciferase gene transcription is similar to the magnitude of
ENaC
mRNA stimulation in H441 cells (Fig. 3A), suggesting
that transcriptional activation of this region is sufficient to account
for the effect of dexamethasone on steady state
ENaC mRNA
levels. A preliminary analysis of the nucleotide sequence in this
region between
289 and
142 showed two imperfect GREs,
TGTcCannnTGTcCT (Up-GRE) and AGAACAnnnTGTcCT (Dn-GRE), which are
candidate cis-elements to mediate this effect.

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Fig. 4.
A, separate promoters drive expression
of transcripts ENaC-1 and ENaC-2 and a dexamethasone-responsive
region maps to a site between 487 and 142 of the human ENaC
gene. Left panel, 5' end of ENaC including the
5'-flanking region showing putative transcription factor binding
motifs. The location and extent of genomic fragments coupled to the
luciferase (luc) coding region are shown. The ENaC
transcription start sites are shown as bent
arrows. Right panel, the genomic constructs were
compared with the empty plasmid pGL3basic and the luciferase assay
corrected for -gal activity is shown. Each construct was tested with
(black bar) and without (white
bar) dexamethasone (100 nM for 24 h). *,
p < 0.002 compared with pGL3basic; #,
p < 0.02 compared with the absence of dexamethasone;
+, p < 0.002 compared with the absence of
dexamethasone. (n = 3-4 determinations).
Panels B and C, mapping of promoters
P1 and P2. B, the minimal sequence for transcriptional
activity of P1 is contained within the 82+44 fragment. *,
p < 0.001 compared with pGL3basic; #,
p < 0.001 compared with 99+44 construct.
C, the minimal sequence for transcriptional activity of P2
is contained within the +476+814 fragment. *, p < 0.002 compared with pGL3basic.
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To define the promoter regions P1 and P2, further deletions of the
constructs
142+44 and +322+814, respectively, were made and tested.
In the case of the P1 promoter, a construct as short as
82 to +44 was
active, suggesting that the minimal promoter was contained within this
region (Fig. 4B). In the case of the P2 promoter, a
construct as short as +476 to +874 was active while the construct
(+596+814) that included just ~70 nt of 5'-flanking sequence was not,
suggesting that the minimal promoter included sequences 5' to this
construct. (Fig. 4C). To determine if the region between
289 and
143 in the 5'-flanking region of the h
ENaC gene could
transduce the glucocorticoid effect, a 146-bp sequence containing these
elements were cloned in reverse orientation (
289
143INV) upstream of
the P1 and P2 promoters and transfected into H441 cells. As expected
for a classic "enhancer," these elements functioned in reverse
orientation to confer glucocorticoid responsiveness to both promoters
(Fig. 5, A and B).
These experiments confirm that the defined region is sufficient to
direct glucocorticoid-regulated gene transcription from P1 and P2. We
also tested this
ENaC genomic fragment with the promoter for
h
ENaC. Our results show that the enhancer region of
ENaC confers
glucocorticoid responsiveness to this heterologous promoter (Fig.
5C).

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Fig. 5.
The glucocorticoid-responsive enhancer tested
with homologous and heterologous promoters. The steroid-responsive
enhancer localized to a 146-bp sequence and functions in a
direction-independent manner. Portions of the h ENaC 5'-flanking
region with the location of two putative glucocorticoid response
elements (GREs) are shown. Panels A and
B, a 146-bp sequence containing both GREs was ligated to a
P1 or P2 construct in reverse orientation and tested in H441 cells
without (ctrl) or with 100 nM dexamethasone
(dex) for 24 h. Panel A, *,
p < 0.01 compared with control; #, p < 0.005 compared with 142+44 construct. Panel B, *,
p < 0.05 compared with control. Panel
C, the same 146-bp sequence ligated to a h ENaC construct
and tested for glucocorticoid responsiveness. *, p < 0.001 compared with control. The results demonstrate that the 146-bp
sequence even when cloned in reverse and coupled to a heterologous
promoter can confer a GC response.
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To further define transcriptional regulatory elements necessary for GC
induction, a new set of 5' deletion constructs were made and tested in
H441 cells (Fig. 6, A and
B). To determine the role of each GRE, single copies of
double-stranded oligonucleotides corresponding to each 15-nt element
were cloned upstream of the h
ENaC-1 promoter and tested in H441
cells (Fig. 6B). These results confirmed that the region
containing the Dn-GRE (
248
142) was sufficient to confer GC
enhancement. First, the
248+44 construct, which excludes the Up-GRE,
is capable of responding to dexamethasone (Fig. 6A);
inclusion of the Up-GRE sequence (
289+44) does not alter either basal
or the GC-enhanced response (Fig. 6B). Second, the construct
that contains only the Dn-GRE permits GC enhancement, but the construct
containing only the Up-GRE does not (Fig. 6B). Finally, a
4-nt mutation that is predicted to disrupt GR binding abolished
GC-mediated luciferase expression from the Dn-GRE (Fig. 6B).
The region immediately 5' to the Dn-GRE also appeared to contain an
inhibitory element as constitutive expression of the
248
142
construct was lower than the
142 +44 construct (Fig. 6A).

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Fig. 6.
Mapping functional GREs.
Panel A, deletional analysis indicates that the
sequence containing the Dn-GRE ( 248 142) is sufficient to confer GC
responsiveness in H441 cells. A constitutive inhibitory element appears
to be present within this site. *, p < 0.001 compared
with control; #, p < 0.01 compared with 142+44.
Panel B, inclusion of the Up-GRE does not confer
dexamethasone enhancement greater than a construct that contains only
the Dn-GRE ( 289+44 versus 248+44). The Up-GRE alone
permits no dexamethasone enhancement while the Dn-GRE does. Thus the
Dn-GRE but not the Up-GRE is functional. A 4-bp mutation in Dn-GRE
(Mut.Dn-GRE) abolishes GC responsiveness. The constitutive inhibitory
element does not correspond to the Dn-GRE but is presumably between
248 and 157. *, p < 0.001 compared with control;
#, p < 0.001 compared with 248+44 control.
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To determine if the identified Dn-GRE could bind GR in a mobility shift
DNA binding assay, oligonucleotides corresponding to these sequences
were synthesized, incubated with purified GR, and then analyzed by PAGE
and autoradiography. To test specificity of interaction, the purified
GR was preincubated with an excess of cold oligonucleotides
corresponding to Dn-GRE, Up-GRE, or a nonspecific oligonucleotide prior
to incubation with labeled oligonucleotide. Our results indicate that
GR binds specifically to the functional Dn-GRE to retard its mobility
(Fig. 7).

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Fig. 7.
GR binds to ENaC
Dn-GRE in gel mobility shift assays. End-labeled ENaC Dn-GRE
incubated with purified GR in the presence or absence of cold
competitor and then analyzed by PAGE. GR binds to Dn-GRE to retard its
mobility in lanes 2 and 3 ("shifted
probe"). The binding of GR is inhibited strongly by a 50-fold excess
of cold Dn-GRE (lane 4), and weakly by an excess
of cold Up-GRE (lane 5) but not by a nonspecific
(NS) competitor (lane 6).
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We next examined amiloride-sensitive Na+ transport and
ENaC gene expression in M-1 cells, a mouse CCD cell line. This cell line, established from an SV40 transgenic mouse, appears to have many
of the characteristics of the native CCD including the presence of
amiloride-sensitive Na+ transport (17). Dexamethasone
treatment leads to marked stimulation of Na+ transport
within 24 h (Fig. 8A). To
determine the mechanism of this effect, we examined the expression of
mouse
ENaC mRNA in these cells when treated with dexamethasone.
We show a potent stimulation of
ENaC mRNA expression by 100 nM dexamethasone in these cells (Fig. 8B). To
determine if the stimulation of m
ENaC mRNA expression occurs at
the level of gene transcription, an
ENaC-luciferase construct that
included the GC-responsive region (
487 +661) was transfected into M-1
cells and then the effect of 24 h of stimulation with 100 nM dexamethasone was studied. As in H441 cells,
dexamethasone increased luciferase gene transcription via
ENaC
genomic fragments about 5-10-fold (Fig. 8C). These
experiments suggest that the GC-mediated induction of
ENaC gene
transcription is similar in airway epithelia and in CCD.

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Fig. 8.
Glucocorticoid-mediated
ENaC gene transcription in M-1 and HT29 cells.
Panel A, amiloride-sensitive Isc in M-1 cells
treated for 24 h with dexamethasone (dex).
Dexamethasone increases Isc in M-1 cells (n = 15-16
determinations ± S.E.). *, p < 0.001 compared
with control. Panel B, M ENaC and -actin
mRNA expression following dexamethasone stimulation for 24 h
in M-1 cells. Dexamethasone increases ENaC mRNA expression
(measured by RPA and expressed as a ratio of -actin levels) in M-1
cells (n = 3 ± S.E.). *, p < 0.02 compared with control. Panel C, the human
ENaC construct  ENaC/ 487+661 or pGL3basic was transfected into
M-1 cells and then exposed to dexamethasone and luciferase activity
measured 24 h later. Dexamethasone stimulates transcription of an
ENaC/luciferase chimeric construct. *, p < 0.001 compared with pGL3basic; #, p < 0.01 compared with
control. Panel D, the human ENaC
construct ENaC/ 487+661 co-transfected with the rat GR in
HT29 cells, then exposed to dexamethasone and luciferase activity
measured 24 h later. The ENaC construct shows constitutive
activity but no further stimulation with dexamethasone unless the GR is
exogenously expressed. p < 0.05 compared with
control.
|
|
To confirm that the GR is required for the GC effect on
ENaC gene
transcription, we studied HT29 cells, a human colonic epithelial cell
line. While these cells express the
ENaC mRNA constitutively (16), we had previously noted that there was no detectable increase in
mRNA expression with GC.2
When the
ENaC gene construct containing the GC-responsive regions (
487 +661) was transfected into these cells, constitutive or basal
activation was seen consistent with the presence of an active
ENaC
promoter (Fig. 8D). When treated with 100 nM
dexamethasone, no further increase was seen unless the GR was
co-transfected with the luciferase construct (Fig. 8D).
These results suggest that these cells lack GR or that the endogenous
GR is not available for binding or trans-activation
following stimulation with GC. Complementation with heterologously
expressed receptor confirms that the GR is required for the GC effect
on
ENaC gene transcription.
 |
DISCUSSION |
Corticosteroids are important physiological regulators of
transepithelial sodium transport in the distal nephron, the distal colon, and the airway epithelia. With the cloning of the ENaC subunits,
the effect of MC and GC on ENaC mRNA expression has been studied in
a variety of epithelial cells and tissues from many species. The
chronic effects of these hormones on Na+ transport coincide
with an increase in mRNA expression of one or more subunits of the
ENaC complex suggesting that synthesis of new ENaC channels may account
for the increase in Na+ transport. However, the exact
mechanism of the corticosteroid regulation of ENaC subunits is unknown.
We examined
ENaC expression in H441 cells, a human lung cell line
where all ENaC subunits are expressed (
,
(Refs. 16 and 19);
(see Footnote 2)). When allowed to grow as a polarized epithelium,
these cells develop an amiloride-sensitive Isc that is enhanced by GC
treatment (Fig. 1). We then used these cells to explore the effect of
GC on
ENaC gene expression. It is important to note that this cell
line expresses all four principal transcripts of h
ENaC described to
date and the transcription start sites are identical to that seen in
human kidney and lung tissue (16). We show that GC increased expression
of both exon 1A- and 1B-initiated transcripts in a
dose-dependent manner and that this effect was blocked by
RU38486 and by actinomycin D. These results suggested that gene
transcription following receptor binding is required for the GC effect
on
ENaC expression in these cells.
To address the mechanisms of transcriptional regulation of the h
ENaC
gene and to identify its promoters, we cloned portions of the
5'-flanking region. Sequence analysis of the region upstream of each
transcription start site did not reveal a canonical TATA box. TATA-less
promoters tend to initiate transcription at multiple sites. Thus, it
seemed possible that a single upstream promoter could initiate
transcription at both
ENaC start sites. Our results show that
genomic fragments directly upstream of each transcription start site
were able to drive luciferase expression in H441 cells (Fig.
4A) consistent with the presence of dual promoters. The approximate limits of each promoter were mapped by deletional analysis
and appear to include 82 nt of 5'-flanking region for
ENaC-1 and 191 nt of 5'-flanking region for
ENaC-2 (Fig. 4, B and
C).
In addition to basal or constitutive activity, we identified
GC-inducible transcriptional activity that mapped to a region upstream
of
ENaC-1 and contained imperfect GREs. Deletional analysis demonstrated that the Dn-GRE but not the Up-GRE was functional (Fig.
6B). Comparison of the GREs showed that the Dn-GRE was an imperfect palindromic repeat with a 3-nt spacer that separates each
hexameric half site and differs from the classic GRE
(AGAACAnnnTGTTCT) by a single nt. The Up-GRE is an imperfect
direct repeat with a 3-nt spacer that differs from the classic GRE by 3 nt. Steroid hormone receptors typically bind as homodimers to bipartite
response elements that are palindromic and separated by 3 nt. However, several natural examples of direct repeats that serve as targets for GR
binding are known including the mouse mouse mammary tumor virus
promoter (20). Unlike palindromic repeats, direct repeats separated by
only 3 nt have poor binding affinity for glucocorticoid and estrogen
receptors but this binding improves when separated by 6 or 9 nt (21).
Our results with transient transfection demonstrating that the
palindromic repeat, Dn-GRE, is functional while the direct repeat,
Up-GRE, is nonfunctional is in keeping with this paradigm.
A search of rodent sequences in GenBank using the Patscan
program at
http://www.mcs.anl.gov/compbio/PatScan/HTML/patscan.html revealed at least two genes whose GREs were functional and identical to
the Dn-GRE in
ENaC (22, 23). One of these, the rat
phenylethanolamine N-methyltransferase (PNMT) gene, has a
functional GRE, AGAACAgagTGTCCT,
513 nt to the transcription start
site (22). In this system the GRE may not act entirely alone. Native
PNMT expression is stimulated 40-fold with dexamethasone but only
5-fold with a reporter construct containing the GRE. In our experiments
in H441 cells, the magnitude of dexamethasone induction of
ENaC
mRNA expression matched that seen with the
248 + 44 construct
(Fig. 6B), suggesting that all the required
cis-elements for glucocorticoid stimulation are present
within this region. Clearly, sequences adjacent to the Dn-GRE (between
248 and
142) are required for the full glucocorticoid effect as
the Dn-GRE alone increased gene transcription by just 3-fold compared
with 15-fold with the
248+44 construct (Fig. 6B).
We have demonstrated by transfection, gel mobility shift, and
trans-activation assays that glucocorticoids acting through their cognate receptor stimulate
ENaC gene transcription via an
imperfect GRE upstream of the h
ENaC gene. GREs can bind MR in
vitro, and so far no distinct mineralocorticoid response elements have been identified (24). Currently, the GREs are thought to be the
natural targets for activated MR. In vivo, in
aldosterone-responsive epithelia the effect of GC on ENaC expression is
very similar to the effect of MC (1) and it is quite likely that the MC effect is also via the
ENaC GRE. It is important to note, however, that the molecular responses to MC and GC have some differences (12,
25). Mechanisms proposed to explain these differences include the
specific metabolism of endogenous GC to an inactive metabolite in
aldosterone-responsive epithelia and the combinatorial regulation by GR
but not MR at composite response elements for steroid receptors and
other trans-acting factors (26, 27).
The effect of corticosteroids on ENaC gene expression in
vivo is tissue-specific with distinct effects on different
subunits in the kidney, colon, and lung. For example, dexamethasone and aldosterone increase
ENaC mRNA in the kidney, but
and
ENaC mRNA in colon (8-10, 28). Under certain culture
conditions, we can induce
ENaC gene transcription in response to
glucocorticoids in a colonic cell line, HT29 (Fig. 8D). The
fact that the
ENaC gene is not induced in the colon in
vivo even though steroid receptors are present implies that the
effect of corticosteroids on these subunits is not a simple consequence
of receptor binding and activation of a GRE. The factors that determine
tissue-specific regulation may include steroid receptor cofactors such
as SRC-1 and SMRT (29, 30) but this remains to be elucidated.
 |
ACKNOWLEDGEMENTS |
We thank Raju Kucherlapati and Kate
Montgomery for sharing information on the SCNN1A locus, David Pearce
and Keith Yamamoto for the GR expression vector, Geza Fejes-Toth for
the M-1 cell line, and Tom Schmidt and Curt Sigmund for helpful
discussions. We acknowledge the DNA synthesis and sequencing services
provided by the University of Iowa DNA core facility.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid from the
American Heart Association and the American Lung Association, by March
of Dimes Foundation Research Grant 6-FY97-0435, and by United States
Public Health Services Grant DK 52617. Portions of this work were
presented at the American Society of Nephrology and the Central Society
for Clinical Research Annual Meetings in Philadelphia, Oct. 25-28,
1998 and in Chicago, Sept. 17-19, 1998, respectively.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.
§
These authors contributed equally to this work.
To whom correspondence should be addressed: Div. of
Nephrology, Dept. of Internal Medicine, University of Iowa Hospitals
and Clinics, 200 Hawkins Dr., Iowa City, IA 52242-1081. Tel.:
319-356-4216; Fax: 319-356-2999; E-mail:
christie-thomas{at}uiowa.edu.
2
C. P. Thomas, unpublished observation.
 |
ABBREVIATIONS |
The abbreviations used are:
ENaC, epithelial
sodium channel;
MC, mineralocorticoid(s);
GC, glucocorticoid(s);
IMCD, inner medullary collecting duct;
Isc, short circuit current;
RT, resistance;
RPA, ribonuclease protection assay;
bp, base pair(s);
nt, nucleotides;
UTR, untranslated region;
GRE, glucocorticoid response element;
GR, glucocorticoid receptor;
MR, mineralocorticoid receptor;
CCD, cortical collecting duct;
PNMT, phenylethanolamine N-methyltransferase;
SRC-1, steroid
receptor co-activator 1;
SMRT, silencing mediator for retinoid and
thyroid hormone receptors;
PAGE, polyacrylamide gel
electrophoresis.
 |
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