Hyperoxia alters the expression and phosphorylation of multiple factors regulating translation initiation

Abstract

Hyperoxia is cytotoxic and depresses many cellular metabolic functions including protein synthesis. Translational control is exerted primarily during initiation by two mechanisms: 1) through inhibition of translation initiation complex formation via sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, with inhibitory 4E-binding proteins (4E-BP); and 2) by prevention of formation and eIF2B activity by phosphorylated eIF2α. In this report, exposure of human lung fibroblasts to 95% O 2 decreased the incorporation of thymidine into DNA at 6 h and the incorporation of leucine into protein beginning at 12 h. The reductions in DNA and protein synthesis were accompanied by increased phosphorylation of eIF4E protein and reduced phosphorylation of 4E-BP1. At 24 h, hyperoxia shifted 4E-BP1 phosphorylation to lesserphosphorylated isoforms, increased eIF4E expression, and increased the association of eIF4E with 4E-BP1. Although hyperoxia did not change eIF2α expression, it increased its phosphorylation at Ser51, but not until 48 h. In addition, the activation of eIF2α was not accompanied by the formation of stress granules. These findings suggest that hyperoxia diminishes protein synthesis by increasing eIF4E phosphorylation and enhancing the affinity of 4E-BP1 for eIF4E. Keywordseukaryotic translation initiation factors; protein synthesis; lung; stress granules Exposure of the developing lung to high concentrations of inspired oxygen is known to inhibit lung growth, both acutely and chronically (7,26). Cell culture and whole animal studies indicate that hyperoxia inhibits not only DNA synthesis but also protein synthesis (2,7,15,23). In adult rats breathing 95% O 2 , the fractional rate of protein synthesis and the rate of protein synthesis per ribosome are depressed by 30% within 6 h (7). Similarly, whole lung slices from rats exposed to hyperoxia for 24 and 48 h show 25% reductions in protein synthetic capacity of both total protein and newly synthesized proteins (15). At the cellular level, Jornot and colleagues (12) revealed that exposure to 95% O 2 inhibited the incorporation of leucine into protein, delayed the ribosomal transit time, and marginally increased the polysome content of cultured endothelial cells. Yet despite the near universality of the inhibitory effect of hyperoxia In mammalian cells, translation is principally regulated by a family of eukaryotic initiation factors (eIF) that control the assembly of the ribosome on the mRNA. The efficiency of ribosome/mRNA binding is governed by the availability of the 5′ m 7 -GpppX (where X is any nucleotide) mRNA cap binding protein, eIF4E, which, together with eIF4G and eIF4A, comprises the heterotrimeric eIF4F initiation complex (reviewed in Refs. 29 and 34). The eIF4F complex, along with eIF3, facilitates the assembly of the 43S ribosomal subunit on the 5′ end of the mRNA. The low abundance of eIF4E relative to the other 4F components renders it rate limiting for eIF4F formation. The level of free eIF4E is regulated by a group o...

Hyperoxia enhances VEGF release from A549 cells via post-transcriptional processes

“…Angiotensin II (ANG II) has been documented to augment VEGF protein expression without altering mRNA levels or mRNA stability via a mechanism involving reactive oxygen species and cap-dependent translation [34,35]. During exposure to hyperoxia, however, both global protein synthesis and cap-dependent translation are depressed [22]. Despite this, VEGF protein expression could be enhanced by a capindependent mechanism involving either the AUG 1039 or CUG 499 start codons regulated by two IRES elements located in the 5'-UTR [36].…”

“…By comparison, hyperoxia lead to a time-dependent increase in secreted VEGF concentrations with respect to time and compared to RA, such that concentrations at 48 hours and during recovery were 2-to 3-fold higher than in RA cells (Fig 1A). To insure that our findings were not unique to the A549 cell line, we also exposed normal, neonatal lung fibroblasts to hyperoxia, cells previously shown to increase VEGF protein expression in response to activated factor VII and TGF-β [20,21] Because these cells are more resistant to oxygen-induced cell death, we exposed them to hyperoxia for the full 72-hour study period [22]. As was observed in the A549 cells, hyperoxia markedly increased secreted VEGF over time and relative to room air-exposed cells (Fig 1B).…”

Roles of mitogen-activated protein kinase signal-integrating kinases 1 and 2 in oxidant-mediated eIF4E phosphorylation

“…Equal amounts of protein were resolved on either 12% Bis-Tris or 3−8% Tris-acetate gels. Resolved proteins were transferred to PVDF membranes and incubated overnight at 4°C with primary antibodies [all at 1:1000, except 4E-BP1 (1:500), α-tubulin (1:2500), active-ERK 1/2 (1:2000) and β-actin (1:5000)] in 5% non-fat dry milk blocking buffer as previously described (Shenberger, Myers, Zimmer, Powell, & Barchowsky, 2005). Following rinsing, membranes were incubated with the corresponding HRP-conjugated secondary antibody (1:5000) and the signal detected by chemiluminescence.…”

“…Alterations in the translational machinery and regulatory pathways is known to influence cell proliferation, survival, and phenotype, any or all of which may lead to aberrant tissue repair and remodeling (Richter & Sonenberg, 2005). Recent reports from our laboratory and others indicate that reactive oxygen species directly modulate translation at the level of initiation (Alirezaei, Marin, Nairn, Glowinski, & Premont, 2001;Duncan, Peterson, & Sevanian, 2005;Shenberger, Adams, & Zimmer, 2002;Shenberger, Myers, Zimmer, Powell, & Barchowsky, 2005). Under basal conditions, translation is principally regulated by a complex of peptide factors that join the mRNA with the ribosome (Gingras, Raught, & Sonenberg, 1999).…”

“…Although it has been clearly demonstrated that ROS enhance eIF4E phosphorylation, the contribution of the individual Mnk isoforms, as well as the significance of phosphorylation to altered translation kinetics, has remained largely unexplored (Alirezaei, Marin, Nairn, Glowinski, & Premont, 2001;Duncan, Peterson, Hagedorn, & Sevanian, 2003;Shenberger, Adams, & Zimmer, 2002;Shenberger, Myers, Zimmer, Powell, & Barchowsky, 2005). For this reason, we sought to characterize the contributions of Mnk1 and Mnk2 to oxidant-mediated changes in eIF4E phosphorylation and to assess the relationship between Mnk expression, eIF4F formation, and the nuclear eIF4E content.…”