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.

Dose-Dependent Effects of Sirolimus on mTOR Signaling and Polycystic Kidney Disease

“…10,32 The effect of mTOR inhibition on cyst volume regression may be caused by increased apoptosis and/or by a potential effect on fluid reabsorption as suggested for fetal lung epithelia. 30,33 Nonetheless, our results support the concept of cyst regression by mTOR inhibitors when used at high enough concentrations.…”

Rapamycin inhibits oncogenic intestinal ion channels and neoplasia in APCMin/+ mice

“…APC Min/ þ mice have an enhanced colonic Na þ absorption that even extends to the proximal colon . Interestingly, a previous report has shown that the expression of ENaC is regulated by a molecule downstream of mTOR, eukaryotic initiation factor (eIF4E) (Otulakowski et al, 2007); mTOR controls the phosphorylation of 4E-BP1, which inhibits eIF4E (Fingar et al, 2002;Koehl et al, 2005;Otulakowski et al, 2007). Remarkably, in our experiments, abnormal expressions of both b-and g-ENaC were reverted to the normal levels found in wt animals by rapamycin treatment (Figure 3a).…”

“…Finally, we examined the enhanced amiloride-sensitive Na þ absorption in the colon of APC Min/ þ animals, which is related to the possible upregulation of b-and g-subunits of ENaC. Interestingly, a previous study on fetal lung epithelia showed a pronounced increase in ENaC expression, which relied on the eukaryotic translation initiation factor eIF4E and activation of the mTOR pathway (Otulakowski et al, 2007). In this study, treatment with rapamycin led to a remarkable downregulation of ENaC expression and Na þ absorption by the colonic epithelium.…”

“…Although a-ENaC is normally constitutively expressed and therefore was not upregulated in APC Min/ þ , rapamycin also significantly reduced the expression of this subunit. As it has been previously shown that electrogenic Na þ absorption by ENaC in fetal lung epithelia is dependent on mTOR (Otulakowski et al, 2007), we measured Na þ transport in the colonic epithelium by applying the ENaC inhibitor amiloride. In these experiments, the effect of 20 mM amiloride on Na þ absorption was enhanced in APC Min/ þ mice when compared with wt animals, but was normalized in rapamycin-treated mice (Figures 3b-d).…”

Glucocorticoid-Mediated Repression of REDD1 mRNA Expression in Rat Fetal Distal Lung Epithelial Cells

“…Both PO 2 and GC have been demonstrated to be important regulators of fetal lung maturation (16). We recently showed that a combination of fetal O 2 levels and DEX inhibited mTOR signaling in primary cultures of rat fetal distal lung epithelia (FDLE), yet this was not associated with increased expression of REDD1 in response to hypoxia and GC as would be predicted (17). Surprisingly, REDD1 was unresponsive to PO 2 and was repressed by 50 nM DEX.…”