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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 250-256, 2002
© 2002 American Thoracic Society

Oxidant-Induced Hypertrophy of A549 Cells Is Accompanied by Alterations in Eukaryotic Translation Initiation Factor 4E and 4E-Binding Protein-1

Jeffrey S. Shenberger, Mary H. Adams and Stephen G. Zimmer

Department of Pediatrics, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire; and Departments of Anatomy/Neurobiology and Microbiology/Immunology, University of Kentucky, Lexington, Kentucky

Address correspondence to: Jeffrey S. Shenberger, M.D., Department of Pediatrics/Neonatology, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756. E-mail: Jeffrey.S.Shenberger{at}Dartmouth.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Control of protein synthesis resides at the level of eukaryotic translation initiation (eIF) complex formation. Complex formation is regulated by the mRNA cap-binding protein, eIF4E, whose activity is influenced by phosphorylation and binding to 4E-binding protein 1 (4E-BP1). To provide a link between alterations in protein synthesis and the pathogenesis of oxidant-mediated lung disease, we investigated the effect of hydrogen peroxide (H2O2) on actively growing A549 cells. Cells were exposed to 200 or 400 µM H2O2 for 4 h and then assessed for changes in proliferation, protein synthesis, and eIF4E and 4E-BP1 status over 72 h. We found that both concentrations of H2O2 inhibited [3H]thymidine incorporation and cell division while inducing a G2/M-predominant growth arrest within 24 h. In addition, H2O2 increased cell size, [3H]leucine incorporation/cell, and total cell protein. Although time had little effect on eIF4E and 4E-BP1 expression and phosphorylation state of control cells, H2O2 induced a 2- to 3-fold increase in eIF4E and 4E-BP1 expression, a 5-fold increase in eIF4E phosphorylation, and a shift in the distribution of 4E-BP1 phosphorylation favoring lesser phosphorylated forms. These findings suggest that oxidant-mediated alterations in protein synthesis and cell morphology occur in concert with changes in factors known to regulate translation kinetics.

Abbreviations: bronchopulmonary dysplasia, BPD • 4E-binding protein 1, 4E-BP1 • enhanced chemiluminescence, ECL • eukaryotic translation initiation, eIF • lactate dehydrogenase, LDH • mitogen-activated protein kinase, MAPK • phosphate-buffered saline, PBS • reactive oxygen species, ROS • trichloroacetic acid, TCA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) are highly toxic byproducts of cellular respiration (1). Under basal conditions, cells are protected from the deleterious effects of ROS by endogenous antioxidants, which reduce them to more inert substances (1). During periods of extreme stress as occur during infection, inflammation, or exposure to environmental oxidants, cellular defenses may be overwhelmed, generating a load of ROS capable of inducing cellular injury (1, 2). Clinically, ROS have been implicated in the pathogenesis of numerous chronic pulmonary diseases, many of which are associated with tissue destruction and/or remodeling. This theory has been corroborated by reports of increased exhaled and urinary H2O2 levels in patients with adult respiratory distress syndrome, bronchiectasis, and chronic obstructive lung disease, and by the finding that alveolar macrophages collected from infants with bronchopulmonary dysplasia (BPD) release large amounts of H2O2 (3–5).

Further delineation of the deleterious effects of H2O2 has evolved from in vitro studies. Brief exposure of fibroblasts to 200–400 µM H2O2 induces a senescent-like state, characterized by the accumulation of neutral ß-galactosidase, nonresponsiveness to mitogenic stimuli, a G1-predominant growth arrest, and increased cell volume and granularity (6). Similar concentrations of H2O2 have been shown to acutely inhibit global protein synthesis, to override the stimulation of protein synthesis induced by insulin, and to enhance protein degradation (7, 8). Given this spectrum of effects, it is conceivable that H2O2 alters protein synthetic processes directly integral to the regulation of proliferation, cell survival, and cell morphology.

Regulation of protein synthesis occurs primarily at the level of initiation (9). This step is controlled via a group of peptides that serve to bind the mRNA to the 40S ribosomal subunit (9). This group of peptides, collectively known as the eukaryotic initiation factor complex (eIF)4F, is comprised of an RNA helicase (eIF4A), a scaffolding protein (eIF4G), an enhancer protein (eIF4B), and a cap binding protein (eIF4E) (9). Under normal conditions, the concentration of eIF4E is low and rate-limiting for complex formation, a concept that is supported by reports that overexpression of eIF4E induces malignant transformation of NIH 3T3 cells and by the observation that expression of antisense eIF4E inhibits cell growth and protein synthesis (10, 11). The level of free eIF4E is governed by its interaction with the inhibitory peptide, 4E-binding protein-1 (4E-BP1) (12, 13). In turn, the activities of eIF4E and 4E-BP1 are regulated by phosphorylation, such that phosphorylation of eIF4E increases the affinity for the 5'-cap and phosphorylation of 4E-BP1 decreases the affinity for eIF4E (14, 15). Thus, phosphorylation of either protein promotes eIF4F formation and translation initiation.

Recent work in quiescent vascular and cardiac smooth muscle cells indicates that H2O2 modifies the phosphorylation of eIF4E and 4E-BP1, thereby providing a link between alternations in protein synthesis and the pathogenesis of oxidant-related diseases such as coronary artery disease (8, 16). To assess the role of protein synthesis in oxidant-mediated lung disease, we studied the effects of oxidant stress on protein synthesis and eIF4E/4E-BP1 in actively growing A549 cells. We hypothesized that exposure to sublethal concentrations of H2O2 would alter protein synthesis in concert with eIF4E and 4E-BP1 phosphorylation and that these changes would coincide with changes in proliferation and cellular morphology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Conditions
Cells from the adenocarcinoma cell line, A549 (American Tissue Culture Collection, Manassas, VA), were grown in Ham's F12 medium (Cellgro/Mediatech, Inc., Herndon, VA) plus 10% fetal bovine serum at 37°C in room air plus 5%CO2. Cells were seeded at a density of 10,000 cells/cm2 and allowed to attach overnight. The following morning, medium was replaced and cells treated with H2O2 (Sigma, St. Louis, MO) at concentrations of 200 and 400 µM. After 4 h of exposure, medium was removed, monolayers rinsed with phosphate-buffered saline (PBS), and medium replaced with fresh Ham's F12. The time upon removal of H2O2 was designated time 0 for experimentation. Plates were then assessed every 24 h for 3 d to determine the effect of H2O2 on proliferation, protein synthesis, cell size, and components of eIF4F.

Proliferation
To measure proliferation, attached cells were trypsinized and counted on a hemacytometer at each time point. Synthesis of DNA was determined by [3H]thymidine incorporation. Briefly, cells were incubated with 2 µCi/ml (final concentration) of [methyl-3H]thymidine (20 Ci/mol; Amersham Pharmacia Biotech, Piscataway, NJ) for 24 h. Monolayers were rinsed twice with ice-cold PBS before a 10-min incubation in 10% trichloroacetic acid (TCA) on ice. Plates were then rinsed with PBS and the precipitate dissolved with 0.5 N NaOH for at least 12 h. After dissolution, radioactivity in the supernatant was measured on a liquid scintillation counter (LS3801; Beckman, Irvine, CA).

Flow Cytometry
Floating and attached cells were rinsed twice with ice-cold PBS and fixed in cold 70% ethanol added dropwise while vortexing. After fixation at 4°C for at least 24 h, cells were stained with propidium–iodine (10 µg/ml) plus RNase A (250 µg/ml; Sigma) for 30 min in the cold. Fixed cells were analyzed within 4 h of staining on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA). DNA histograms were modeled using ModFit (Verity, Topsham, ME). Cell size was determined using forward side light scatter.

Cell Injury/Cell Death
Cell injury/death was determined by measuring released lactate dehydrogenase (LDH) in the medium as a function of total well LDH. Cellular LDH was determined on cells scraped into one half of the medium. Both samples were sonicated on high power for 30 s. Medium-derived and cellular LDH were measured colorimetrically using pyruvate as a substrate and NAD/oxidase as a detector (Sigma). The percent LDH released was determined by the following equation: (2 x [media]/[media + cells]) x 100%.

The ability of cells to exclude trypan blue was assessed on attached cells to assure the viability of cells used to analyze the effects of H2O2 on the components of the eIF4F complex. Trypsinized monolayers were incubated with 0.4% trypan blue (50:50 vol/vol; Grand Island Biologicals, Grand Island, NY) for 2 min. Cells found to be permeable to the dye under light microscopy were deemed nonviable.

Protein Synthesis/Content
Protein synthesis was measured using [3H]leucine incorporation. Plates were pulsed with 3 µCi/ml [4,5-3H]leucine (51 Ci/mmol; Amersham) for 24 h. Monolayers were then rinsed twice with ice-cold PBS and protein precipitated with 10% TCA on ice for 10 min. After removal of the TCA, precipitates were rinsed again with PBS before dissolution in 0.5 N NaOH. Radioactivity was measured by scintillation counting and the values normalized to cell number from matched wells.

To determine cell protein content, cells were trypsinized, counted on a hemacytometer, centrifuged, and resuspended in 0.5 N NaOH. Following dissolution of the pellet, total protein content was quantitated by the bicinchoninic acid assay (Pierce Chemical, Rockford, IL) and normalized back to cell number.

Western Blotting
Cell monolayers were rinsed twice with PBS and trypsinized with 0.05% trypsin/EDTA (Cellgro/Mediatech). Cell number was determined on a hemacytometer, after which the suspensions were centrifuged at 1,800 rpm for 10 min at 4°C. The pellet was washed with PBS and then suspended in RIPA extraction buffer (0.15 M NaCl, 0.01 M Tris-HCl pH 8.0, 0.1% NP-40, 0.1 g/ml deoxycholic acid, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride [final concentrations]) at a ratio of 2,000 cells/µl. Protein extracts from equal cell numbers were resolved on 12.5% SDS-polyacrylamide gels. Resolved proteins were then transferred to PVDF membranes. Membranes were incubated overnight at 4°C with antibodies to eIF4E (1:500; Transduction Laboratories, Lexington, KY), phospho(ser209)-eIF4E (1:1,000; Cell Signaling Technology, Beverly, MA), 4E-BP1 (1:1,000; Zymed Laboratories, San Francisco, CA), or ß-actin (1:5,000; Sigma) in blocking buffer. After rinsing, membranes were incubated with the corresponding horseradish peroxidase–conjugated secondary antibody for 2.5 h at room temperature, rinsed, and detected using enhanced chemiluminescence (ECL; Amersham). Relative band intensity was determined by densitometry (Alpha Innotech, San Leandro, CA) and normalized to ß-actin expression.

Statistics
All studies, except Western blots, were performed a minimum of three times with intra-experiment replicates of 3–6. Western blots were repeated four times and results averaged. Data was analyzed using repeated measures ANOVA with Neuman-Keuls pairwise comparison or Fisher's LSD testing to determine individual differences when appropriate. Statistical analysis was performed using GBStat v7.05 software (Dynamic Microsystems, Inc., Silver Spring, MD). Data are listed as mean ± SE and the level of significance set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of H2O2 on Proliferation, Cell Cycle Distribution, and Cell Death
The addition of H2O2 to actively growing A549 cells dramatically inhibited proliferation (Figures 1A and 1B) . Differences in cell number and [3H]thymidine between treated and nontreated cells became apparent by 48 h, with the 200-µM concentration of H2O2 producing a shorter duration of growth suppression than that of 400 µM. As shown in Table 1 , the loss of proliferative capacity was also associated with specific derangements in the cell cycle. The most notable change was the prolongation of G2/M. The accrual of cells in this phase was maximal at 24 h, with 200 and 400 µM H2O2-treated cultures containing 3- and 4-fold more cells, respectively, than controls (control: 10.6 ± 1.6; 200 µM: 34.0 ± 2.2; 400 µM: 45.5 ± 7.7%, P < 0.01). The accumulation of cells in G2/M was accompanied by a decrease in the percentage of cells in G0/G1 and S phases (P < 0.001). None of the samples, in any group, displayed a pre-G0/G1 peak indicative of apoptosis.



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  		Figure 1. The effect of H2O2 on cell number (A) and [3H]thymidine (B) incorporation in A549 cells. H2O2 inhibited cell number expansion and thymidine incorporation over time compared with controls (P < 0.0001). Both concentrations of H2O2 behaved similarly. Individual differences between H2O2 and controls were noted at 48 and 72 h (*P < 0.01). Data are expressed as the mean (n = 6) ± SEM. Solid bars, control; open bars, 200 µM H2O2; shaded bars, 400 µM H2O2.

 

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  		TABLE 1 The effect of H2O2 on cell cycle distribution

 
To assess the magnitude of cell injury induced by H2O2, we measured the release of cellular LDH. In control cells, the %LDH released declined steadily over time (Time 0: 26.7 ± 0.3% versus 72 h: 6.2 ± 0.7%, P < 0.01) owing to the large increase in cellular LDH. Hydrogen peroxide, on the other hand, increased %LDH released over time in 400 µM–treated, but not in 200 µM–treated cells. When compared with control cells, 200 µM– and 400 µM–treated cells released 3- and 6-fold more LDH, respectively, at 72 h (control: 6.2 ± 0.7; 200 µM: 17.7 ± 0.9; 400 µM: 37.7 ± 2.2%, P < 0.01).

Effect of H2O2 on Protein Synthesis, Cellular Protein Content, and Cell Size
The rate of [3H]leucine incorporation/cell remained constant in control cells throughout the experiment. Treatment of A549 cells with H2O2, on the other hand, produced a marked increase in [3H]leucine incorporation/cell within 48 h. By 72 h, H2O2-treated cells approximately doubled the rate of protein synthesis compared with control cells (Figure 2A) .



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  		Figure 2. The effect of H2O2 on [3H]leucine incorporation (A) and cellular protein content (B) in A549 cells. H2O2 increased leucine incorporation (P < 0.05) and total cell protein (P < 0.01) over time compared with controls. Differences in leucine incorporation between H2O2-treated cells and controls were noted at 24–72 h (*P < 0.05). As shown in B, H2O2 also increased total cell protein compared with controls at 48 and 72 h (*P < 0.05). At 72 h, 400 µM H2O2 (shaded bars) increased cell protein compared with both 200 µM H2O2 (open bars) and controls (solid bars) (**P < 0.05). Data are expressed as the mean (n = 6) ± SEM.

 
Total cell protein content decreased over time in the control cells (Figure 2B). This finding was reproducible and may have represented the alterations in cell cycle distribution observed in the near-confluent control cells. By contrast, treatment of the cells with H2O2 increased total cell protein, although differences were not observed until 48–72 h (72 h: control: 12.5 ± 0.7; 200 µM: 33.1 ± 1.0; 400 µM: 49.5 ± 2.3 µg/105 cells, P < 0.05). For the 400 µM concentration of H2O2, the effect was still significant even when compared with the higher baseline level of cell protein (control at Time 0: 29.2 ± 7.7; 400 µM at 72 h: 49.5 ± 2 µg/105 cells, P < 0.01).

Because H2O2-treated cells appeared larger than controls, we semiquantitatively determined cell size using forward light scatter. Although the control cells displayed minor variations in median light scatter between trials secondary to alterations in instrument calibration, we did not detect differences in light scatter over time within each trial. Exposure of matched cells to H2O2, however, consistently shifted median light to the right, indicating an increase in cell size (Figure 3) . This effect was apparent for both 200 µM– and 400 µM–treated cells and was detectable from 24 through 72 h after exposure. Plotting of forward light scatter as a function of fluorescence (propidium iodine) revealed that all phases of the cell cycle increased in cell size beginning at 24 h (data not shown).



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  		Figure 3. The effect of H2O2 on cell size. Ethanol-fixed cells were assessed for changes in forward light scatter using flow cytometry. A rightward shift in light scatter is indicative of an increase in cell size. In control cells, we observed inter-trial shifts in light scatter values secondary to differences in flow cytometer calibration, but no alteration in light scatter values over time within each trial. Treatment of cells with H2O2, however, induced a rightward shift in forward light scatter at both 200 and 400 µM concentrations. This effect appeared at 24 h and was maintained through 72 h.

 
Effect of H2O2 on Translation Initiation Factors
In control A549 cells, no changes were observed in the level of eIF4E or 4E-BP1 or in their degree of phosphorylation. When cells were exposed to H2O2, however, the levels of both eIF4E and 4E-BP1 increased. For eIF4E, the changes were modest, ranging from 2.5- to 3-fold at 72 h (Figure 4) . More dramatic changes were observed in the degree of eIF4E phosphorylation of these proteins. Compared with unstimulated cells at time 0, H2O2 increased eIF4E phosphorylation within 4 h (control: 1 ± 0; 200 µM: 4.2 ± 1.0; 400 µM: 5.4 ± 1.0* arbitrary units, *P < 0.05). At 72 h, there was a tendency for 200 µM to induce greater phosphorylation than 400 µM H2O2, although there was a great deal of variation between individual trials (Figure 5) .



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  		Figure 4. The effect of H2O2 on eIF4E expression. Cells were exposed to 200 (open bars) or 400 (shaded bars) µM H2O2 for 4 h and cell lysates assayed for eIF4E by SDS-PAGE. Expressed values were normalized to ß-actin levels. Top panel illustrates a representative Western blot of eIF4E migrating at 25 kD. The expression of eIF4E in control cells did not change over time. Hydrogen peroxide increased the expression of eIF4E compared with controls (solid bars) with individual differences detected at 48 and 72 h (*P < 0.05). There were no differences between the expression of cells treated with 200 or 400 µM H2O2. Data are expressed as the mean of four separate experiments ± SEM.

 


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  		Figure 5. The effect of H2O2 on phosphorylated eIF4E expression. Cells were exposed to 200 (open bars) or 400 (shaded bars) µM H2O2 for 4 h and cell lysates assayed for phospho(ser 209)-eIF4E by SDS-PAGE. Expressed values were normalized to ß-actin levels. Top panel illustrates a representative Western blot of phospho-eIF4E migrating at 25 kD. The expression of phospho-eIF4E in control cells did not change over time. Hydrogen peroxide increased the expression of eIF4E compared with controls (*P < 0.05). At 72 h, there was also a tendency for 200 µM to induce greater phosphorylation than controls (solid bars) and 400 µM H2O2 (**P < 0.05), although there was a great deal of variation between trials. Data are expressed as the mean of four separate experiments ± SEM.

 
Eukaryotic initiation factor 4E-BP1 migrated as four distinct bands, each indicative of a unique phosphorylation pattern. These bands were designated as {alpha} (least or nonphosphorylated) through {delta} (most phosphorylated). Incubation of cells with 200 or 400 µM H2O2 increased total 4E-BP1 expression within 24 and 48 h, respectively. By 72 h, the increase was 3- to 4-fold greater than at baseline (Figure 6) . Characterization of the individual phosphorylation bands contributing to total 4E-BP1 expression revealed that H2O2 increased the expression of {alpha}, ß, and {chi} bands, but not {delta} bands (Figure 7) . Thus, H2O2 increased total 4E-BP1 expression primarily through enhanced expression of non-phosphorylated and under-phosphorylated species.



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  		Figure 6. The effect of H2O2 on 4E-BP1 expression. Cells were exposed to 200 (open bars) or 400 (shaded bars) µM H2O2 for 4 h and cell lysates assayed for 4E-BP1 by SDS-PAGE. Top panel illustrates a representative Western blot of 4E-BP1 migrating as four distinct bands at 16–19 kD. Total 4E-BP1 expression values were normalized to ß-actin levels. The expression of 4E-BP1 in control cells did not change over time. Hydrogen peroxide increased the expression of 4E-BP1 compared with controls (solid bars) (*P < 0.01). At 72 h, there was also a tendency for 200 µM to induce greater phosphorylation than controls and 400 µM H2O2 (**P < 0.05), although there was a great deal of variation between trials. Data are expressed as the mean of three separate experiments ± SEM.

 


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  		Figure 7. The effect of H2O2 on 4E-BP1 phosphorylation. Cells were exposed to 200 or 400 µM H2O2 for 4 h and cell lysates assayed for 4E-BP1 by SDS-PAGE. Insets in upper left-hand corner of each graph denote the phosphorylation bands of 4E-BP1, with {alpha} representing the least, and {delta} representing the most, phosphorylated forms. Individual phosphorylation bands normalized to ß-actin are expressed in arbitrary units. Treatment with H2O2 increased the expression of {alpha}, ß, and {chi} bands (P < 0.05). Effects were seen within 24 h in {alpha} and ß bands and at 48 h in {chi} bands (*P < 0.05). Incubation with 200 µM H2O2 increased the expression of ß and {chi} bands compared with both controls and 400 µM H2O2 at 72 h only (**P < 0.05). Data are expressed as the mean of three separate experiments ± SEM. Open bars, Time 0; solid bars, 24 h; darkly shaded bars, 48 h; lightly shaded bars, 72 h.

 
To exclude the possibility that H2O2–treated cell extracts contained an increased quantity of dead or dying cells, we assessed the viability of trypsinized monolayers using trypan blue. Our results revealed that the vast majority of attached cells in both treated and untreated groups were viable (94–97%) and that H2O2 had no time-dependent or -independent effects on the viability of the attached cells utilized to make cell extracts. This finding, coupled with information collected from cell cycle analysis and LDH studies, indicates that throughout the 72-h study period, H2O2 induced significant cell injury at a sublethal level.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Exposure of cells to heightened oxidative stress is known to contribute to the pathogenesis of diseases characterized by aberrant cell growth. The current study demonstrates that H2O2-mediated growth arrest of A549 cells occurs in concert with increases in protein synthesis, net protein accretion, and cell size, a pattern characterizing hypertrophy. These morphologic changes are associated with alterations in the expression and phosphorylation of eIF4E and 4E-BP1, key components in the rate-limiting step of translation initiation. This report, therefore, provides novel clues to the potential involvement of translational regulation in oxidant-mediated cellular hypertrophy in the lung.

The primary locus of translational regulation resides at the level of eIF4F complex formation, a process controlled by the low abundance of eIF4E (14). Regulation of eIF4E activity is multifaceted, occurring directly through changes in expression and phosphorylation, and indirectly through binding to the repressor protein, 4E-BP1 (12). Under basal conditions, eIF4E is bound to 4E-BP1 at the eIF4G-binding site (12). As a result, 4E-BP1 prevents the association of eIF4E with eIF4G, thereby inhibiting the binding of the 43S pre-initiation complex to mRNA (17). The affinity of 4E-BP1 for eIF4E is inversely related to its degree of phosphorylation (18). Stimulation with growth factors sequentially activates phosphoinositol-3-kinase (PI-3-kinase), Akt/protein kinase B, and mammalian target of rapamycin, which initiate the phosphorylation of 4E-BP1 (13, 14, 18). Hyperphosphorylation of 4E-BP1 favors the dissolution of eIF4E/4E-BP1 complexes (18, 19). Unbound eIF4E is then free to bind to eIF4G and facilitate formation of the 48S ribosomal subunit. The affinity of eIF4E for the 7-methyl-guanosine mRNA cap is also enhanced via phosphorylation of serine-209 by mitogen-activated protein kinase (MAPK)-interacting protein kinase-1 (19). In general, therefore, stimuli that increase eIF4E and 4E-BP1 phosphorylation favor protein synthesis.

The present study, however, demonstrates that the regulation of eIF4E activity during hypertrophy is likely to be complex and involve multiple, overlapping pathways. In our model, H2O2 enhanced the expression and phosphorylation of eIF4E and increased the expression of nonphosphorylated and lesser-phosphorylated forms of 4E-BP1, processes that oppose one another in the regulation of initiation. These findings are analogous to those observed in etoposide-treated Chinese hamster ovary cells, where G2 cell cycle arrest is accompanied by increased expression of eIF4A and its competitive translational repressor, NAT1 (20). Further credence is added to our findings by reports that indicate that H2O2 is capable of phosphorylating both eIF4E and 4E-BP1. In growth-arrested vascular smooth muscle cells, for example, H2O2 induced a rapid increase in eIF4E phosphorylation which was unaccompanied by changes in protein synthesis (16). Hydrogen peroxide also shifted 4E-BP1 to lesser-phosphorylated forms, augmented eIF4E/4E-BP1 binding, and decreased [3H]phenylalanine incorporation into protein in cardiac myocytes (8). Although no previous study has simultaneously examined eIF4E and 4E-BP1 phosphorylation in response to oxidant stress, the phosphodiesterase inhibitor, SQ20006, has been shown to inhibit protein synthesis without altering eIF4E or 4E-BP1 phosphorylation or eIF4E/4E-BP1 binding (21). In addition, recently published reports also indicate that cap-dependent translation can proceed despite 4E-BP1 dephosphorylation and the lack of eIF4E phosphorylation (22, 23). Therefore, although it is clear that oxidants alter eIF4E and 4E-BP1 phosphorylation, the process whereby these modifications influence translation kinetics remains to be determined.

In addition to the alterations in phosphorylation, we also observed an increase in eIF4E and 4E-BP1 expression. Modified levels of eIF4E and 4E-BP1 have been shown to affect proliferation and protein synthesis in malignant and ras-transformed cell lines in vitro (11, 12). Although changes in expression in response to oxidant stress have not been reported, H2O2 has been found to increase the expression of eukaryotic elongation factors (24). Nevertheless, the magnitude and timing of eIF4E and 4E-BP1 expression correlate more closely with changes in total cell protein than protein synthesis. Given that altered eIF4E/4E-BP1 expression has not been shown to regulate translation under normal physiologic conditions, the increased levels of eIF4E and 4E-BP1 appear to be more likely a consequence of, rather than a cause of, increased protein synthesis (25).

The enhanced eIF4E expression and phosphorylation would be expected to selectively promote the efficient translation of mRNA with complex, highly structured, 5'-untranslated region (UTR) (9, 25). Growth regulatory genes are five times more likely to possess complex 5'-UTR than other native genes (26). The list of translationally regulated genes includes transforming growth factor ß, vascular endothelial growth factor, fibroblast growth factor, ornithine decarboxylase, Fas/Apo-1, Mdm-2, and cyclin D1 (24, 27). Because peroxides activate the G1/S and G2/M checkpoints through the inhibition of cyclinE/cdk2 and the dephosphorylation of Cdc2, augmented translation of growth regulatory genes may avert activation of the apoptotic cell death cascade (3, 28, 29). This concept is supported by work in rat embryo fibroblasts that shows that eIF4E amplification protects cells from Myc-induced apoptosis via a cyclin D1-dependent process (27). Indeed the current data indicate that the sublethal cell injury induced by 200–400 µM H2O2 is accompanied by suppression of DNA synthesis and a G2/M-predominant form of growth arrest. Thus, alterations in eukaryotic translation initiation factors may serve to "switch" protein synthesis from a proliferative to a cytoprotective mode, resulting in a profile of cellular proteins that suppresses apoptosis and promotes cellular repair (30).

Although the data generated in this study are consistent with the involvement of eIF4E in the translational regulation of hypertrophy, they do not prove causality. Changes in protein accumulation could represent changes in turnover, potentially via inhibition of the ubiquitin pathway. Additionally, increases in protein synthesis could reflect alterations in the activity of translational regulators other than eIF4E. The phosphorylation of eIF2{alpha}, for example, is known to serve a critical role in the control of cell proliferation (31). Enhanced phosphorylation of eIF2{alpha} has been observed in conjunction with growth arrest, decreased protein synthesis, and entrance into G0 (31). More recently, eIF2{alpha} phosphorylation has been correlated with diminished protein synthesis during the G2 to M phase transition (32). Elongation pathways have also been shown to regulate protein synthesis in response to stress. In cumene hydroperoxide–treated rats, for example, the declination in liver protein synthesis has been found to involve principally elongation and to coincide with decreased elongation factor–2 activity secondary to oxidation and fragmentation (33). Taken together, these studies suggest that drawing conclusions regarding the direct involvement of eIF4E in oxidant-induced cellular hypertrophy would be premature without the analysis of the activities of translational regulators other than eIF4E, the modification of eIF4E activity through site-directed mutagenesis, and the confirmation of the current observations in normal lung cells.

From a clinical perspective, the present findings support the concept that alterations in protein synthesis are integral to the pathogenesis of various forms of oxidant-mediated lung disease. The recent delineation of the histology of BPD in the post-surfactant era indicates that the pathogenesis involves arrest of lung development, leading to a paucity of alveoli and alveolar crests (34, 35). Inflammatory-mediated oxidant stress has the potential to induce senescence and/or senescent-like phenotypes characterized by increased cell volume and permanent loss of replicative capacity, findings similar to the ones noted in H2O2-treated A549 cells (6). Alterations in cap-dependent translation, therefore, may not only contribute to the hypocellularity and abnormal cell phenotype, but also to the generalized cellular dysfunction characteristic of the disease. Future work aimed at correlating derangements in protein synthesis and translational regulation with structural and functional pulmonary abnormalities will promote a greater understanding of these pathogenic processes.


   Acknowledgments
 
This report is supported in part by a National Institutes of Health Research Grant #CA77614 (S.G.Z.).

Received in original form November 21, 2001

Received in final form March 19, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stogner, S. W., and D. K. Payne. 1992. Oxygen toxicity. Ann. Pharmacol. 26:1554–1562.[Abstract]
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