p38α mediates cell survival in response to oxidative stress via induction of antioxidant genes: effect on the p70S6K pathway

Abstract

We reveal a novel pro-survival role for mammalian p38α in response to H(2)O(2), which involves an up-regulation of antioxidant defenses. The presence of p38α increases basal and H(2)O(2)-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) H(2)O(2)-induced SOD-2 expression through a direct regulation of transcription mediated by activating transcription factor 2 (ATF-2) and (ii) H(2)O(2)-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 H(2)O(2), 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.

FIGURE 1.

p38α protects from H₂O₂-induced cell death. MEFs (WT and p38α^−/−) maintained in the presence of serum were treated with H₂O₂ when indicated. A, p38α expression increases cell viability in cells treated with H₂O₂ (0.1 or 0.5 mm) for 24 h. B, p38α reconstitution in p38α^−/− cells (Rec) rescues cells from cell death upon treatment with H₂O₂ (1 mm) for 6 h. The data correspond to cell viability expressed as percentages, and p38α expression was determined by Western blot and normalized with tubulin. C, loss of p38α increases the number of apoptotic nuclei in cells treated with H₂O₂ (0.5 mm) for 3 h. *, p < 0.05 and ***, p < 0.001, p38α^−/− versus WT MEFs upon treatment with H₂O₂.

FIGURE 2.

p38α MAPK positively regulates p70S6K through an Akt independent mechanism. MEFs (WT and p38α^−/−) maintained in the presence of serum were treated with H₂O₂ (0.1–1 mm in A, 1 mm in B and C) for 20 min when indicated. Western blot analysis of the levels of Thr(P)-389-p70S6K (P-Thr389-p70S6K), Ser(P)-939-TSC-2 (P-Ser939-TSC-2), Ser(P)-473-Akt (P-Ser473-Akt), Thr(P)-172-AMPK (P-Thr172-AMPK), Ser(P)-79-acetyl-CoA-carboxylase (P-Ser79-ACC), Thr/Tyr(P)-180/182-p38 MAPK (P-Thr/Tyr-180/182-p38 MAPK) (P-p38) Ser (P)-189/207-MKK3/MKK6 (P-Ser-189/207-MKK3/MKK6) (P-MKK3/6), as well as total levels of p70S6K, Akt, TSC-1, and p38α normalized with tubulin are shown. A and B, effect of p38α expression on the activation of Akt and p70S6K in response to H₂O₂. p38α-deficient cells show a decrease activation of p70S6K as compared with WT or p38α^−/− with reconstitution of p38α (Rec). C, analysis of the activation of the p70S6K pathway by p38α showing p70S6K phosphorylation by mTOR and the activation of different upstream regulators (positive and negative). P-p70/tubulin represents the relative value resulting from the densitometric analysis of Thr(P)-389-p70S6K versus tubulin levels multiplied by 10.

FIGURE 3.

mTORC1/p70S6K does not mediate p38α-dependent survival in response to H₂O₂. MEFs (WT and p38α^−/−) maintained in the presence of serum were treated with H₂O₂ (0.1, 0.5, or 1 mm) when indicated. A, effect of mTORC1 inhibition by rapamycin (Rapa) on H₂O₂-induced activation p70S6K and Akt. Western blot analysis of the levels of Thr(P)-389-p70S6K (P-Thr389-p70S6K) and Ser(P)-473-Akt (P-Ser473-Akt) normalized with tubulin upon treatment with H₂O₂ for 20 min. B, effect of rapamycin on H₂O₂-induced apoptosis (left panel) and cell viability (right panel) after 24 h. The results show the percentage of hypodiploid cells (apoptotic cells) analyzed by flow cytometry and the percentage of viable cells determined by crystal violet staining. *, p < 0.05, p38α^−/− versus WT MEFs treated with 0.1 mm H₂O₂. No significant differences were found in the presence of rapamycin. C, effect of the expression of transfected WT p70S6K and p70Δ29–46ΔCT104 active mutant on cell viability. *, p < 0.05, p38α^−/− versus WT MEFs treated with H₂O₂. No significant differences were found upon expression of WT p70S6K and p70Δ29–46ΔCT104. Western blot analysis shows HA expression in cells transfected with WT p70S6K and p70Δ29–46ΔCT104 active mutant. Arrows indicate the migration of 80- and 50-kDa molecular weight markers.

FIGURE 4.

ROS accumulation upon H₂O₂ treatment is higher in cells lacking p38α. MEFs (WT and p38α^−/−) were incubated with DCFH and treated with H₂O₂ (1 mm for A, and 0.1 and 1 mm for B) for 15 min. A, analysis of DCFH-positive cells using an inverted fluorescence microscope. B, percentage of DCFH-positive cells. **, p < 0.01, p38α^−/− versus WT MEFs treated with the same dose of H₂O₂.

FIGURE 5.

Loss of p38α reduces the expression and activity of antioxidant enzymes. MEFs (WT and p38α^−/−) maintained in the presence of serum were treated with H₂O₂ (0.5 mm) for the indicated time periods. A, catalase and SOD-2 protein levels determined by Western blot analysis and normalized with tubulin. B, rescue of catalase protein expression by p38α reconstitution in p38α^−/− cells (Rec) treated with H₂O₂ for 2 h. Western blot analysis of catalase and p38α normalized with tubulin. 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 < 0.05; **, p < 0.01; ***, p < 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 < 0.05; ***, p < 0.001.

FIGURE 6.

ATF-2 is an important mediator of p38α in the induction of SOD-2 expression and cell viability upon H₂O₂ treatment. MEFs (WT and p38α^−/−) maintained in the presence of serum were treated with H₂O₂ (0.1 and 0.5 mm in A or 0.5 mm in B–D) for different time periods, as indicated. A, Western blot analysis of the levels of P-ATF-2 normalized with β-actin. B, effect of ATF-2 siRNA on SOD-2 mRNA levels. Top panel, Western blot analysis of ATF-2 levels normalized with GAPDH; lower panel, histograms showing SOD-2 mRNA levels. *, p < 0.05; ***, p < 0.001. C, ChIP analysis of P-ATF-2 binding to SOD-2 promoter. PCR analysis of DNA immunoprecipitated by a P-ATF-2 antibody and of input DNA. D, effect of ATF-2 siRNA decreasing cell viability of WT MEFs treated with H₂O₂ for 24 h. **, p < 0.01.

FIGURE 7.

The presence of p38α stabilizes catalase protein. MEFs (WT and p38α^−/−) maintained in the presence of serum were treated with H₂O₂ (0.5 mm) in the presence or absence of actinomycin D (ActD) or MG-132 for the indicated time periods. A, analysis of catalase mRNA levels by RT-quantitative PCR in the presence or absence of actinomycin D. Catalase mRNA expression was normalized using GAPDH (catalase C_t − GAPDH C_t = ΔC_t) and then referred to WT control values to calculate the RQ value (2^−ΔΔCt). The histograms show the mean values ± S.E. *, p < 0.05; +, p < 0.05; **, p < 0.01; n = 4. B, effect of the proteasome inhibitor MG-132 on catalase protein expression. Western blot analysis of catalase normalized with tubulin. Catalase/tubulin represents the relative value resulting from the densitometric analysis of catalase versus tubulin levels multiplied by 10.

FIGURE 8.

Treatment with the antioxidant, N-acetyl cysteine, or catalase protects from ROS-induced apoptosis and allows activation of p70S6K by mTOR. MEFs (WT and p38α^−/−) maintained in the presence of serum were treated with H₂O₂ (1 mm) for 20 min (B) or 3 h (A). When indicated, the cells were pretreated for 1 h with NAC (2.5 mm) or catalase. A, effect of NAC and exogenous catalase on H₂O₂-induced apoptosis. The results show the percentage of apoptotic nuclei. ***, p < 0.001, p38α^−/− versus WT MEFs upon treatment with H₂O₂; and ++, p < 0.01, as compared with p38α^−/− MEFs treated with H₂O₂ plus NAC or catalase. B, effect of NAC on H₂O₂-induced p70S6K activation. C, effect of catalase on H₂O₂-induced p70S6K activation. B and C, Western blot analysis of the levels of Thr(P)-389-p70S6K (P-Thr 389-p70S6K) and Ser(P)-473-Akt (P-Ser 473-Akt) normalized with tubulin. P-p70/tubulin represents the relative value resulting from the densitometric analysis of Thr(P)-389-p70S6K versus tubulin levels multiplied by 10.

FIGURE 9.

The up-regulation of antioxidant genes by p38α MAPK promotes cell survival and allows p70S6K activation. Model showing the effect of p38α MAPK increasing the levels of the antioxidant enzymes SOD and catalase in response to H₂O₂ through different mechanisms, which leads to the removal of ROS. As a consequence, ROS levels decrease, which allows cell survival and mTOR/p70S6K activation.