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Published ahead of print on December 12, 2003, doi:10.1165/rcmb.2003-0381OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 862-870, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0381OC

Differential Translational Efficiency of ENaC Subunits During Lung Development

Gail Otulakowski, Bijan Rafii and Hugh O'Brodovich

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

Address correspondence to: Gail Otulakowski, Ph.D., Programme in Lung Biology Research, Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: gail.otulakowski{at}sickkids.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The amiloride-sensitive epithelial Na+ channel (ENaC), the rate-limiting step in epithelial Na+ transport, consists of three subunits: {alpha}, ß, and {gamma}. The abundance of mRNA encoding the {alpha}-subunit far surpasses the amount for other subunits, and considerably exceeds the predicted subunit protein stoichiometry. We evaluated 5'-untranslated region (UTR) expression and found that fetal rat lung uses alternative 5'UTRs for {alpha}-ENaC during development. Sucrose density gradient analysis of postnuclear supernatants from fetal rat lung homogenates demonstrated that all three ENaC subunits were associated with high molecular weight polysomes, indicating active translation of the mRNAs, but translational efficiency was much lower for the {alpha}-subunit. Sucrose density gradient distributions were comparable for the endogenously expressed {alpha}-ENaC 5'UTRs in rat lung at Fetal Day 20 or Postnatal Day 1 using Northern analysis. Although birth resulted in a global decrease in lung mRNA translation, the loading of ribosomes on ENaC subunit mRNAs was largely unaffected. Evaluation of cytokeratin 18 and vimentin mRNAs in these gradients suggested a cell-specific effect. We conclude that there are different translational efficiencies for ENaC subunits and that perinatal processes globally modulate lung mRNA translation.

Abbreviations: cytokeratin, CK • endoplasmic reticulum, ER • epithelial sodium channel, ENaC • eukaryotic initiation factor, eIF • polymerase chain reaction, PCR • protein S6 kinase, p70S6k • rapid amplification of cDNA ends, RACE • ribonuclease protection assay, RPA • RNA-ligation mediated, RLM • untranslated region, UTR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The amiloride-sensitive epithelial sodium channel (ENaC) is found in the apical membrane of salt-absorbing epithelia lining the distal nephron, distal colon, and lung, where it constitutes the rate limiting step in transepithelial Na+ absorption (for reviews see Refs. 1–3). ENaC consists of three homologous subunits ({alpha}, ß, and {gamma}) initially cloned from rat distal colon (4, 5); the subunits share a common topology of two membrane-spanning domains, a large extracellular loop containing multiple potential N-glycosylation sites, and short intracellular NH2 and COOH termini (6–8). In heterologous expression systems (e.g., Xenopus oocytes), coexpression of all three subunits is necessary for maximal channel activity, although small currents arise when {alpha}-ENaC is expressed alone or paired with either ß- or {gamma}-ENaC (4). The channel subunits are believed to preferentially assemble into a heterotetrameric structure consisting of two {alpha}-subunits separated by one ß- and one {gamma}-subunit (9, 10). A nonameric stoichiometry has also been proposed (11).

In the kidney, ENaC contributes to electrolyte balance and plays an important role in the regulation of systemic blood pressure. Gain-of-function mutations result in an inherited form of hypertension termed Liddle's syndrome (12, 13). Loss-of-function mutations result in the genetic hypotensive salt-wasting syndrome known as pseudohypoaldosteronism type I, which is associated with a mild dysfunction in lung fluid absorption (14–16). In mouse knockout models, inactivation of the ß- or {gamma}-subunit of the mouse ENaC resulted in severe defects in renal Na+ and K+ transport, leading to death from hyperkalemia within 2 d of birth (17, 18). In the lung, ENaC is important in controlling the amount of liquid in the lung airspace; {alpha}-ENaC, but not ß- or {gamma}-ENaC, knockout mice die within 2 d of birth, are unable to clear their fetal lung fluid, and exhibit severe neonatal respiratory distress (19). These models highlight the critical role of the {alpha}-subunit in ENaC-mediated control of lung fluid clearance at birth, the importance of which had been previously demonstrated pharmacologically via instillation of amiloride in the lungs of newborn guinea pigs, which resulted in hypoxemia, respiratory distress, and a failure to clear airspace fluid in the subject animals (20).

At present there is no explanation for the data arising from Northern analysis, in situ hybridization, and quantitative reverse transcription–polymerase chain reaction, which all indicate that {alpha}-ENaC mRNA is expressed at markedly greater levels than ß- and {gamma}-ENaC mRNA in respiratory tract epithelia (21–25) and far in excess of the predicted protein stoichiometry. Mapping of the transcriptional start sites of the ENaC subunits has indicated that the rat, mouse, and human {alpha}-ENaC mRNAs each possess an unusually long (up to 750 nt) 5' untranslated region (UTR), with evidence for multiple transcription start sites, out of frame upstream AUG codons, and (in the human) alternative splicing (26–29). Our laboratory has published evidence that the {alpha}-ENaC 5'UTR contains distinct elements capable of modulating translation of the attached open reading frame (ORF) in both reticulocyte lysates and transiently transfected cell lines (30). In contrast, the ß- and {gamma}-mRNAs contain short (< 170 nt) 5'UTRs more typical of well-translated mammalian mRNAs (31–34).

There has been considerable progress in recent years in understanding the mechanisms involved in regulating ENaC transcription, trafficking, and cell surface stability, particularly in response to hormones (26, 35, 36). Assembly of ENaC subunits in the endoplasmic reticulum (ER) appears to be an important factor influencing successful maturation of the complex; maturation seems to be particularly low in heterologous systems, especially those expressing only a single subunit or pairs of subunits (37, 38). The complex mechanisms involved in the assembly of multimeric channels are not completely understood, and both the rate of peptide synthesis and the steady-state levels of immature protein subunits in the ER may be important factors. We hypothesized that {alpha}-ENaC may be less efficiently translated than ß- and {gamma}-ENaC, but that physiologic regulation of {alpha}-subunit translation may be exerted via its 5'UTR. Regulation at the level of translation could influence channel assembly and thus trafficking to the membrane, playing a role in enabling ENaC-expressing tissues to respond quickly to physiologic needs, such as the clearance of lung fluid at birth to allow the transition to air-breathing. Most changes in the translational efficiency of a given mRNA can be assessed by determining the percentage of that mRNA associated with actively translating polysomes of various sizes. Under most circumstances, the rate-limiting step in protein translation is the initiation step, and it is often the target of regulatory mechanisms that may be either general (via eukaryotic translation initiation factors) or gene-specific, via 5'UTR elements (39–42). We therefore analyzed the relative expression of alternative 5'UTRs for {alpha}-ENaC during lung development, and the distribution of {alpha}-, ß-, and {gamma}-ENaC mRNAs following sucrose density gradient fractionation of polysomes from fetal and newborn rat lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNase Protection Assay
Cloned rat genomic {alpha}-ENaC DNA fragments (26) were used to construct templates in pBluescript II KS- (Stratagene, LaJolla, CA) for RNase protection assay (RPA) analysis of transcription start site usage (Figure 1A). Four overlapping probes were constructed. (i) MA324 was produced by cloning a MscI-ApaI {alpha}-ENaC fragment into EcoRV-ApaI digested vector. Antisense cRNA (346 nucleotides [nt], of which 324 nt are homologous to {alpha}-ENaC) was synthesized in vitro using T3 RNA polymerase following linearization of the plasmid with EcoRI. (ii) EH3 consists of a HinfI-EarI fragment cloned into the SmaI site of the vector. Antisense cRNA (360 nt, of which 296 nt are homologous) was synthesized in vitro using T7 RNA polymerase following linearization with EcoRI. (iii) PvS340 consisted of a 334-bp PvuII-SacI fragment. Antisense cRNA (355 nt, of which 334 nt are homologous) was synthesized in vitro using T7 RNA polymerase following linearization of the vector with HindIII. (iv) LRPAM was generated by polymerase chain reaction (PCR). The construct was sequenced to exclude possible sequence errors introduced by PCR amplification. Antisense cRNA (565 nt, of which 509 are homologous) was synthesized in vitro using T3 RNA polymerase following linearization with XbaI.



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  		Figure 1. RPA analysis of 5' end of {alpha}-ENaC transcripts in rat lung. (A) Genomic DNA regions used to generate RPA probes. The upper, heavy horizontal line represents the region of cloned rat genomic DNA containing the 5' end of {alpha}-ENaC from which probe sequences were generated to assess transcriptional start sites. The open rectangle indicates genomic sequences encoding {alpha}-ENaC protein; numbering (+1) is from the start of the open reading frame. The bent solid arrows indicate transcriptional start sites reported earlier by 5'RACE and primer extension (26). The dotted arrows represent additional start sites characterized in the present study. The horizontal lines in the lower part of the figure represent DNA sequences used to generate RPA probes; they are labeled to the left as described in MATERIALS AND METHODS. (B) Ontogeny of {alpha}-ENaC transcript expression in the developing rat lung. Total RNA was isolated from whole lung of fetal (F, gestational ages 17–21 d) and postnatal (P, age 1 d, 7 d, and Adult) rat and subjected to RPA using probe EH3. Intact probe (360 nt) is seen in the control (yeast RNA) lane in the absence of RNase digestion. Some undigested probe remains in the rat lung samples. Protected fragments (arrows) are seen in rat lung RNA hybridizations at 244 nt, 192 nt, and 110 nt. Markers are radiolabeled in vitro transcribed 100 nt-RNA ladder (Ambion). Exact sizes of protected fragments were determined by co-electrophoresis of manual sequencing reactions alongside the RPA samples and RNA ladder. (C) Quantitative analysis of {alpha}-ENaC 5' UTR usage in rat lung. Optical densities of autoradiographic bands from protected fragments from four different experiments (as in B) were determined using NIH Image software. Band density was corrected for fragment length, and expressed as a percentage of the 516 nt UTR in adult rat lung (y axis). Ages shown are 20 and 21 d gestational fetal lung (FD20, FD21), postnatal 1 d, 7 d (P1, P7), and adult (Ad). Bars represent mean of four experiments from separate litters. *P < 0.01 compared with 516 nt UTR at all earlier ages. #P < 0.05 compared with shorter UTRs at same age, and P < 0.01 compared with 650 nt UTR at FD20 and P7.

 
Total RNA was prepared from fetal and adult lung from Wistar rats using Trizol (Life Technologies, Burlington, ON, Canada) at ages as indicated in RESULTS. All 32P-labeled cRNA probes were gel purified before use. Probes were hybridized overnight with 20 µg of sample RNA or yeast RNA, using the RPAIII kit (Ambion, Austin, TX) according to the manufacturer's directions. Hybridized samples were digested with RNase and analyzed by denaturing polyacrylamide gel electrophoresis.

5' Rapid Amplification of cDNA Ends
Adult rat lung RNA was prepared as described above. RNA ligation-mediated (RLM) 5' rapid amplification of cDNA ends (5'RACE) was used to amplify cDNAs from the rat {alpha}-ENaC mRNA (First Choice RLM-RACE kit; Ambion). The manufacturer's protocol was followed with the gene-specific primers AHSP2: 5'-CGCGGATCCGTCTTCATGCGGTTGTGTTT, or RACE2: 5'-CTGCCTGGCTTAGCGTCTCT, with the kit's outer adapter primer. The amplified cDNAs were cloned into pGEM-Teasy for sequence analysis of multiple subclones.

Sucrose Density Gradient Analysis of Polysomes
Timed gestation Wistar rats were used in all studies. Lungs from fetal (gestational age 20 d, term = 22 d) or newborn (1 d) rats were rapidly removed and snap-frozen in liquid nitrogen. Approximately 200 mg of frozen lung was pulverized under liquid nitrogen. All subsequent procedures were performed on ice or at 4°C. The powder was homogenized in 3 vols of ice-cold polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 7.4, 100 µg/ml cycloheximide, 0.5% Triton X-100, 0.5% sodium deoxycholate, 250 mM sucrose, and 1,000 U/ml placental RNase inhibitor) with eight passes of a Dounce B pestle followed by six passes with the A pestle. Nuclei were removed by two sequential centrifugations at 12,000 x g for 5 min. The resulting supernatant was layered on a linear 15–45% (wt/vol) sucrose gradient in polysome gradient buffer (100 mM KCl, 5 mM MgCl2, and 10 mM HEPES, pH 7.4), and gradients were centrifuged at 35,000 rpm for 2 h in a Beckman SW41 rotor. The gradients were 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. Each sample was diluted with an equal volume of proteinase K solution (0.2 M Tris-HCl, pH 7.5, 25 mM EDTA, 0.3 M NaCl, 2% SDS, and 250 µg/ml proteinase K). Following incubation for 30 min at 45°C, samples were extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, vol/vol) and the aqueous phase precipitated with 0.1 vol of 3.0 M sodium acetate, pH 5.2, and 2.5 vols of ethanol, with glycogen as a coprecipitant. The entire RNA pellet from each fraction was subjected to electrophoresis on a 1% agarose 2.2 M formaldehyde gel in 1x MOPS buffer (1x MOPS = 20 mM 3-N[Morpholino] propane sulfonic acid, 5 mM sodium acetate, 1 mM EDTA) and transferred to a nylon membrane (Hybond-N+; Amersham, Baie d'Urfe, PQ, Canada). 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}-, ß-, and {gamma}-ENaC as described (43), for mouse cytokeratin 18 (CK18), consisting of nt 440–810, and for rat vimentin, consisting of nt 844–1294. A probe specific to the extreme 5' end of the rat {alpha}-ENaC mRNA transcribed from the most upstream start site ({alpha}5p) was generated by PCR using the primers p4406 (5'-AATGAGGCTTCTGTCGCT) and p4540R (5'-TCCGCTGTGACTGGTTCCTTTCCA) and labeled with 32P using hot asymmetric PCR to generate a single-stranded antisense probe. Hybridized membranes were washed in 0.2 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0) and 0.1% SDS for 1 h at 50°C. Quantitative analysis of hybridized probes was performed using a Molecular Dynamics PhosphorImager equipped with ImageQuant software.

Statistical Analysis
Data in bar graphs are presented as mean ± SE, and statistical significances were calculated using one-way ANOVA. A P < 0.05 was considered to be statistically significant. Statistical analysis was performed using GraphPad InStat version 3.01 (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcription Start Site Analysis
In our earlier publication describing the structure of the gene encoding rat {alpha}-ENaC (26), we identified two transcriptional start sites, using 5'RACE and primer extension. These sites were 454 bp apart and would result in alternative, overlapping 5'UTRs of 516 or 62 nt. Because results from 5'RACE experiments suggested that alternative 5'UTR expression may be developmentally regulated in lung, we undertook a quantitative characterization of {alpha}-ENaC transcript expression in fetal rat lung using RPA. Four overlapping probes were generated (Figure 1A), extending over a region from 186 nt upstream of the putative first transcriptional start site to 236 nt downstream of the putative second transcriptional start site.

We predicted that rat lung RNA would protect two bands from probe MA324, one of 237 nt, corresponding to transcripts initiating at the downstream start site (bent arrow at –62 in Figure 1A) and one full length of 324 nt, corresponding to transcripts initiating at the upstream start site (bent arrow at –516 in Figure 1A). However, only the full-length protected fragment was detected in RPA experiments using rat RNA from adult lung, adult kidney, fetal lung (gestational age 20 d), or fetal distal lung epithelial cells (aged 20 d) (data not shown). Similarly, hybridization of probe PvS340 (Figure 1A) to RNA from these four sources also yielded a single protected fragment corresponding in size to the full length of the homologous region (data not shown).

Probe EH3, in addition to protecting a band at 110 nt as predicted, also yielded longer protected fragments at 192 and 244 bp (Figure 1B). The faint bands at ~ 290 nt are present in control reactions of undigested probe hybridized to yeast RNA, and are therefore classified as background. Preliminary RPA experiments with probe LRPAM yielded protected fragments at ~ 370 and 500 nt and a weak band at ~ 450 nt, in agreement with the pattern obtained from EH3 (data not shown). To confirm whether these additional bands were transcriptional start sites or splicing junctions, we performed RLM-RACE experiments, a modification of the 5'-RACE procedure designed to amplify cDNA only from full-length, capped mRNA. We performed independent experiments using gene-specific primers located within the {alpha}-ENaC open reading frame (primer AHSP2) and within the 5'UTR (primer RACE2). Cloning of the RACE products and sequencing of multiple independent subclones revealed that all were contiguous with the known genomic sequence and confirmed the existence of transcriptional start sites located 516, 598, and 650 nt upstream of the initiation codon as indicated by RPA. No subclones corresponding to the transcriptional start at –62 nt were found.

We used the EH3 probe to characterize the ontogeny of {alpha}-ENaC transcript start site usage in fetal rat lung. Total RNA from fetal (gestational ages 17–21 d, term = 22 d) and postnatal (1 d, 1 wk, and adult) rat lung was subjected to RPA in four independent experiments (Figure 1B). Quantitation of the protected fragments, expressed as a percentage of the 516 nt 5'UTR in adult lung (Figure 1C), revealed that the increase in {alpha}-ENaC mRNA expression around the time of birth consists mainly of transcripts initiating at the most upstream site. The fall in {alpha}-ENaC mRNA levels on Postnatal Day 7 has been noted previously using Northern analysis, and correlates with glucocorticoid resistance of rat during the first week after birth (44, 45). Transcripts initiating at the –516 site are upregulated to comparable levels only in the adult, whereas transcripts corresponding to the 192 nt protected band (598 nt UTR in Figure 1C) are expressed at much lower levels throughout lung development.

{alpha}-ENaC mRNA Is Associated with High Molecular Weight Polysomes in Fetal Rat Lung
Although distal lung epithelial cells isolated from developing rat lung clearly express functional ENaC in primary culture (46), expression of ENaC in the whole fetal lung has been limited to studies of the mRNAs (25, 45, 47, 48). We used sucrose gradient sedimentation to evaluate the association of {alpha}-ENaC mRNA with ribosomes in the developing rat whole lung as an indication of the efficiency of protein translation from this mRNA. Postnuclear supernatants from Fetal Day 20 rat lungs were sedimented on 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.

A representative profile is shown in Figure 2A with a numbered grid to illustrate the position of collected fractions, and results from Northern analysis below. A prominent 80S monosome peak was present in fraction 4, with free 60S and 40S ribosomal subunits detected as shoulders in fractions 2 and 3. Disomes, trisomes, and larger polysomes were distributed in denser fractions; typically 8–10 distinct species could be observed. Assignment of ribosomal particles (40S, 60, 80S) to peaks was confirmed by isolation of total RNA from each fraction and observation of the intensity of 28S and 18S rRNA staining with ethidium bromide on denaturing agarose gels (as in Figure 3C). Northern blots using a cDNA probe directed against the protein coding region of the {alpha}-ENaC mRNA indicated that {alpha}-ENaC mRNA was most abundant in fractions 9, 10, and 11, indicating an association with polysomes. To verify that the hybridization observed in the polysomal portion of the gradient was truly due to an active association between mRNA and ribosomes, postnuclear supernatants were prepared and sedimented in the presence of 10 mM EDTA (Figure 2B). Chelation of Mg2+ with EDTA dissociates mRNA and ribosomes, such that the dissociated molecules remain in the upper regions of the gradient. As indicated in Figure 2B, Northern analysis of fractions derived from a gradient prepared in the presence of EDTA indicated that {alpha}-ENaC mRNA was present almost exclusively in the nontranslated region of the gradient (fraction 3).



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  		Figure 2. Association of {alpha}-ENaC mRNA with polysomes in fetal rat lung. (A) Postnuclear supernatant from Fetal Day 20 rat lung was sedimented through a 15–45% linear sucrose gradient in the presence of 5 mM Mg2+ as described in MATERIALS AND METHODS. The upper tracing denotes the absorbance at 254 nm of the gradient, monitored continuously during collection, the direction of sedimentation being from left to right. The position of the 80S monosome peak is indicated (M). Location of recovered fractions is indicated (numbered 1–13) and the results of Northern blot detection of {alpha}-ENaC mRNA in each fraction are illustrated below. (B) The same procedure was performed following addition of 10 mM EDTA to both the supernatants and the gradients, to dissociate polysomes, 80S ribosomal subunits, and 40S ribosomal/mRNA preinitiation complexes.

 


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  		Figure 3. Sucrose gradient distribution of ENaC mRNAs in fetal rat lung. (A) Representative ultraviolet absorbance profile of fractions from rat lung extract sedimented through a 15–45% linear sucrose gradient. On the tracing, the density of the gradient increases from left to right, and the position of the 80S monosome peak is indicated (M). Polysome species up to n = 8 ribosomes/mRNA were resolved in this particular gradient. (B) Relation of polysome size (n) to peak position on A254 profile chart. Location of polysomes with n = 9 and n = 10 determined by extrapolation of line drawn by linear regression analysis of resolved polysome species. (C) Northern blot analysis of RNA prepared from sucrose gradient fractions. Upper panel, ethidium bromide–stained gel. Note that only 5S tRNA is visible in fraction 1, whereas fraction 2 contains 18 s rRNA but no 28S rRNA, indicating the presence of free 40S ribosomal subunits in this fraction. The assignment of the monosome peak to fraction 4 is confirmed by the relative intensities of the 28S and 18S subunits in this fraction. Lower panels, autoradiographic images of the corresponding Northern blot following sequential hybridizations to 32P-labeled cDNA probes directed against {alpha}-, ß-, and {gamma}-ENaC. (D) ENaC subunit mRNA distribution through sucrose density gradients. Quantitative analysis of Northern blot phosphorimages was performed to calculate the amount of each subunit mRNA in each fraction as a percentage of the total for the entire gradient. Fractions were grouped to represent untranslated mRNAs, and small, medium, and large polysomes as described in the text for analysis. Columns represent mean ± SE for n = 6 gradients derived from 4 litters.

 
{alpha}-ENaC mRNA Is Less Efficiently Translated than ß- and {gamma}-ENaC mRNAs
To assess the relative translational efficiency of the three ENaC subunits, we performed sucrose density gradient analyses on postnuclear supernatants from whole lungs derived from multiple litters of Fetal Day 20 rats. Figure 3A illustrates a representative A254 nm absorbance profile with the positions of the monosome peak and recovered fractions indicated. Using log-log plots of ribosomes per mRNA (n) versus distance migrated (Figure 3B), we determined the positions of polysome species with n = 9 and n = 10 when these species were not adequately resolved, by extrapolation from the sedimentation behavior of polysomes with n >= 4. The theory and use of this extrapolation has been described in detail for species up to n = 17 (49).

Northern blots prepared from total RNA isolated from the gradient fractions were sequentially hybridized with cDNA probes for {alpha}-, ß-, and {gamma}-ENaC (Figure 3C). All three subunits were associated with polysomal fractions, indicating that all are actively translated in Fetal Day 20 lung. However, the {alpha}-subunit mRNA (but not ß- or {gamma}-) consistently showed a hybridization signal in fraction 3, suggesting that a portion of this mRNA is not being translated (monosomes appear in fraction 4). In addition, ß- and {gamma}-ENaC mRNA distributions consistently peaked in a denser fraction than {alpha}-ENaC mRNA. To define these differences as accurately as possible, we quantitated the distribution of each ENaC subunit across the 13 fractions of six independent gradients from four different litters. Figure 3D presents the summarized results as the percentage of each mRNA associated with untranslated mRNAs (gradient fractions 1–3, n = 0), monsomes and "small" polysomes (fractions 4–6, n = 1–4), "medium" polysomes (fractions 7–10, n = 5–10), and "large" polysomes (fractions 11–13, n > 10). {alpha}-ENaC mRNA was the only subunit found in significant amounts in the untranslated fractions of the gradient, and its distribution peaked in medium polysomes, leveling off in large polysomes. In contrast, fractions with > 10 ribosomes per mRNA contained the majority of ß-ENaC (78%) and {gamma}-ENaC (56%) mRNAs, with a significantly smaller proportion of the {alpha}-ENaC mRNA (41%, P < 0.001).

Gradient Distribution of {alpha}-ENaC mRNAs with Extended 5'UTRs
As noted earlier, the fetal rat lung expresses {alpha}-ENaC mRNAs with 5'UTRs of various lengths that appear to be developmentally regulated. Different UTRs might have different effects on translational efficiency, or different responses to physiologic stress. Because the UTRs are overlapping in sequence, it was possible to specifically probe Northern blots only for the longer species. Accordingly, we prepared a short (~ 130 nt) probe extending from –650 to –517 nt upstream of the protein coding region. This probe ({alpha}5p) is homologous to the 650-nt and 598-nt UTRs expressed in lung, but contains no sequence corresponding to 516 nt UTR (Figure 1). Sucrose density gradients were prepared from Fetal Day 20 and Postnatal Day 1 rat lungs, followed by sequential hybridization of Northern blots with probes directed against the protein coding region or against the extended UTR. Gradient distributions of the {alpha}-ENaC mRNAs with extended 5'UTRs were virtually indistinguishable from the distribution of the total pool of {alpha}-ENaC mRNA (Figure 4). Similar results were obtained in independent gradients from four sets of litters at each age. Thus, although the transcriptional usage of different 5'UTRs is influenced by development, there is no effect of development on translational efficiency of the various 5'UTRs.



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  		Figure 4. Sucrose gradient distribution of {alpha}-ENaC mRNAs in fetal and postnatal rat lung. Gradients and Northern blots were prepared and analyzed as described in Figure 3, from Fetal Day 20 (FD20) and Postnatal Day 1 (P1) rat lungs. Northern blots were hybridized sequentially with cDNA probes homologous to all {alpha}-ENaC mRNAs ({alpha}, solid line) or specific to the extended 5'UTR ({alpha}5p, dotted line). Blots were stripped and re-exposed to storage phosphor screen between hybridizations to confirm that signal had returned to background levels. Amount of {alpha}-ENaC mRNA in each fraction is expressed as a percent of the total in that gradient.

 
Changes in Polysome Profiles in Postnatal Lung
Ultraviolet absorbance profiles from fetal and postnatal lungs revealed changes in the distribution of RNA from heavy to lighter polysome fractions after birth (Figures 5A and 5B). Quantitation of the distribution of 18S rRNA in this experiment (Figure 5C) indicated that slightly over 50% of the ribosomal subunits in fetal lung were associated with high molecular weight polysomes in fractions 9–13, with 29% associated with small polysomes in fractions 4–6. This ratio was completely reversed in the postnatal lung studied in this experiment. Redistribution from denser to lighter fractions was consistently seen in paired gradient experiments from independent fetal and postnatal litters, although the extent of the change varied among different postnatal litters. The degree of redistribution was maintained in independent gradient analyses from littermates. The example shown in Figure 5 is one of the most dramatic examples, selected to highlight effects on mRNA distributions described below. However, the same pattern was seen in all experiments.



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  		Figure 5. Sucrose density gradient analysis of fetal versus postnatal rat lung. Ultraviolet absorbance profiles of postnuclear supernatants prepared from (A) Fetal Day 20 (FD20) and (B) Postnatal Day 1 (P1) rat lung and sedimented through a 15–45% sucrose gradient. Direction of sedimentation is from left to right. The 80S monosome peaks are indicated (M). Equal amounts of supernatant (crude A260 units) were loaded on each gradient. Note the change in the shape of the profiles in the denser portions of the gradient indicated by arrows. (C) Distribution of 18S rRNA in fractions recovered from sucrose density gradients from fetal day 20 (dotted lines) and postnatal Day 1 (solid lines) rat lung extracts. Distribution of 18S rRNA was calculated from intensity of ethidium bromide staining on the agarose gel and quantitated using NIH Image.

 
The distribution of ENaC mRNAs throughout the density gradient showed little or no change from fetal to postnatal lung, although there was a slight shift in {alpha}-ENaC in cases such as the one shown, in which ribosomal redistribution was very pronounced (Figure 6, upper panels). The shift in {alpha}-ENaC distribution was much less than that seen for 18S rRNA in the same gradient. To determine if this effect was specific to ENaC, we further hybridized the Northern blots with probes for CK18 as a marker for epithelial cells, and for vimentin as a marker for mesenchymal cells. As the lower panels in Figure 6 clearly show, the epithelial-specific CK18 mRNA distribution in the gradient was essentially unchanged in the postnatal lung, but fibroblast-specific vimentin mRNA underwent a shift in distribution toward lighter fractions of the gradient very similar to what was seen for 18S rRNA. These data indicate that translational efficiency of mRNA in the lung is influenced by development and/or birth, but can differ between cell types and proteins.



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  		Figure 6. Distribution of specific mRNAs ({alpha}-ENaC, ß-ENaC, {gamma}-ENaC, CK18, and vimentin) in fractions recovered from sucrose gradient sedimentation of Fetal Day 20 (dotted lines) and Postnatal Day 1 (solid lines) rat lung extracts, as determined by Northern blot hybridization with 32P-labeled cDNA probes followed by phosphorimage analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our experiments are the first to provide a potential explanation for the markedly different levels of mRNA encoding the ENaC subunits in lung, specifically that {alpha}-ENaC mRNA, relative to ß- and {gamma}-ENaC, is less efficiently translated into protein. We also found that although development influences the lung use of different 5'UTRs for {alpha}-ENaC, the resultant mRNAs are equally efficiently translated before and shortly after birth. Finally, we found that there are marked differences in global ribosome loading onto mRNAs before and after birth, which likely vary among different proteins and between different cell types.

Regulation of gene expression at the level of translation initiation is an important developmental mechanism (50). It has long been known that many maternal mRNAs are stored in the oocyte as inactive, masked ribonucleoproteins (mRNPs), which become translationally activated at specific times and locations within the oocyte and developing embryo. More recently, it has become clear that regulation of translation initiation via elements of the 5'UTR is used to control the synthesis of specific proteins late in development also; for example, the insulin-like growth factor II gene generates multiple mRNAs with different 5'UTRs that are translated in a differential manner during development. This effect is mediated by a family of mRNA-binding proteins that are able to specifically bind to the 5'UTR of the insulin-like growth factor II leader 3 mRNA (51). Other 5'UTR elements can also achieve developmental regulation of protein expression. Upstream AUG codons present in the extended 5'UTR of the embryonic-expressed proinsulin mRNA tightly repress synthesis of the hormone by initiating translation of an overlapping, out-of-frame ORF; these upstream AUGs are missing from the actively translated pancreatic form of the mRNA (52).

We investigated the possible role of the 5'UTR of {alpha}-ENaC in regulating translation of the mRNA into protein in vivo. Our RPA and RLM-RACE analyses have confirmed that rat {alpha}-ENaC mRNAs use multiple start sites and possess unusually long 5'UTRs (516, 598, and 650 nt). Using these methods, we were unable to detect {alpha}-ENaC mRNAs with a 62 nt 5'UTR as predicted by our earlier work in rat (26), although an alternative transcriptional start site at approximately this position has also been reported in mouse by others (27). It is possible that secondary structure in this region inhibited reverse transcriptase progression in earlier 5'RACE and primer extension approaches. Multiple, long 5'UTRs have also been reported for the {alpha}-ENaC mRNA in humans, although the situation is even more complex with alternative splicing leading to four different mRNAs (29). In addition, we have shown that the ontogeny of {alpha}-ENaC mRNA expression is such that the "peak" commonly shown around the time of birth and believed to be important in switching the fetal lung from a fluid-secreting to an absorbing organ (45) consists primarily of the mRNA with the most extended 5'UTR. In adult lung, a somewhat shorter mRNA becomes relatively abundant. Thus, the {alpha}-ENaC mRNA structure has the potential to be the subject of translational regulation via its 5'UTR during lung development.

We employed sucrose density gradient fractionation to examine the loading of ribosomes on rat {alpha}-, ß-, and {gamma}-ENaC mRNAs in developing rat lung to compare the translation efficiency of the subunits. All three subunits were actively translated in Fetal Day 20 rat lung in vivo; however, a comparison of distribution of each of the three ENaC subunit mRNAs across the gradient demonstrated that the loading of ribosomes per mRNA was in the order ß >> {gamma} > {alpha}. The efficiency of ribosome loading was lowest for the {alpha}-subunit, as expected for an mRNA possessing a long 5'UTR. Interestingly, translational efficiency for the three ENaC subunit mRNAs was inverted relative to their abundance in rat lung ({alpha} > {gamma} > ß). It would be useful to assess ribosome loading in human respiratory epithelium, where the relative mRNA abundance of the ß- and {gamma}-subunits is reversed compared with rodents (i.e., {alpha} > ß > {gamma}). If in human respiratory epithelium ribosomes were most efficiently loaded on {gamma}-ENaC mRNA, it would raise the question of whether mechanisms exist by which translation of differentially expressed subunit mRNAs can be coordinately regulated to deliver appropriate amounts of protein to the ER for correct channel assembly. This is potentially important for ENaC, because only heteromultimeric channels containing all three subunits are efficiently processed and trafficked to the cell surface. For example, excessive delivery of nascent {alpha}-ENaC peptides to the ER by efficient translation of its very abundant mRNA might favor formation of homomultimeric {alpha}4 channels. These channels may be preferentially targeted for degradation (36), but it has also been suggested that at least part of the diversity of Na+-permeable channels in lung arises from the assembly of different combinations of ENaC subunits to form channels with different biophysical properties and different mechanisms for regulation (53).

A number of physiologic mechanisms underlie perinatal lung liquid absorption, including effects triggered by ß-adrenergic agonists, oxygen, glucocorticoids, and thyroid hormones (54). 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 (55). The key Na+ transport proteins affected are the Na+,K+-ATPase and ENaC. Although direct transcriptional effects have been documented for some of these pathways (e.g., glucocorticoid activation of {alpha}1- and ß1-ATPase promoters [56] and of the {alpha}-ENaC promoter [26]), there is evidence that translation is also specifically regulated. Like {alpha}-ENaC, the subunit mRNAs of the Na+,K+-ATPase have been shown to possess long 5'UTRs rich in G/C sequences that predict complex and stable mRNA secondary structure (57–59) and impair translation (60). It has recently been shown that glucocorticoids can directly enhance Na+,K+-ATPase translation in vitro in a subunit-specific manner via a putative modulatory element in the 5'UTR of the {alpha}-subunit mRNAs (61) and that ß-agonists regulate Na+,K+-ATPase via protein S6 kinase (p70S6k) in transfected alveolar epithelial cells (62). p70S6k 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 also phosphorylates eIF4B, a protein regulating the helicase activity of eIF4A, important for the initiation of translation on mRNAs containing long and structured 5'UTRs. We have shown that the {alpha}-ENaC mRNA is translated less efficiently than the ß- and {gamma}-ENaC mRNAs in vivo (Figure 3), and that its 5'UTR contains elements that influence translation (30). The potential influence of hormones on ENaC translation has not yet been investigated, but could represent a potential mechanism for coordinate regulation of Na,K-ATPase and ENaC at the translational level by hormones involved in fetal lung maturation and the labor/birth process.

We were unable to demonstrate a difference in ribosome loading on the longest {alpha}-ENaC mRNAs compared with total {alpha}-ENaC mRNA in fetal or neonatal rat lung in these experiments. The sensitivity of this approach was limited by the inability to specifically probe for the shorter 5'UTR on Northern blots. Because the majority of the {alpha}-ENaC mRNAs are of the extended 5'UTR form at this point in development, it may not be possible to differentiate the profile of the extended 5'UTR from the total pool. A more productive approach in future may be to analyze the polysome distribution of the {alpha}-ENaC 5'UTRs in adult lung, where the longest and shortest forms are equally abundant.

The relative and absolute efficiency of translation of ENaC subunit mRNAs into protein appears to be maintained in the early postnatal period, despite a global decrease in the proportion of ribosomes associated with heavy polysomes. The basis of this phenomenon is unclear, but preliminary characterization of ribosome loading on CK18 and vimentin mRNAs suggests that it may be based on cell-specific effects (epithelial versus mesenchymal) rather than gene-specific effects. It cannot be determined from the present data whether this decrease in translation efficiency in the lung between Fetal Day 20 and Postnatal Day 1 is due to lung maturation, the birth process, or postnatal adaptations such as the change in oxygen tension or the need to establish independent glucose homeostasis. A variety of stresses, including ischemia/reperfusion, osmotic stress, oxidative stress, as well as defects in glucose homeostasis, can cause rapid inhibition of global protein synthesis in eukaryotic cells. For example, perturbations that alter ER homeostasis lead to accumulation of unfolded proteins in the lumen, which limit the availability of chaperone proteins, leading to phosphorylation of the translation initiation factor eIF2{alpha} and reducing formation of translation initiation complexes. This phenomenon has been termed the unfolded protein response (41, 63–65). It will be of interest to determine whether this mechanism underlies the decrease in ribosome loading in postnatal lung, to investigate whether various epithelial cells and mRNAs are truly able to avoid or shorten the associated decrease in translational efficiency, and if so whether this plays a role in lung development in general and specifically its adaptation at birth.

Most studies agree that both 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 (36). Thus ongoing peptide synthesis of the ENaC subunits must be a requirement for lung fluid clearance and a successful transition to air-breathing at birth. Little is known about the effects of labor, birth, and early postnatal metabolic adaptations on global protein synthesis rates in the neonate, although it has been shown that tissue protein synthesis rates in humans, rats, and pigs respond much more markedly to feeding in the neonatal period (66). The mechanism of this effect varies among tissues; for example, stimulation of skeletal muscle protein synthesis by feeding is primarily insulin-mediated, but stimulation of liver protein synthesis seems to be largely a function of amino acid concentration (66). Fractional protein synthesis in neonatal rat lung approximately doubled in fed animals 24 h after birth compared with fasted animals 4 h after birth (67), but no information is available regarding mechanism or the effect on synthesis of specific lung proteins. Thus it will be of interest to examine the sucrose density gradient distribution of mRNAs encoding proteins that would be expected to be important immediately after birth, such as surfactant protein-B and -C, as well as non-ENaC components of the ion transport pathway such as the Na+,K+-ATPase subunits. The effects of nutritional parameters such as glucose and amino acids, hormones such as insulin, glucocorticoids and ß-agonists, as well as physiologically relevant environmental changes such as the shift from fetal to postnatal oxygen tensions are all possible triggers for the changes in translational efficiency seen in postnatal rat lung. Their effects on translation initiation should be examined both in the intact animal and in lung epithelial and fibroblast cells in primary culture to determine which of these factors underlies the effects we have described, and to confirm the existence of cell-specific responses.


   Acknowledgments
 
The authors express appreciation to Celia Taha and Sam Dougaparsad for assistance with sucrose density gradient protocols. This work was supported by the Canadian Institutes of Health Research (CIHR) Operating Grant MGP-25046 and Group Grant in Lung Development.

Received in original form October 24, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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G. Otulakowski, W. Duan, S. Gandhi, and H. O'Brodovich
Steroid and Oxygen Effects on eIF4F Complex, mTOR, and ENaC Translation in Fetal Lung Epithelia
Am. J. Respir. Cell Mol. Biol., October 1, 2007; 37(4): 457 - 466.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
A. Dagenais, R. Frechette, M.-E. Clermont, C. Masse, A. Prive, E. Brochiero, and Y. Berthiaume
Dexamethasone inhibits the action of TNF on ENaC expression and activity
Am J Physiol Lung Cell Mol Physiol, December 1, 2006; 291(6): L1220 - L1231.
[Abstract] [Full Text] [PDF]

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 Am. J. Respir. Crit. Care Med.Home page
O. Helve, S. Andersson, T. Kirjavainen, and O. M. Pitkanen
Improvement of Lung Compliance during Postnatal Adaptation Correlates with Airway Sodium Transport
Am. J. Respir. Crit. Care Med., February 15, 2006; 173(4): 448 - 452.
[Abstract] [Full Text] [PDF]