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

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

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 -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 -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 -ENaC mRNA, and decreased amiloride-sensitive short-circuit current, but no change in ribosomal loading onto any of - or -ENaC or cytokeratin 18 mRNAs. We speculate that at birth increased PO₂ acts with GC through an mTOR-related pathway to increase -ENaC protein synthesis, thereby promoting lung fluid absorption.

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

CLINICAL RELEVANCE 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.