1 Département de Médecine, Centre de Recherche, Centre Hospitalier de l'Université de Montréal-Hôtel-Dieu, Université de Montréal, Montreal, Quebec H2W 1T8; and 2 Centre for Molecular Medicine, Ottawa General Hospital Research Institute, Ottawa, Ontario K1H 8L5, Canada
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ABSTRACT |
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cAMP and
dexamethasone are known to modulate Na+ transport in
epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and
dexamethasone modulate the mRNA expression of two key elements of the
Na+ transport system in isolated rat alveolar epithelial
cells: -,
-, and
-subunits of the epithelial Na+
channel (ENaC) and the
1- and
1-subunits
of Na+-K+-ATPase. The cells were treated for up
to 48 h with DBcAMP or dexamethasone to assess their long-term
impact on the steady-state level of ENaC and
Na+-K+-ATPase mRNA. DBcAMP induced a twofold
transient increase of
-ENaC and
1-Na+-K+-ATPase mRNA that peaked
after 8 h of treatment. It also upregulated
- and
-ENaC mRNA
but not
1-Na+-K+-ATPase mRNA.
Dexamethasone augmented
-ENaC mRNA expression 4.4-fold in cells
treated for 24 h and also upregulated
- and
-ENaC mRNA. There was a 1.6-fold increase at 8 h of
1-Na+-K+-ATPase mRNA but no
significant modulation of
1-Na+-K+-ATPase mRNA expression.
Because DBcAMP and dexamethasone did not increase the stability of
-ENaC mRNA, we cloned 3.2 kb of the 5' sequences flanking the mouse
-ENaC gene to study the impact of DBcAMP and dexamethasone
on
-ENaC promoter activity. The promoter was able to
drive basal expression of the chloramphenicol acetyltransferase (CAT)
reporter gene in A549 cells. Dexamethasone increased the activity of
the promoter by a factor of 5.9. To complete the study, the
physiological effects of DBcAMP and dexamethasone were investigated by
measuring transepithelial current in treated and control cells. DBcAMP
and dexamethasone modulated transepithelial current with a time course
reminiscent of the profile observed for
-ENaC mRNA expression.
DBcAMP had a greater impact on transepithelial current (2.5-fold
increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of
-ENaC and Na+-K+-ATPase gene expression is one of the
mechanisms that regulates Na+ transport in alveolar
epithelial cells.
sodium channel; adenosine 3',5'-cyclic monophosphate; steroid; sodium-potassium-adenosinetriphosphatase; transepithelial current; epithelial sodium channel
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INTRODUCTION |
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VECTORIAL SODIUM TRANSPORT
from alveoli to the interstitium plays an important role in the removal
of fetal lung liquid at birth (39) and the clearance of
liquid in pulmonary edema (4, 32). Recent experimental
data have allowed us to better define the structure and function of the
major systems involved in this transepithelial transport.
Na+ entry occurs in part by amiloride-sensitive
Na+ channels located at the apical surface of the cells and
is extruded by the sodium pump (Na+-K+-ATPase)
located at the basolateral surface (5, 32). The Na+ channel involved in this system, the epithelial
Na+ channel (ENaC), has been cloned and consists of three
subunits, -,
-, and
-ENaC (8). In situ
hybridization and immunohistochemical staining have shown that ENaC
subunits are expressed in alveolar epithelial cells (18,
31). The physiological role of
-ENaC in the lung has been
demonstrated in a mouse model where the
-ENaC gene was deleted by
targeting a transgene by homologous recombination (24).
Unable to clear liquid from their lungs, these mice die shortly after
birth (24). The other major component of the
transepithelial Na+ transport system is
Na+-K+-ATPase, consisting of two subunits. The
-subunit, the catalytic component of the complex, is involved in
Na+ extrusion, K+ influx, and ATPase activity
(15). The
-subunit is a highly glycosylated protein,
the role of which is not well understood but seems to be an important
regulatory component of the sodium pump (15, 17). The
1- and
1-isoforms are the subunits that have been detected in the lungs (41, 43). Inhibition of
Na+-K+-ATPase with ouabain greatly reduces
solute and water transport in alveoli (3) and the
short-circuit current (Isc) of alveolar epithelial cells (12). Enhanced expression of either the
1 (16, 17)- or
1- and
1-subunits (53) in the lung protects against pulmonary edema and enhances lung liquid clearance.
Several studies have evaluated the changes in -ENaC and
1-Na+-K+-ATPase expression in
physiological or pathophysiological conditions where lung liquid
clearance is important. Modulation of
-ENaC (40) and
-Na+-K+-ATPase mRNA (41) has
been reported at birth when Na+ transport allows clearance
of the alveolar spaces (39). The expression of
-ENaC
(63) and Na+-K+-ATPase (21,
36, 65) is also modulated in the lungs and alveolar epithelial
cells during the induction or resolution of lung injury. Although it
has been suggested that modulation of Na+ transport could
be an important therapeutic avenue in treatment of lung injury
(5, 55), there is little information regarding the
pharmacological regulation of
-ENaC and
Na+-K+-ATPase expression. We observed recently
that sustained treatment of alveolar epithelial cells with the
-agonist terbutaline enhances
-ENaC and
Na+-K+-ATPase expression (34),
suggesting that cAMP could be involved in their regulation.
Dexamethasone, a synthetic steroid, is known to increase
-ENaC mRNA
expression in the fetal lung (56) and in cultured fetal
epithelial cells (10) where it raises the amiloride-sensitive current (10) and amiloride-sensitive
alveolar fluid clearance in adult rats (20). Recent
findings have shown that functional glucocorticoid regulatory elements
(GRE) are present in the promoter of
-ENaC (44, 51),
indicating that dexamethasone could act on ENaC-mediated
Na+ transport by affecting the expression of the channel at
the gene level. Dexamethasone also upregulates the expression of the
1-Na+-K+- ATPase subunit in
alveolar epithelial cells (2).
Although modification of ENaC and Na+-K+-ATPase
gene expression could be important for lung liquid clearance, the best
pharmacological strategy to enhance the expression of the
Na+ channel and pump in lung epithelial cells has still not
been found. In the present work, we studied the regulation of ENaC (,
, and
) and Na+-K+-ATPase
(
1 and
1) gene expression by cAMP and
dexamethasone in alveolar epithelial cells isolated from the adult rat
lung. After evaluating the time course of ENaC and
Na+-K+-ATPase mRNA after treatment with
dibutyryl-cAMP (DBcAMP) or dexamethasone, we
investigated the role of transcription and translation in the modulation of the
-subunits of these genes by exposing the cells to
actinomycin D and cycloheximide. Because DBcAMP and dexamethasone did
not increase the stability of
-ENaC mRNA, we cloned and sequenced 2.7 kb of mouse
-ENaC 5'-flanking DNA and tested the activity of the
promoter in A549 lung epithelial cells. Finally, we assessed the
physiological impact of DBcAMP and dexamethasone on transepithelial current (Ite) generated at times when ENaC mRNAs
are upregulated.
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MATERIALS AND METHODS |
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Alveolar epithelial cell isolation and experimental conditions. Alveolar epithelial cells were isolated from male Sprague-Dawley rats as described previously (19). Perfused lungs were digested with elastase, and the cells were purified by a differential adherence technique on bacteriological plastic plates coated with rat IgG (19). The cells were maintained in MEM (Life Technologies, Burlington, Ontario, Canada) containing 10% FBS (GIBCO BRL, Life Technologies), 0.08 mg/l gentamicin, 0.2% NaHCO3, 0.01 M HEPES, and 2 mM L-glutamine. Cells were plated at 4 × 105 cells/cm2 in 25-cm2 flasks and cultured at 37°C with 5% CO2 in a humidified incubator. The medium was replaced every 2-3 days.
The effect of DBcAMP and dexamethasone was tested onNorthern blotting.
Total RNA from alveolar epithelial cells was extracted by a
modification of the guanidinium-phenol technique (14),
except for the determination of -ENaC mRNA stability, where RNA was purified with Trizol reagent according to the manufacturer's protocol (Life Technologies). For Northern blotting, 10 µg of total RNA were
electrophoresed on a 1% agarose-formaldehyde gel and transferred to
GeneScreen nylon membranes (NEN, Boston, MA) by overnight blotting with
10× saline-sodium citrate. Hybridization was performed, as reported
previously, in 0.5 M sodium phosphate, pH 7.2, 7% SDS (wt/vol),
and 1 mM EDTA, pH 8 (14). Blots were exposed to Kodak X-AR
film, using an intensifying screen, or to a phosphorimager (Molecular
Dynamics, Sunnyvale, CA) for densitometric analysis. The nylon
membranes were hybridized successively with different cDNA probes
(
-,
-, and
-ENaC,
1- and
1-Na+-K+-ATPase,
-actin, and
18S rRNA).
-ENaC mRNA was detected with a-764 bp mouse
-ENaC cDNA
(His445 to the stop codon), which has a high homology with
rat
-ENaC cDNA (14).
- and
-ENaC mRNAs were
detected with the complete cDNA clone, a gift from Dr. B. C. Rossier (Institut de Pharmacologie et de Toxicologie, Université
de Lausanne, Lausanne, Switzerland). The
1- and
1-Na+-K+-ATPase probes were
gifts from Dr. J. Orlowski (Physiology Department, McGill University,
Montreal, Quebec, Canada). The
1-Na+-K+-ATPase probe is the
same as that used previously (14) and consisted of a
NarI-StuI 332-bp cDNA fragment coding from
nuclear transcript (nt) 89 to 421 [from the 5'-untranslated region
(5'-UTR) to Arg61]. The
1-Na+-K+-ATPase probe consisted
of a NcoI-SspI 750-bp cDNA fragment that encompasses the entire coding region (62). For
quantitative study,
-ENaC mRNA expression was normalized to
-actin expression to ensure that the same amount of RNA was present
on each lane. The
-actin probe was a gift from Dr. P. Hamet (Centre
Hospitalier de l'Université de Montréal-Hôtel-Dieu,
Montreal, Quebec, Canada) and consisted of a PstI 1.5-kb
cDNA fragment coding for rat brain
-actin (38). Because
actinomycin D stops the transcription of all RNA, including the
-actin transcript, in studies involving actinomycin D treatment, we
chose to normalize the amount of RNA loaded on the gel with 18S rRNA.
The 18S rRNA probe consisted of a 640-bp cDNA fragment that had been
amplified by RT-PCR between nt 852 and 1492 of the rat 18S rRNA
sequence (11). For hybridization, the probe was labeled by
random priming, and the membrane was hybridized in a plastic container
closed with a tight cap. The reproducibility of 18S hybridization was
also tested by hybridization with an 18S oligonucleotide
(5'-GTTATTGCTCAATCTCGGTGG-3') labeled with [
-32P]ATP
by T4 polynucleotide kinase, and the membrane was hybridized in a
plastic container as described above. The relative amount of 18S rRNA
from lane to lane was similar to that found with cDNA hybridization.
For reproducibility and statistical reasons, Northern blotting was
repeated several times with RNA extracted from cells isolated from
different animals. The numbers (n) in the legends for Figs.
1-7 refer to experiments performed in different animals.
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Cloning and sequencing of the mouse -ENaC gene.
To determine the nature of the regulatory sequences that could drive
-ENaC gene expression, we screened a mouse genomic library kindly
provided by Drs. A. Reaume and R. Zirngibl (7). This library was produced by Sau3A partial digestion of genomic
DNA from a 129-Sv mouse ligated into BamHI-cut
DASH II
vector (Stratagene, La Jolla, CA). Plaques (1 × 106)
were probed with two mouse
-ENaC cDNAs (nt 76-1676 and mouse
-ENaC nt 1333-2097; see Ref. 14) according to
standard procedures (47). A single 20-kb DNA clone was
isolated. From this clone, a 3,228-bp BamHI fragment that
strongly hybridized to rat
-ENaC nt 0-223 probe
(14) was subcloned into pBluescript KS (Stratagene) and
sequenced in the presence of DMSO by the dideoxy technique (Amersham
Pharmacia Biotech, Baie d'Urfé, Quebec, Canada; see Ref.
43), using the T3 and T7 primers of the vector.
The full sequence of 3.2-kb BamHI genomic DNA was determined
with a set of nested deletions generated by exonuclease III digestion
(Erase-a-base system; Promega, Madison, WI). A computer search for
putative regulatory elements of the mouse
-ENaC promoter was
undertaken with GeneWorks software (Intelligenetics, Mountain View, CA)
and Transcription Element Search Software (TESS) developed by J. Schug and G. C. Overton of the Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania (URL: http://agave.humgen.upenn.edu/tess/index.html).
5'-Rapid amplification of cDNA ends.
The transcription initiation site was determined by 5'-rapid
amplification of cDNA ends (RACE) from RNA purified from the CD1 mouse
lung and kidney. Total RNA (20 µg) was heated for 5 min at 65°C and
kept on ice for 3 min. cDNA was synthesized with 62.5 units of avian
myeloblastosis virus RT (Roche Molecular Biochemicals, Laval, Quebec,
Canada) using 500 pmol of random dNTP hexamer (Amersham Pharmacia
Biotech) as primer (22) and incubated for 60 min at 55°C
in reverse transcription buffer (50 mM Tris · HCl, pH 8.5, 8 mM
MgCl2, 30 mM KCl, and 1 mM dithiothreitol) with a mixture of dNTPs (1 mM each) and human placental RNase inhibitor (25 units; Life Technologies). After a 10-min incubation at 65°C, the cDNA was
purified over a G-50 Sephadex spun column, dried by lyophilization, and
resuspended in 19 µl of sterile H2O. Tailing of
deoxyadenosine 5'-triphosphate (0.3 mM) was performed in 100 mM
potassium cacodylate, pH 7.2, 1 mM CoCl2, and 0.1 mM
dithiothreitol with 30 units of terminal transferase (Life
Technologies) for 1 h at 37°C followed by a 10-min incubation at
70°C. For PCR amplification, 20 pmol (0.4 µM) of the sense primer
5'-GGA ATT CTC GAG ATC GAT GCT T(16)-3' containing an EcoRI site, 50 pmol (1 µM) of the sense
primer 5'-GGA ATT CTC GAG ATC GAT GCT, and 50 pmol (1 µM) of the
antisense primer 5'-CGG GAT CCT TGC ATG GGC AGA GGA GGA C-3'
corresponding to nt 191-211 of the rat -ENaC gene linked to a
BamHI site were used as described (14). The PCR
conditions were 35 cycles for 1 min at 94°C for denaturation, 1 min
at 52°C for annealing, and 2 min at 72°C for extension followed by
a 7-min final extension at 72°C. RACE DNA was digested with
BamHI and EcoRI and ligated to the corresponding
sites in pBluescript KS (Stratagene).
Transient transfection and chloramphenicol acetyltransferase
assay.
For the reporter gene assay, a BamHI-MscI 2.9-kb
fragment of the mouse -ENaC genomic clone was linked upstream of the
chloramphenicol acetyltransferase (CAT) gene in pjfCAT vector (a gift
from Dr. Pierre-André Bédard, York University, North York,
Ontario, Canada) at an Msc I site 161 nt upstream of the
open reading frame but within the 5'-UTR downstream of the first
transcription initiation start site. For transfection, the plasmid was
purified with the QIAEX II kit (QIAGEN, Mississauga,
Ontario, Canada) according to the manufacturer's protocol. A549 cells
were a generous gift from Dr. André Cantin (Pneumology Division,
Département de Médecine, Université de Sherbrooke).
The cells were cultured in DMEM with 10% FBS in the presence of
penicillin (50 U/ml) and streptomycin (50 µg/ml). The day before
transfection, 8 × 105 cells were seeded in 60-mm
dishes and cultured for 24 h to reach 80% confluency. Plasmid (6 µg; 4 µg of ENaC-CAT plasmid and 2 µg of pSV-
-galactosidase)
mixed with 12 µl of transfection reagent (Superfect;
QIAGEN) were incubated for 10 min at 21°C in 150 µl of
culture medium devoid of antibiotic and serum. The DNA complex was
added to cells in 1 ml of culture medium (complete with FBS and
antibiotic) and incubated for 3 h at 37°C. After transfection (48 h), the cells were collected, resuspended in 40 µl of 250 mM
Tris · HCl at pH 8, and lysed by three cycles of freeze-thawing (dry ice-ethanol for 5 min at 37°C). CAT assay and
-galactosidase assay were performed as described (47, 60). After the cell debris was pelleted by centrifugation (13,000 rpm) and the supernatant was heated for 10 min at 65°C, 20 µl of the cell lysate were
incubated with 2 µl of 200 µCi/ml
[14C]chloramphenicol, 20 µl of 4 mM acetyl-CoA, 32.5 µl of 1 M Tris · HCl, pH 7.5, and 75.5 µl of
H2O for 1 h at 37°C. CAT was removed by extraction
with 2 volumes of tetramethylpentadecane-xylene (2:1) by vigorous
shaking, and the top organic phase was counted by scintillation in a
-counter (1). A549 cells were transfected at
least four different times in duplicate.
-Galactosidase activity was
used to normalize the differences in transfection efficiency arising
from plate to plate. CAT activity was reported in arbitrary units
consisting of the 14C counts per minute of the sample minus
the 14C counts per minute of untransfected cells.
Electrophysiology.
For electrophysiological studies, two techniques were employed. Because
we wanted to evaluate the long-term effects of DBcAMP and dexamethasone
in most experiments, potential differences across the monolayers (PD;
mV) and transepithelial resistance (Rte;
· cm2) were measured successively with an
epithelial voltohmmeter (EVOM; World Precision Instruments, Sarasota,
FL). Alveolar cells plated at a density of 1 × 106
cells/cm2 on polycarbonate membranes (1.0 cm2;
Costar Transwell, Toronto, Ontario, Canada) were cultured for 3 days
until the cells reached confluence and were then treated with 1 mM
DBcAMP, 100 nM dexamethasone, or a combination of 1 mM DBcAMP plus 100 nM dexamethasone added on their apical and basolateral sides. EVOM
measurements were performed at selected times (0, 8, 24, 32, and
48 h) after the initiation of treatment. Ite across these monolayers was calculated by
the following formula: Ite = PD/Rte. Measurements were done in triplicate for
each time point and treatment condition in cells purified from three
different animals. To quantify the amount of amiloride-sensitive
current generated by alveolar cells after an 8-h treatment with DBcAMP, dexamethasone, or both agents, Ite was
determined successively in the absence and presence of 1 µM amiloride
after a 5-min incubation at 37°C (n = 15 from 5 animals). At this concentration, amiloride is a specific inhibitor of ENaC.
Statistics.
The data are presented as means ± SE. Comparisons between groups
were analyzed by unpaired t-test, ANOVA, and post hoc
comparison using Statview software (SAS Institute, Cary, NC).
P < 0.05 was considered to be significant. The decay
curves of -ENaC mRNA were compared by multiple regression analysis.
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RESULTS |
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Modulation of -ENaC and
1-Na+-K+-ATPase mRNA expression
by DBcAMP and dexamethasone.
To investigate the effect of DBcAMP and dexamethasone on
-ENaC and
1-Na+-K+-ATPase mRNA expression,
alveolar epithelial cells were treated after 3 days of culture when
they reached confluence and were capable of vectorial Na+
transport resulting in the formation of domes. DBcAMP modulated
-ENaC and
1-Na+-K+-ATPase
mRNA expression similarly in these cells (Fig.
1, A and B). There
was a twofold transient rise in the level of
-ENaC and
1-Na+-K+-ATPase mRNA, which
reached a maximum after 8 h of treatment (Fig. 1, A and
B). Forskolin (20 µM), an activator of adenylate cyclase that increases cytosolic cAMP, elevated by 1.9-fold the
-ENaC steady-state mRNA level after 8 h of treatment, demonstrating that
cAMP and not butyrate modulated
-ENaC and
1-Na+-K+-ATPase (data not
shown). When alveolar cells were cultured on permeable membranes,
DBcAMP increased by 1.7-fold the amount of
-ENaC mRNA with 8 h
of treatment, showing that DBcAMP regulation of
-ENaC mRNA is
independent of culture conditions.
- and
-ENaC mRNAs were
also upregulated by DBcAMP.
-ENaC mRNA was regulated similarly to
-ENaC mRNA by DBcAMP, with a 1.9-fold increment after 8 h of
treatment.
-ENaC mRNA expression was modulated differently by DBcAMP
and did not present any increase after 8 h of treatment. There
was, however, a 1.5-fold elevation after 24 h.
1-Na+-K+- ATPase mRNA was not
modulated by DBcAMP. There was a good correlation between
-ENaC and
1-Na+-K+-ATPase mRNA expression
in cells treated with DBcAMP (r = 0.889).
Effect of concurrent treatment with DBcAMP and dexamethasone on
-ENaC and
1-Na+-K+-ATPase
mRNA expression.
To determine if concurrent treatment with DBcAMP and dexamethasone
could have an additive effect on
-ENaC and
1-Na+-K+-ATPase mRNA expression,
alveolar epithelial cells were treated for 8 h with 1 mM DBcAMP,
100 nM dexamethasone, or a combination of the two agents. Densitometric
quantitation of Northern blots showed that cells treated with DBcAMP
and dexamethasone presented a statistically significant increase in
-ENaC and
1-Na+-K+-ATPase
mRNA expression (Fig. 2) compared with
cells treated with either agent alone. This increase was additive for
1-Na+-K+-ATPase mRNA expression
(3.3×) and slightly more than additive for
-ENaC mRNA expression
(6.6×).
Role of transcription and translation in the modulation of -ENaC
and
1-Na+-K+-ATPase mRNA
expression by DBcAMP and dexamethasone.
Next, we investigated the role of transcription and translation in the
modulation of
-ENaC and
1-Na+-
K+-ATPase mRNA expression by DBcAMP and dexamethasone. The
transcription inhibitor actinomycin D (5 µg/ml) abolished the
increase of
-ENaC mRNA detected after 8 h of treatment with
DBcAMP or dexamethasone (Fig.
3A and Table
1). It also diminished the increase of
1-Na+-K+-ATPase mRNA in
DBcAMP-treated cells (Fig. 3A and Table 1). The translation
inhibitor cycloheximide (2.5 µg/ml) decreased the basal level of
-ENaC mRNA expression by 75% (Fig. 3B and Table 1).
However, it did not abolish the increase in
-ENaC mRNA elicited by
dexamethasone (Fig. 3B and Table 1). Although the level of
-ENaC mRNA expression in DBcAMP plus cycloheximide-treated cells was
56% of the untreated control value, DBcAMP in these cells still
doubled the expression of the gene compared with cycloheximide treatment alone (Table 1). The basal expression level of
1-Na+-K+-ATPase transcripts was
not decreased by cycloheximide. However, cycloheximide inhibited the
increase in
1-Na+-K+-ATPase mRNA
evoked by DBcAMP (Fig. 3B and Table 1).
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Cloning of the 5'-flanking region of mouse -ENaC gene.
Although the promoter of the
1-Na+-K+-ATPase gene has been
cloned and characterized (52, 54), the
-ENaC promoter
has been cloned only recently (30, 51, 57). To determine
the nature of the regulatory sequence that could drive
-ENaC gene
expression, a mouse genomic DNA library was probed with the following
two mouse
-ENaC cDNAs: mouse
-ENaC nt 76-1676 and mouse
-ENaC nt 1333-2097 (14). A 3.2-kb BamHI
fragment consisting of part of exon 1 comprising the start of
translation (ATG), the 5'-UTR, and 2.7 kb of the 5'-flanking sequence
was cloned and sequenced (Fig. 5).
Several potential transcription initiation sites were found by 5'-RACE
for lung and kidney RNA (Fig. 5). No TATA box or CCAAT box was seen.
Numerous GRE half-sites and one progesterone receptor were noted (Figs.
5 and 6). Only one GRE at positions
718
to
732 bp of the first potential transcription site had the proper
orientation and spacing to be a functional GRE (Fig. 5). Two consensus
sequences for activating transcription factor (ATF)/cAMP responsive
element binding (CREB) transcription factors at positions
1756 and
+572 bp of the longer transcription site could modulate cAMP
transcription of the gene (Fig. 5). A CREB/c-jun binding
site was also detected at position
1695 bp. Other consensus sequences
for the binding of activator protein (AP)-1, AP-2, Sp1, Ets, GATA-1,
and PEA3 transcription factor were also seen with a degenerate nuclear
factor-
B (Fig. 5).
Activity of the mouse -ENaC promoter in A549 cells.
To test the activity of the mouse
-ENaC promoter, a
BamHI-MscI 2.9-kb fragment linked to a CAT
reporter gene was transfected in A549 lung epithelial cells. The
fragment encompasses 2.45 kb of the sequence upstream to the first
transcription initiation start site (Fig. 6A). Although CAT
expression driven by the promoter was modest, it was statistically
different from cells transfected with the pjfCAT vector alone
(P < 0.05; Fig. 6B). Treatment with DBcAMP
for 8 h caused nonsignificant changes in the expression of the CAT
gene compared with those in untreated cells (Fig. 6B). Dexamethasone treatment for 24 h increased CAT activity in these cells by a factor of 5.9 (Fig. 6B).
Impact of DBcAMP and dexamethasone on Ite.
To determine if changes in ENaC and
Na+-K+-ATPase mRNA expression have an impact on
ion transport, we measured PD and Rte with EVOM
and calculated the Ite of alveolar epithelial
cells grown on permeable filters treated with DBcAMP, dexamethasone, or
a combination of the two agents (Fig. 7).
There was a significant increase of Ite 8 h
after the beginning of treatment with DBcAMP (Fig. 7A) or
DBcAMP plus dexamethasone (Fig. 7C). This heightened Ite decreased gradually toward control values
over 48 h. Dexamethasone also induced an increase of
Ite that was smaller than with combined treatment and reached its maximum at 24 h (Fig. 7B).
Forty-nine to fifty-eight percent of Ite
determined after 8 h of treatment for stimulated and unstimulated
cells was suppressed with 1 µM amiloride, a specific inhibitor of
ENaC at this concentration (Table 2). To
confirm the modulation of Ite detected with
EVOM, the Isc of cells treated for 8 h with
DBcAMP and dexamethasone was recorded with the Ussing system. Treated
cells showed a marked increase in Isc compared
with untreated cells (Fig. 7D).
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DISCUSSION |
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In this study, we found that both DBcAMP and dexamethasone
modulate ENaC and Na+-K+-ATPase gene expression
in alveolar epithelial cells. The modulation of -ENaC and
1-Na+-K+-ATPase mRNAs by these
two agents involves different pathways and shows a different
physiological response for the two treatments.
Treatment of alveolar epithelial cells with 1 mM DBcAMP modified the
steady-state mRNA level of the three ENaC subunits and the
1- but not the
1-subunits of
Na+-K+-ATPase. The expression of
-ENaC and
1-Na+-K+-ATPase mRNAs doubled
transiently after 8 h of treatment before returning to control
values after 24 h (Fig. 1, A and B).
Although DBcAMP modulated the steady-state level of mRNA with a similar time course, the mechanisms involved in the upregulation of the two
genes were probably different. This increase in
-ENaC and
1-Na+-K+-ATPase mRNA was
probably related to changes in transcription since actinomycin D
inhibited the elevation of
-ENaC and
1-Na+-K+-ATPase mRNA detected
after treatment with DBcAMP (Fig. 3 and Table 1). There was a
difference, however, in response to cycloheximide between
-ENaC and
1-Na+-K+-ATPase mRNAs in
DBcAMP-treated cells. The increase of
-ENaC mRNA was not inhibited
by cycloheximide, suggesting that DBcAMP could directly stimulate the
expression of the transcript in the absence of protein synthesis. In
contrast, the
1-Na+-K+-ATPase
mRNA increase brought by DBcAMP was sensitive to cycloheximide, indicating that the cAMP effect was not direct and required the secondary synthesis of some factor (Fig. 3 and Table 1). One unexpected
finding was that cycloheximide downregulated basal
-ENaC mRNA
expression in alveolar epithelial cells (Fig. 3B). Such an
observation has also been reported for
-,
-, and
-ENaC mRNA in
cultured human fetal lung explants (59). The inhibition of
expression elicited by cycloheximide is not unique to ENaC since other
genes are affected similarly (13). It suggests that a
short-lived labile factor could be important for the basal
transcription or stability of
-ENaC mRNA. The upregulation of
-ENaC mRNA by DBcAMP in alveolar epithelial cells has not been
reported previously.
Dexamethasone differentially regulated the ENaC and
Na+-K+-ATPase expression in alveolar epithelial
cells. The three ENaC subunits were upregulated by dexamethasone.
-ENaC mRNA expression increased markedly after 8 h (3-fold),
24 h (4.4-fold), and 48 h of treatment (Fig. 1C).
The elevation of the
-ENaC steady-state mRNA level by dexamethasone
was sensitive to actinomycin D (Fig. 3B), suggesting again
that regulation in the transcription level of the gene was likely to be
involved in this process. Although dexamethasone has been shown to
modulate
-ENaC mRNA in fetal lungs and cells (56, 59),
in adult rat lungs after adrenalectomy (45), and in a
bronchial epithelial cell line (27), the results reported here are the first to confirm that it augments
-ENaC mRNA expression in adult alveolar epithelial cells. As for DBcAMP, the data with cycloheximide indicate that dexamethasone does not require protein synthesis to affect
-ENaC mRNA accumulation. In contrast to
-ENaC, dexamethasone did not alter
1-Na+-K+- ATPase mRNA
expression in alveolar epithelial cells. There was, however,
upregulation of
1-Na+-K+- ATPase mRNA. These
results are in agreement with several reports showing that
dexamethasone does not increase
1-Na+-K+-ATPase mRNA expression
in alveolar epithelial cells (2) or in the fetal lung
during development (25, 56) but can upregulate the
1-Na+-K+-ATPase mRNA level
(2). There are some differences, however, with other
studies that demonstrated recently that dexamethasone elevates the
expression of
1-Na+-K+-ATPase
mRNA in a rat pre-type II cell line (FD18; see Ref. 9). This discrepancy could be related in part to the fact that the experiments were conducted in a fetal cell line (9) with a different dexamethasone concentration and at different time points.
The results presented above indicate that dexamethasone and DBcAMP
modulate the expression of the -ENaC and
Na+-K+-ATPase genes through distinct pathways.
We therefore tested if combined treatment with DBcAMP and dexamethasone
would have a different impact on
-ENaC and
Na+-K+-ATPase mRNA expression than treatment
with a single agent. Treatment of alveolar epithelial cells with 100 nM
dexamethasone and 1 mM DBcAMP for 8 h had an additive effect on
-ENaC mRNA, raising the expression of these transcripts 6.6-fold.
This is different from the modulation found in 20- to 24-wk fetal lung
explants where 8-bromo-cAMP had no impact on
-ENaC mRNA expression
and did not further increase the heightened expression evoked by
dexamethasone (59). These results suggest again that the
regulatory mechanism may differ between adult and fetal cells. The
presence of dexamethasone also enhanced the increased expression of
1-Na+-K+-ATPase mRNA seen after
8 h of treatment with DBcAMP. This is somewhat surprising since
dexamethasone alone did not heighten the expression of the
1-subunit (Fig. 1D). Although we do not have
a specific explanation for this observation, it is possible that
dexamethasone induces a small increase of the
1-subunit, but we could not identify it because of a lack of power in our protocol. It is also possible that cAMP could activate a factor that is
stimulated directly or indirectly by dexamethasone. Further experiments
will be needed to explore this question.
The steady-state mRNA level of a given gene is a balance between
transcription of new RNA on the one hand and mRNA stability on the
other hand. To determine the mechanisms involved in the modulation of
ENaC mRNA by DBcAMP and dexamethasone, we first studied -ENaC mRNA
stability in control and DBcAMP- and dexamethasone-treated alveolar
cells. We calculated that
-ENaC mRNA had a long half-life (15.1 h),
longer than the half-life reported in the parotid gland (8 h; see Ref.
64) but shorter than the half-life in fetal distal lung
epithelial cells (22 h; see Ref. 44). DBcAMP and
dexamethasone did not increase the stability of the
-ENaC
transcript, suggesting that regulation in the transcription of the gene
and not an increase in mRNA stability could be involved in the
modulation brought by these two products (Fig. 3). Other factors
besides transcriptional change could be involved, however, in the
control of
-ENaC mRNA accumulation. This hypothesis is suggested by
the significant decrease of
-ENaC mRNA stability elicited by
dexamethasone (Fig. 3) in alveolar epithelial cells, an interesting
observation that should be investigated further.
Because transcription could be involved in the modulation of -ENaC
mRNA expression, we sought to identify putative regulatory elements in
the
-ENaC promoter that could participate in the change produced by
DBcAMP and dexamethasone. We cloned and sequenced 2.9 kb of the
5'-flanking region of the mouse
-ENaC gene, which presents some
similarities and differences with the human
-ENaC gene
(57). The sequence of 5'-RACE products is identical to that of the genomic clone, showing that in the mouse, as demonstrated in the rat (44), the 5'-UTR and start of the open-reading
frame (ATG) are part of exon 1. This is in contrast to the human
-ENaC gene (57) where a 665- to 190-nt intron has been
found in conjunction with alternative splicing between some of the
initiation start sites and exon 2. As for human
- and
-ENaC genes
(57, 58) and the rat
-ENaC gene, several potential
transcription initiation sites with two major sites at
606 and
72
bp of ATG have been identified in the mouse by 5'-RACE. These multiple
transcription start sites may be associated with the lack of TATA or
CCAAT boxes in the 5'-flanking sequence of ENaC genes. The 5'-flanking
sequence of the mouse
-ENaC gene, as for other genes devoid of a
TATA box, is very rich in GC content. It lacks, however, GC boxes and GC box homologous motifs identified for human
- and
-ENaC genes (57, 58). Sp1-binding elements used in some TATA-less
promoters for transcription initiation (42) are
nevertheless found at 10-13 bp of some transcription initiation
sites (
606 and
287 bp). In this promoter region, we have also
identified multiple regulatory sites that could be important for the
modulation of ENaC expression by cAMP (CREB/c-jun
1689 bp
and ATF/CREB
1751 bp) or dexamethasone (glucocorticoid receptor
binding sequence
718 bp).
To determine if these putative sites could play a role in ENaC
expression, we measured the impact of DBcAMP and dexamethasone on
-ENaC promoter activity in A549 cells. This cell line was chosen
because it has some characteristics of alveolar epithelial cells,
expresses
-ENaC mRNA (28, 29), and has been used by others to study ENaC promoter activity (44, 61). The
-ENaC genomic clone acts as an active promoter, driving the
expression of the CAT reporter gene in A549 cells. The activity of the
promoter in unstimulated cells is very weak and reflects the low basal expression of
-ENaC mRNA in these cells (data not shown). Upon stimulation with dexamethasone, however, there was a sixfold increase in the activity of the promoter compared with that in unstimulated cells. Thus, in A549 cells, dexamethasone significantly augmented the
transcription driven by the
-ENaC promoter. Similar results have
been obtained recently for the rat and human
-ENaC promoter (30, 44, 51). The
718- to
732-bp GRE that we find in
the mouse
-ENaC promoter (AGAACAgaaTGTCCT) is identical in sequence and position, relative to ATG, to the functional GRE detected in the
rat promoter (30, 44). Although the profile of
-ENaC mRNA elicited by DBcAMP (transient activation followed by
fading expression) is reminiscent of a gene regulated by a cAMP
response element (CRE) (35, 50), we could not stimulate
the activity of the promoter with DBcAMP. Several hypotheses could be
put forward to explain these results. It is possible that the 2.9-kb
fragment tested does not contain an active CRE or that the
-ENaC
gene promoter is regulated differently in A549 and alveolar epithelial cells. Finally, because
-agonists and DBcAMP have been observed to increase intracellular calcium concentration
([Ca2+]i) in alveolar epithelial cells
(37, 49), we cannot exclude the possibility that the rise
in [Ca2+]i could be involved in the
modulation of transcription, as demonstrated for other genes (26,
46).
The modulation of ENaC and Na+-K+-ATPase mRNA
expression observed upon treatment of alveolar epithelial cells with
DBcAMP or dexamethasone might not necessarily result in a concomitant
rise in the activity of the channel or the sodium pump. For this
reason, we tested the physiological effect of DBcAMP and/or
dexamethasone on alveolar epithelial cells by recording
Ite. DBcAMP modulated Ite, with a maximum effect occurring after
8 h of stimulation, whereas with dexamethasone, a maximum was
reached after 24 h. Interestingly, the changes in current parallel
the changes in - and
-ENaC expression. The maximal increase in
- and
-ENaC expression with DBcAMP stimulation occurred at 8 h, whereas the maximal changes with dexamethasone occurred at 24 h. These results are also supported by data in other systems in which
there was a parallel between the changes in ENaC expression induced by
dexamethasone (10, 23) or aldosterone (33)
and Ite. Despite these results, more information
will be needed to establish a causal relationship between the two
phenomena. First, we should be able to demonstrate that ENaC mRNA
synthesis precedes the increase in Ite. This
seems to be the case since we have some preliminary data that already show a significant rise in
-ENaC mRNA after 6 h of treatment with DBcAMP. Second, it will be important to demonstrate an increase in
the active channel at the membrane. Finally, it will be important to
establish if the individual or combined modulation of any of the ENaC
subunits has an impact on the Ite.
Although an increase in ENaC expression might be important for the
modulation of the Ite with DBcAMP and
dexamethasone, these results also indicate that other factors are
important for stimulating Ite. Besides ENaC,
DBcAMP stimulates a parallel increase in
1-Na+-K+-ATPase expression,
suggesting a possible role of Na+-K+-ATPase in
the generation of higher Ite. We have shown
previously that chronic stimulation with DBcAMP leads to enhanced
Na+-K+-ATPase activity (34).
Furthermore, it has been demonstrated that
-adrenergic stimulation,
which elevates intracellular cAMP, can result in increased membrane
insertion of
1-Na+-K+-ATPase
(6). Taken together, these results indicate that, besides changes in
-ENaC expression, an increase in the expression or activity of Na+-K+-ATPase could also be
required to augment Na+ transport in DBcAMP-treated cells.
The data obtained after dexamethasone stimulation also support this
hypothesis. Dexamethasone, a more potent inducer of ENaC mRNA than of
DBcAMP, fails to stimulate Ite to the same level
as DBcAMP. Its influence is also negligible on the modulation of
Ite induced by combined treatment with
dexamethasone and of DBcAMP (Fig. 7C and Table 2). Although
it is possible that translation of ENaC protein might not correlate
with the level of ENaC mRNA detected in stimulated cells, these results suggest that an increase in the expression or activity of
Na+-K+-ATPase could be required to augment
Na+ transport in alveolar epithelial cells. The absence of
1-Na+-K+-ATPase modulation and
the modest increment brought by dexamethasone to
1-Na+-K+-ATPase mRNA could
explain the low impact of 100 nM dexamethasone on
Ite. It is possible, however, that at other
concentrations and at other time points, dexamethasone could have a
more potent impact on
1-Na+-K+-ATPase expression and
sodium pump membrane activity as reported by Barquin et al.
(2). Other experimental results suggest that Na+-K+-ATPase expression is important for
Na+ transport in the lung. Recently, it has been shown that
enhanced Na+-K+-ATPase expression by gene
therapy increases lung liquid clearance and decreases edema formation,
two events linked to Na+ and fluid transport (17,
53). Overall, these results suggest that, in lung epithelial
cells, unlike kidney epithelial cells, the modulation of
Na+-K+-ATPase synthesis and activity in
parallel to changes in ENaC could play an important complementary role
in transepithelial Na+ and fluid transport.
In summary, we found that DBcAMP and dexamethasone can regulate the
steady-state mRNA levels of ENaC and
Na+-K+-ATPase subunits in isolated alveolar
epithelial cells. DBcAMP affects the expression of the -,
-, and
-ENaC subunits and
1-Na+-K+- ATPase mRNAs in
alveolar epithelial cells, whereas dexamethasone modulates the
expression of ENaC subunits and
1-Na+-K+-ATPase mRNA but not of
1-Na+-K+-ATPase. Treatment with
actinomycin D and mRNA demonstrated that the elevation of
-ENaC mRNA
probably derives from an increase in gene transcription and not from
stabilization of
-ENaC mRNA. Cloning of the mouse
-ENaC promoter
and testing of its activity demonstrated that regulatory elements
present in the 2.9-kb 5'-flanking sequence allow the regulation of
transcription by dexamethasone. There is a parallel increase in the
expression of
-ENaC and
1-Na+-K+-ATPase mRNA produced by
DBcAMP treatment and the Ite recorded in
alveolar epithelial cells. Clearly, a change in the expression of ENaC
and Na+-K+-ATPase mRNA is part of the mechanism
that regulates Na+ transport in alveolar cells.
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ACKNOWLEDGEMENTS |
---|
Special thanks to Nancy Léveillée for technical assistance. We acknowledge the editorial work done on this manuscript by Ovid Da Silva, éditeur/rédacteur of the Research Support Office of the CHUM Research Center.
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FOOTNOTES |
---|
Y. Berthiaume is a chercheur-boursier clinicien from Fonds de la Recherche en Santé du Québec. This work was supported in part by Grant MT-1203 from the Medical Research Council of Canada, The Canadian Cystic Fibrosis Foundation, and the Association Pulmonaire du Québec.
Address for reprint requests and other correspondence: A. Dagenais, Centre de Recherche CHUM-Hôtel-Dieu, 3840 St-Urbain, Montreal, Quebec, Canada H2W 1T8 (E-mail: andre.dagenais.chum{at}ssss.gouv.qc.ca).
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.
Received 28 March 2000; accepted in final form 9 February 2001.
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