p38α Mediates Cell Survival in Response to Oxidative Stress via Induction of Antioxidant Genes

Background: p38α MAPK is activated by stress stimuli, which can regulate cell death. Results: In response to H2O2, p38α MAPK increases SOD and catalase levels, impairs ROS accumulation, and leads to cell survival. Conclusion: p38α MAPK signals survival under moderate oxidative stress through up-regulation of antioxidant defenses. Significance: To know how p38α regulates ROS levels is important for cell homeostasis. We reveal a novel pro-survival role for mammalian p38α in response to H2O2, which involves an up-regulation of antioxidant defenses. The presence of p38α increases basal and H2O2-induced expression of the antioxidant enzymes: superoxide-dismutase 1 (SOD-1), SOD-2, and catalase through different mechanisms, which protects from reactive oxygen species (ROS) accumulation and prevents cell death. p38α was found to regulate (i) H2O2-induced SOD-2 expression through a direct regulation of transcription mediated by activating transcription factor 2 (ATF-2) and (ii) H2O2-induced catalase expression through regulation of protein stability and mRNA expression and/or stabilization. As a consequence, SOD and catalase activities are higher in WT MEFs. We also found that this p38α-dependent antioxidant response allows WT cells to maintain an efficient activation of the mTOR/p70S6K pathway. Accordingly, the loss of p38α leads to ROS accumulation in response to H2O2, which causes cell death and inactivation of mTOR/p70S6K signaling. This can be rescued by either p38α re-expression or treatment with the antioxidants, N-acetyl cysteine, or exogenously added catalase. Therefore, our results reveal a novel homeostatic role for p38α in response to oxidative stress, where ROS removal is favored by antioxidant enzymes up-regulation, allowing cell survival and mTOR/p70S6K activation.

The intracellular redox state is tightly regulated because it is essential for the control of cell fate. High levels of ROS 5 can lead to molecular damage and cell death, whereas low ROS levels can be essential second messengers (1). Pro-oxidant and antioxidant systems are involved in this regulation, preventing an excessive accumulation of ROS.
Different members of the MAPK family, such as ERKs, JNKs, and p38, can be activated by ROS (1). This activation leads to a great variety of biological responses, including cell death or survival. Hence, although the stress MAP kinases can induce apoptosis in response to oxidative stress (2,3), differences in the duration and magnitude of the oxidative stress might be directly proportional to the state of activation of these kinases, and this might determine cell death or survival.
p38␣ MAPK plays an important role in the coordination of cellular stress responses to signals such as ROS. In fact, it is well known that p38␣ MAPK plays an important role in mediating apoptosis (4) and/or senescence induced by different stimuli, including ROS (5,6). For example, ROS generated by oncogenic H-Ras induces apoptosis through p38␣ activation, inhibiting tumor initiation (7). In contrast, low levels of oxidative stress can also induce cell cycle arrest (8) or cell survival (9, 10) through p38. Initially, stress signaling mechanisms are pro-survival systems because they tend to repair damage before committing cells to death or senescence. Interestingly, p38␣ can mediate survival upon activation with H 2 O 2 (10), and p38␣ and ␤ can have pro-survival roles (11)(12)(13) such as during quiescence of dormant tumor cells (14). However, the precise mechanisms by which p38 signaling achieves cell survival are poorly understood.
We have previously demonstrated that Akt activity is negatively regulated by p38␣ (15), and recent data from Nogueira et al. (16) showed that Akt activation sensitized cells to oxidative stress through down-regulation of ROS scavengers leading to the accumulation of intracellular ROS and cell death. Therefore, we hypothesized that p38␣, through inhibition of Akt, might allow a proper expression of antioxidant genes and cell survival. Thus, we analyzed the precise function of p38␣ in the regulation of the cell fate using nontransformed WT and p38␣ Ϫ/Ϫ MEFs exposed to oxidative stress. Furthermore, we explored the mechanisms involved in the regulation of antioxidant responses, in the context of Akt/mTOR signaling, as well as other pathways linked to ROS level regulation.

EXPERIMENTAL PROCEDURES
Cell Lines, Culture Conditions, and Inhibitors-WT and p38␣-deficient MEFs, immortalized either by passages or by LTAg (Large T Antigen) expression, were grown in DMEM supplemented with 10% FBS (Invitrogen) at 37°C in a humidified atmosphere with 5% CO 2 . For signaling experiments, confluent cells were stimulated with 0.1-1 mM H 2 O 2 for 20 min. For cell death analysis, growing cells were treated with 0.1-1 mM H 2 O 2 for 6 -24 h. The mTORC1 inhibitor, rapamycin was used at a concentration of 1-10 M.
Treatment with Antioxidants, Actinomycin D, and MG-132-The cells were treated with the following antioxidants to decrease intracellular levels of ROS and/or to metabolize H 2 O 2 : 50 units/ml of catalase (Sigma; C-1345) and 2.5 mM N-acetyl cysteine (Sigma; A-9165). The antioxidants were added 1 h before H 2 O 2 treatment. To inhibit transcription, the cells were treated with actinomycin D (Sigma; A9415) at 5 g/ml. To block proteasome-dependent proteins degradation cells were treated with the proteasome inhibitor MG-132 at 1 M.
ATF-2 RNA Interference-Transfections of cells with siRNA targeting ATF-2 (Cell Signaling; 6433) or a control scrambled siRNA (Ambion) diluted in medium without serum at a final concentration of 50 nM were performed using siPORT NeoFX transfection reagent (Ambion) following the manufacturer's instructions. Trypsinized cells were resuspended and overlaid onto the transfection complexes. After 24 h under normal cell culture conditions, protein and RNA was isolated, or cell viability was quantified.
SOD-2 Luciferase Analysis-To assess SOD-2 promoter activity upon H 2 O 2 treatment in WT and p38␣ Ϫ/Ϫ MEFs, the cells were cotransfected with a construct containing the SOD-2 promoter coupled to luciferase reporter (kindly provided by Daret St. Clair, Kentucky University) and a plasmid-encoding Renilla luciferase (Clontech) (100 -500 ng). Then cells were treated with H 2 O 2 0.5 mM for 4 and 8 h and lysed using the passive lysis buffer from Promega. Luciferase activity was detected with a luminometer (Molecular Devices Spectramax M5E) using a dual luciferase reporter kit from Promega following the manufacturer's instructions. Luciferase activity was normalized to Renilla luciferase activity.
Catalase and SOD Activity Assays-Catalase activity was measured by quantification of peroxide decomposition in a 50 mM phosphate buffer at pH 7 containing 3 mM H 2 O 2 . This was monitored spectrophotometrically at 240 nm. SOD activity from cell extracts was quantified using a kit (BioVision, reference number K335-100), where WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfo-phenyl)-2H tetrazolium, monosodium salt) is the substrate. WST-1 produces a watersoluble formazan dye upon its reduction with superoxide anion, which can be monitored spectrophotometrically at 450 nm. The rate of the reduction is linearly related to the xanthine oxidase activity and inhibited by SOD, so the IC 50 of SOD is determined as a measure of SOD activity.
Analysis of Cell Viability and Apoptosis-Cell viability was assayed through staining of adhered (viable) cells with crystal violet. The cells were washed with PBS, incubated with a crystal violet solution (0.2%, w/v) for 20 min, washed, and dried. Stained cells were lysed in 1% SDS, and absorbance at 560 nm was measured.
Apoptotic cells were quantified by flow cytometric analysis of the cell cycle. The cells were trypsinized, washed with PBS, and fixed with cold ethanol (70% v/v). Then they were washed, resuspended in PBS, and incubated with RNase (25 g/10 6 cells) for 30 min at 37°C. After the addition of 0.05% propidium iodide, the cells were analyzed in the cytometer. The percentage of cells in sub-G 1 was determined and considered as apoptotic.
To analyze condensed and fragmented nuclei, characteristic of apoptosis, they were stained with propidium iodide (5 g/ml in PBS, 0.1% Triton X-100, 0.1 M EDTA supplemented with 5 g/ml RNase) (19) and visualized by fluorescent microscopy. Apoptotic indices were calculated after counting 500 -1,000 cells/treatment in an inverted fluorescence microscope (Eclipse TE300; Nikon).
Statistical Analysis-The data are represented as the means Ϯ S.E. The comparisons were made between two experimental groups. An unpaired Student's t test was used, and alternatively, an analysis of variance test was carried out for comparisons of more than two experimental groups.

Loss of p38␣ Sensitizes Cells to H 2 O 2 -induced Cell Death-
We first tested viability of WT and p38␣ Knock-out MEFs in response to a level of oxidative stress able to generate damage but not massive toxicity. As shown in Fig. 1A, WT MEFs exhibited a higher resistance to H 2 O 2 (0.1-0.5 mM) than p38␣-deficient MEFs. This suggests that p38␣ allows cells to survive under mild levels of oxidative stress. This was confirmed by p38␣ reconstitution in p38␣ Ϫ/Ϫ cells, which increased cell viability in response to H 2 O 2 up to the levels observed in WT cells (Fig. 1B). The morphological analysis of nuclei showed a significantly higher number of condensed and/or fragmented nuclei in p38␣-deficient MEFs than in WT cells upon H 2 O 2 treatment (Fig. 1C), which suggests that cells would be dying by apoptosis. These data demonstrate that p38␣ protects from low levels of H 2 O 2 -induced cell death. Loss of p38␣ MAPK Impairs mTOR/p70S6K Activation in Response to H 2 O 2 through Akt-independent Mechanisms-Akt is involved in the activation of mTORC1 through the phosphorylation and inactivation of TSC-2, which inhibits mTOR through inhibition of Rheb (20,21). We have previously shown that Akt activity is negatively regulated by p38␣ (15), so that Akt is hyperactivated in p38␣ Ϫ/Ϫ cells, leading to increased survival in response to serum deprivation (15). However, whether this has any effect on ROS sensitivity remains unknown. Recently, Nogueira et al. (16) showed that Akt activation sensitized cells to oxidative stress through down-regulation of ROS scavengers, which increased intracellular ROS. This led us to hypothesize that p38␣, through inhibition of Akt, might allow a proper expression of antioxidant genes. Thus, we next tested the activation of the Akt/mTOR/p70S6K pathway in response to H 2 O 2 in WT and p38␣-deficient MEFs. As shown in Fig. 2A, Akt phosphorylation in response to H 2 O 2 (0.1-1 mM) was higher (particularly, at 0.5 mM) in p38␣ Ϫ/Ϫ than in WT MEFs. Surprisingly, this activation was uncoupled from mTOR-mediated p70S6K phosphorylation in Thr-389, because phosphorylation at this site was markedly reduced in p38␣-deficient cells. Under these conditions, p38␣ was activated by H 2 O 2 in WT cells, whereas in p38␣ Ϫ/Ϫ MEFs another p38 isoform with a higher mobility was slightly activated (Fig. 2A). Therefore, these data suggest that in the absence of p38␣, mTOR/p70S6K pathway activation becomes uncoupled from that of Akt and is impaired in response to H 2 O 2 treatment. This was confirmed by p38␣ reconstitution in p38␣ Ϫ/Ϫ cells, which rescued the levels of p70S6K activation (see Thr(P)-389-p70S6K levels; Fig. 2B), reaching a level comparable with WT cells treated with H 2 O 2 .
To gain further insight into the mechanisms involved in the regulation of mTOR/p70S6K pathway by p38␣, we analyzed whether the regulators of mTORC1 were differentially modulated in the absence of p38␣. As shown in Fig. 2C, the high level of Akt phosphorylation in p38␣-deficient cells was correlated with Akt-mediated TSC-2 phosphorylation in Ser-939 (an inhibitory site) in response to H 2 O 2 , whereas TSC-1 and TSC-2 levels remained unchanged in all cases. Nevertheless, this did not result in a high activation of mTOR/p70S6K pathway. Hence, we measured activation of AMPK, an inhibitor of mTORC1 pathway. However, phosphorylation of AMPK (in Thr-172) and its substrate, acetyl-CoA-carboxylase in response to H 2 O 2 was enhanced in WT MEFs (Fig. 2C). As a consequence, these changes in AMPK activity in WT and p38␣-deficient cells did not explain the higher activation of mTOR/ p706SK in WT cells exposed to oxidative stress. Therefore, neither TSC-1/2 nor AMPK appear to mediate the inhibition of mTOR signaling observed in p38␣ Ϫ/Ϫ cells, which indicates that other mechanisms might be involved. However, it is clear that Akt and AMPK signaling is uncoupled from mTOR/ p70S6K activation under these conditions.
We considered the possibility that the higher mTOR/ p70S6K activation in cells expressing p38␣ could be responsible for the increased survival in response to H 2 O 2 . Thus, we inhibited this pathway with rapamycin to evaluate it. As shown in Fig. 3A, treatment with rapamycin completely abolished p70S6K phosphorylation by mTOR in response to H 2 O 2 . However, rapamycin did not sensitize WT cells to ROS-induced cell death (Fig. 3B). Thus, the higher activation of mTOR/p70S6K appears not to be responsible for the p38␣-mediated increased survival. Nevertheless, rapamycin was able to decrease cell size in WT cells but not in those deficient in p38␣ (data not shown), suggesting a potential function for the p38␣-mTOR pathway promoting and/or maintaining cell size and homeostasis under oxidative stress conditions. This is in agreement with recent published results (22). Moreover, transfection of either an active p70S6K (p70⌬29 -46 ⌬CT104 mutant) or a WT p70S6K construct did not increase the cell viability of p38␣-deficient MEFs treated with H 2 O 2 (Fig. 3C). In contrast, basal viability was slightly reduced upon expression of these p70S6K constructs.
On the other hand, Akt inhibition with the specific chemical inhibitor, A443354 (23) had no effect on H 2 O 2 -induced cell death, either in WT or p38␣ Ϫ/Ϫ MEFs (supplemental Fig. S1), which suggests that the Akt pathway does not play a major role in p38␣-mediated survival. We also tested other pathways that could be potentially involved in cell death, such as JNKs. We found a higher JNK activation in p38␣ Ϫ/Ϫ MEFs in response to H 2 O 2 and under basal conditions (supplemental Fig. S2). However, JNK inhibition with SP600125 did not decrease cell death, which indicates that JNK is not responsible for the enhanced cell death observed in p38␣-deficient cells.

Activation of p38␣ Prevents the Accumulation of ROS upon H 2 O 2 Treatment via Induction of Antioxidant Enzymes-We
next explored alternative mechanisms by which p38␣ might protect cells from ROS damage, allowing cell survival. Although p38␣ MAPK is known to mediate cell death in response to oxidative stress (5-7), p38 has been shown to mediate the expression of antioxidant enzymes (24,25). This led us to hypothesize that p38␣ through the regulation of the antioxi-dant response might maintain low ROS intracellular levels, leading to cell survival.
As shown in Fig. 4, the percentage of cells with detectable levels of ROS (positive for DCFH) was slightly higher in untreated p38␣-deficient cells and was highly increased upon treatment with H 2 O 2 in a dose-dependent manner. This suggests that upon treatment with H 2 O 2 , p38␣ Ϫ/Ϫ cells are unable to scavenge ROS, leading to a high and progressive accumulation of ROS (Fig. 4B).
This high ROS accumulation in p38␣-deficient cells could be a consequence of a deficiency in the activation of antioxidant mechanisms. Thus, we analyzed the expression of relevant antioxidant enzymes such as SOD or catalase. As shown in Fig. 5A, p38␣ Ϫ/Ϫ cells expressed lower protein levels of SOD-2 and catalase than WT cells under basal conditions. Moreover, upon treatment with H 2 O 2 , these cells were either unable to efficiently induce the expression of these enzymes, as observed for

p38␣ Mediates Survival in Response to Oxidative Stress
SOD-2, or had a delay and a reduced induction, as happens for catalase. These results suggest that impaired or delayed induction of antioxidant enzymes in p38␣ Ϫ/Ϫ MEFs could be responsible for its higher sensitivity to H 2 O 2 -induced cell death. This was supported by the fact that p38␣ reintroduction in p38␣deficient cells led to an increase in catalase protein levels after 2 h of treatment with H 2 O 2 (Fig. 5B). In addition, basal and H 2 O 2 -induced catalase and SOD activities were significantly higher in WT than in p38␣ Ϫ/Ϫ MEFs (Fig. 5, C and D, respectively). Therefore, all of these data indicate that the presence of p38␣ highly increases the antioxidant activity of cells.
To get further insight into the role of p38␣ as a regulator of antioxidant enzyme expression, we measured SOD-1 and SOD-2 mRNAs levels by RT-PCR. The levels were significantly lower in p38␣ Ϫ/Ϫ cells under basal conditions, and H 2 O 2 only induced an increase in SOD-1 mRNA after 20 -60 min, whereas SOD-2 mRNA remained unchanged (Fig. 5E).
To better understand the regulation of SOD-2 expression by p38␣ in response to H 2 O 2 , we analyzed SOD-2 promoter activity using luciferase as a reporter. As shown in Fig. 5F, H 2 O 2 treatment induced a significant increase in SOD-2 reporter activity in WT cells after 8 h, but not in p38␣ Ϫ/Ϫ MEFs. This lack of activation of SOD-2 promoter in the absence of p38␣ could be a consequence of the lower activation of the transcription factor ATF-2 (Fig. 6A). To address this issue, ATF-2 knockdown experiments were performed using an ATF-2 siRNA (which markedly reduced ATF-2 protein levels, Fig. 6B), and results showed a high decrease in SOD-2 mRNA levels in WT cells, either untreated or treated with H 2 O 2 (Fig. 6B). Moreover, ChIP assays revealed a significant binding of P-ATF-2 to SOD-2 promoter in WT MEFs treated with H 2 O 2 , whereas in p38␣ Ϫ/Ϫ cells, there was no detectable binding (Fig.  6C). In addition, ATF-2 activation was required for p38␣-mediated cell survival in the presence of H 2 O 2 , so its knockdown induced death of WT cells (Fig. 6D). Therefore, these results indicate that p38␣ through ATF-2 regulation induces SOD-2 expression and resistance to H 2 O 2 treatment.
We next explored the mechanisms involved in the regulation of catalase expression. Catalase mRNA levels were increased by H 2 O 2 progressively at 2 and 4 h in both WT and p38␣-deficient cells, but to a higher extend in WT cells at 4 h (Fig. 7A). However, after 8 h of treatment, catalase mRNA highly decreased to the level of control in p38␣ Ϫ/Ϫ cells, whereas in WT cells, these levels remained above control values. Inhibition of transcription by actinomycin D abolished the increase in catalase mRNA levels observed at 2 and 8 h, regardless of the presence of p38␣. However, at 4 h it was just a partial decrease in catalase mRNA upon actinomycin D treatment, specially, in WT cells. This would suggest that increases in the catalase mRNA level are only partially dependent on transcription, and p38␣ might stabilize catalase mRNA at 4 -8 h of treatment. In addition, the fact that the proteasome inhibitor MG-132 increased catalase protein levels only in p38␣-deficient cells (Fig. 7B) strongly suggests that catalase protein would be also stabilized by p38␣. Hence, based on these results, p38␣ might be a positive regulator of catalase through protein stabilization and mRNA expression and/or stabilization.
Treatment with Either the Antioxidant N-Acetyl Cysteine or Catalase Protects from ROS-induced Cell Death: Effect on mTOR/p70S6K Pathway-Our data indicate that p38␣ activation in MEFs can protect from H 2 O 2 -induced cell death through a mechanism that reduces ROS accumulation via induction of antioxidant enzymes. Thus, we next studied the effect of the antioxidant N-acetyl cysteine (NAC) on H 2 O 2induced cell death.
As shown in Fig. 8A, NAC significantly decreased the number of apoptotic nuclei induced by H 2 O 2 in p38␣ Ϫ/Ϫ cells. This correlates with a decrease in Ser(P)-18-p53 levels (supplemental Fig. S3), so p53 could be an important mediator of this process of cell death. Moreover, NAC not only protected from cell  A and B, catalase/tubulin and SOD-2/tubulin represents the relative value resulting from the densitometric analysis of catalase or SOD-2, respectively, versus tubulin levels multiplied by 10. C and D, catalase and SOD activities, respectively, are shown as a fold increase of that of WT untreated cells (9.35 milliunits/mg protein for catalase and 7.32 milliunits/mg protein for SOD). *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001. E, RT-PCR analysis of the expression of SOD-1 and SOD-2 mRNAs. F, SOD-2 reporter activity quantification using luciferase as reported. The results are expressed using arbitrary units. *, p Ͻ 0.05; ***, p Ͻ 0.001.

p38␣ Mediates Survival in Response to Oxidative Stress
death but also induced an increase in Thr(P)-389-p70S6K levels, which was particularly strong in p38␣-deficient cells, reaching a similar level to that found in WT cells (Fig. 8B). In contrast, Akt and p38␣ phosphorylation decreased in NAC-treated cells. All of these results suggest that H 2 O 2 -induced cell death would be a consequence of the damage induced by ROS accumulation, which is higher in p38␣ Ϫ/Ϫ MEFs. Hence, because catalase levels were lower in these cells, we added exogenous catalase to mimic WT cells situation. As shown in Fig. 8A, catalase highly decreased the number of apoptotic nuclei in p38␣deficient MEFs treated with H 2 O 2 . In addition, a parallel increase in P-Thr389-p70S6K levels was observed (Fig. 8C). In contrast, pretreatment with the cell-permeable SOD mimetic, Mn-TBAP, which is a superoxide scavenger, neither protected p38␣ Ϫ/Ϫ MEFs from H 2 O 2 -induced cell death nor protected from mTOR/p70S6K inactivation (supplemental Fig. S4). Therefore, only the selective H 2 O 2 scavenger, catalase, which is down-regulated in p38␣-deficient cells, allows p70S6K activation in the presence of H 2 O 2 . Moreover, in agreement with the idea that a high accumulation of ROS impairs p70S6K activation, we observed that treatment of WT MEFs with a very high dose of H 2 O 2 (5 mM) impaired p70S6K activation (supplemental Fig. S5), which indicates that a very high accumulation of ROS either prevents activation or inactivates the mTOR/ p70S6K pathway.

DISCUSSION
p38␣ plays an important role as a mediator of apoptosis in response to different stress stimuli (4,15,26), including oxidative stress (5,7). For example, p38␣ functions as a tumor suppressor in the transformation induced by oncogenic H-Ras (7), because of its ability to induce apoptosis upon generation of ROS. However, we have recently found a survival effect of p38␣ in response to H 2 O 2 (10), which agrees with some data from other groups (8,9,27). Therefore, we have further evaluated the mechanisms by which p38␣ might protect cells from oxidative stress.
Here, we reveal a pro-survival function of p38␣, which is dependent on the regulation of the antioxidant response. In addition, we found that although this response is associated with reduced Akt activity and enhanced mTOR/p70S6K signal-  ing, these cascades are not immediately responsible for the p38␣-mediated survival effect, but instead their modulation is a consequence of the ROS content in the cells. In contrast, the enhanced mTOR/p70S6K signaling caused by p38 activation appears to favor cell size maintenance during stress, a function recently identified in Drosophila melanogaster cells (22). Hence, the loss of p38␣ induces ROS accumulation, which leads to cell death and inactivation of the mTOR/p70S6K pathway. Although we did not measure this process, autophagic cell death could contribute to the loss of cells observed in p38␣deficient cells treated with H 2 O 2 .
In agreement with the proposed role for p38␣ as a positive regulator of antioxidant enzymes expression, it was previously reported that p38 MAPK up-regulated catalase (24) and heme oxygenase-1 (25) mRNAs in response to H 2 O 2 . We have now characterized the precise role of p38␣ in the regulation of the expression and activity of different antioxidant enzymes, as well as the mechanisms involved. Our data reveal a novel function for p38␣ controlling (i) H 2 O 2 -induced SOD-2 expression through direct regulation of transcription via ATF-2 activation and (ii) basal and H 2 O 2 -induced catalase expression through regulation of mRNA expression and/or stability and protein stability (Fig. 9).
It is worth highlighting that we describe for the first time a p38␣-ATF-2-dependent transcriptional regulation of SOD-2 in response to H 2 O 2 . Previously, it had been shown that low levels of H 2 O 2 induced ATF-2 expression (8) or activation through p38␣ MAPK (29), leading to growth arrest. A role for JNKs-ATF-2 and p38 MAPK in the regulation of heme oxygenase-1 in response to oxidative stress was also demonstrated (25). However, we now show that the p38␣ MAPK-ATF-2 cascade also mediates SOD-2 up-regulation and cell survival in response to low levels of oxidative stress.
In contrast to our results demonstrating that mTOR/p70S6K cascade is not responsible for the p38␣-mediated survival effect in response to H 2 O 2 , there are a number of data in the literature proposing that p38 MAPK activation and/or mTOR inhibition are required for H 2 O 2 -induced cell death in other cell types (1, 5, 30 -32). For example, in keratinocytes activation of p38 by AMPK contributes to H 2 O 2 -induced apoptosis, as well as to mTOR activity down-regulation (30). In PC12 cells, AMPKmediated mTOR inhibition was shown to be partially responsible for the apoptotic cell death induced by H 2 O 2 (32). Our findings are not incompatible with these results. They simply highlight the complexity of the mechanisms and that the function of these pathways may depend on the cell type, H 2 O 2 dose, and duration of the stress signal.
Based on our results, it is unclear that Akt has any potential role in the response to H 2 O 2 mediated by p38␣ in MEFs. The expression of SOD and catalase is higher in WT than in p38␣ Ϫ/Ϫ cells under basal conditions, when Akt activation is quite similar in both cell lines, and the Akt inhibitor does not affect survival. Hence, these findings do not support the involvement of Akt antagonizing the antioxidant response in these cells, as found in other systems (16).
Regarding the regulation of mTOR/p70S6K by H 2 O 2 , there are also conflicting data in the literature. Some data indicate that mTOR can be inhibited by H 2 O 2 treatment through different mechanisms such as up-regulation of REDD1 (regulated in development and DNA damage response 1) (33) or inhibition of PDK1 and Akt, accompanied by AMPK activation (32). In addition, AMPK through p38-dependent and -independent mechanisms can decrease mTOR activation by H 2 O 2 (30), mediating cell death (30,32). In contrast, our results indicate that p38␣ do not act as a negative regulator of mTOR/p70S6K as previously suggested (26). This would be in agreement with previous data demonstrating a pro-survival role of p38␣ and a p38␣-dependent activation of mTOR/p70S6K signaling in response to different types of stresses (22,28). Hence, in agreement with the results of Cully et al. (22), we also found that in cells treated with H 2 O 2 at a low dose, mTORC1 activation is dependent on p38␣. We additionally found that p38␣ regulates mTORC1/p70S6K in an indirect way, which is dependent on ROS accumulation. Therefore, in cells lacking p38␣, a high accumulation of ROS is produced, which impairs activation of this pathway. However, in the presence of either catalase or N-acetyl cysteine, which prevents a high ROS accumulation, mTOR/p70S6K can be activated in p38␣-deficient cells treated with H 2 O 2 .
However, the mechanisms controlling mTOR by p38 can be different depending on the context. Hence, in quiescent tumor cells, activation of the transcription factor ATF6 via p38␣ up-

p38␣ Mediates Survival in Response to Oxidative Stress
regulates Rheb, which in turn activates mTOR and survival through an Akt-independent mechanism (28).
We conclude that p38␣ has a pro-survival function because of its ability to up-regulate antioxidant genes expression, preventing from a high accumulation of ROS upon exposure to low or moderate doses of H 2 O 2 (Fig. 9). In this way, cell damage can be overcome, allowing cell survival and mTOR/p70S6K pathway activation. In contrast, in the absence of p38␣ the antioxidant defense is not properly activated, leading to ROS accumulation and high cell damage. As a consequence, the mTOR/ p70S6K pathway is inactivated, which might avoid protein synthesis and cell growth of damaged cells. In agreement with other studies (21,33), we also found that mTOR/p70S6K activation is dependent on p38␣ activation in an oxidative microenvironment. We believe this could be critical for other cellular responses such as autophagy that can also control cell size and to maintain cellular homeostasis and function.