help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on June 7, 2007, doi:10.1165/rcmb.2007-0055OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2007-0055OCv1
37/4/457    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Otulakowski, G.
Right arrow Articles by O'Brodovich, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Otulakowski, G.
Right arrow Articles by O'Brodovich, H.
American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 457-466, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2007-0055OC

Steroid and Oxygen Effects on eIF4F Complex, mTOR, and ENaC Translation in Fetal Lung Epithelia

Gail Otulakowski1,2, Wenming Duan1, Shephali Gandhi1 and Hugh O'Brodovich1,2,3

1 CIHR Group in Lung Development, and Program in Physiology and Experimental Medicine, Hospital for Sick Children Research Institute; and Departments of 2 Paediatrics and 3 Physiology, University of Toronto, Toronto, Ontario, Canada

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


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Fetal distal lung epithelium (FDLE) must increase amiloride-sensitive epithelial Na+ channel (ENaC) activity during the perinatal period to increase Na+ transport and fluid clearance. Glucocorticosteroid (GC) levels increase, there is a 7-fold increase in PO2 at birth, and we have previously shown that dexamethasone (DEX)-induced {alpha}-ENaC mRNA is efficiently translated only under postnatal (21%) O2 (Otulakowski et al., AJRCMB 2006;34:204–212). Translation of mRNAs with long GC-rich 5'UTRs, such as {alpha}-ENaC mRNA, are sensitive to the amount of eIF4F, the mRNA 5'-cap binding complex composed of eIF4E and eIF4G. We now show, by Western blotting and m7GTP-Sepharose pull-down experiments, that in FDLE cultured under 3% O2, DEX decreases formation of eIF4F and increases association of eIF4E with its inhibitor 4E-BP by changing 4E-BP phosphorylation. Conversely, FDLE cultured at 21% O2 expressed lower levels of 4E-BP and maintained eIF4E-eIF4G association independent of DEX. Phosphorylation of 4E-BP is regulated by the kinase mTOR. Under 3% O2, DEX decreased abundance of phosphorylated forms of the mTOR effectors, S6 kinase and ribosomal protein S6. Neither effect was associated with changes in REDD1, an upstream regulator of mTOR. When mTOR was inhibited (3 nM rapamycin) there was reduced 4E-BP phosphorylation, fewer ribosomes on {alpha}-ENaC mRNA, and decreased amiloride-sensitive short-circuit current, but no change in ribosomal loading onto any of beta- or {gamma}-ENaC or cytokeratin 18 mRNAs. We speculate that at birth increased PO2 acts with GC through an mTOR-related pathway to increase {alpha}-ENaC protein synthesis, thereby promoting lung fluid absorption.

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



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This investigation demonstrates a novel mechanism of regulation of Na+ transport relevant to the clearance of lung fluid at birth. An understanding of this process may lead to insights in lung pathophysiology during the immediate post-partum period.

 
The developing fetal lungs actively secrete fluid into the airspaces, a process which is essential for normal lung development, but this fluid must be cleared at birth to allow effective gas exchange and a successful transition to air breathing. Impaired clearance of this fetal lung liquid results in transient tachypnea of the newborn (TTN), and if combined with immaturity of the surfactant system, infants will suffer from neonatal respiratory distress syndrome (nRDS) (1–3). This switch from fluid secretion to fluid absorption at birth requires the developmental regulation of membrane transport proteins responsible for transepithelial Na+ transport, including effects triggered by beta-adrenergic agonists, oxygen, glucocorticoids (GC), and thyroid hormones (1). These factors have been demonstrated to interact with each other; for example, GC and thyroid hormones given concurrently advance the maturation of the absorptive response to epinephrine in fetal lamb lungs (4), and more recently it has been shown that oxytocin-induced labor augments IL-1beta–stimulated distal lung fluid absorption in fetal guinea pig lungs (5). The key Na+ transport membrane proteins involved are the basolateral Na+,K+-ATPase and the apical membrane's amiloride sensitive epithelial Na channel (ENaC), which under most circumstances represents the rate-limiting step in Na+ absorption (6, 7).

ENaC consists of homologous {alpha}-, beta-, and {gamma}-subunits (8, 9). The subunits share a common topology, and in Xenopus oocytes, a heterologous expression system, one requires co-expression of all three subunits for maximal channel activity, although small currents arise when {alpha}-ENaC is expressed alone or paired with either beta- or {gamma}-ENaC (8). As such, our previous work has focused on the integrated effects of dexamethasone (DEX) and changes in PO2 on {alpha}-ENaC expression and function. In primary cultures of rat fetal distal lung epithelia (FDLE), exposure to GC increases {alpha}-ENaC mRNA levels (10) via an increase in transcription mediated by a GC-responsive element in the 5' flanking region of the {alpha}-ENaC gene (11–13). There is also evidence that GC increase apical membrane ENaC expression in the short term by augmenting ENaC trafficking and membrane retention via the serum- and glucocorticoid-regulated kinase (sgk) (14, 15). Single-channel recordings from cultured adult alveolar type II epithelia (ATII) have shown that the presence of GC and an air–liquid interface promotes the expression of low-conductance, highly Na+-selective channels in contrast to the nonselective cation channels that are predominant in primary cultures of alveolar type I epithelia grown under comparable conditions (16) or ATII grown in submersion culture in the absence of steroids (17).

Recently, we have demonstrated that DEX-induced {alpha}-ENaC mRNA was efficiently translated into protein in primary cultures of FDLE only under postnatal (21%) O2 (18). Our sucrose density gradient analyses of polysomes showed changes in {alpha}-ENaC mRNA distribution consistent with specific modulation of {alpha}-ENaC translation initiation; specifically, DEX treatment of cells under fetal (3%) O2 decreased the association of {alpha}-ENaC mRNA with large polysomes, whereas shifting DEX-treated cells to 21% O2 restored {alpha}-ENaC mRNA association with large polysomes. These effects were specific to {alpha}ENaC: polysome distribution of beta and {gamma}ENaC was not affected. These data indicated that in the mature fetal lung, DEX can induce a pool of {alpha}-ENaC mRNA that is then available for rapid protein synthesis via gene-specific translational regulation when PO2 subsequently increases at birth. Since {alpha}-ENaC is less efficiently translated than the beta- and {gamma}-subunits (19), such a mechanism could alter the relative amounts of {alpha}-, beta-, and {gamma}-ENaC subunit proteins and thereby alter stoichiometry and/or have a direct or indirect effect on protein folding and hence channel assembly in the endoplasmic reticulum (ER). Protein translocation and folding has been demonstrated to occur co-translationally for a variety of membrane proteins, including the cystic fibrosis transmembrane regulator (CFTR) (20).

Translation initiation is a complex process that is regulated by both global and gene-specific mechanisms (reviewed in Refs. 21, 22). The cap structure at the 5' end of most mRNAs facilitates ribosome binding to the mRNA through an interaction with a cap-binding complex (eIF4F) consisting of eukaryotic initiation factors eIF4G and eIF4E. The availability of eIF4E is limited by the amount that is bound to its repressor, 4E-BP. Hypoxia is known to inhibit protein synthesis via repression of the initiation step of mRNA translation, limiting availability of both eIF4F and of the ternary complex bearing the initiating methionyl-tRNA (23). GC have also been shown to attenuate mRNA translation by a variety of mechanisms (24), including decreased phosphorylation of the ribosomal protein S6 kinase (p70S6K) and of 4E-BP (25–27). The serine-threonine kinase, mammalian target of rapamycin (mTOR) is a central integrator of environmental signals, such as nutrients, growth factors, hormones and PO2, and mTOR promotes mRNA translation by phosphorylating p70S6K and 4E-BP (28). The mTOR pathway thus is potentially capable of controlling translation initiation by integrating signals from exposure to GC and changes in PO2.

The {alpha}-ENaC gene uses multiple transcription start sites to express mRNAs with alternative, long 5' untranslated regions (UTRs) with considerable potential for secondary structure (11, 29–31). These are features that are typical of mRNAs, which require increased levels of eIF4F for efficient translation (21). In our present study, we therefore hypothesized that the decreased polysomal association of {alpha}-ENaC mRNA contained within primary cultures of FDLE maintained under 3% O2 atmosphere and exposed to DEX was mediated via the mTOR pathway and availability of eIF4F. We also examined the effects of PO2 and DEX treatment on eIF4F complex formation and on phosphorylation of 4E-BP and p70S6K, and present data demonstrating changes consistent with a decrease in translation initiation for eIF4F-sensitive mRNAs. Finally, we showed that, similar to the effects seen with DEX treatment, rapamycin-induced inhibition of mTOR activity resulted in a specific decrease in loading of ribosomes on FDLE {alpha}-ENaC mRNA.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
Rapamycin Insolution was purchased from Calbiochem (San Diego, CA) and stored frozen in single-use aliquots, protected from light. Antibody to REDD1 (Regulated in Development and DNA damage responses, also known as RTP801) was purchased from ProteinTech Group (Chicago, IL). Antibody to eIF4G was from Bethyl Laboratories (Montgomery, TX); all other primary antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Secondary antibody (goat anti-rabbit, horseradish peroxidase conjugated) was purchased from Pierce (Rockford, IL). Cell culture media was purchased from Invitrogen (Burlington, ON, Canada). All other reagents were from Sigma (Oakville, ON, Canada) unless otherwise indicated.

Cell Isolation and Culture
FDLE from 20-d gestation rat fetuses were isolated and grown in primary culture as previously described (32). All animal procedures were reviewed and approved by the Hospital for Sick Children Animal Care committee. FDLE were seeded at a density of 1 x 106 cells/cm2 onto 0.4-µm pore size Snapwell for Ussing chamber studies or 0.5 x 106 cells/cm2 on plastic dishes for biochemical studies. All cells were grown as submersion cultures in Dulbecco's modified Eagle's medium–high glucose (4.5 g/L glucose with 2 mM L-glutamine and 110 mg/L sodium pyruvate) supplemented with 10% fetal bovine serum (FBS, Cansera, Rexdale, ON, Canada), 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulphate. The culture media was replaced 24 hours after seeding to remove unattached cells, at which time media containing hormone-depleted FBS (stripped with charcoal and ion exchange resin) (33) was used, supplemented as indicated with 50 nM DEX, and cells were placed in incubators containing either 3% O2-5% CO2-balance N2 ("fetal" atmosphere) or 5% CO2-balance room air ("postnatal" atmosphere) for a 48-hour period before analysis. Cells were fed with fresh media 90 minutes before harvest, using solutions which had been pre-equilibrated at the appropriate PO2 (48 h) in the "fetal" or "postnatal" tissue culture incubators and handling "fetal" cells in a hypoxic workstation.

Lung epithelial cell lines (A549, H441, and MLE-15) for REDD1 studies were grown to confluence in "postnatal" atmosphere, after which media were changed as above for FDLE and cells were allocated to "fetal" or "postnatal" incubators for 48 hours, followed by a final media change and harvest as above.

Western Blots
To harvest the cellular protein, the tissue culture dishes were placed on ice and washed twice with ice-cold PBS, then lysed by direct addition of whole cell extract buffer (150 mM NaCl; 50 mM Tris pH7.4; 5 mM EDTA; 0.1% SDS; 20 mM beta-glycerophosphate; 10mM NaF; 0.25 mM Na orthovanadate; 1x Roche complete protease inhibitor cocktail [Roche Applied Science, Laval, PQ, Canada]). Protein concentrations were determined using the BioRad RC DC protein assay kit (BioRad Laboratories, Mississauga, ON, Canada). Proteins were size fractionated on SDS-PAGE gels and transferred to nitrocellulose membrane. After blocking using 5% nonfat dry milk in TBST buffer (20 mM Tris pH7.6; 140 mM NaCl; 0.1% [wt/vol] Tween20), membranes were incubated overnight with primary antibody (antibody to 4E-BP, eIF4G at 1:2,000, all others at 1:1,000) in 5% bovine serum albumin in TBST buffer. Secondary antibody was diluted 1:20,000 or 1:40,000 in blocking buffer and incubated for 1 hour at room temperature. All washes were in TBST. Enhanced chemiluminescence detection reagents were purchased from GE Healthcare (Baie d'Urfé, PQ, Canada) and used according to the manufacturer's recommendations. Blots were stripped in 61.5 mM Tris, pH 6.8, 2% SDS and 100 mM beta-mercaptoethanol at 55°C for 1 hour before being blocked and re-probed for actin according to standard protocols.

m7GTP Affinity Purification
FDLE plated on 100-mm-diameter dishes were placed on ice, washed twice with ice-cold PBS, and harvested by scraping into 800 µl per plate of pulldown buffer (20 mM HEPES pH7.4; 50 mM KCl; 0.2 mM EDTA; 25 mM beta-glycerophosphate; 0.5 mM Na-orthovanadate; 0.5% Triton X-100; 15 mM beta-mercaptoethanol; 50 mM NaF; 1x Roche complete protease inhibitor cocktail). After remaining on ice for 10 minutes, cells were thoroughly homogenized by passage through a 27-gauge syringe needle and then centrifuged for 10 minutes at 10,000 x g at 4°C. Extract (400 µg) was then incubated with 25 µl (packed volume) m7GTP-conjugated Sepharose beads (GE Healthcare) at 4°C for 2 hours on a rocking platform. After three washes in pulldown buffer and collection using low-speed centrifugation, beads were re-suspended in SDS-PAGE loading buffer and heated 10 minutes at 95°C. Heated supernatants then underwent electrophoresis on SDS-PAGE gels alongside 50 µg of unpurified lysates, followed by Western blotting as described above, probing for eIF4G and 4E-BP. After stripping, blots were re-probed for total eIF4E for normalization (34).

Sucrose Density Gradient Fractionation of Polysomes
Polysome profiles were prepared as previously described (18). Briefly, six 10-cm plastic dishes containing confluent FDLE were used for each gradient. Cells were harvested on ice 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, pH7.4, 100 µg/ml cycloheximide, 0.5% Nonidet P-40, and 1,000 U/ml placental RNase inhibitor). The lysates were scraped to a 1.5-ml microcentrifuge tube and passed three to four times through a 27-gauge needle to ensure the lysis of all cells. Nuclei were pelleted by two sequential centrifugations at 12,000 x g for 5 minutes at 4°C. The resulting supernatants (10–12 A260nm units per gradient) were layered on linear 15 to 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 hours in a Beckman SW41 rotor (Beckman Coulter, Mississauga, ON, Canada) 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 stored at –80°C.

Northern Blot Analysis
RNA was isolated from individual sucrose density fractions by proteinase K digestion, followed by phenol/chloroform extraction and ethanol precipitation as described (18). 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 MOPS pH 7.0, 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 (18). Hybridized membranes were washed at high stringency and analyzed via PhosphorImager (GE Healthcare).

FDLE Bioelectric Properties
Culture media were replaced 24 hours after seeding, at which time media containing hormone-depleted FBS was used, supplemented as indicated with 3 nM rapamycin, and cells were placed in an incubator containing 3% O2-5% CO2-balance N2 ("fetal" atmosphere) for a 48-hour period before analysis. Epithelial cells were studied in Ussing chambers at 37°C maintained under open circuit conditions and then switched to short circuit current (Isc) with transepithelial potential difference (PD), Isc, and transepithelial resistances (R) determined intermittently with voltage/current clamps (35, 36) Addition of amiloride (final concentration = 10–4 M) to the apical side of the monolayer yielded amiloride-sensitive and -insensitive Isc.

Statistical Analysis
Data are presented as mean ± SEM. Statistical significances were calculated using one-way ANOVA, with P < 0.05 being considered as statistically significant. Statistical analysis was performed using GraphPad Instat version 3.01 and GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effects of PO2 and DEX on Translational Regulation Pathways
As a preliminary indication of which pathway was regulating {alpha}-ENaC mRNA translation in DEX-treated FDLE cultured under 3% O2 atmosphere (see DISCUSSION), we assessed the relative abundance of phosphorylated forms of 4E-BP and eIF2{alpha} in FDLE cultured for 48 hours in 3% or 21% O2 in the presence or absence of 50 nM DEX (Figure 1). DEX treatment resulted in a decrease in the relative abundance of 4E-BP phosphorylated on Ser65 in cells cultured at low PO2, an effect that was not observed in cells cultured at 21% O2. It was also noted that the abundance of 4E-BP protein was increased in FDLE cultured in 3% O2. Phosphorylation at Ser65 is critical to prevent interaction of this inhibitor with eIF4E. These results suggest that incubation under fetal O2 levels induces expression of 4E-BP protein, but that this protein would have limited ability to repress translation due to its phosphorylation at Ser65. Addition of DEX under these conditions, however, results in reduced levels of hyperphosphorylated 4E-BP, and presumably results in an increase in sequestration of eIF4E. These results are consistent with the hypothesis that this pathway may be responsible for our previous observation of decreased translation of {alpha}ENaC mRNA in FDLE cultured under 3% O2 and treated with DEX, a phenomenon that can be relieved by switching to 21% O2 (18). In contrast to the changes in 4E-BP, no significant change in phosphorylated eIF2{alpha} at Ser51 were seen under the different culture conditions. Thus, the eIF2{alpha} regulatory pathway seems to be an unlikely mechanism for changes in lung epithelial {alpha}ENaC translational regulation under these conditions. As such, the remainder of our experiments focused on interactions within the 4E-BP pathway.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Figure 1. In fetal distal lung epithelium (FDLE), the combined effect of fetal (3%) O2 atmosphere and dexamethasone (DEX) increases levels of hypophosphorylated 4E-BP. FDLE were cultured in the presence or absence of 50 nM DEX for 48 hours under fetal (3% O2) or postnatal (21% O2) atmospheres, and whole cell extracts were Western-blotted for total or phosphorylated (Ser65) 4E-BP (left panel) or total and phosphorylated (Ser51) eIF2{alpha} (right panel). Minimal effects on eIF2{alpha} were seen. Results are representative of at least three independent preparations.

 
Combining 3%O2 and DEX Reduces the Availability of the Translation Initiation Complex eIF4F
4E-BP phosphorylation regulates its association with its target, eIF4E (37). The data on 4E-BP in Figure 1 suggest that there would be changes in 4E-BP association with eIF4E, and hence in the association of eIF4E with eIF4G. To evaluate this possibility, we then used m7GTP-conjugated Sepharose to affinity-purify eIF4E-containing complexes from FDLE lysates (Figure 2). DEX treatment of FDLE cultured in 3% O2 atmosphere significantly increased the association of 4E-BP with eIF4E and reciprocally decreased association of eIF4G with eIF4E (compared with cells in hormone-free conditions), indicative of a translationally repressed state. In contrast, when cells were cultured under 21% O2 atmosphere, there was neither a statistically significant change in 4E-BP within the pulldown fraction nor a significant decrease in the association of eIF4G. These data indicate that DEX treatment did not repress translation when FDLE are grown in a postnatal PO2 environment. It should also be noted that the abundance of total 4E-BP protein in the total lysate immunoblot is increased by 3% O2, independent of DEX, but that the changes in eIF4E complex formation occur only when DEX is added, consistent with our observation of increased abundance of the hyperphosphorylated form of 4E-BP in the 3% O2, hormone-free culture condition (as seen in Figure 1). Thus the formation of active eIF4F translation initiation complexes (eIF4E/eIF4G complexes) in primary cultures of rat FDLE is regulated by combined effects of PO2 and GC.


Figure 2
View larger version (25K):
[in this window]
[in a new window]

  		Figure 2. Under 3% O2 atmosphere, DEX suppresses association of eIF4G with eIF4E and enhances association of eIF4E with its repressor 4E-BP. (A) FDLE were cultured in the presence or absence of 50 nM DEX for 48 hours under fetal (3%) or postnatal (21%) O2 atmospheres. Upper panel: 400 µg of cell extract were affinity purified with m7GTP-conjugated Sepharose beads and Western blotted for proteins eIF4G, 4E-BP, and eIF4E (PD, pulldown). Lower panel: 50 µg of cell extract was directly loaded on duplicate gels and Western blotted as above (Total Lysate). (B) Quantitation of pulldown results indicates that only the combination of 3% O2 and 50 nM DEX resulted in a significant increase in 4E-BP and decrease of eIF4G pulled down with eIF4E. Data shown are mean and standard error of six (4E-BP) or five (eIF4G) independent experiments, *P < 0.01 relative to hormone-free control.

 
We further evaluated the effects of O2 on 4E-BP expression by examining the steady-state levels of 4E-BP mRNA (Figure 3). Consistent with changes in 4E-BP protein levels, FDLE cultured under a 21% O2 atmosphere contained decreased levels of 4E-BP mRNA. 4E-BP mRNA expression was not affected by the presence or absence of DEX (Figure 3).


Figure 3
View larger version (30K):
[in this window]
[in a new window]

  		Figure 3. Postnatal (21%) O2 environment decreases steady-state levels of mRNA encoding 4E-BP, while DEX exposure does not affect 4E-BP mRNA levels. FDLE were cultured in the presence or absence of 50 nM DEX for 48 hours under fetal (3% O2) or postnatal (21% O2) atmospheres. (A) Total RNA was analyzed by denaturing agarose gel electrophoresis and Northern blotting with a probe specific for 4E-BP. (B) After stripping and reprobing with 18S rRNA, data was quantitated and 4E-BP signal expressed relative to 18S rRNA signal (data are mean and standard error of four independent experiments,*P < 0.05 relative to 3% O2 condition).

 
Effects of PO2 and DEX on Signal Transduction via mTOR
4E-BP phosphorylation is regulated by the serine-threonine kinase mTOR (38), a central regulator of cell growth and proliferation that integrates a wide variety of environmental cues, including nutrient/energy availability, PO2, and hormonal and growth factor signaling. In addition to regulating 4E-BP activity, the other major substrate for mTOR-mediated regulation of translation is p70S6K and its downstream target ribosomal protein S6. To investigate whether the observed changes in 4E-BP phosphorylation state in response to DEX in FDLE cultured at 3% O2 were due to changes in mTOR activity, we tested the effects of pO2 and DEX on p70S6K and S6 (Figure 4A). Consistent with the changes in 4E-BP, we observed that FDLE cultured under 3% O2 in hormone-free conditions showed increased levels of p70S6K phosphorylated at Thr398 (pp70S6K) and of S6 phosphorylated on Ser235/236 (pS6) compared with cells grown under 21% O2. In both p70S6k and S6, this phenomenon was blocked by the presence of DEX. As with 4E-BP, phosphorylation of S6K also enhances formation of the translation initiation complex and cap-dependent translation (39). Together, the data on 4E-BP, p70S6K, and S6 phosphorylation suggest enhanced mTOR activity in cells cultured for 48 hours under 3% O2, and that this enhancement is inhibited by DEX. In contrast, DEX appears not to affect relative phosphorylation levels of p70S6K or S6 in cells cultured under 21% O2.


Figure 4
View larger version (32K):
[in this window]
[in a new window]

  		Figure 4. Under 3% O2 atmosphere, DEX decreases phosphorylation of downstream targets of mTOR (mammalian target of rapamycin), phospho-p70S6K, and phospho-ribosomal protein S6. FDLE were cultured in the presence or absence of 50 nM DEX for 48 hours under fetal (3%) or postnatal (21%) O2 atmospheres. (A) Whole cell extracts were Western blotted for phosphorylation of downstream targets of mTOR: pp70S6K, phospho-p70 S6 kinase(Thr398), and pS6, phospho-ribosomal protein S6(Ser235/236). Blots were also probed with antibodies recognizing the respective proteins independent of phosphorylation (p70S6K, S6) and actin as controls. mTOR signaling through the p70S6K to S6 pathway is decreased by DEX only under 3% O2. Note: antibodies against pp70S6K also recognize the nuclear-localized p85 isoform of that protein. (B) Whole cell extracts were Western blotted for phosphorylation of the central controller of cell growth and translation, mTOR on Ser2448 (pmTOR), for total protein level of mTOR, and actin (control). Neither DEX nor O2 modified phosphorylation of mTOR at Ser2448, the site of phosphorylation via the Akt pathway.

 
Growth factors, mitogens, and hormones activate the PI3K/Akt/mTOR signaling pathway, and mTOR is itself phosphorylated at residue Ser2448 by Akt, although the exact significance of this phosphorylation site on mTOR activity is uncertain, since substitution of Ser2448 with Ala does not affect mTOR activity (38). In addition to well-understood genomic effects, GC can also exert effects in a nongenomic manner and mediated by PI3K and Akt phosphorylation (40). Therefore, we assessed the phosphorylation state of mTOR at Ser2448 using antibodies specific to the Ser2448 phosphorylation site (pmTOR, Figure 4B). Our results demonstrated that relative phosphorylation of this site on mTOR was not affected by PO2 or DEX in FDLE, indicating that the PI3K/Akt pathway is not involved in regulating mTOR activity in response to PO2 and DEX in the primary cell culture model system that we used.

We next examined whether the translational repression that we observed in FDLE when they were cultured under the combination of fetal O2 atmosphere and DEX treatment was associated with a change in expression of REDD1, an upstream inhibitor of mTOR. Surprisingly, and in contrast to previous reports in cell lines (41–44), when primary cultures of FDLE were exposed to different pO2 levels there was no significant effect on REDD1 mRNA or protein levels. In addition, although REDD1 has been reported to be induced by DEX in lymphoid and muscle cells (45, 46), 50 nM DEX treatment did not increase either REDD1 mRNA or protein levels but again to our surprise significantly decreased REDD1 in FDLE cultured at 21% O2 (Figure 5). Using an identical approach, we then confirmed that several lung epithelial cell lines (A549, H441, MLE-15) do increase REDD1 levels when exposed to changes in PO2 and DEX in an additive fashion (data not shown). Thus although DEX treatment resulted in inhibition of mTOR activity in primary cultures of FDLE, as assessed by decreased phosphorylation of mTOR targets 4E-BP and p70S6K, it appears this was not mediated via induction of the upstream mTOR inhibitor, REDD1.


Figure 5
View larger version (53K):
[in this window]
[in a new window]

  		Figure 5. REDD1 expression is not increased by hypoxia or DEX in primary cultures of FDLE. REDD1, an upstream repressor of mTOR, has been reported to be transcriptionally induced by hypoxia and by GC in permanent cell lines. In contrast, FDLE cultured in the presence of 50 nM DEX for 48 hours showed significantly decreased expression of REDD1 under postnatal (21%) O2 atmosphere. Changes in PO2 failed to significantly affect REDD1 levels whether in presence or absence of DEX. (A) Total RNA was analyzed by denaturing agarose gel electrophoresis and Northern blotting with a probe specific for REDD1. (B) After stripping and reprobing with 18S rRNA, data was quantitated and REDD1 signal expressed relative to 18S rRNA signal (data are mean and standard error from four independent cell preparations, *P < 0.05 relative to hormone-free control). (C) Whole cell extracts were Western blotted for REDD1 (arrow).

 
Inhibition of mTOR Inhibits {alpha}-ENaC Translation and Amiloride-Sensitive Isc
To determine whether the observed changes in 4E-BP and p70S6K phosphorylation state were responsible for the decrease in ribosome loading on {alpha}-ENaC mRNA in DEX-treated FDLE, we undertook to demonstrate that direct inhibition of mTOR by rapamycin would induce similar changes in {alpha}ENaC mRNA distribution in polysome gradients. In order to determine the concentration of rapamycin that would most closely mimic the effects of DEX, we tested different rapamycin concentrations and also determined the levels of p70S6K and 4E-BP phosphorylation state relative to FDLE cultured in media alone and in media supplemented with 50 nM DEX. Phosphorylation state of p70S6K was assessed using an antibody specific to phosphorylation of Thr389, a site phosphorylated by mTOR, and normalized to total p70S6K in the lysate (Figure 6A). 4E-BP is phosphorylated on six sites, which can be resolved into three bands ({alpha}, beta, and {gamma}) on SDS-PAGE, with the band of slowest mobility representing the fully phosphorylated form (Figure 6B). Comparison of the p70S6K and 4E-BP results in Figure 6 demonstrates that p70S6K phosphorylation state was more sensitive to rapamycin than 4E-BP—treatment of FDLE with 0.8 nM rapamycin for 90 minutes resulted in decreased abundance of p70S6K phosphorylated on (Thr389), comparable to the effects of 48 hours of exposure to 50 nM DEX. In contrast, a concentration of 3.0 nM rapamycin was required to decrease the relative expression of hyperphosphorylated 4E-BP ({gamma}-form) to a level comparable to the effects of 50 nM DEX.


Figure 6
View larger version (42K):
[in this window]
[in a new window]

  		Figure 6. Effect of different concentrations of rapamycin on p70S6K and 4E-BP phosphorylation. FDLE cultured in hormone-free conditions for 48 hours under fetal (3%) O2 atmosphere were exposed to different concentrations of rapamycin for 90 minutes before harvest of whole cell lysates for Western blotting, in order to determine the concentration most closely mimicking the effects of 50 nM DEX on downstream mTOR effectors. (A) Western blots were probed with antibody that recognizes p70S6K only when phosphorylated on Thr389, stripped, and reprobed with antibody that recognizes p70S6K independent of phosphorylation. Phosphorylation level is expressed as the ratio of the signals from the two probings. (B) Western blots were probed with an antibody which recognizes 4E-BP independent of phosphorylation. 4E-BP can be detected as up to three bands ({alpha}, beta, {gamma}) of different mobilities, corresponding to different levels of phosphorylation. Phosphorylation level is expressed as the ratio of the slowest migrating ({gamma}, most highly phosphorylated form) relative to the total of all bands in the lane (n = 3–8 determinations from a minimum of three independent cell preparations).

 
Since 4E-BP phosphorylation state controls the availability of eIF4F, we used the higher concentration of rapamycin to treat FDLE before polysome gradient analysis. FDLE maintained at 3% O2 in the absence of DEX were treated with 3 nM rapamycin for 90 minutes before fractionation of postnuclear supernatants on 15 to 45% sucrose gradients. The optical density at 254 nm was recorded during recovery of the gradient to determine the distribution of ribosomal RNAs. Representative profiles, with monosomes recovered in fractions 4 and 5, are shown in Figure 7A. 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 7A, lower panels). Both the A254 profiles and the ethidium bromide staining of the distribution of 28 and 18S rRNA indicate that 3 nM rapamycin treatment did not markedly alter the overall distribution of ribosomes between monosomes and polysomes.


Figure 7
View larger version (45K):
[in this window]
[in a new window]

  		Figure 7. Rapamycin treatment decreases ribosome loading on {alpha}ENaC mRNA in FDLE. FDLE cultured in hormone-free conditions for 48 hours under fetal (3% O2) atmosphere were exposed to 3 nM rapamycin (rapa) for 90 minutes before preparation of cytoplasmic extracts for fractionation on 15 to 45% linear sucrose gradients, and the distribution of ENaC and cytokeratin 18 (CK18) mRNAs were analyzed. Control cells were not exposed to rapamycin. (A) Representative ultraviolet absorbance profiles of FDLE lysates sedimented through sucrose gradients. 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 mRNAs in fractions recovered from sucrose density gradients, expressed as percent of total for that mRNA in the gradient, as determined by Northern blot. Percentage of mRNA in gradient fractions was integrated for untranslated/monosomes (fractions 1–4), lighter polysomes (fractions 5–10) and heavier polysomes (fractions 11–13). Rapamycin (3 nM) caused a statistically significant shift of {alpha}-ENaC mRNA from the heaviest to the lighter polysomes (*P < 0.05 relative to control). mRNAs encoding the housekeeping gene CK18 or the beta- or {gamma}-ENaC subunits were not significantly redistributed between the two polysome fractions in response to rapamycin. Results shown for specific mRNAs are mean and standard error of four ({alpha}-ENaC, CK18) or three (beta-ENaC, {gamma}-ENaC) independent experiments.

 

Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		Figure 8. Rapamycin treatment inhibits amiloride-sensitive Isc in FDLE FDLE were cultured in hormone-free conditions for 48 hours under fetal (3% O2) atmosphere in the absence (control) or presence of 3 nM rapamycin (rapa) before bioelectric measurements. ASC, amiloride-sensitive Isc; AIC, amiloride-insensitive Isc. (*P < 0.0001 between control and rapa-treated cells, n = 12 monolayers from three independent cell preparations).

 
Quantitation, using densitometric scanning of the Northern analysis of mRNAs in fractions recovered from the gradients (Figure 7B), showed that 3 nM rapamycin treatment of FDLE maintained under fetal PO2 resulted in redistribution {alpha}-ENaC mRNA from the heaviest polysomes (fractions 11–13, n > 8 ribosomes per mRNA) toward lighter polysome fractions (fractions 5–10, n = 2–8 ribosomes per mRNA). There was no change in the amount of {alpha}-ENaC mRNA associated with monosomes/pre-initiation complexes (fractions 1–4). There was no significant change in the distribution of mRNAs encoding the epithelial cell marker CK18, or the beta- or {gamma}-ENaC subunits, confirming that the change seen in {alpha}-ENaC distribution is specific to this one ENaC subunit's mRNA (Figure 7B). The distributions shown in Figure 7 represent the means of four fractionations from independent preparations of epithelia grown in primary culture.

In order to demonstrate whether the magnitude of this change in ribosome loading was sufficient to modulate ENaC function, we measured Isc in FDLE monolayers cultured for 48 hours under 3% O2 and treated with 3 nM rapamycin (Figure 8). Rapamycin decreased amiloride-sensitive Isc by approximately 65%, without significantly affecting amiloride-insensitve Isc.


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Glucocorticosteroids promote the maturation of several different essential pathways in the fetal lung, including an increase in {alpha}-ENaC mRNA levels (10), which is needed for the newborn lung to clear the fetal lung liquid that fills its airspaces (1–3). The increase in {alpha}-ENaC mRNA levels occurs from an increase in transcription mediated, at least in part, by a glucocorticoid-responsive element in the 5' flanking region of the {alpha}ENaC gene (11–13). We have shown that if FDLE are cultured under fetal (3%) O2 conditions, despite 50 nM DEX increasing {alpha}-ENaC mRNA levels there is no associated increase in either {alpha}-ENaC protein synthesis or function (18). A decrease in the translation efficiency of the {alpha}-ENaC mRNA is at least one of the mechanisms (18), and this low PO2 effect can be relieved by switching the cells to a postnatal (21%) O2 environment. We now show in FDLE cultured at 3% O2 that 50 nM DEX limits the availability of the cap-binding factor eIF4F.

Among the complex series of interactions involved in assembly of an actively translating ribosome on eukaryotic mRNA, two sets of initiation factor interactions are the primary targets of regulatory signaling. These include 4E-BP, which competes with eIF4G for binding to the cap-binding protein eIF4E, and eIF2{alpha}, a component of the ternary complex (eIF2/Met-tRNA/GTP) which is left in an inactive, GDP-bound state at the end of each round of initiation and must be reactivated by its guanine-nucleotide exchange factor, eIF2B. Both of these pathways are regulated by phosphorylation. Sequential hyperphosphorylation of 4E-BP on several sites, culminating in phosphorylation of Ser65, prevents sequestration of eIF4E by 4E-BP and hence promotes translation initiation (21), while phosphorylation of eIF2{alpha} in response to specific stress conditions inhibits its interaction with eIF2B and thus impairs translation initiation (47). Our initial experiments indicate that changes in 4E-BP, rather than eIF2{alpha}, are a likely candidate for the regulation of {alpha}-ENaC mRNA translation by PO2 and DEX in primary cultures of FDLE.

The low PO2 effect occurs from an induction of the mRNA and protein encoding the translational repressor 4E-BP, combined with a DEX-induced decrease in the relative abundance of the hyperphosphorylated form of the 4E-BP protein. In addition to changes in 4E-BP phosphorylation state, PO2 and DEX also influenced phosphorylation state of additional downstream targets of mTOR, consistent with inhibition of signaling via mTOR in the cells exposed to DEX under a 3% O2 atmosphere. Direct inhibition of mTOR signaling by using rapamycin at a dose which mimicked the DEX-induced change in hyperphosphorylated 4E-BP abundance was capable of specifically reducing {alpha}-ENaC translation, without affecting translation of either the other ENaC subunit mRNAs or an epithelial-specific mRNA encoding for a cytoskeleton protein, CK18. The proportion of {alpha}-ENaC mRNAs in polysomes of n > 8 decreased from 63% to 56% under rapamycin treatment. This is comparable to the decrease from 68% to 55% reported in this polysome subpopulation by Otulakowski and coworkers in response to 50 nM DEX treatment (18). In addition, this rapamycin treatment was sufficient to markedly inhibit amiloride-sensitive Na+ transport in FDLE monolayers.

DEX down-regulates protein synthesis in rat lungs in vivo (48) and induces a net decrease in the abundance of phosphorylated forms of 4E-BP and p70S6K in cultured myoblasts (25, 49); this is similar to our results in FDLE. Although the upstream mTOR inhibitor, REDD1, has been reported to be induced by either low PO2 and/or DEX (41, 45) and to mediate the DEX-induced repression of translation initiation in L6 myoblasts (46), we found that REDD1 expression was not affected by culturing FDLE at 3% versus 21% O2, nor was it induced by 50 nM DEX. Although many of the studies reporting induction of REDD1 involved even lower PO2 than we used (41, 44), we found that using a switch to 3% O2, in a manner that was identical to what we used in FDLE, was sufficient to increase REDD1 mRNA in two different human lung epithelial cancer lines (A549, H441) and a murine lung (MLE15) cell line (data not shown). This suggests that our results are due to differences in the responses of primary cell cultures vs. transformed cells to low PO2. Similarly, although DEX has been demonstrated to up-regulate REDD1 in cell lines, it failed to do so in FDLE, and in fact decreased REDD1 expression in cells cultured in a 21% O2 atmosphere. The promoter region of REDD1 has not yet been described in the literature, so it is not known if it contains a functional glucocorticoid-responsive element or whether DEX-induced up-regulation of REDD1 in cell lines is direct or indirect. Since FDLE are known to contain glucocorticoid receptor, it would appear that up-regulation of REDD1 may be indirect.

If not via REDD1, DEX may be inhibiting translation of {alpha}-ENaC via other post-transcriptional and nongenomic pathways (reviewed by Stellato [24]). GC have been shown to exert effects on protein synthesis by regulating both translation and mRNA turnover, mediated via a variety of signaling pathways including mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38 (also known as SAPK2), and the I{kappa}B kinases. For example, DEX has been shown to rapidly induce nitric oxide synthase activity in human endothelial cells via a transcription-independent pathway which involves activation of phosphatidyl inositol 3-kinase and Akt (50). However, our results suggest that DEX is not affecting Akt-mediated phosphorylation of mTOR in FDLE cultured at 3% O2. Thus the pathway by which DEX represses mTOR signaling in this system will require ongoing investigation.

Hypoxia decreases translation both via inhibition of mTOR and its targets (51), and by triggering ER stress and phosphorylation of eIF2{alpha} via the kinase PERK (52). A recent study using HeLa and other cell lines indicated that translational control in response to acute versus prolonged hypoxia operates through distinct mechanisms (23). Early inhibition, which reached a maximum in 1 to 2 hours, was due to phosphorylation of eIF2{alpha}, while chronic hypoxic exposure activated a second, eIF2{alpha}-independent pathway that was characterized by disruption of eIF4F and sequestration of eIF4E due to increased expression of its inhibitor 4E-BP. Our results are consistent with these observations; in FDLE cultured for 48 hours under 3% O2, we observed very minimal change in eIF2{alpha} phosphorylation (Figure 1) but an increase in 4E-BP levels. In FDLE, in contrast to the reported study in HeLa cells, this increase in 4E-BP only resulted in sequestration of eIF4E when DEX was present. However, 4E-BP appears to be much more highly phosphorylated in our primary cultures of FDLE, such that sequestration of eIF4E occurred only when DEX treatment induced dephosphorylation.

Although specialized mRNA binding factors confer gene-specific regulation in some cases, general translation factors can specifically promote or repress translation initiation on some mRNAs depending on features of their UTRs, such as length and secondary structure of the 5' UTR (reviewed by Dever [53]). The {alpha}-ENaC mRNA possesses a long, complex 5' UTR that is highly GC-rich immediately adjacent to the 5' cap, consistent with a high requirement for eIF4F. The beta- and {gamma}-ENaC mRNAs, in contrast, have short 5' UTRs as does CK18. Our results, demonstrating specific inhibition of ribosome loading on {alpha}-ENaC mRNA in FDLE treated with rapamycin, are consistent with a model in which {alpha}-ENaC translation is limited via eIF4F availability.

Appropriate ENaC activity is critical for the normal transition from fetal to postnatal life. Although many factors, including GC, thyroid hormones, PO2, and beta-agonists have all been implicated in regulating ENaC function during the perinatal period, the ways in which these cues interact has not been defined. We have now demonstrated that changes in PO2 and GC interact to regulate {alpha}-ENaC mRNA translation in primary cultures of rat FDLE, most likely via their impact on mTOR signaling pathways. mTOR is a central controller of cell growth and protein synthesis, integrating not only hormonal and O2 signals, but also amino acid and energy availability, which would also be expected to fluctuate during the birth process and immediate neonatal period. Thus an understanding of translational regulation of Na+ transport proteins may lead to insights in normal lung physiology and pathophysiology during the immediate post-partum period of life.


   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.2007-0055OC on June 7, 2007

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 February 21, 2007

Accepted in final form April 9, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Barker PM, Olver RE. Invited review: clearance of lung liquid during the perinatal period. J Appl Physiol 2002;93:1542–1548.[Abstract/Free Full Text]
  2. O'Brodovich H. Deficient Na+ channel expression in newborn respiratory distress syndrome. Pediatr Pulmonol 2004;37:141–142.[CrossRef]
  3. Olver RE, Walters DV, Wilson M. Developmental regulation of lung liquid transport. Annu Rev Physiol 2004;66:77–101.[CrossRef]
  4. Barker PM, Markiewicz M, Parker KA, Walters DV, Strang LB. Synergistic action of triiodothyronine and hydrocortisone on epinephrine-induced reabsorption of fetal lung liquid. Pediatr Res 1990;27:588–591.[Medline]
  5. Nair PK, Li T, Bhattacharjee R, Ye X, Folkesson HG. Oxytocin-induced labor augments IL-1{beta}-stimulated distal lung fluid absorption in fetal guinea pig lungs. Am J Physiol Lung Cell Mol Physiol 2005;289:L1029–L1038.[Abstract/Free Full Text]
  6. Folkesson HG, Matthay MA. Alveolar epithelial ion and fluid transport: recent progress. Am J Respir Cell Mol Biol 2006;35:10–19.[Free Full Text]
  7. Matthay MA, Folkesson HG, Clerici C. Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev 2002;82:569–600.[Abstract/Free Full Text]
  8. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 1994;367:463–467.[CrossRef][Medline]
  9. Canessa CM, Horisberger J-D, Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 1993;361:467–470.[CrossRef][Medline]
  10. Tchepichev S, Ueda J, Canessa C, Rossier BC, O'Brodovich H. Lung epithelial Na channel subunits are differentially regulated during development and by steroids. Am J Physiol Cell Physiol 1995;269:C805–C812.[Abstract/Free Full Text]
  11. Otulakowski G, Rafii B, Bremner HR, O'Brodovich H. Structure and hormone responsiveness of the gene encoding the {alpha}-subunit of the rat amiloride-sensitive epithelial sodium channel. Am J Respir Cell Mol Biol 1999;20:1028–1040.[Abstract/Free Full Text]
  12. Chow YH, Wang Y, Plumb J, O'Brodovich H, Hu J. Hormonal regulation and genomic organization of the human amiloride-sensitive epithelial sodium channel {alpha} subunit gene. Pediatr Res 1999;46:208–214.[Medline]
  13. Sayegh R, Auerbach SD, Li X, Loftus RW, Husted RF, Stokes JB, Thomas CP. Glucocorticoid induction of epithelial sodium channel expression in lung and renal epithelia occurs via trans-activation of a hormone response element in the 5'-flanking region of the human epithelial sodium channel {alpha} subunit gene. J Biol Chem 1999;274:12431–12437.[Abstract/Free Full Text]
  14. Itani OA, Liu KZ, Cornish KL, Campbell JR, Thomas CP. Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5'-flanking region. Am J Physiol Endocrinol Metab 2002;283:E971–E979.[Abstract/Free Full Text]
  15. Itani OA, Auerbach SD, Husted RF, Volk KA, Ageloff S, Knepper MA, Stokes JB, Thomas CP. Glucocorticoid-stimulated lung epithelial Na(+) transport is associated with regulated ENaC and sgk1 expression. Am J Physiol Lung Cell Mol Physiol 2002;282:L631–L641.[Abstract/Free Full Text]
  16. Johnson MD, Bao HF, Helms MN, Chen XJ, Tigue Z, Jain L, Dobbs LG, Eaton DC. Functional ion channels in pulmonary alveolar type I cells support a role for type I cells in lung ion transport. Proc Natl Acad Sci USA 2006;103:4964–4969.[Abstract/Free Full Text]
  17. Jain L, Chen XJ, Ramosevac S, Brouwn LA, Eaton DC. Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions. Am J Physiol Lung Cell Mol Physiol 2001;280:L646–L658.[Abstract/Free Full Text]
  18. Otulakowski G, Rafii B, Harris M, O'Brodovich H. Oxygen and glucocorticoids modulate alphaENaC mRNA translation in fetal distal lung epithelium. Am J Respir Cell Mol Biol 2006;34:204–212.[Abstract/Free Full Text]
  19. Otulakowski G, Rafii B, O'Brodovich H. Differential translational efficiency of ENaC subunits during lung development. Am J Respir Cell Mol Biol 2004;30:862–870.[Abstract/Free Full Text]
  20. Kleizen B, van Vlijmen T, de Jonge HR, Braakman I. Folding of CFTR Is Predominantly Cotranslational. Mol Cell 2005;20:277–287.[CrossRef][Medline]
  21. Richter JD, Sonenberg N. Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 2005;433:477–480.[CrossRef][Medline]
  22. Gebauer F, Hentze MW. Molecular mechanism of translational control. Nat Rev Mol Cell Biol 2004;5:827–835.[CrossRef][Medline]
  23. Koritzinsky M, Magagnin MG, van den Beucken T, Seigneuric R, Savelkouls K, Dostie J, Pyronnet S, Kaufman RJ, Weppler SA, Voncken JW, et al. Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control. EMBO J 2006;25:1114–1125.[CrossRef][Medline]
  24. Stellato C. Post-transcriptional and nongenomic effects of glucocorticoids. Proc Am Thorac Soc 2004;1:255–263.[Abstract/Free Full Text]
  25. Shah OJ, Kimball SR, Jefferson LS. Among translational effectors, p70S6k is uniquely sensitive to inhibition by glucocorticoids. Biochem J 2000;347:389–397.[CrossRef][Medline]
  26. Shah OJ, Iniguez-Lluhi JA, Romanelli A, Kimball SR, Jefferson LS. The activated glucocorticoid receptor modulates presumptive autoregulation of ribosomal protein S6 protein kinase, p70 S6K. J Biol Chem 2002;277:2525–2533.[Abstract/Free Full Text]
  27. Long W, Wei L, Barrett EJ. Dexamethasone inhibits the stimulation of muscle protein synthesis and PHAS-I and p70 S6-kinase phosphorylation. Am J Physiol Endocrinol Metab 2001;280:E570–E575.[Abstract/Free Full Text]
  28. Martin DE, Hall MN. The expanding TOR signaling network. Curr Opin Cell Biol 2005;17:158–166.[CrossRef][Medline]
  29. Dagenais A, Denis C, Vives MF, Girouard S, Masse C, Nguyen T, Yamagata T, Grygorczyk C, Kothary R, Berthiaume Y. Modulation of alpha-ENaC and alpha(1)-Na(+)-K(+)-ATPase by cAMP and dexamethasone in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 2001;281:L217–L230.[Abstract/Free Full Text]
  30. Kohler S, Pradervand S, Verdumo C, Merillat AM, Bens M, Vandewalle A, Beermann F, Hummler E. Analysis of the mouse Scnn1a promoter in cortical collecting duct cells and in transgenic mice. Biochim Biophys Acta 2001;1519:106–110.[Medline]
  31. Thomas CP, Auerbach S, Stokes JB, Volk KA. 5' Heterogeneity in epithelial sodium channel {alpha}-subunit mRNA leads to distinct NH2-terminal variant proteins. Am J Physiol Cell Physiol 1998;274:C1312–C1323.[Abstract/Free Full Text]
  32. Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM, O'Brodovich H. Pulmonary oedema fluid induces non-alpha-ENaC-dependent Na(+) transport and fluid absorption in the distal lung. J Physiol 2002;544:537–548.[Abstract/Free Full Text]
  33. Samuels HH, Stanley F, Casanova J. Depletion of L-3,4,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 1979;105:80–85.[Abstract]
  34. Wang X, Beugnet A, Murakami M, Yamanaka S, Proud CG. Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins. Mol Cell Biol 2005;25:2558–2572.[Abstract/Free Full Text]
  35. Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM, O'Brodovich H. Pulmonary oedema fluid induces non-{alpha}-ENaC-dependent Na+ transport and fluid absorption in the distal lung. J Physiol 2002;544:537–548.[Abstract/Free Full Text]
  36. Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM, O'Brodovich H. Pulmonary oedema fluid induces non-{alpha}-ENaC-dependent Na+ transport and fluid absorption in the distal lung. J Physiol 2002;544:537–548.[Abstract/Free Full Text]
  37. Gingras AC, Raught B, Gygi SP, Niedzwiecka A, Miron M, Burley SK, Polakiewicz RD, Wyslouch-Cieszynska A, Aebersold R, Sonenberg N. Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev 2001;15:2852–2864.[Abstract/Free Full Text]
  38. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 2004;18:1926–1945.[Abstract/Free Full Text]
  39. Mamane Y, Petroulakis E, LeBacquer O, Sonenberg N. mTOR, translation initiation and cancer. Oncogene 2006;25:6416–6422.[CrossRef][Medline]
  40. Song IH, Buttgereit F. Non-genomic glucocorticoid effects to provide the basis for new drug developments. Mol Cell Endocrinol 2006;246:142–146.[CrossRef][Medline]
  41. Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, Hafen E, Witters LA, Ellisen LW, Kaelin WG Jr. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev 2004;18:2893–2904.[Abstract/Free Full Text]
  42. Corradetti MN, Inoki K, Guan KL. The stress-inducted proteins RTP801 and RTP801L are negative regulators of the mammalian target of rapamycin pathway. J Biol Chem 2005;280:9769–9772.[Abstract/Free Full Text]
  43. Schwarzer R, Tondera D, Arnold W, Giese K, Klippel A, Kaufmann J. REDD1 integrates hypoxia-mediated survival signaling downstream of phosphatidylinositol 3-kinase. Oncogene 2005;24:1138–1149.[CrossRef][Medline]
  44. Shoshani T, Faerman A, Mett I, Zelin E, Tenne T, Gorodin S, Moshel Y, Elbaz S, Budanov A, Chajut A, et al. Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol Cell Biol 2002;22:2283–2293.[Abstract/Free Full Text]
  45. Wang Z, Malone MH, Thomenius MJ, Zhong F, Xu F, Distelhorst CW. Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals. J Biol Chem 2003;278:27053–27058.[Abstract/Free Full Text]
  46. Wang H, Kubica N, Ellisen LW, Jefferson LS, Kimball SR. Dexamethasone represses signaling through the mammalian target of rapamycin in muscle cells by enhancing expression of REDD1. J Biol Chem 2006;281:39128–39134.[Abstract/Free Full Text]
  47. Proud CG. eIF2 and the control of cell physiology. Semin Cell Dev Biol 2005;16:3–12.[CrossRef][Medline]
  48. Fussell JC, Kelly FJ. Effects of dexamethasone on lung protein turnover. Biochem J 1991;273:93–97.[Medline]
  49. Shah OJ, Kimball SR, Jefferson LS. Glucocorticoids abate p70(S6k) and eIF4E function in L6 skeletal myoblasts. Am J Physiol Endocrinol Metab 2000;279:E74–E82.[Abstract/Free Full Text]
  50. Hafezi-Moghadam A, Simoncini T, Yang Z, Limbourg FP, Plumier JC, Rebsamen MC, Hsieh CM, Chui DS, Thomas KL, Prorock AJ, et al. Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nat Med 2002;8:473–479.[CrossRef][Medline]
  51. Arsham AM, Howell JJ, Simon MC. A novel hypoxia-inducible factor-independent hypoxic response regulating mammalian target of rapamycin and its targets. J Biol Chem 2003;278:29655–29660.[Abstract/Free Full Text]
  52. Koumenis C, Naczki C, Koritzinsky M, Rastani S, Diehl A, Sonenberg N, Koromilas A, Wouters BG. Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha. Mol Cell Biol 2002;22:7405–7416.[Abstract/Free Full Text]
  53. Dever TE. Gene-specific regulation by general translation factors. Cell 2002;108:545–556.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
H. O'Brodovich, P. Yang, S. Gandhi, and G. Otulakowski
Amiloride-insensitive Na+ and fluid absorption in the mammalian distal lung
Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L401 - L408.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2007-0055OCv1
37/4/457    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Otulakowski, G.
Right arrow Articles by O'Brodovich, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow <