title: Steroid and oxygen effects on eIF4F complex, mTOR, and ENaC translation in fetal lung epithelia.
abstract: 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 Po(2) at birth, and we have previously shown that dexamethasone (DEX)-induced alpha-ENaC mRNA is efficiently translated only under postnatal (21%) O(2) (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 m(7)GTP-Sepharose pull-down experiments, that in FDLE cultured under 3% O(2), 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% O(2) 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% O(2), 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 Po(2) acts with GC through an mTOR-related pathway to increase alpha-ENaC protein synthesis, thereby promoting lung fluid absorption.
authors.affiliation: CIHR Group in Lung Development, Program in Physiology and Experimental Medicine, Hospital for Sick Children Research Institute, Toronto, ON, Canada. gail.otulakowski@sickkids.ca
authors.forename: Gail
authors.initials: G
authors.lastname: Otulakowski
authors.forename: Wenming
authors.initials: W
authors.lastname: Duan
authors.forename: Shephali
authors.initials: S
authors.lastname: Gandhi
authors.forename: Hugh
authors.initials: H
authors.lastname: O'brodovich
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D053503:Epithelial Sodium Channels / Q000096:biosynthesis* / Q000235:genetics; D039562:Eukaryotic Initiation Factor-4F / Q000378:metabolism*; D005333:Fetus / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005786:Gene Expression Regulation / Q000187:drug effects; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010100:Oxygen / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects*; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors
chemical_list: D004268:DNA-Binding Proteins; C000632146:Ddit4 protein, rat; D053503:Epithelial Sodium Channels; D039562:Eukaryotic Initiation Factor-4F; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D003907:Dexamethasone; D011494:Protein Kinases; C546845:mTOR protein, rat; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D010100:Oxygen; D020123:Sirolimus