INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (I_sc) 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 (I_te) generated at times when ENaC mRNAs are upregulated.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

Northern 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.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Modulation of -epithelial Na+ channel (ENaC; A and C) and 1-Na+-K+-ATPase mRNA (B and D) expression by dibutyryl cAMP (DBcAMP; A and B) and dexamethasone (C and D) detected by Northern blot quantitation. Alveolar epithelial cells were cultured for 3 days and then treated for 1, 4, 8, 24, or 48 h with 1 mM DBcAMP or 100 nM dexamethasone. There was a 2-fold increment in the level of -ENaC and 1-Na+-K+-ATPase mRNA after 8 h of DBcAMP treatment. In dexamethasone-treated cells, -ENaC mRNA increased gradually to a maximum 4.5-fold expression after 24 h (C). Data are presented as percentages ± SE of expression relative to time-matched untreated controls (ctrl). mRNA expression was corrected to -actin expression. n, No. of animals from different experiments [for DBcAMP treatment: 1 h, n = 4; 4 h, n = 7; 8 h, n = 18; 24 h, n = 8; 48 h, n = 8; for dexamethasone treatment: 1 h, n = 10; 4 h, n = 13; 8 h, n = 14; 24 h, n = 10; 48 h, n = 10]. *P < 0.05, 4 h and 8 h; 8 h and 24 h (A-C); 1 h and 8 h (D) by post hoc analysis (Fisher's protected least significant difference).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Representative Northern blot (C) and corresponding densitometric quantitation showing the effect of combined treatment with DBcAMP and dexamethasone (Dex) on the expression of -ENaC (A) and 1-Na+-K+-ATPase (B) mRNA. Alveolar epithelial cells were cultured for 3 days and treated for 8 h with 1 mM DBcAMP, 100 nM dexamethasone, or a combination of both agents. Combined treatment with DBcAMP and dexamethasone had an additive effect on -ENaC and 1-Na+-K+-ATPase mRNA. Data are presented as percentages of expression compared with time-matched controls. The amount of mRNA was corrected with the level of -actin mRNA. Scheffé's post hoc analysis shows that the differences of -ENaC and 1-Na+-K+-ATPase mRNA expression are significant for all of the conditions studied. DBcAMP, n = 18; dexamethasone, n = 12; DBcAMP + dexamethasone, n = 3.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Representative Northern blots showing the role of transcription and translation in the modulation of -ENaC and 1-Na+-K+-ATPase mRNA expression after treatment with DBcAMP or dexamethasone. Alveolar cells were treated for 8 h with 1 mM DBcAMP or 100 nM dexamethasone in the presence and absence of the transcription inhibitor actinomycin D (Act D; 5 µg/ml; A) or the translation inhibitor cycloheximide (Cyclo; 2.5 µg/ml; B). Actinomycin D depressed the increase of -ENaC mRNA detected after treatment with DBcAMP or dexamethasone and inhibited the elevation of 1-Na+-K+-ATPase mRNA in DBcAMP-treated cells. Cycloheximide had an impact on -ENaC mRNA expression, decreasing the basal expression level of the transcripts, but did not inhibit the increase of -ENaC mRNA evoked by DBcAMP and dexamethasone. Cycloheximide suppressed the elevation of 1-Na+-K+-ATPase mRNA elicited by DBcAMP.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   -ENaC mRNA stability was determined in control cells (A) and cells treated with 1 mM DBcAMP (B) or 100 nM dexamethasone (C). Cells were treated with 5 µg/ml actinomycin D for 0, 2, 4, 6, 8, or 10 h in the presence and absence of 1 mM DBcAMP or 100 nM dexamethasone. 18S rRNA was used to normalize the amount of RNA loaded on each well. Four different experiments were combined to generate the mRNA decay slope. The calculated half-life (T1/2) is shown.

View larger version (85K): [in this window] [in a new window]   Fig. 5.   Sequence of 5'-flanking DNA of the mouse -ENaC gene showing different 5'-untranslated regions (UTR) resulting from different transcription initiation sites and putative transcription factor-binding sites defined with the TESS program. Several potential transcription initiation sites were detected by 5'-RACE. The sequences are numbered to the more distal transcription initiation site. Arrows show the 2 major transcription initiation sites found (filled arrow: lung; open arrow: kidney), and arrowheads show the minor start site cloned (filled arrowhead: lung; open arrowhead: kidney). The first codon is labeled in bold. Putative transcription factor-binding sequences are shown in boxes. The glucocorticoid receptor binding sequences (GR) are underlined. GenBank accession number AF228802. AP, activator protein; Sp, small protein; CREB, cAMP responsive element binding; PPAR, peroxisome proliferator-activated receptor; NK-B deg, degenerate nuclear factor-B; IL-6, interleukin-6.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Activity of the -ENaC promoter in A549 cells. A: map of the 3.2-kb -ENaC mouse genomic clone showing the position of the putative glucocorticoid regulatory element (GRE) transcription factor-binding sequences. Arrows indicate the 2 major transcription initiation sites detected by 5'-RACE in the lung. The rectangle depicts the part of exon 1 encompassed in the clone with the 5'-UTR (open box) and coding sequence (open reading frame; filled box). For the reporter gene assay, a BamHI-MscI 2.9-kb fragment was linked upstream of a chloramphenicol acetyltransferase (CAT) gene in the pjfCAT vector. The genomic fragment ends at the 5'-UTR downstream of the first transcription initiation site. B: activity of the CAT reporter gene transfected in A549 cells expressed as arbitrary units (counts/min of sample  counts/min of untransfected cells). The pjfCAT vector alone (n = 10) has very low background expression. The BamHI-MscI -ENaC genomic clone is able to drive basal expression of the gene in control untreated cells (n = 9) and 8-h DBcAMP-treated cells (n = 8). Treatment with dexamethasone for 24 h (n = 10) increases the basal activity of the promoter by a factor of 5.9. P < 0.05, Dex 24 h compared with CTRL () and CTRL compared with pjfCAT (*) by unpaired t-test.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Impact of DBcAMP and dexamethasone treatment on transepithelial current. Alveolar epithelial cells were grown for 3 days on semipermeable membranes and were treated with 1 mM DBcAMP (A), 100 nM dexamethasone (B), or a combination of 1 mM DBcAMP plus 100 nM dexamethasone (C and D) added at the apical and basolateral sides of the cells. Treatment of alveolar epithelial cells with DBcAMP or dexamethasone raised transepithelial current with a time course similar to -ENaC mRNA expression. Potential differences across the monolayers (mV) and transepithelial resistance ( · cm2) were recorded with an epithelial voltohmmeter (EVOM) at 0, 8, 24, 32, and 48 h of treatment, and transepithelial current was determined as reported. Data are presented as percentages of current compared with time-matched controls. The effect of each type of treatment was measured in triplicate for each time point, and the whole procedure was repeated 3 times with alveolar cells isolated from different animals. P < 0.05, 0-8 h, 0-24 h, and 0-32 h (A and C); *P < 0.05, 0-24 h (B); and P < 0.05, 8-24 h and 8-32 h (C) by post hoc analysis (Bonferroni-Dunn). To confirm that EVOM is sensitive enough to detect the modulation of transepithelial current in DBcAMP- + dexamethasone-treated cells, measurements were also performed in the Ussing chamber (D). With both methods, treated cells show a marked increase in short-circuit current (Isc) compared with untreated cells. Although Isc recording with Ussing is more sensitive in detecting current variations, the difference was not statistically significant between EVOM and Ussing measurements (unpaired t-test; n = 5 filters for Ussing and n = 14 filters for EVOM).

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 (R_te;  · 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. I_te across these monolayers was calculated by the following formula: I_te = PD/R_te. 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, I_te 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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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).

View this table: [in this window] [in a new window]   Table 1.   -ENaC and 1-Na+-K+-ATPase mRNA expression on treatment with actinomycin D and cycloheximide

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 I_te. To determine if changes in ENaC and Na⁺-K⁺-ATPase mRNA expression have an impact on ion transport, we measured PD and R_te with EVOM and calculated the I_te 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 I_te 8 h after the beginning of treatment with DBcAMP (Fig. 7A) or DBcAMP plus dexamethasone (Fig. 7C). This heightened I_te decreased gradually toward control values over 48 h. Dexamethasone also induced an increase of I_te that was smaller than with combined treatment and reached its maximum at 24 h (Fig. 7B). Forty-nine to fifty-eight percent of I_te 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 I_te detected with EVOM, the I_sc of cells treated for 8 h with DBcAMP and dexamethasone was recorded with the Ussing system. Treated cells showed a marked increase in I_sc compared with untreated cells (Fig. 7D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 I_te. DBcAMP modulated I_te, 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 I_te. 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 I_te. 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 I_te.

Although an increase in ENaC expression might be important for the modulation of the I_te with DBcAMP and dexamethasone, these results also indicate that other factors are important for stimulating I_te. 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 I_te. 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 I_te to the same level as DBcAMP. Its influence is also negligible on the modulation of I_te 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 I_te. 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 I_te 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.