Hyperoxia inhibits protein synthesis and increases eIF2α phosphorylation in the newborn rat lung

Abstract

Prolonged exposure to hyperoxia contributes to aberrant lung growth in premature infants. Of the deleterious effects induced by hyperoxia, alterations in protein synthesis are likely to be of great importance to the developing lung. Regulation of mRNA translation occurs predominantly at the level of initiation via control of mRNA/ribosome binding by proteins known as eukaryotic initiation factors (eIF). Although hyperoxia is known to suppress mRNA translation in adult lungs, little is known regarding the effects in newborns or the involved mechanism. This study was performed to determine the effect of exposure to 95% O2 on pulmonary protein synthesis in 4-day-old Sprague-Dawley rat pups. We found that hyperoxia suppressed the incorporation of [ 3 H]phenylalanine into lung protein over time, resulting in a 23% reduction after 72 h compared with pups reared in room air. This effect was preceded by a shift in total lung RNA to lower order polysomes. Hyperoxia increased eIF4G-eIF4E binding, a surrogate maker of eIF4F complex assembly, and initially activated, then suppressed, the phosphorylation of ribosomal S6 kinase 1 and ribosomal S6 protein, downstream targets of mammalian target of rapamycin. Exposure to 95% O2 enhanced the phosphorylation of the translational repressor eIF2␣ in whole lung extracts and the immunoreactivity of phosphorylated eIF2␣ in epithelial cells. Cell culture studies further demonstrated that hyperoxia increases eIF2␣ phosphorylation in lung epithelial cells, but not in lung fibroblasts. These findings illustrate that hyperoxia-induced suppression of mRNA translation in the newborn lung is accompanied by increased phosphorylation of eIF2␣ in the epithelium. oxygen toxicity; mRNA translation; eukaryotic initiation factors; lung injury; mammalian target of rapamycin; bronchopulmonary dysplasia ADMINISTRATION OF HIGH CONCENTRATIONS of oxygen (hyperoxia) is a potentially life-saving therapy for the treatment of premature infants with surfactant deficiency. Direct exposure of the immature lung to this atmosphere, however, is potentially injurious. In animal models, hyperoxia severely hinders lung alveolarization and disrupts pulmonary vascularization, alterations that adversely affect pulmonary function and gas exchange (24). In clinical practice, hyperoxia is a well-known contributor to the pathogenesis of bronchopulmonary dysplasia (BPD) and the reduction in alveolar surface area (4). At the cellular level, hyperoxia generates reactive oxygen species capable of influencing vital cellular processes regulating cell division, survival, and gene expression (24). Of the many deleterious effects induced by exposure to hyperoxia, alter-

VARA attenuates hyperoxia-induced impaired alveolar development and lung function in newborn mice

“…tein synthesis or stability may be involved with, and possibly regulated by, retinoids (29). Alveolar development is coordinated by multiple paracrine interactions between the fibroblastic, epithelial, and microvascular lung components, all of which also interact with the extracellular matrix (11).…”

Hyperoxia-induced alterations in the pulmonary proteome of juvenile rats

“…Given that hyperoxia is known to suppress global protein synthesis in the lung, proteomics provides a means to discover genes containing sequence and structural elements capable of enhancing translational efficiency during periods of stress [6]. The length, GC content, and specific regulatory nucleotide sequences contribute to the complexity of secondary structure of the 5'-UTR that hinders recruitment of the ribosome, the rate-limiting step in translation initiation.…”

“…These highly struetured nueleotide sequenees promote 40S ribosome binding independent of the 5'-^methyl-GTP eap strueture, thereby providing an alternate meehanism to initiate translation. Cap-independent mRNA translation may be advantageous for expression of adaptive or stress-responsive proteins during periods of generalized eap-dependent translational repression sueh as oeeurs during hyperoxie exposure [6]. Although AnxA6 protein and mRNA expression data are eonsistent with a funetional 1RES, RNA-binding proteins and 1RES trans-aeting faetors (ITAF) are eommonly required for aetivation, making generalization to all eells less likely [34].…”

“…Whole right upper lobes were homogenized in CHAPS lysis buffer as previously described [6]. Equal amounts of protein were electrophoretically separated on 10% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes.…”

“…Since that time, a multitude of studies illustrate that elevated levels of ambient or inspired O2 diminish the fractional rate of protein synthesis, global protein synthesis, and protein synthetic efficiency in isolated cells and intact lungs [5][6][7]. Prolonged hyperoxic exposure activates the integrated stress response (ISR), a coordinated adaptive signal transduction program which promotes 108 W. M. Konsavageefa/.…”