Oxidant Stress Stimulates Phosphorylation of eIF4E without an Effect on Global Protein Synthesis in Smooth Muscle Cells LACK OF EVIDENCE FOR A ROLE OF H 2 O 2 IN ANGIOTENSIN II-INDUCED HYPERTROPHY*

Reactive oxygen species (ROS) are implicated in the pathogenesis of several proliferative diseases, including atherosclerosis and cancer. Eukaryotic translation initiation factor 4E (eIF4E) plays an important role in cell proliferation and differentiation. To gain insight into molecular mechanisms by which ROS influence the pathogenesis of these diseases, I have studied the effect of H 2 O 2 , a ROS, on eIF4E phosphorylation. H 2 O 2 induced eIF4E phosphorylation in a dose- and time-dependent manner in growth-arrested smooth muscle cells (SMC). H 2 O 2 -induced eIF4E phosphorylation occurred on ser- ine residues. PD098059, a specific inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase inhibited ERK activities but had no significant effect on eIF4E phosphorylation induced by H 2 O 2 . Similarly, SB203580, a specific inhibi- tor of p38 MAPK, although inhibiting H 2 O 2 -induced p38 MAPK activity, had no effect on H 2 O 2 -induced eIF4E phosphorylation. Calphostin C, a specific inhibitor of protein kinase C, also had no effect on H 2 O 2 -induced eIF4E phosphorylation. In contrast, trifluoperazine, an antagonist of calcium/calmodulin kinases, completely blocked H 2 O 2 -induced eIF4E phosphorylation. In addi- tion, intracellular and extracellular Ca 2 1 chelators significantly inhibited H 2 O 2 -induced eIF4E phosphorylation.

Reactive oxygen species (ROS), 1 superoxide anion (O 2 . ), hy-drogen peroxide (H 2 O 2 ), and hydroxyl radicals ( ⅐ OH) are produced by all aerobic organisms as a consequence of inevitable oxygen consumption and respiratory metabolism (1)(2)(3). ROS are toxic to cells. Cells are protected from these toxic ROS by their removal or conversion into inert molecules such as H 2 O and oxygen via defense systems involving superoxide dismutase, glutathione peroxidase, catalase, vitamin C, and vitamin E (3). Under some unusual circumstances such as infections, chronic inflammation, and deficient scavenging systems, cellular levels of ROS can be increased, leading to generation of local or global oxidant stress (3,4). In response to oxidant injury, genetic remodeling may take place, causing cells to switch from their normal physiological function to an adaptative protective state. This adaptative genetic reprogramming may be a triggering event in the pathogenesis of diseases such as atherosclerosis and cancer (1)(2)(3)(4). Studying cellular signaling events that are responsive to oxidant stress may ultimately unravel the underlying mechanisms of early genetic events, which may be important in disease processes in which oxidant stress is implicated. In view of this goal, many laboratories including ours have previously demonstrated that oxidants stimulate several early growth response events such as the stimulation of protein tyrosine phosphorylation (5)(6)(7)(8)(9), the activation of extracellular signal-regulated kinases (ERKs) (5,7), and the induction of expression of the protooncogenes c-fos, c-jun, and c-myc in various cell types (10 -15). At nontoxic levels, these oxidants also induced growth in some cell types (13,16,17). Numerous additional studies have reported that oxidants are, in fact, produced by and active in growth factorinduced mitogenic signaling events in several cell types (18 -21). Translational processes are important in the manifestation of gene regulation (22)(23)(24)(25). The important step in this process is the binding of mRNA to ribosomes, which is facilitated by an initiation complex, eIF4F (22)(23)(24)(25). eIF4F consists of three polypeptides: eIF4E, the cap-binding protein; eIF4A, an RNA helicase; and eIF4G, a bridging protein for eIF4E and eIF4A (26). Via its subunit eIF4E, eIF4F binds to mRNA by interacting with the cap structure present in the 5Ј end of eukaryotic mRNAs (22,25). eIF4F in combination with another initiation factor, eIF4B, is thought to unwind mRNA secondary structure, thereby rendering it capable of binding to ribosomes (22). Because eIF4E is crucial for mRNA binding to ribosomes, a rate-limiting step in polypeptide synthesis, eIF4E is considered to be a regulator of protein synthesis (22,25,27). Indeed, a large body of data supports this notion. Overexpression of * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 1 The abbreviations used are: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; eIF4E, eukaryotic translation initiation factor 4E; SMC, smooth muscle cell(s); ATF-2, activating transcriptional factor-2; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis(acetoxymethyl ester); APE-1, apurinic/apyrimidinic endonuclease-1; CaM, calmodulin; HSP70, 70,000-Da heat shock protein; MAPK, mitogen-activated protein kinase; AII, angiotensin II; GST, glutathione S-transferase. eIF4E causes malignant transformation in NIH 3T3 cells (28) and aberrant growth in HeLa cells (29). Expression of antisense eIF4E inhibits protein synthesis and reverses the transformation induced by Ras (30,31). Furthermore, eIF4E is a phosphoprotein that is present in small amounts compared with other initiation factors, a characteristic feature of acute regulation by agonists (32,33). In addition, eIF4E activity is regulated by its binding proteins, 4E-BP1 and 4E-BP2 (34 -37). These proteins negatively modulate eIF4E activity. Analogous to retinoblastoma protein, 4E-BP1/2 with underphosphorylation binds with eIF4E and prevents the phosphorylation of eIF4E by agonists. Conversely, upon phosphorylation these proteins dissociate from eIF4E and facilitate the phosphorylation and activation of eIF4E by agonists (35,36). A recent report demonstrated that overexpression of 4E-BP1/2 downregulates cell growth (38). Together, these findings clearly support the concept that translation plays an important role in the regulation of cell growth (27,39,40).
To elucidate the role of oxidants in proliferative diseases, I have studied the effect of H 2 O 2 on the phosphorylation state of eIF4E. I found that 1) H 2 O 2 induces the phosphorylation of eIF4E in a dose-and time-dependent manner in growth-arrested SMC; 2) induced eIF4E phosphorylation by H 2 O 2 is Ca 2ϩ -and Ca 2ϩ /CaM kinase-dependent and ERK-, p38 MAPK-, and protein kinase C-independent; and 3) although H 2 O 2 had no significant effect on global protein synthesis, a correlation was found between enhanced eIF4E phosphorylation and translation of a small subset of mRNAs such as c-fos, c-jun, and HSP70 mRNAs in H 2 O 2 -treated SMC.
Cell Culture-SMC were isolated from the thoracic aortae of 200 -250-g male Harlan Sprague-Dawley rats by enzymatic digestion as described previously (13). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Cultures were maintained at 37°C in a humidified 5% CO 2 atmosphere.
Determination of eIF4E Phosphorylation-SMC were plated onto 60-mm dishes (cells were plated onto 100-mm dishes for phosphoamino acid analysis) and grown in Dulbecco's modified Eagle's medium containing 10% calf serum. At 70 -80% confluence, cells were growtharrested by incubating for 72 h in Dulbecco's modified Eagle's medium containing 0.1% calf serum. Growth-arrested SMC were labeled with 200 Ci/ml [ 32 P]orthophosphate for 4 h and treated with and without various concentrations of H 2 O 2 for 30 min at 37°C. In the case of time course experiments, labeled SMC were treated with and without 200 M H 2 O 2 for the indicated times at 37°C. After treatments, medium was removed, and cells were rinsed with cold phosphate-buffered saline and lysed in 500 l of lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EGTA, 80 mM ␤-glycerophosphate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride) for 20 min on ice. The cell lysates were collected onto 1.5-ml Eppendorf tubes and cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Cell lysates containing equal amounts of protein from control and each treatment were incubated with 5 l of eIF4E polyclonal antibodies for 2 h on ice with gentle rocking (41,42). Forty microliters of 10% (w/v) protein A-Sepharose beads were added, and the incubation was continued for another 2 h. The beads were collected by centrifuga-tion at 4000 rpm for 2 min at 4°C and washed five times with cold lysis buffer and once with cold phosphate-buffered saline. The beads were heated in 40 l of Laemmli sample buffer at 95°C for 5 min. The proteins were resolved by electrophoresis on 0.1% SDS, 10% polyacrylamide gel under reducing conditions. The gel was dried and exposed to Kodak X-Omat AR x-ray film with an intensifying screen for 1 day at Ϫ80°C.
Phosphoamino Acid Analysis-Phosphoamino acid analysis was performed according to the method of Hildebrandt and Fried (43). Briefly, after separation on SDS-polyacrylamide gel, eIF4E was transferred from the gel onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA) by electroblotting in 25 mM Tris-Cl, pH 8.3, 192 mM glycine, and 20% (v/v) methanol for 4 h at 100 mA and autoradiographed. The region of the polyvinylidene difluoride membrane corresponding to the eIF4E band was excised and exposed to vapors of 5.7 M HCl at 110°C for 4 h in a Reactivial (Pierce). Following acid hydrolysis, one drop of methanol was placed on the membrane patches, and amino acids and peptide fragments were extracted with 1 ml of H 2 O. A mixture of phosphoserine, phosphothreonine, and phosphotyrosine (100 nmol each) was added to the aqueous extract before freeze drying over NaOH. Samples were dissolved in 20 l of H 2 O and spotted on a 20 ϫ 20-cm thin layer cellulose plate (J. T. Baker Inc.). The phosphoamino acids were separated by one-dimensional electrophoresis in 7.8% acetic acid, 2.2% formic acid (pH 1.9) at 500 V for 4 h. Authentic phosphoamino acids were identified by ninhydrin reaction, and the TLC plate was exposed to Kodak X-Omat AR x-ray film with an intensifying screen for 5-7 days at Ϫ80°C.
ERK Assay-ERK activity was measured using an in-gel kinase assay as described previously (44). In brief, cell lysates containing equal amounts of protein (150 g) from control and each treatment were incubated with 1 g of rabbit anti-ERK1/2 antibodies for 2 h with gentle rocking on ice. Forty microliters of 10% (w/v) protein A-Sepharose beads were added, and the incubation was continued for an additional 2 h on ice. The immunoprecipitates were resolved on 0.1% SDS, 10% polyacrylamide gel that was copolymerized with 300 g/ml myelin basic protein.
The gel was washed twice for 30 min with 150 ml of 50 mM Tris-Cl buffer (pH 8.0) containing 20% isopropyl alcohol and twice for 30 min with 150 ml of 50 mM Tris-Cl buffer (pH 8.0) containing 5 mM ␤-mercaptoethanol (Buffer A). After incubation for 1 h in 150 ml of 6 M guanidine hydrochloride in Buffer A at room temperature, the gel was washed repeatedly in Buffer A containing 0.04% Tween 20 at 4°C. The kinase reaction was performed by incubating the gel in 30 ml of 40 mM Hepes buffer (pH 8.0) containing 10 mM MgCl 2 , 0.5 mM EGTA, 2 mM dithiothreitol, 50 M ATP, and 5 Ci/ml [␥-32 P]ATP for 1 h at room temperature. The gel was washed several times with 200 ml of 5% trichloroacetic acid, 1% sodium pyrophosphate until the counts/min were at background levels; then the gel was dried and subjected to autoradiography.
p38 MAPK Assay-p38 MAPK activity was measured using an immunocomplex kinase assay as described previously (44). Briefly, cell lysates containing equal amounts of protein (500 g) from control and each treatment were incubated with 1 g of rabbit anti-p38 MAPK antibodies for 2 h with gentle rocking on ice. Forty microliters of 10% (w/v) protein A-Sepharose beads were added, and the incubation was continued for an additional 2 h on ice. The beads were washed three times with lysis buffer and once with kinase buffer (20 mM Tris-Cl, pH 7.4, 20 mM NaCl, and 10 mM MgCl 2 ). The beads were suspended in 25 l of kinase buffer containing 1 mM dithiothreitol, 20 M ATP, 2 g of GST⅐ATF-2 fusion protein, and 5 Ci of [␥-32 P]ATP and incubated for 30 min at 30°C. The reaction was stopped by adding an equal volume of 2ϫ Laemmli sample buffer and heating for 5 min. The mixtures were separated by electrophoresis on 0.1% SDS, 12% polyacrylamide gels. The dried gel was subjected to autoradiography.
Determination of New Protein Synthesis-Growth-arrested SMC were treated with and without H 2 O 2 (200 M) or AII (100 nM) for the indicated times. Cells were labeled with 1 Ci/ml [ 14 C]leucine for the last 2 h of each treatment period. After labeling, cells were washed with cold phosphate-buffered saline and lysed in lysis buffer. Protein concentration was determined using BCA protein assay reagent according to the instructions of the manufacturer (Pierce). Equal amounts of protein from each sample were precipitated with cold 10% (w/v) trichloroacetic acid on ice for 30 min. The mixture was passed through a glass fiber filter (GF/F, Whatman). The filter was washed once with cold 5% trichloroacetic acid and once with cold 70% (v/v) ethanol. The filter was dried and placed in a liquid scintillation vial containing the scintillant fluid, and the radioactivity was measured in a liquid scintillation counter (Tracor Analytic Delta 300).
Determination of Cellular [ 14  The cell pellet was suspended in cold 10% (w/v) trichloroacetic acid and vortexed vigorously to lyse cells. After standing on ice for 60 min, the mixture was cleared by centrifugation at 12,000 rpm for 30 min at 4°C. The soluble fraction was extracted five times with ethyl ether to remove the trichloroacetic acid. The residue was dissolved in 50 l of water, spotted onto a 20 ϫ 20-cm cellulose thin layer plate, and resolved by one-dimensional chromatography using n-butyl alcohol:acetic acid:water (13:3:5) as a solvent system (45). Amino acids were identified by spraying the TLC plate with 0.2% ninhydrin in acetone. The spot on the TLC plate corresponding to leucine was scraped and extracted with ethanol containing 0.3% ninhydrin for 30 min. The optical density reading of the extract was measured in a spectrocolorimeter at 540 nm, and the radioactivity was measured in a liquid scintillation counter as described above. A standard graph generated using cold leucine was used as a reference for quantitation of the cellular leucine levels.

RESULTS
To determine the phosphorylation state of eIF4E in response to oxidant stress, growth-arrested and [ 32 P]orthophosphatelabeled SMC were treated with and without various concentrations of H 2 O 2 for 30 min, and eIF4E phosphorylation was measured by immunoprecipitation using its specific antibodies followed by SDS-polyacrylamide gel electrophoresis and autoradiography (41,42). H 2 O 2 induced eIF4E phosphorylation in a concentration-dependent manner with a near maximum effect of a 4.5-fold increase at 200 M (Fig. 1, upper panel) (Fig. 1, middle panel). After 15 and 30 min of H 2 O 2 (200 M) treatment, eIF4E phosphorylation was increased 1.8-and 3.7-fold, respectively, as compared with control. The increases in eIF4E phosphorylation by H 2 O 2 were sustained for at least 2 h although at a level (2-fold) that is lower than at 30 min of treatment. AII (100 nM), which was used as a positive control, induced eIF4E phosphorylation 4-fold by 15 min as compared with control, and this result is consistent with our previous observations (42). To find out whether the observed increases in eIF4E phosphorylation by H 2 O 2 were due to corresponding increases in eIF4E levels, equal amounts of protein from control and each treatment were analyzed by Western blotting for eIF4E levels using its specific antibodies. As shown in Fig. 1 (bottom panel), no significant changes were observed in eIF4E levels between control and H 2 O 2 or AII treatments. In response to growth factors and phorbol esters, eIF4E was phosphorylated on serine residues (41, 42, 46 -48). To examine whether H 2 O 2 -induced phosphorylation of eIF4E occurs on serine residues, growth-arrested and [ 32 P]orthophosphate-labeled SMC were treated with and without H 2 O 2 (200 M) or AII (100 nM), and eIF4E was immunoprecipitated and subjected to phosphoamino acid analysis. As shown in Fig. 2, serine is the only amino acid residue phosphorylated in eIF4E by H 2 O 2 . Consistent with our previous results, AII caused phosphorylation of eIF4E on serine residues (42).
Recent work from several laboratories showed that the ERK and p38 MAPK groups of MAPKs play a role in the enhanced phosphorylation of eIF4E in response to different stimulants in various cell types (35, 49 -51). In addition, several laboratories including ours have previously reported that H 2 O 2 activates both the ERK and p38 MAPK in SMC (5,7,52). To study the role of these MAPKs in H 2 O 2 -induced eIF4E phosphorylation, I tested the effect of PD098059 and SB203580, specific inhibitors of MEK1/2 (53) and p38 MAPK (54) Cell lysates containing equal amounts of protein from control and each treatment were immunoprecipitated with rabbit anti-eIF4E antibodies, and the immunoprecipitates were separated by electrophoresis on 0.1% SDS, 10% polyacrylamide gel and subjected to autoradiography. Growth-arrested SMC were treated with and without 200 M H 2 O 2 for the indicated times, and equal amounts of protein from control and each treatment were analyzed by Western blotting for eIF4E levels using its specific antibodies (bottom panel).

FIG. 2. H 2 O 2 induces eIF4E phosphorylation on serine residues.
Growth-arrested and [ 32 P]orthophosphate-labeled SMC were treated with and without H 2 O 2 (200 M) or AII (100 nM) for 30 min, and cell lysates were prepared. Cell lysates containing equal amounts of protein from control and each treatment were immunoprecipitated with rabbit anti-eIF4E antibodies, and the immunoprecipitates were separated by electrophoresis on 0.1% SDS, 10% polyacrylamide gel. The proteins were transferred from the gel to a polyvinylidene difluoride membrane and autoradiographed. The spot corresponding to eIF4E on the membrane was cut and acid-hydrolyzed, and the hydrolysate was analyzed by chromatography on a silica gel TLC plate. Authentic phosphoamino acids were identified by reaction with ninhydrin, and the TLC plate was subjected to autoradiography. PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine.
in-gel kinase assay using myelin basic protein as a substrate, and p38 MAPK activity was determined by an immunocomplex kinase assay using ATF-2 as a substrate. H 2 O 2 induced activation of ERKs 6.2-fold as compared with control (Fig. 3, middle panel). PD098059 at a 50 M concentration completely inhibited the H 2 O 2 -induced activation of ERKs. Similarly, H 2 O 2 induced activation of p38 MAPK 8-fold as compared with control, and SB203580 at a 10 M concentration completely inhibited this effect (Fig. 3, bottom panel). These results indicate that both PD098059 and SB203580 at the concentrations used were effective in the inhibition of the activities of ERKs and p38 MAPK in SMC. Together these results suggest that H 2 O 2 induces eIF4E phosphorylation independent of the activation of ERKs and p38 MAPK.
We next studied the role of protein kinase C and Ca 2ϩ /CaM kinases on H 2 O 2 -induced eIF4E phosphorylation. We have previously reported that calphostin C, a potent and specific inhibitor of protein kinase C, at a 200 nM concentration inhibits the AII-induced eIF4E phosphorylation in SMC (42). As shown in Fig. 4 (upper panel), calphostin C (200 nM) did not affect H 2 O 2induced eIF4E phosphorylation. Similarly, bisindolylmaleimide, which inhibits both Ca 2ϩ -dependent and Ca 2ϩ -independ-ent protein kinase C isozymes at a 1 M concentration (55), had no significant effect on H 2 O 2 -induced eIF4E phosphorylation (data not shown). In contrast, trifluoperazine, a specific antagonist of Ca 2ϩ /CaM kinases (56), at a 25 M concentration completely inhibited H 2 O 2 -induced eIF4E phosphorylation (Fig. 4,  upper panel). These results suggest a role for Ca 2ϩ /CaM kinases in H 2 O 2 -induced eIF4E phosphorylation. Ca 2ϩ /CaM kinases require Ca 2ϩ for their activity (56). Therefore, to examine the role of Ca 2ϩ in H 2 O 2 -induced eIF4E phosphorylation, growth-arrested and [ 32 P]orthophosphate-labeled SMC were treated with and without H 2 O 2 in the presence and absence of EGTA (5 mM) or BAPTA-AM (50 M), extracellular and intracellular Ca 2ϩ chelators, respectively, and eIF4E phosphorylation was measured. Both EGTA and BAPTA-AM significantly (Ͼ90%) inhibited H 2 O 2 -induced eIF4E phosphorylation (Fig. 4, lower panel).
Earlier reports from other laboratories suggested a role for H 2 O 2 in AII-induced hypertrophy in SMC (52,57,58). To relate enhanced phosphorylation of eIF4E by H 2 O 2 to global protein synthesis and understand the role of H 2 O 2 in AII-induced hypertrophy, I first studied the effect of H 2 O 2 and AII on protein levels. Growth-arrested SMC were treated with and without H 2 O 2 (200 M) or AII (100 nM) for 6 or 12 h, and protein levels were determined. H 2 O 2 had no significant effect on protein levels as compared with control (Fig. 5, upper panel). In contrast, AII treatment for 6 or 12 h increased SMC protein levels by 21 and 29%, respectively. Second, I tested the effect of H 2 O 2 and AII on the rates of new protein synthesis in SMC. Growtharrested SMC were treated with and without H 2 O 2 (200 M) or AII (100 nM) for 6 or 12 h, and the rates of new protein synthesis were measured by pulse-labeling the cells for the last 2 h in each treatment period with 1 Ci/ml [ 14 C]leucine and counting the trichloroacetic acid-insoluble radioactivity. H 2 O 2 either had no effect or decreased the new protein synthesis rates in SMC (Fig. 5, lower panel). In contrast, treatment of SMC with AII for 6 and 12 h caused 19 and 15% increases, respectively, in the rate of new protein synthesis. To find out whether the observed differences in [ 14 C]leucine incorporation were due to its altered cellular pool size, SMC were treated with and with- Cell lysates containing equal amounts of protein from control and each treatment were analyzed for eIF4E phosphorylation as described in the legend to Fig. 1. Middle panel, growth-arrested SMC were treated with and without H 2 O 2 (200 M) in the presence and absence of PD098059 (50 M) for 10 min, and cell lysates were prepared. Cell lysates containing equal amounts of protein from control and each treatment were immunoprecipitated with rabbit anti-ERK1/2 antibodies; the immunoprecipitates were separated by electrophoresis on 0.1% SDS, 10% polyacrylamide gel that was copolymerized with 300 g/ml myelin basic protein, and then the immunoprecipitates were subjected to an in-gel kinase assay. Bottom panel, growth-arrested SMC were treated with and without H 2 O 2 (200 M) in the presence and absence of SB203580 (10 M) for 10 min, and cell lysates were prepared. Cell lysates containing equal amounts of protein from control and each treatment were immunoprecipitated with rabbit anti-p38 MAPK antibodies, and the immunoprecipitates were subjected to an immunocomplex kinase assay using GST⅐ATF-2 fusion protein as a substrate.  (Fig. 6). DISCUSSION The significant findings in the present study are that H 2 O 2 , an ROS and a cellular oxidant, induces phosphorylation of eIF4E in SMC and that this activity is dependent on Ca 2ϩ and Ca 2ϩ /CaM kinases. In addition, H 2 O 2 -induced eIF4E phosphorylation is independent of ERKs, p38 MAPK, and protein kinase C. Although H 2 O 2 mimicked hormones such as AII and insulin in the stimulation of eIF4E phosphorylation, it differed from them in the induction of global protein synthesis. H 2 O 2 had no effect or decreased global protein synthesis. This could be due to the lack of effect of H 2 O 2 on signaling events that, in conjunction with eIF4E phosphorylation, are important in induced global protein synthesis and that are activated by those hormones. On the other hand, besides enhancing eIF4E phosphorylation, H 2 O 2 may also be activating the signal transduction pathways involved in the negative regulation of protein synthesis. However, in contrast to the present observations, in recent years it was reported that diphenyleneiodonium, a potent inhibitor of the flavin-containing enzymes including NADH/NADPH oxidase, attenuates AII-induced global protein synthesis in SMC (57). Furthermore, these authors have demonstrated that SMC overexpressing catalase fail to respond to AII in the induction of global protein synthesis (58). Considering these findings, a role for H 2 O 2 in AII-induced hypertrophy in SMC was suggested. However, these studies did not address the direct effect of H 2 O 2 on protein synthesis, a critical component of hypertrophy. Because the present studies showed a lack of inducing effect of H 2 O 2 on global protein synthesis, it is unlikely that this oxidant is involved in AII-induced hypertrophy in SMC.
It is intriguing to note that agents such as arsenite and anisomycin that inhibit protein synthesis and cause cellular stress have also been reported to stimulate eIF4E phosphorylation (51,59). Although the physiological significance of induced eIF4E phosphorylation by cellular stress is not clear at the present time, this event may well be an adaptative response to cellular injury. Enhanced eIF4E phosphorylation by various stimulants of cellular stress may be associated with translation of a few mRNA species whose products are critical in cell survival. Indeed, H 2 O 2 induced the expression of c-Fos, c-Jun, and HSP70, which all play an important role in cell survival. The effect of H 2 O 2 on the induction of expression of these proteins appears to be selective because it did not affect the APE-1 expression or global protein synthesis. APE-1 plays an important role in cleaving off abasic sites in the DNA that result because of the action of DNA glycosylases on the damaged bases. Earlier studies have reported an induction of expression of APE-1 in response to genotoxicity caused by oxidants in several cell types (60). The lack of effect of H 2 O 2 on APE-1 expression in SMC further confirms our earlier findings that H 2 O 2 is not genotoxic to this cell type (13). Considering these findings, it is likely that H 2 O 2 -induced eIF4E phosphorylation is associated with translation of a small subset of mRNAs such as those listed above in oxidant-treated SMC.
Previous studies from several laboratories including ours showed that protein kinase C plays a role in the phosphorylation of eIF4E induced by AII, insulin, and phorbol esters (42,61). In contrast, H 2 O 2 -induced eIF4E phosphorylation was found to be protein kinase C-independent. This finding further supports the lack of a role for H 2 O 2 in AII-induced hypertrophy in SMC. The interesting observation made in the present study is that trifluoperazine, an antagonist of Ca 2ϩ /CaM kinases, completely blocked H 2 O 2 -induced eIF4E phosphorylation. This result suggests that Ca 2ϩ /CaM kinases are important in oxidant-induced eIF4E phosphorylation, at least in SMC. However, enhanced eIF4E phosphorylation by other cellular stressors such as arsenite was reported to be mediated by p38 MAPK (51). In view of these findings, it is conceivable that several kinases including Ca 2ϩ /CaM kinases and p38 MAPK mediate eIF4E phosphorylation induced by different cellular stressors. It is also important to note that H 2 O 2 was reported to induce ribosomal protein S6 phosphorylation in mouse epidermal JB6 cells in a manner that is dependent on Ca 2ϩ and Ca 2ϩ /CaM kinases (56), findings which are analogous to the present observations. The present findings along with those of Larson and Cerutti (56) clearly provide evidence for the ability of oxidants, particularly H 2 O 2 , to activate translation machinery.
Initial studies assigned serine 53 to be the amino acid phosphorylated on eIF4E in response to agonists (48). However, later studies using a mutant in which serine 53 was replaced by alanine revealed that eIF4E is phosphorylated on a different site, which is distinct from serine 53 (62). Subsequent studies have identified this phosphorylation site as serine 209 (63,64). Although the present findings clearly showed that eIF4E is phosphorylated on serine residues in response to H 2 O 2 , future studies are required to identify the serine residue(s) that are phosphorylated in response to oxidants.
ERKs, a group of MAPKs, are believed to be important in cell proliferation (65,66). It was reported that ERKs modulate eIF4E activity by phosphorylating eIF4E binding proteins 4E-BP1 and 4E-BP2, thereby relieving the inhibitory constraint of these proteins on eIF4E (35,49). It was also reported that insulin induces eIF4E phosphorylation in an ERK-dependent manner (67). Recent studies have further identified serine/ threonine kinases known as MAPK-interacting kinases 1/2, Mnk1/2, that are downstream to ERKs and p38 MAPKs and are able to phosphorylate eIF4E in vitro (68,69). Although, H 2 O 2 is capable of activating ERKs and p38 MAPK in SMC, H 2 O 2 -induced eIF4E phosphorylation appears to be independent of activation of these kinases because PD098059 and SB203580, specific inhibitors of ERK1/2 and p38 MAPK, respectively, failed to block the effect. In fact, recent reports have shown that growth factor-induced eIF4E phosphorylation occurs independently of activation of the "ERK MAPK" pathway (70,71). Together, these observations lead us to infer that eIF4E can be phosphorylated by various serine/threonine kinases including Ca 2ϩ /CaM kinases, MAPKs, and protein kinase C, perhaps depending on cell type and agonist.
In summary, the present study demonstrates the ability of oxidants to induce the phosphorylation of eIF4E, an important regulator of translation, in SMC. The present findings also show for the first time a role for Ca 2ϩ and Ca 2ϩ /CaM kinases in the enhanced phosphorylation of eIF4E, at least in response to oxidants such as H 2 O 2 in SMC.