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Published ahead of print on October 6, 2005, doi:10.1165/rcmb.2005-0273OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 204-212, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0273OC

Oxygen and Glucocorticoids Modulate {alpha}ENaC mRNA Translation in Fetal Distal Lung Epithelium

Gail Otulakowski, Bijan Rafii, Michael Harris and Hugh O'Brodovich

CIHR Group in Lung Development, Programme in Lung Biology Research, Hospital for Sick Children Research Institute, and Departments of Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada

Correspondence and requests for reprints should be addressed to Gail Otulakowski, Ph.D., Programme in Lung Biology Research, Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail: gail.otulakowski{at}sickkids.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 RESULTS
 DISCUSSION
 References
 
Glucocorticoid hormones play an important role in fetal lung maturation. It is unknown how they interact with changes in O2 tension, which play an important role in converting the lung from a fluid-secreting to a fluid-absorbing organ at birth. Airspace fluid absorption arises from active transepithelial Na+ transport with the amiloride-sensitive epithelial Na channel (ENaC), consisting of {alpha}, beta, and {gamma} subunits, representing the rate-limiting step under nonpathologic conditions. We investigated the individual and combined effects of dexamethasone (DEX) and PO2 on {alpha}ENaC mRNA levels, rate of {alpha}ENaC protein synthesis, and amiloride-sensitive short-circuit current in primary cultures of rat fetal distal lung epithelial cells. DEX significantly induced {alpha}ENaC mRNA in fetal (3%) and postnatal (21%) O2, but increases in {alpha}ENaC protein synthesis and function occurred only when epithelia were grown under a postnatal PO2. Sucrose density gradient analyses showed that DEX treatment of cells cultured at 3% O2 decreased the association of {alpha}ENaC mRNA with large polysomes and enhanced the association with small polysomes. Conversely, incubation of DEX-treated cells in 21% O2 restored {alpha}ENaC mRNA association with large polysomes. No significant changes were seen in the overall polyribosome profiles or in the distribution of mRNAs encoding beta and {gamma} subunits of ENaC or cytokeratin 18, indicating specific modulation of {alpha}ENaC mRNA translation. These data suggest that postnatal O2 exposure may be important for efficient translation of the {alpha}ENaC mRNA.

Key Words: ion transport • translational regulation • postnatal gas exchange • fluid absorption


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 RESULTS
 DISCUSSION
 References
 
Fluid secretion by lung epithelia into the developing lungs' lumen is essential for normal lung development, but this fluid must be cleared at birth so that effective gas exchange can occur. Impaired clearance of this fetal lung fluid results in transient tachypnea of the newborn, and infants with impaired clearance combined with immaturity of the surfactant system suffer from neonatal respiratory distress syndrome (1). Clearance of airspace fluid results from active transepithelial Na+ transport, which induces a paracellular movement of Cl–, with fluid following (2). A number of physiologic mechanisms underlie perinatal lung fluid absorption, including effects triggered by beta-adrenergic agonists, oxygen, glucocorticoids, and thyroid hormones (3). These factors interact with each other; for example, glucocorticoid and thyroid hormones are capable of advancing maturation of the epinephrine response in fetal lamb lungs (4). The key Na+ transport proteins involved are the basolateral Na+,K+-ATPase, which provides the driving force, and the apical epithelial Na+ channel (ENaC), which under normal conditions is the rate-limiting step in transepithelial Na+ absorption.

One way in which glucocorticoids promote maturation of the fetal lung phenotype is via an increase in {alpha}ENaC mRNA levels (5), achieved via an increase in transcription mediated by a glucocorticoid-responsive element in the 5' flanking region of the {alpha}ENaC gene (6–8). There is also evidence that glucocorticoids increase apical membrane ENaC expression in the short term by augmenting ENaC trafficking and membrane retention via the serum- and glucocorticoid-regulated kinase (sgk) (9, 10). Direct transcriptional effects of glucocorticoids have also been shown on the {alpha}1 and beta1 subunits of the Na+,K+-ATPase (11), providing the possibility for coordinate regulation of the basolateral pump and apical channels in response to these hormones.

The change in oxygen concentration from the fetal (~ 3%) to postnatal (21%) environment has been shown to increase Na+ transport and ENaC mRNA levels in fetal distal lung epithelial cells (FDLE) (12–14). A direct activation of the {alpha}ENaC promoter in response to a shift from 3% to 21% O2 has been demonstrated using promoter/reporter constructs in transfected cells (15), but the transcriptional effect and mRNA increase were temporally delayed relative to the increase in Na+ transport (12, 14). This suggests that the initial effects of the O2 increase are not caused by direct increases in {alpha}ENaC transcription, and other recent studies have provided additional evidence for post-transcriptional mechanisms. First, in cultured adult rat alveolar epithelial type II cells, exposure to 3% O2 reduced amiloride-sensitive sodium channel activity via a reduction in localization of ENaC subunits to the apical membrane without reducing ENaC subunit mRNA or protein levels (16). Second, under certain serum-free media conditions, O2-evoked increases in apical Na+ conductance occurred in FDLE without corresponding increases in ENaC mRNA abundance (17).

There is limited information regarding the integration of glucocorticoid and oxygen signals on FDLE. Thome and colleagues (18) showed that corticosterone increased ENaC function regardless of O2 concentration, despite the observation that hormone treatment of hypoxic cells resulted in reduced ENaC protein levels. In addition, single-channel recordings from cultured alveolar epithelial cells have shown that the presence of steroids and air interface promotes expression of low-conductance, highly Na+-selective channels rather than the nonselective cation channels predominant in cells grown submerged in the absence of steroids (19).

Recent research suggests that there is translational regulation of Na+ transport proteins, including the Na+,K+-ATPase subunit mRNAs (20, 21) and {alpha}ENaC (22, 23). Post-transcriptional mechanisms could provide a highly effective strategy to produce the rapid increase in Na+ transport required at birth. We speculated that the prenatal surge in glucocorticoids may create a pool of {alpha}ENaC mRNA, whose translation is stimulated by the change in oxygen tension as air breathing begins. We used primary cultures of rat FDLE to examine the integrated effects of dexamethasone (DEX) and changes in PO2 on {alpha}ENaC. We quantitated changes in the mRNA level, rate of {alpha}ENaC synthesis (immunoprecipitation of pulse-labeled cells), and function (amiloride-sensitive Isc across primary cultures of epithelia in an Ussing chamber). As an indicator of translational regulation, sucrose density gradient analyses of postnuclear supernatants from FDLE were used to evaluate changes in the distribution of {alpha}ENaC mRNA across the polysome profile in cells treated with DEX or shifted to postnatal O2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
RESULTS
 DISCUSSION
 References
 
Cell Isolation and Culture
FDLE from 20-d gestation rat fetuses were isolated and grown in primary culture as previously described (24). All animal procedures were reviewed and approved by the Hospital for Sick Children Animal Care committee. FDLE were seeded at 1 x 106 cells/cm2 onto 0.4-µm pore size Snapwell cell culture inserts (Corning Costar, Cambridge, MA) for bioelectric studies or at 0.5 x 106 cells/cm2 on 24-mm-diameter, 0.4-µm pore size Transwell inserts for metabolic labeling and RNA isolation. All cells were grown as submersion cultures in Dulbecco's modified Eagle medium (4.5 g/l glucose with 2 mM L-glutamine and 110 mg/l sodium pyruvate) supplemented with 10% FBS (Cansera, Rexdale, Ontario, Canada), 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate. The culture medium was replaced 24 h after seeding to remove unattached cells, at which time medium containing hormone-depleted FBS (stripped with charcoal and ion exchange resin [25]) was used. The medium was supplemented with 50 nM DEX, and cells were placed in incubators containing 3% O2-5% CO2-balance N2 ("fetal" atmosphere) or 5% CO2-balance room air ("postnatal" atmosphere) for 48–72 h before electrophysiologic or biochemical analysis.

Measurement of FDLE Monolayer Bioelectric Properties
The bioelectric properties of the FDLE monolayers were determined as previously described with modified Ussing chambers. The cells were bathed in 37°C Hank's balanced salt solution (Gibco, Burlington, ON, Canada) supplemented with 1.8 g/l of sodium bicarbonate and equilibrated with a 5% CO2-balance air gas mixture (24, 26). Monolayers were maintained under short-circuit conditions, and Isc was monitored continuously with a voltage-current clamp (VCC600; Physiological Instruments, San Diego, CA). Once the bioelectric properties stabilized, amiloride-sensitive Isc was determined by the addition of 0.1 mM amiloride to the apical side of monolayers. Transepithelial resistance was calculated by dividing the transepithelial potential difference by the Isc.

RNA Analysis
RNA was extracted from FDLE monolayers using RNeasy (Qiagen, Mississauga, ON, Canada) according to the manufacturer's instructions and quantitated through standard UV absorbance measurements. {alpha}ENaC mRNA expression was quantitated by slot blot analysis. Briefly, 3-µg aliquots of FDLE mRNA were applied to Hybond-N+ membranes (GE Healthcare, Baie d'Urfé, PQ, Canada) and fixed by UV crosslinking. Blots were hybridized to a 32P-labeled cDNA probe to rat {alpha}ENaC and washed as previously described (26). Quantitative analysis of probe hybridization was performed using a Molecular Dynamics PhosphorImager equipped with ImageQuant software (Amersham Biosciences, Baie d'Urfe, PQ, Canada).

Metabolic Labeling and Immunoprecipitation
Biosynthetic labeling of FDLE grown on permeable supports with [35S]methionine followed by immunoprecipitation of {alpha}ENaC subunits was adapted from procedures published for renal cells (27, 28). Cells were fed with fresh medium 90 min before labeling, using solutions that had been pre-equilibrated at the appropriate PO2 (>= 24 h) in the "fetal" or "postnatal" tissue culture incubators and handling "fetal" cells in a hypoxic workstation. Cells were rinsed and incubated briefly in serum-free, methionine-free medium, and 250 µl of methionine-free medium containing 1 mCi/ml [35S]methionine was placed on the basolateral surface of inverted filters for 30 min at 37°C at the indicated PO2. The labeling medium was removed, and cells were rinsed twice with ice-cold phosphate-buffered saline, followed by a 10-min incubation on ice in 250 µl/well homogenization buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid [EDTA], 1% Triton X-100, 0.2% BSA, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). Cells were harvested by scraping, freeze-thawed, and passed three times through a 29-gauge needle. Insoluble material was removed by a 2-min centrifugation at 15,000 x g. [35S]methionine incorporation was determined by trichloroacetic acid precipitation of 5-µl aliquots of the supernatants. Equal amounts of counts per minute (cpm) were submitted to immunoprecipitation.

Immunoprecipitation of {alpha}ENaC was performed on denatured proteins. SDS was added to the lysates to a final concentration of 1%, and the samples were heated at 95°C for 5 min. The denatured protein preparation was diluted with 1 vol of homogenization buffer, then pre-cleared with normal rabbit sera (1:20) and 50 µl Pansorbin for 1 h at 4°C. Cleared lysates were incubated overnight at 4°C with a rabbit polyclonal antibody we raised against a glutathione-S-transferase–{alpha}ENaC (rat) fusion protein (1:20). Protein A-Sepharose (25-µl packed beads) was added, incubated for 1 h at 4°C, and recovered by centrifugation. The beads were washed three times with homogenization buffer and then four times with wash buffer (10 mM Tris-HCl [pH 7.5], 2 mM EDTA, 0.1% [wt/vol] SDS). The immunoprecipitated proteins were recovered by heating in Laemmli sample buffer and separated on 7.5% acrylamide SDS-polyacrylamide gels. Gels were fixed, incubated in Amplify (GE Healthcare), and dried for analysis on a Molecular Dynamics PhosphorImager equipped with ImageQuant software.

Sucrose Density Gradient Fractionation of Polysomes
Polysome profiles were prepared using a method modified from Otulakowski and colleagues (23) for perinatal lung tissue (23). Briefly, six 10-cm plastic dishes or 75-mm-diameter Transwells of highly confluent FDLE were used for each gradient. After the 48-h treatment with or without 50 nM DEX at the indicated PO2, cells were fed with fresh medium using solutions that had been pre-equilibrated at the appropriate PO2 (>= 24 h) in the "fetal" or "postnatal" tissue culture incubators and handling "fetal" cells in a hypoxic workstation. Cells were harvested on ice 90 min later by washing twice with ice-cold PBS containing 100 µg/ml cycloheximide, followed by lysis in 100 µl per dish of polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES [pH 7.4], 100 µg/ml cycloheximide, 0.5% Nonidet P-40, and 1,000 U/ml placental RNase inhibitor). The lysates were scraped into a 1.5-ml microcentrifuge tube and passed three to four times through a 27-gauge needle to ensure lysis. Nuclei were pelleted by two sequential centrifugations at 12,000 x g for 5 min at 4°C. The resulting supernatants (10 A260nm units per gradient) were layered on linear 15–45% (wt/vol) sucrose gradients in polysome gradient buffer (100 mM KCl, 5 mM MgCl2, and 10 mM HEPES [pH 7.4]). Gradients were centrifuged at 35,000 rpm for 2 h in a Beckman SW41 rotor and recovered in 13 equal fractions using a Brandel gradient fractionator equipped with an ISCO UA-6 flow cell set to 254 nm (Brandel, Gaithersberg, MD). Fractions were snap-frozen and stored at –80°C.

Northern Blot Analysis of Polysome Gradient Fractions
RNA was isolated from individual sucrose density fractions by proteinase K digestion followed by phenol/chloroform extraction and ethanol precipitation as described (23). The entire RNA pellet from each fraction was subjected to electrophoresis on a 1% agarose 2.2 M formaldehyde gel in 1x MOPS buffer (20 mM 3-N[Morpholino] propane sulfonic acid, 5 mM sodium acetate, 1 mM EDTA) and transferred to Hybond-N+ membrane. Membranes were prehybridized and hybridized in Expresshyb solution (Clontech, Palo Alto, CA) at 65°C using 32P-labeled random primed cDNA probes for rat {alpha}-, beta-, and {gamma}ENaC and for mouse cytokeratin 18 (CK18) as described (23). Hybridized membranes were washed at high stringency and analyzed via PhosphorImager as described previously.

Statistical Analysis
Data are presented as mean ± SE. Statistical significances were calculated using one-way analysis of variance, and P < 0.05 was considered to be statistically significant. Statistical analysis was performed using GraphPad Instat version 3.01 and GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 RESULTS
DISCUSSION
 References
 
Effect of Oxygen on Induction of ENaC by DEX in FDLE
To analyze the effect of PO2 on glucocorticoid induction of ENaC activity in FDLE, we measured amiloride-sensitive Isc in monolayers cultured for 48 h in 3% or 21% O2 in the presence or absence of 50 nM DEX (Figure 1A). Incubation at postnatal PO2 significantly increased amiloride-sensitive Isc by 80–100% relative to cells maintained at fetal PO2; these results were consistent with previously published work (12, 14, 17, 26, 29), and this increase was independent of glucocorticoid supplementation. DEX increased amiloride-sensitive Isc under 21%, but not 3%, O2 conditions (Figure 1A). Total transepithelial Isc showed the same induction pattern in response to O2 and DEX as amiloride-sensitive Isc (Figure 1B); transepithelial resistance was not different among groups (Figure 1C). Because DEX is known to directly activate {alpha}ENaC gene transcription, we quantitated {alpha}ENaC mRNA levels by slot blot in FDLE monolayers cultured under identical conditions (Figure 1D). This dose of DEX was sufficient to increase {alpha}ENaC mRNA levels severalfold under both O2 conditions, suggesting that the failure of DEX to increase amiloride-sensitive Isc in cells maintained at 3% O2 was due to translational or post-translational regulation.


Figure 1
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  		Figure 1. O2 modulates the effect of DEX on ENaC expression in FDLE. FDLE monolayers were cultured in the absence or presence of 50 nM DEX on permeable membranes under fetal (3% O2) or postnatal (21% O2) O2 conditions. (A) Amiloride-sensitive short circuit current (ASC) was measured in these monolayers after 48 h exposure to the indicated conditions. ASC was significantly induced in FDLE cultured under postnatal O2 atmosphere in the absence and presence of DEX (*P < 0.01 versus 3% O2). DEX treatment significantly induced ASC under 21% O2 ({dagger}P < 0.05 versus hormone-free) but not under 3% O2. n = 14 monolayers/group. (B) Effects of DEX and O2 on total Isc paralleled observations of amiloride-sensitive Isc (*P < 0.05 versus 3% O2; {dagger}P < 0.05 versus hormone-free, n = 14 monolayers/group). (C) Resistance was not significantly different among groups (n = 14 monolayers/group). (D) mRNA extracted from parallel monolayers was immobilized on a slot blot, and levels of {alpha}ENaC mRNA were quantitated after hybridization to a radiolabeled cDNA probe. Representative slot blot autoradiograph for {alpha}ENaC mRNA is shown below the graphed quantitative data. DEX treatment significantly induced {alpha}ENaC mRNA under both O2 conditions (*P < 0.001 versus hormone-free, n = 10 monolayers). The effect of postnatal O2 exposure was significant in the presence of 50 nM DEX ({dagger}P < 0.001 versus 3% O2, n = 10 monolayers).

 
Effect of Oxygen on {alpha}ENaC Protein Synthesis in FDLE
We developed an immunoprecipitation approach to assess the rate of {alpha}ENaC protein synthesis in FDLE cultured on permeable supports. FDLE were pulse-labeled using [35S]methionine for 30 min and harvested without any chase period. The specificity of the antiserum is shown in Figure 2A, in which a single specific band can be seen migrating at ~ 90 kD. Immunoprecipitation from metabolically labeled lung epithelial H441 and from the mouse kidney epithelial cell line mpkCCDcl4 also yielded a single band migrating at 90 kD (data not shown). A nonspecific band at ~ 30 kD is precipitated by the antiserum and normal rabbit serum. When FDLE maintained under various culture conditions were pulse-labeled and precipitated with the {alpha}ENaC antiserum, a maximal amount of {alpha}ENaC protein synthesis was seen in FDLE cultured at 21% O2 in media containing DEX (Figure 2B).


Figure 2
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  		Figure 2. Immunoprecipitation of {alpha}ENaC from metabolically labeled FDLE. (A) Rat FDLE cultured on permeable membranes were pulse-labeled with [35S]methionine. Labeled proteins were immunoprecipitated with anti-{alpha}ENaC antiserum (AS) or with normal rabbit serum (NS). Position of molecular weight markers (Benchmark, Invitrogen) is indicated on the left (kD). Arrow indicates position of {alpha}ENaC, migrating at ~ 90 kD. (B) Effects of DEX and O2 on the rate of {alpha}ENaC protein synthesis. Confluent FDLE cultured on filters were maintained under 3% or 21% O2, without (–) or with (+) 50 nM DEX for 48 h. {alpha}ENaC was immunoprecipitated from equal amounts of incorporated [35S] in lysates from cells cultured and pulse-labeled under the indicated conditions. Arrow, {alpha}ENaC.

 
To quantitate the relative amounts of {alpha}ENaC protein synthesis under the four experimental conditions, equal amounts of newly synthesized, pulse-labeled protein were immunoprecipitated and subjected to SDS-PAGE. Incorporation of [35S]methionine into the 90-kD band was quantitated using a PhosphorImager. DEX increased {alpha}ENaC protein synthesis under 21%, but not 3%, O2 conditions (Figure 3A). Similarly, the effect of postnatal relative to fetal PO2 reached significance only in cells treated with the hormone. Although glucocorticoids are major catabolic hormones regulating protein metabolism and hypoxia is known to affect protein translation (30), the effects in shown in Figure 3A should represent specific regulation of {alpha}ENaC protein synthesis because equal amounts of pulse-labeled protein were submitted to immunoprecipitation. Although DEX and maintenance in 3% O2 decreased uptake of radiolabel from the media into the cells (data not shown), there were no significant effects on percent incorporation of [35S]methionine into trichloroacetic acid–precipitable peptides (Figure 3B), suggesting that global protein synthesis was not grossly inhibited under the conditions tested.


Figure 3
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  		Figure 3. Effects of O2 and DEX on {alpha}ENaC and total protein synthesis in FDLE. FDLE monolayers were cultured in the absence or presence of 50 nM DEX on permeable membranes under fetal (3% O2) or postnatal (21% O2) O2 conditions. (A) Results of quantitation of immunoprecipitated {alpha}ENaC protein from pulse-labeled FDLE. Equal amounts of incorporated radiolabel from FDLE lysates cultured under the indicated conditions were immunoprecipitated with {alpha}ENaC-specific antiserum and resolved on SDS-PAGE, and the intensity of the 90-kD band corresponding to {alpha}ENaC was quantitated. DEX treatment significantly increased {alpha}ENaC protein synthesis onlyunder postnatal O2 conditions (*P < 0.001 versus hormone-free, n = 14–21 monolayers/group). Similarly, postnatal O2 significantly increased {alpha}ENaC protein synthesis only in the presence of DEX (*P < 0.001 versus 3% O2, n = 14–21 monolayers/group). (B) Effects of O2 and DEX on total protein synthesis. Aliquots of pulse-labeled FDLE lysates were precipitated with trichloroacetic acid, filtered, and quantitated using a liquid scintillation counter. Percent incorporation of [35S]met into protein was calculated as precipitable counts per minute (cpm) relative to total cpm in an equal aliquot of lysate directly spotted onto an identical filter. Although 50 nM DEX tended to decrease incorporation of radiolabel into precipitable cpm under both O2 conditions, this effect was not statistically significant (n = 20–22 monolayers/group).

 
Table 1 summarizes the individual and combined effects of PO2 and 50 nM DEX on ENaC expression and function. The fold increase in mRNA, protein synthesis, and amiloride-sensitive Isc, induced by the four culture conditions, was calculated from each of the experiments summarized in Figures 1 and 3. A switch to postnatal O2 under hormone-free conditions did not result in a statistically significant increase in {alpha}ENaC mRNA levels or protein synthesis, although there was a trend to increase these two measurements 40–50%. Although a type II error may be the source of the lack of statistical significance, the fold increase in amiloride-sensitive Isc at 80% was significantly greater than the trend to increase RNA and protein, again supporting a post-translational level of regulation in O2-mediated increases in ENaC activity under hormone-free conditions. In contrast, cells switched from 3% to 21% O2 in the presence of DEX responded with a consistent 2-fold increase in all three {alpha}ENaC measurements. The effects of DEX on ENaC expression were also dependent on O2 environment. DEX robustly induced {alpha}ENaC mRNA 4- to 6-fold under both O2 environments. However, induction of protein synthesis was weaker. Under 3% O2, DEX showed only a trend to increase ENaC protein synthesis by 50%, which did not reach statistical significance (Figures 2B and 3A) and was highly statistically different from the fold increase in mRNA (P < 0.001). Under 21% O2, a significant (P < 0.05 versus hormone-free) increase in ENaC protein synthesis (~ 3-fold) was induced by DEX; this induction was modestly lower than the mRNA induction (~ 5-fold). These data suggest a block in {alpha}ENaC translation when cells cultured under 3% O2 are treated with DEX, which is at least partially relieved in cells cultured under 21% O2.


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  		TABLE 1. EFFECTS OF DEXAMETHASONE AND O2 ATMOSPHERE ON {alpha}ENAC EXPRESSION AND FUNCTION IN FDLE

 
Effect of DEX on the Distribution of {alpha}ENaC mRNA in Polysome Profiles from FDLE Maintained Under Fetal PO2
Because the increased expression of {alpha}ENaC mRNA seen in cells treated with DEX under 3% O2 did not result in a significant increase in {alpha}ENaC protein, we examined the association of {alpha}ENaC mRNA with actively translating polysomes. Postnuclear supernatants from FDLE maintained at 3% O2 in the absence or presence of DEX were fractionated on 15–45% sucrose gradients and recovered in 13 fractions. The optical density at 254 nm was recorded during recovery of the gradient to determine the distribution of ribosomal RNAs. Representative profiles are shown in Figure 4A. Monosomes were recovered in fraction 4, with prominent free 40S and 60S peaks recovered in fractions 2 and 3. Assignment of peaks was confirmed by observation of the intensity of 28S and 18S rRNA bands on denaturing agarose gel electrophoresis of RNA from each fraction (Figure 4A, lower panels). The A254 profiles and quantitation of the distribution of 18S rRNA (Figure 4B, 18S rRNA panel) indicate that DEX treatment did not alter the overall distribution of ribosomes between monosomes and polysomes.


Figure 4
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  		Figure 4. Dexamethasone decreases ribosome loading on {alpha}ENaC mRNA in FDLE cultured under fetal O2 concentrations. FDLE monolayers were cultured under 3% O2 for 48 h in the absence (hormone-free) or presence (50 nM DEX) of hormone. Cytoplasmic extracts were prepared and fractionated on 15–45% linear sucrose gradients, and the distribution of various RNA species was analyzed. (A) Representative ultraviolet absorbance profiles of FDLE lysates sedimented through sucrose gradient. On the tracings, the density of the gradient increases from left to right, and the position of the 80S monosome peak is indicated (M). Location of the recovered fractions (1–13) and total RNA from each fraction analyzed by agarose gel electrophoresis and ethidium bromide staining is shown below each tracing. (B) Distribution of specific RNAs in fractions recovered from sucrose density gradients, expressed as percent of total for that RNA in the gradient. 18S rRNA was calculated from the intensity of ethidium bromide staining on the agarose gel; specific mRNAs for ENaC and CK18 distributions were determined from Northern blots hybridized with 32P-labeled cDNA probes followed by phosphorimage analysis. Results shown are mean and standard error of three independent experiments.

 
Quantitation of the Northern analysis of RNA in fractions recovered from the gradients (Figure 4B) showed that DEX treatment of FDLE maintained under fetal PO2 resulted in redistribution of {alpha}ENaC mRNA from the heaviest polysomes (fractions 11–13) toward lighter polysome fractions (fractions 6–8). There was no change in the amount of {alpha}ENaC mRNA associated with monosomes/preinitiation complexes (fractions 1–4). There was no change in the distribution of mRNAs encoding the epithelial cell marker CK18 or the other two ENaC subunits, confirming that the change seen in {alpha}ENaC distribution is specific to this mRNA. The distributions shown in Figure 4B represent the means of three fractionations from independent preparations of epithelia grown in primary culture.

Incubation at Postnatal Po2 Restores Efficient Ribosome Loading on {alpha}ENaC mRNA in DEX-Treated FDLE
To determine whether exposure to 21% O2 could improve the efficiency of translation of the abundant {alpha}ENaC mRNA in DEX-treated cells, we analyzed paired polysome gradients from hormone-treated cells grown under the different O2 conditions (Figure 5). Increasing O2 to postnatal levels did not greatly alter the overall polysome profiles (Figure 5A) or change the distribution of small ribosomal subunits (Figure 5B, 18S rRNA panel). In contrast, it did result in a shift of {alpha}ENaC mRNA from smaller polysomes (fractions 6–8) into the heaviest polysomes (fractions 11–13), reversing the change seen in Figure 4B. There was no change in distribution of CK18 or of beta- or {gamma}ENaC mRNA, indicating that the effect of postnatal PO2 was specific to {alpha}ENaC mRNA translation.


Figure 5
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  		Figure 5. Postnatal O2 increases ribosome loading on {alpha}ENaC mRNA in FDLE cultured in 50 nM DEX. FDLE monolayers were cultured in the presence of 50 nM DEX for 48 h under fetal (3% O2) or postnatal (21% O2) atmospheres. Cytoplasmic extracts were prepared and fractionated on 15–45% linear sucrose gradients, and the distribution of various RNA species was analyzed. (A) Representative ultraviolet absorbance profiles of FDLE lysates sedimented through sucrose gradient. On the tracings, the density of the gradient increases from left to right, and the position of the 80S monosome peak is indicated (M). Location of the recovered fractions (1–13) and total RNA from each fraction analyzed by agarose gel electrophoresis and ethidium bromide staining is shown below each tracing. (B) Distribution of specific RNAs in fractions recovered from sucrose density gradients, expressed as percent of total for that RNA in the gradient. 18S rRNA was calculated from the intensity of ethidium bromide staining on the agarose gel; specific mRNAs for ENaC and CK18 distributions were determined from Northern blots hybridized with 32P-labeled cDNA probes followed by phosphorimage analysis. Results shown are mean and standard error of three independent experiments.

 
For statistical analysis of the changes in {alpha}ENaC translation, fractions were grouped into "untranslated/monosomes" (fractions 1–4), "lighter polysomes" (fractions 5–9), and "heavier polysomes (fractions 10–13). Integrations of the {alpha}ENaC mRNA distribution patterns in polysome profiles from FDLE at fetal PO2 showed a statistically significant increase in {alpha}ENaC mRNA associated with the lighter polysome fraction and reciprocal decrease in association with the heavier polysome fraction induced by DEX (Figure 6A). An exact reversal of this pattern was seen when FDLE cultured in DEX-supplemented media were shifted from 3% to 21% O2 (Figure 6B).


Figure 6
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  		Figure 6. Redistribution of {alpha}ENaC mRNA between light and heavy polysome fractions in response to DEX and O2. Percentage of {alpha}ENaC mRNA in gradient fractions from Figures 4 and 5 was integrated for untranslated/monosomes (fractions 1–4), lighter polysomes (fractions 5–9), and heavier polysomes (fractions 10–13). (A) Treatment of FDLE cultured under 3% O2 with 50 nM DEX for 48 h resulted in a significant increase in {alpha}ENaC mRNA in fractions 5–9 and a decrease in fractions 10–13 (*P < 0.05, n = 3). (B) FDLE in DEX-supplemented media were maintained in 3% or 21% O2 for 48 h. Higher O2 resulted in a decrease in {alpha}ENaC mRNA in fractions 5–9 and an increase in fractions 10–13 (*P < 0.05, n = 3). Data shown are means and standard error. Data for 3% O2 plus 50 nM DEX in parts A versus B were collected from completely separate sets of experiments.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 RESULTS
 DISCUSSION
References
 
The induction of an absorptive phenotype in the perinatal lung epithelium is crucial to the successful transition to air-breathing at birth. This change depends on regulation of key Na+ transport proteins by hormones such as glucocorticoid and increases in ambient O2 tensions (3). Barker and Gatzy (31) used distal lung explants from fetal rats to demonstrate that a shift from fetal to postnatal O2 resulted in a reduction in lung wet-to-dry weights and in the number and size of fluid-filled cysts in late gestation explants but not in explants from early gestations. The effect of O2 on fluid secretion could be induced in immature explants by hormone treatment. Direct activation of the {alpha}ENaC gene promoter by glucocorticoid receptors has been documented by several investigators (6–8). Although postnatal O2 environments increase ENaC mRNA levels in FDLE, the mechanism by which O2 exerts its effect is less clear because time-course studies indicate that increases in ENaC mRNA levels lag the increase in amiloride-sensitive Isc (12), and it has been shown that O2 can raise FDLE Na+ conductance without changes in ENaC mRNA abundance (17).

Using primary cultures of rat FDLE cultured in submersion on permeable membranes, we found that 50 nM DEX alone did not induce a statistically significantly increase in amiloride-sensitive Isc under fetal O2. This was initially surprising because Thome and colleagues (18) had reported that corticosterone treatment increased amiloride-sensitive Isc under any O2 condition. However, several differences exist between the experimental systems used, including the choice of hormone, the inclusion of cultures maintained at 5% O2 by Thome and colleagues, the use of serum-free versus stripped-serum medium, and the use of Cl–-free versus normal saline in the Ussing experiments, which may have resulted in our differing observations. In addition, Richard and colleagues (17) have shown that choice of growth medium affects the actions of hormones and O2 on FDLE Na+ conductance and ENaC mRNA levels. In agreement with our study, they also showed minimal increases in apical Na+ conductance when cells in minimal defined serum-free medium were treated with 200 nM DEX and 10 nM thyroid hormone at fetal PO2. Nevertheless, 50 nM DEX treatment significantly induced {alpha}ENaC mRNA under both O2 conditions in our experiments.

We also noted that O2 significantly increased amiloride-sensitive Isc under hormone-free conditions in the absence of significant changes in {alpha}ENaC mRNA or protein synthetic rates. This may be due to O2-dependent modulation of channel maturation, trafficking to the membrane, cell-surface stability and recycling, or regulation of apical membrane channel activity. The signal transduction pathways controlling these aspects of apical Na+ transport in alveolar epithelial cells under physiologic or pathologic conditions have not been elucidated in detail. Proteolytic cleavage of ENaC by furin during channel maturation correlates with channel activity (32), and extracellular serine proteases (e.g., channel-activating protease) modulate lung epithelial cell ENaC currents in vivo and in vitro with no change in protein abundance (33). The effects, if any, of O2 conditions on these processes have not been investigated. Trafficking of ENaC to the membrane and cell-surface stability are the targets of hormonal regulation; there is evidence for mediation via serum- and glucocorticoid-regulated kinase and Nedd4 pathways (10, 34, 35) and via phosphoinositide pathways (36). Planes and colleagues (16) have demonstrated the O2 dependence of ENaC trafficking to the apical membrane in response to beta-agonists in cultured ATII cells. In addition, single-channel electrophysiologic characteristics can be modulated via hormones and oxygen (19, 37, 38), and reactive oxygen and nitrogen species modulate ion-channel function at multiple levels (39). Thus, O2 may regulate ENaC activity at the post-translational level via a variety of mechanisms, which we have not investigated further at this time.

Our study is unique in reporting immunoprecipitation of endogenous {alpha}ENaC protein from metabolically labeled primary FDLE. Intracellular and cell-surface pools of ENaC subunit proteins turn over rapidly (t1/2 ~ 1–3 h) (34). We immunoprecipitated newly synthesized {alpha}ENaC subunits from FDLE pulse-labeled for 30 min with no chase period; therefore, our results should represent changes in the rate of {alpha}ENaC protein synthesis under the four culture conditions with relatively little contribution from changes in protein stability. Pulse-chase experiments would permit measurement of the protein's half-life; however, the {alpha}ENaC signal is not sufficiently high in our basal conditions (hormone-free, 3% O2) to permit accurate quantitation of its decay.

Analysis of the data presented in Figures 1 and 3 and Table 1 show that, under fetal O2 environments, the increased intracellular pool of {alpha}ENaC mRNA induced by DEX has little effect on protein synthetic rate or amiloride-sensitive Isc, providing strong evidence for inhibition of {alpha}ENaC mRNA translation under these conditions. Increasing O2 to postnatal levels seems to at least partially release this block, although the increase in protein synthesis induced by DEX at 21% O2 (~ 3 fold) lags the increase in mRNA level (5- to 6-fold).

Initiation is usually the rate-limiting step in protein translation, and it is often the target of regulatory mechanisms that involve elements of a specific mRNA's 5' untranslated region (UTR). Changes in translational efficiency can be assessed by the distribution of mRNA from heavy (i.e., many ribosomes/mRNA) to lighter (i.e., few ribosomes/mRNA) polysome fractions. Polysome distribution analysis indicated changes in the efficiency of loading of ribosomes on the {alpha}ENaC mRNA. Under fetal O2, addition of DEX shifted {alpha}ENaC mRNA away from the heavier polysomes and toward lighter fractions, although there was no change in the proportion of untranslated message. This is consistent with a model in which the larger pool of {alpha}ENaC mRNAs induced by DEX failed to result in an increased rate of {alpha}ENaC protein synthesis because each mRNA is translated by fewer ribosomes. In contrast, shifting the DEX-supplemented cells from a fetal to a postnatal O2 environment shifted the {alpha}ENaC mRNA back into the heavy polysome fractions (i.e., increased the number of ribosomes translating each {alpha}ENaC mRNA molecule), which would result in an increase in the rate of protein synthesis consistent with the immunoprecipitation data. These shifts were specific to {alpha}ENaC mRNA ribosome loading; no changes were seen in ribosome loading onto beta- or {gamma}ENaC mRNAs or onto the housekeeping gene CK18.

Density-gradient analysis allowed us to resolve polysome species up to n = 8 or 10 ribosomes/mRNA in a typical experiment. Thus, the "lighter polysomes" in fractions 5–9 contain mRNAs with as few as two ribosomes/polysome to as many as eight ribosomes/polysome. Resolution of individual polysome species in the heavier fractions is not possible; however, on average, ribosomes in polysomes occur once every 80–100 nucleotides, with a limit of one per 30–40 nucleotides due to packing constraints (40). For a 3.5-kb mRNA such as {alpha}ENaC, one would predict at least 35 ribosomes/mRNA, consistent with {alpha}ENaC's peak distribution in fractions 11 and 12. Thus, a redistribution of {alpha}ENaC mRNAs between the "heavier" and "lighter" polysomes as described in Figure 6 would represent a profound change in translational efficiency, with roughly a tenfold change in the number of actively translating ribosomes on those individual mRNA molecules. Inspection of the distribution curves for {alpha}ENaC mRNA in Figures 4 and 5 along with the quantitative data in Figure 6 suggests that a subset of ~ 15% of the total pool of {alpha}ENaC mRNA undergoes this dramatic change in ribosome loading, rather than the entire pool undergoing a slight change in ribosome loading. Inhibition of translation on only 15% of the pool of {alpha}ENaC mRNA is less than might have been anticipated based on the mRNA and immunoprecipitation results. We cannot rule out the possibility that regulation of peptide chain elongation or termination steps of translation may also play a role in {alpha}ENaC protein synthesis in our experimental model.

Understanding the complex process of translation initiation (41, 42) has helped elucidate how gene-specific protein production can be regulated by modifying the general translation machinery (43) and by specific regulatory elements within the 5'-UTR of specific mRNAs (44). The {alpha}ENaC gene uses multiple transcription start sites to express mRNAs with alternative, long 5' UTRs with considerable potential for secondary structure (6, 45–47), in contrast to the short, simple 5' UTRs found in typical mammalian mRNAs including betaENaC, {gamma}ENaC, and CK18. Thus, the {alpha}ENaC mRNA is a good potential candidate for translational regulation via the 5' UTR; for example, long 5' UTRs with secondary structure are particularly sensitive to limitations in the availability of the translation initiation complex eIF4F, which is responsible for unwinding secondary structure. DEX and hypoxia have the potential to inhibit translation initiation factors in ways that would preferentially affect {alpha}ENaC mRNA via its unusual 5' UTR. Our observations are consistent with the possibility that the combined effects of DEX and hypoxia are necessary to inhibit {alpha}ENaC translation.

Glucocorticoids attenuate mRNA translation at two levels: translational efficiency (i.e., translation initiation) and translational capacity (i.e., ribosome biogenesis) in lung (48) and in muscle (49) where this effect seems to be mediated via inhibition of the ribosomal protein S6 kinase (50, 51). S6 kinase targets the S6 protein, part of the 40S ribosomal complex, to regulate the translation of mRNAs containing 5'-terminal oligopyrimidine tracts (mostly ribosomal proteins and elongation factors) and phosphorylates the eukaryotic initiation factor eIF4B, a protein regulating eIF4A. The helicase activity of eIF4A, a component of the 5'-cap binding complex eIF4F, is important for the initiation of translation on mRNAs containing long and structured 5' UTRs (52). This pathway could provide a mechanism for the specific regulation of {alpha}ENaC translation in response to DEX observed in our study because the resulting limitation of eIF4F would be expected to preferentially impede translation initiation on {alpha}ENaC mRNA rather than betaENaC, {gamma}ENaC, or CK18 mRNAs. Direct glucocorticoid inhibition of the translation initiation mechanism might also explain the difference between our results and those of Thome and colleagues (18) if the latter's use of the less potent glucocorticoid (corticosterone) did not elicit this inhibitory effect.

Hypoxia is also known to inhibit protein synthesis via repression of the initiation step of mRNA translation, limiting the availability of eIF4F and of the ternary complex bearing the initiating methionyl-tRNA (30). Much of the work on hypoxia and mRNA translation has demonstrated severe repression of overall translation initiation, but in contrast to the study reported here, these studies used severe hypoxia (< 1.5% O2) or anoxia. The polysome profiles in our study (Figure 5) showed no significant overall decrease in translation in fetal cells adapted long-term to mild hypoxia; yet, as discussed previously for glucocorticoid effects, the long, complex 5' UTR of {alpha}ENaC might provide the potential for "fine-tuning" of translation initiation on this mRNA via moderate changes in the availability of the initiation complexes.

Most studies agree that the intracellular and the cell-surface pools of ENaC subunit proteins turn over rapidly, with a half-life of the order of 1–3 h (34). Therefore, ongoing peptide synthesis of the ENaC subunits must be a requirement for lung fluid clearance and a successful transition to air-breathing at birth. The present study is the first to demonstrate specific translational regulation of {alpha}ENaC subunits in response to physiologic regulators such as steroid hormones and O2. In addition to regulation at the levels of gene transcription and membrane trafficking, control of translation initiation may be a further level of control on ENaC expression in the perinatal lung, allowing lung epithelial cells to "stockpile" {alpha}ENaC mRNA induced by the prenatal surge in glucocorticoids.


   Acknowledgments
 
The authors gratefully acknowledge assistance from Dr. Olivier Staub and Dr. Daniela Rotin in preparation of the {alpha}ENaC antiserum used in these studies.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research Operating Grant MGP-25046 and Group Grant in Lung Development.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0273OC on October 6, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 19, 2005

Received in final form September 27, 2005


   References
Top
 Abstract
 Introduction
 Materials and Methods
 RESULTS
 DISCUSSION
 References
 

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