Article11 November 2004free access FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK Marieke AG Essers Marieke AG Essers Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Sanne Weijzen Sanne Weijzen Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Alida MM de Vries-Smits Alida MM de Vries-Smits Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Ingrid Saarloos Ingrid Saarloos Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Nancy D de Ruiter Nancy D de Ruiter Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Johannes L Bos Johannes L Bos Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Boudewijn M T Burgering Corresponding Author Boudewijn M T Burgering Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Marieke AG Essers Marieke AG Essers Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Sanne Weijzen Sanne Weijzen Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Alida MM de Vries-Smits Alida MM de Vries-Smits Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Ingrid Saarloos Ingrid Saarloos Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Nancy D de Ruiter Nancy D de Ruiter Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Johannes L Bos Johannes L Bos Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Boudewijn M T Burgering Corresponding Author Boudewijn M T Burgering Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Author Information Marieke AG Essers1,‡, Sanne Weijzen1,‡, Alida MM de Vries-Smits1,‡, Ingrid Saarloos1, Nancy D de Ruiter2, Johannes L Bos1 and Boudewijn M T Burgering 1 1Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands 2Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA, USA ‡These authors contributed equally to this work *Corresponding author. Department of Physiological Chemistry & Centre for Biomedical Genetics, University Medical Center Utrecht, Stratenum, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Tel.: +31 30 253 8918; Fax: +31 30 253 9035; E-mail: [email protected] The EMBO Journal (2004)23:4802-4812https://doi.org/10.1038/sj.emboj.7600476 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Forkhead transcription factors of the FOXO class are negatively regulated by PKB/c-Akt in response to insulin/IGF signalling, and are involved in regulating cell cycle progression and cell death. Here we show that, in contrast to insulin signalling, low levels of oxidative stress generated by treatment with H2O2 induce the activation of FOXO4. Upon treatment of cells with H2O2, the small GTPase Ral is activated and this results in a JNK-dependent phosphorylation of FOXO4 on threonine 447 and threonine 451. This Ral-mediated, JNK-dependent phosphorylation is involved in the nuclear translocation and transcriptional activation of FOXO4 after H2O2 treatment. In addition, we show that this signalling pathway is also employed by tumor necrosis factor α to activate FOXO4 transcriptional activity. FOXO members have been implicated in cellular protection against oxidative stress via the transcriptional regulation of manganese superoxide dismutase and catalase gene expression. The results reported here, therefore, outline a homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI-3K/PKB) and positive (Ras/Ral) inputs. Introduction Reactive oxygen species (ROS) are oxygen free radicals that are highly reactive toward cellular constituents including protein, lipid and DNA. Formation of ROS can be caused by exogenous sources such as UV or ionizing radiation or by endogenous sources such as normal aerobic metabolism or by pathological conditions such as ischemia. Also in normal cell signalling, ROS are generated by growth factor-induced activation of enzyme complexes such as NADH oxidase (reviewed in Finkel, 2000). Furthermore, cellular levels of ROS fluctuate throughout the cell cycle and in fact ROS are required for cell proliferation (Clopton and Saltman, 1995; Shackelford et al, 2001). Nevertheless, cells have developed numerous antioxidant systems to prevent excess generation of ROS as the highly reactive nature of ROS will easily result in ROS-induced adverse modifications of protein, lipid or DNA. Normally, ROS-induced modification or damage is either repaired, as in the case of chromosomal DNA damage, or removed by degradation and subsequent resynthesis. In the case of excessive damage or ineffective repair, cell death (apoptosis) is triggered. Thus, cells require both a stringent homeostasis mechanism for ROS and efficient repair to tolerate levels of ROS required for normal cell function that will otherwise result in certain cell death. Consistent with its role in normal growth factor-induced signalling, ROS generation by exogenous sources such as H2O2 treatment has been shown to trigger most of the signalling pathways downstream of growth factor receptors (reviewed in Finkel, 2000). For example, ROS have been shown to activate members of the JNK/p38 stress kinase family, MAPKs, PI-3K signalling, NF-κB and many more. Activation by ROS of some of these signalling cascades has been implicated primarily in the induction of apoptosis (JNK/p38), whereas others have been implicated in cell survival (PI-3K). Protein kinase B (PKB/c-Akt) mediates many of the antiapoptotic effects of PI-3K signalling. A large number of PKB substrates have been implicated in the regulation of cellular survival (reviewed in Lawlor and Alessi, 2001). However, little is known as to how PI-3K/PKB signalling may regulate the cellular level of ROS. Recently, we and others have shown that the PKB-regulated Forkhead transcription factor FOXO3a can reduce the level of cellular oxidative stress by directly increasing mRNA and protein levels of manganese superoxide dismutase (MnSOD) and catalase (reviewed in Burgering and Medema, 2003). PKB-mediated phosphorylation of FOXO results in translocation of FOXO from the nucleus to the cytosol. Consequently, PKB activation decreases MnSOD and catalase levels and this is likely to contribute to an increase in cellular ROS. As cell cycle progression requires increased ROS level, this is in agreement with the role of PI-3K signalling in stimulating cell proliferation. Because of the inverse relationship between PI-3K/PKB signalling and FOXO activity, we were interested whether an increase in ROS could regulate FOXO activity and oppose the effect of PI-3K/PKB signalling. Here, we show that oxidative stress induced by treatment of cells with H2O2 results in the activation of the small GTPase Ral. Activation of Ral results in the phosphorylation and activation of JNK and JNK-mediated phosphorylation of FOXO4 on T447 and T451. Phosphorylation of these residues is critical to FOXO4 transcriptional activity. Thus, H2O2 can induce FOXO4 transcriptional activity and this is further confirmed by the observation that H2O2 treatment results in translocation of FOXO4 from the cytosol to the nucleus. In addition, we show that tumor necrosis factor α (TNFα), a ligand known to increase cellular H2O2 levels, also activates FOXO4 transcriptional activity and that this involves cellular ROS, Ral and JNK. These results indicate that FOXOs can function in a negative feedback loop to control the cellular level of oxidative stress in a cell and therefore these results start to outline a novel homeostasis mechanism of ROS control. Results To investigate whether FOXO could function in a feedback mechanism to control cellular redox, we first analyzed the possibility that, similar to insulin signalling, cellular oxidative stress generated by H2O2 treatment of cells could induce FOXO phosphorylation. Cells transiently expressing HA-FOXO4 were labelled with [32P]orthophosphate and treated with various concentrations of H2O2. At the lowest concentration tested (20 μM), H2O2 treatment induced phosphorylation of FOXO4 (Figure 1A). Phosphorylation by H2O2 did not correlate with H2O2-induced PKB activation, as increased PKB phosphorylation in these cells was only observed at the highest concentration of H2O2 (200 μM; Figure 1A). By mutational analysis, we have previously defined two residues within FOXO4, threonine 447 (T447) and threonine 451 (T451), that can be phosphorylated independently of PKB activation (De Ruiter et al, 2001) and recently, we confirmed phosphorylation of these residues on FOXO4 by mass spectrometry (data not shown). To study regulation of T447/451 phosphorylation, we obtained phosphospecific antibodies against both phosphorylated T451 (T451P) and T447 (T447P). H2O2 treatment induced both T451 and T447 phosphorylation (Figure 1B). The T451P antibody did not recognize HA-FOXO4-T451A and the T447P antibody did not recognize HA-FOXO4-T447A isolated both from untreated and H2O2-treated cells, indicating their specificity. The T447P antibody also did not recognize HA-FOXO4-T451A, suggesting that T451 is an essential part of the epitope for the T447P antibody. Moreover, we analyzed phosphorylation of endogenous FOXO4. Mouse C2C12 cells expressing endogenous FOXO4 were treated with H2O2 and displayed increased T447 phosphorylation (Figure 1C). As T451 is not conserved between human and mouse FOXO4, we could not test endogenous T451 phosphorylation in these cells. These results show that in vivo FOXO4 becomes phosphorylated at T447 and T451 following treatment of cells with H2O2. The T451P antibody is of better quality compared to the T447P antibody. Therefore, the results with the T451P antibody are shown in the following figures, and similar results were obtained using the T447P antibody. Figure 1.H2O2 induced phosphorylation of FOXO4 on T447 and T451. (A) A14 cells, transfected with HA-FOXO4, were labelled with [32P]orthophosphate for 3 h and left untreated or treated for 60 min with indicated H2O2 concentrations. Cells were lysed and HA-FOXO4 was immunoprecipitated. Following exposure to the film, the blot was probed with 12CA5 monoclonal antibody to ensure equal expression of HA-FOXO4 in each lane. H2O2 treatment induced a 2.5-fold increase in phosphorylation of FOXO4. In parallel, samples were analyzed on Western blot for Ser473 phosphorylation of PKB (lower panel). (B) 293T cells, transfected with HA-FOXO4, HA-FOXO4-T447A or HA-FOXO4-T451A, were left untreated or treated with 100 μM H2O2 for 60 min. HA-FOXO4 were immunoprecipitated and analyzed on Western blot for Thr447 or Thr451 phosphorylation. Same results were obtained with 200 and 400 μM H2O2. (C) Mouse C2C12 cells were left untreated or treated with 100 μM of H2O2 for 60 min. Endogenous FOXO4 was analyzed on blot for T447 phosphorylation. Same results were obtained using 200 or 400 μM H2O2. Download figure Download PowerPoint In insulin signalling, phosphorylation of T447 and T451 occurs in a Ral-dependent manner (De Ruiter et al, 2001). Thus, we analyzed whether H2O2-induced phosphorylation of T447 and T451 was dependent on activation of the small GTPase Ral. Therefore, we first analyzed whether H2O2 could induce the activation of Ral. Cells treated for various periods of time with H2O2 were lysed and the level of active Ral (Ral-GTP) was determined by a pull-down assay (Wolthuis et al, 1998). H2O2 treatment induced a rapid and time-dependent increase in RalGTP levels (Figure 2A). To determine whether this H2O2-induced Ral activation mediated the phosphorylation of T451, we expressed HA-FOXO4 in the presence or absence of dominant-negative Ral (RalN28). Expression of dominant-negative Ral completely blocked phosphorylation of T451 (Figure 2B), indicating the involvement of Ral in H2O2-induced phosphorylation. Subsequently, we examined whether activation of endogenous Ral could mediate T451 phosphorylation. Activation of endogenous Ral, both through the expression of active Ras (RasV12) and through the expression of active Ral guanine nucleotide exchange factors (RlfCAAX and RalGEF2), but not the expression of a control in which the catalytic domain was mutated (RlfCAAXΔGEF), resulted in increased T451 phosphorylation (Figure 2C). Taken together, these data demonstrate that H2O2 treatment of cells results in the activation of the small GTPase Ral, which is necessary and sufficient to induce phosphorylation of T451 on FOXO4. Figure 2.H2O2 induces Ral activation and Ral activation is necessary and sufficient for H2O2-induced T451 phosphorylation. (A) A14 cells were treated with 100 μM of H2O2 for the indicated time, and Ral GTP levels were analyzed on Western blot using a Ral pull-down assay (upper panel). The lower panel shows endogenous Ral protein levels. Same results were obtained using 200 and 400 μM H2O2. (B) 293T cells, transfected with HA-FOXO4 and HA-RalN28 or a control construct, were treated with 100 μM H2O2 for the indicated time and T451 phosphorylation was analyzed on Western blot. Same results were obtained using 200 or 400 μM H2O2. (C) 293T cells were transfected with myc-FOXO4 together with the indicated constructs. T451 phosphorylation was analyzed. Download figure Download PowerPoint To investigate which kinase could mediate Ral-induced phosphorylation of T451, we treated cells with a variety of kinase inhibitors prior to H2O2 treatment. The MEK inhibitor PD98059, the PI-3K inhibitor LY294002 or the p38 inhibitor SB203580 could not inhibit H2O2-induced phosphorylation of T451 and T447 on FOXO4 (data not shown). These results indicate that T451 phosphorylation is not mediated by PI-3K, MAPK or p38. To study a potential involvement of JNK, we used immortalized mouse embryo fibroblasts (MEFs) derived from JNK1,2−/− mice, since there is no specific JNK inhibitor available. As these MEFs also do not express JNK3, they lack any JNK activity (Sabapathy et al, 1999). H2O2 treatment of JNK1,2−/− MEFs did not induce phosphorylation of T451, whereas H2O2 treatment of control MEFs (wild-type (wt) MEFs) did, strongly indicating that in vivo JNK mediates T451 phosphorylation (Figure 3A). To further confirm this, we rescued JNK expression in JNK1,2−/− MEF cells by coexpression of either JNK1 or JNK3. This restored H2O2-induced JNK activity and the induction of T451 phosphorylation (Figure 3B). JNK is often observed bound to its potential substrates. We therefore analyzed the binding between JNK and FOXO4. Treatment of cells with increasing concentrations of H2O2 induced the binding of JNK1 (data not shown) and JNK3 to FOXO4 (Figure 3C). Consistent with the in vivo data, active JNK1, but not p38α, could efficiently phosphorylate T451/447 of FOXO4 in vitro (Figure 3D). Thus, we conclude that JNK phosphorylates FOXO4 in vitro and in vivo at T451 and that this phosphorylation can be induced by H2O2 treatment. Figure 3.JNK is involved in the H2O2-induced Ral-mediated phosphorylation of T451 and T447 on FOXO4. (A) JNK1,2−/− MEFs, transfected with HA-FOXO4 together with JNK1, JNK3 or an empty vector, were treated with 100 μM H2O2 for the indicated time, and T451 phosphorylation was analyzed on Western blot. wt MEFs were included as control. Similar results were obtained using 200 or 400 μM H2O2. (B) JNK1,2−/− MEFs, wt MEFs and JNK−/− cotransfected with either JNK1 or JNK3, transfected with HA-FOXO4, were left untreated or treated with 100 μM of H2O2 for 60 min. T451 phosphorylation was analyzed. In parallel, a GST-Jun pull-down was performed to measure JNK activity (lower panel). Same results were obtained using 200 or 400 μM H2O2. (C) 293T, transfected with myc-FOXO4 and HA-JNK3, were treated with different concentrations of H2O2 for indicated times. HA-JNK3 was immunoprecipitated and binding of myc-FOXO4 to HA-JNK3 was analyzed on Western blot (upper panel). The lower panels show expression of the constructs. (D) Purified bacterially expressed GST-FOXO4(C) and GST-FOXO4-T447/451A(C) were incubated in the presence (+) or absence of active JNK or active p38α. P indicates pretreatment with either active JNK or p38α in the presence of unlabelled rATP. Prephosphorylation by JNK or p38α did not enhance the ability of p38α or JNK to subsequently phosphorylate GST-FOXO4. MBP substrate was included as control for the activity of active JNK and p38α. (E) 293T cells transfected with HA-JNK3 with or without HA-RalN28 were treated with 100 μM of H2O2 for increasing periods of time. JNK phosphorylation was analyzed on western blot. Similar results were obtained using 200 or 400 μM of H2O2. (F) 293T cells were transfected with HA-JNK3 together with indicated constructs. JNK phosphorylation was analyzed on Western blot. (G) 293T cells transfected with HA-FOXO4 or HA-JNK3 with or without HA-RalN28 were untreated or treated with 10 μg/ml anisomycin for 30 or 60 min. FOXO4-T451 and JNK phosphorylations were analyzed on Western blot. The 60 min treatment of anisomycin induced a three-fold increase in FOXO4-T451 phosphorylation, both in the presence and absence of RalN28. Download figure Download PowerPoint Our results thus far suggest a role for Ral in mediating H2O2-induced JNK activation in vivo. To test this directly, we expressed HA-JNK1 (not shown) or HA-JNK3 either in the absence or the presence of dominant-negative RalN28 and stimulated JNK activity by H2O2 treatment. Dominant-negative Ral inhibited, especially at later time points, the induction of JNK phosphorylation and activation by H2O2 (Figure 3E). Again, we tested whether activation of endogenous Ral would be sufficient to increase JNK activity. Indeed, as was shown for T451 phosphorylation, coexpression of active RalGEFs but not that of the inactive GEF increased JNK activity (Figure 3F). To analyze the specificity of the involvement of Ral in H2O2-induced JNK activation, we also tested whether Ral is involved in anisomycin-induced JNK activation. Consistent with a role for JNK in mediating T447/451 phosphorylation of FOXO4, anisomycin treatment also induced T451 phosphorylation. However, RalN28 did not block the anisomycin-induced phosphorylation of both JNK and T451 (Figure 3G). Therefore, we conclude that FOXO4 is phosphorylated by JNK at T447 and T451 and that JNK is differentially regulated following cellular stress: JNK activation following oxidative stress as generated by H2O2 treatment is mediated by the small GTPase Ral, whereas JNK activation following ER stress as generated by anisomycin treatment occurs independently of Ral. We and others have previously shown that insulin signalling results in the translocation of FOXO from the nucleus to the cytosol (Biggs et al, 1999; Brownawell et al, 2001). Thus, we analyzed the effect of increased oxidative stress on FOXO4 localization and transcriptional activity. As was shown by others (Brunet et al, 2004), treatment of cells with H2O2 cultured in the presence of serum, when FOXO4 is predominantly localized in the cytosol, induced relocalization of FOXO4 from the cytosol to the nucleus (Figure 4A). Translocation induced by H2O2 appeared to be stochastic. For reasons that are not clear, cells appear to respond in an all-or-none fashion. An example of this is shown in Figure 4A (middle panel). To analyze the effect on transcriptional activity, we performed reporter assays using FOXO responsive promoters. Interestingly, we only observed a small but reproducible increase of FOXO transcriptional activity at low H2O2 concentrations, as measured by an increase in activity on the MnSOD promoter construct (Figure 4B) (Kim et al, 1999). Similar results were obtained using the 6xDBE or p27 promoter constructs, both of which are FOXO responsive promoters (Furuyama et al, 2000; Medema et al, 2000; data not shown). The decrease in FOXO transcriptional activity following overnight H2O2 treatment at higher concentrations is often accompanied by a decrease in HA-FOXO4 expression (Figure 4B, loading control). Whether this decrease in FOXO transcriptional activity observed at higher H2O2 concentrations is also due to PKB/c-Akt signalling, which is switched on at high concentrations of H2O2, or that other H2O2-induced modifications inhibit FOXO activity and/or expression in a dominant fashion, is at present unknown. Figure 4.H2O2 induces nuclear translocation and activation of FOXO4. (A) DLD1 cells, transfected with HA-FOXO4, were maintained in the presence of serum and left untreated (left panel) or treated with 100 μM of H2O2 for 60 min (middle and right panels). Cells were fixed and HA-FOXO4 was stained. The middle panel shows differential response to oxidative stress with respect to localization. Same results were obtained using 200 or 400 μM of H2O2. (B) DLD1 cells, transfected with pSODLUC-3340 in the absence or presence of HA-FOXO4, were treated with indicated concentrations of H2O2 for 16 h and subjected to luciferase assays. Equal expression was tested by Western blot. Data represent the average of three independent experiments. (C) DLD1 cells were transfected with 6xDBE-luciferase together with the indicated constructs, and subjected to luciferase assays as described in panel B. (D) DLD-1 cells were transfected with pSODLUC-3340 in the absence or presence of HA-FOXO4 and absence or presence of dominant-negative Ral (RalN28), and subjected to luciferase assays as described in panel B. (E) A14 cells were transfected with 6xDBE-luciferase in the presence of HA-FOXO4 or the indicated mutant constructs and either in the presence or absence of dominant-negative Ral (RalN28), and subjected to luciferase assays as described in panel B. (F) JNK1,2−/− MEFs and wt MEFs were transfected with pSODLUC-3340 in the absence or presence of HA-FOXO4, HA-JNK1 or both, and subjected to luciferase assays as described in panel B. (G) JNK1,2−/− MEFs, transfected with pSODLUC-3340 with or without JNK3, were treated with indicated concentrations of H2O2 for 16 h, and subjected to luciferase assays as described in panel B. (H) wt MEFs and JNK1,2−/− were transfected with 6xDBE-luciferase together with indicated amounts of HA-RlfCAAX, and subjected to luciferase assays as described in panel B. (I) A14 cells were transfected with the indicated constructs and a puromycin selection vector. At 36 h after transfection, cells were put on 2 μg/ml puromycin and either cultured in medium containing FCS with glucose or medium with FCS lacking glucose. After 48 h of glucose deprivation, cells were harvested, stained with rhodamine-1,2,3 and analyzed for mitochondrial membrane stability. Download figure Download PowerPoint To further demonstrate that phosphorylation of either T447 or T451 results in activation of FOXO4, we tested a series of T447 and T451 mutants that can no longer be phosphorylated at these sites or could mimic phosphorylation (Figure 4C). Mutating either T447 or T451 to alanine was already sufficient to almost completely block transcriptional activity. Importantly, both the phospho-mimicking T447E and the T451E mutant displayed enhanced transcriptional activity compared to wt FOXO4. Thus, these data suggest that phosphorylation of either T447 or T451 is sufficient to activate FOXO4 transcriptional activity. In agreement with a role for Ral, introduction of the dominant-negative RalN28 completely blocks FOXO4 activity (Figure 4D), whereas the phospho-mimicking mutants were either partially (T447E and T451E) or not inhibited by RalN28 coexpression (T447/451E; Figure 4E). These data indicate that the effect of Ral on FOXO4 transcriptional activity is entirely through the regulation of T447 and T451 phosphorylation. To confirm the role of JNK in transcriptional activation via T447/451 phosphorylation, we analyzed FOXO transcriptional activity in JNK1,2−/− cells. In these cells, FOXO activity was lower compared to wt MEFs, and reintroducing JNK, clearly enhanced FOXO4 transcriptional activity (Figure 4F). H2O2-induced activation of FOXO activity was also reduced in JNK1,2−/− MEFs. However, reintroduction of JNK3 in these cells restored stress-induced FOXO4 activity (Figure 4G). Previously, we reported that the effect of RlfCAAX on FOXO4-mediated transcription was sensitive to the amount of RlfCAAX used (De Ruiter et al, 2001). Here, we show that the effect of RlfCAAX on FOXO activity is also dependent on JNK, since RlfCAAX-induced FOXO activity is lowered in JNK−/− cells (Figure 4H). The dose-dependent effect of RlfCAAX shows similarities with the dose-dependent effect of H2O2 treatment. Next, we tested whether the loss of transcriptional activity as a result of the T447/451A mutation in FOXO4 as measured by the reporter assays resulted in a change in FOXO function. Previously, we have shown that FOXOs can protect cells from glucose deprivation-induced mitochondrial membrane instability (Kops et al, 2002) and thus we tested whether the FOXO4 mutants were compromised in this respect. Inhibition of Ral signalling and expression of the T447/451A mutant reduced the ability of FOXO4 to protect cells from glucose deprivation, and consistent with this the phospho-mimicking T447/451D mutant displayed slightly enhanced protection (Figure 4I). Thus, from these data, we conclude that H2O2 induces a translocation of FOXO4 from the cytosol to the nucleus and that this translocation is part of the mechanism whereby H2O2 induces transcriptional activation of FOXO4. Mutant analysis shows that this transcriptional activation involves T447 and T451 phosphorylation and is dependent on the presence of JNK. Finally, we tested whether stimuli other than H2O2 that are known to influence intracellular ROS levels could activate FOXO transcriptional activity. TNFα is a cytokine that has been shown to increase intracellular H2O2 levels and likely concomitantly to increase cellular oxidative stress (Goossens et al, 1995). This increase in cellular ROS may mediate the cytotoxic action of TNFα, although the exact mechanism is largely unknown. For example, overexpression of antioxidants including catalase to reduce cellular H2O2 has been shown to enhance (Bai and Cederbaum, 2000) or reduce TNFα cytotoxicity (Wong et al, 1989). There are also reports that demonstrate unaltered TNFα cytotoxicity after overexpression of antioxidants (O'Donnell et al, 1995). Interestingly, TNFα has also been shown to increase the expression of MnSOD (Wong et al, 1989) and FOXOs can induce MnSOD expression (Kops et al, 2002). A14 cells are sensitive to TNFα, as measured by activation of NF-κB, but show no obvious signs of TNFα-induced cell death (data not shown). Treatment of A14 cells with increasing concentrations of TNFα induced a dose-dependent increase in FOXO4 transcriptional activity (Figure 5D). Similar to the H2O2-induced FOXO4 transcriptional activity, the TNFα-induced increase is mediated by Ral, as it is inhibited by expression of the dominant-negative RalN28. Moreover, it is mediated by JNK, as it is absent in JNK1,2−/− cells and can be restored by reintroducing JNK in JNK1,2−/− cells (Figure 5B). Importantly, TNFα-induced JNK activation involved an increase in cellular oxidative stress, as pretreatment with N-acetyl-L-cysteine (NAC), which enhances the scavenging of oxygen radicals, reduced TNFα-induced JNK activation (Figure 5C). Figure 5.TNFα induces FOXO4 transcriptional activity. (A) A14 cells were transfected with the HA-FOXO4 with or without HA-RalN28, together with 6xDBE-luciferase and Tk-renilla. Cells were treated with increasing concentrations of TNFα (5, 10 and 20 ng/ml). Pretreatment with NAC was performed by adding 10 mM of NAC 16 h before treatment with TNFα. At 16 h after TNFα treatment, luciferase activity was measured. Data represent the average of three independent experiments. (B) JNK1,2−/− MEFs were transfected with HA-FOXO4 with or without HA-JNK1 together with 6xDBE-luciferase and Tk-renilla, and treated for 16 h with TNFα (20 ng/ml). Luciferase activity was measured as described in panel B. (C) A14 cells were treated with 10 mM NAC and the next day treated with TNFα (20 ng/ml) or H2O2 (100 μM) for the indicated time points. JNK activity was measured by a GST-Jun pull-down. (D) A14 cells were treated