Basal levels of eIF2alpha phosphorylation determine cellular antioxidant status by regulating ATF4 and xCT expression

Abstract

eIF2alpha is part of a multimeric complex that regulates cap-dependent translation. Phosphorylation of eIF2alpha (phospho-eIF2alpha) is induced by various forms of cell stress, resulting in changes to the proteome of the cell with two diametrically opposed consequences, adaptation to stress or initiation of programmed cell death. In contrast to the robust eIF2alpha phosphorylation seen in response to acute insults, less is known about the functional role of basal levels of eIF2alpha phosphorylation. Here we show that mouse embryonic fibroblasts expressing a nonphosphorylatable eIF2alpha have enhanced sensitivity to diverse toxic insults, including amyloid beta-(1-42) peptide (Abeta), a key factor in the pathogenesis of Alzheimer disease. This correlates with impaired glutathione metabolism because of down-regulation of the light chain, xCT, of the cystine/glutamate antiporter system X(-)(c). The mechanistic link between the absence of phospho-eIF2alpha and xCT expression is nuclear factor ATF4. Consistent with these findings, long term activation of the phospho-eIF2alpha/ATF4/xCT signaling module by the specific eIF2alpha phosphatase inhibitor, salubrinal, induces resistance against oxidative glutamate toxicity in the hippocampal cell line HT22 and primary cortical neurons. Furthermore, in PC12 cells selected for resistance against Abeta, increased activity of the phospho-eIF2alpha/ATF4/xCT module contributes to the resistant phenotype. In wild-type PC12 cells, activation of this module by salubrinal ameliorates the response to Abeta. Furthermore, in human brains, ATF4 and phospho-eIF2alpha levels are tightly correlated and up-regulated in Alzheimer disease, most probably representing an adaptive response against disease-related cellular stress rather than a correlate of neurodegeneration.

FIGURE 1.

Defective eIF2α phosphorylation increases the sensitivity of mouse embryonic fibroblasts to system X^-_c inhibition and oxidative stress. Embryonic fibroblasts derived from homozygous eIF2α-S51A mice (A/A) and wild-type embryonic fibroblasts (S/S) were plated at a density of 1 × 10⁴ cells per well in 96-well plates. After 24 h of culture, cells were treated with the system X^-_c inhibitors glutamate (A) and homocysteic acid (HCA) (B); glutamate cysteine ligase (GCL) inhibitor buthionine sulfoximide (BSO)(C); SIN-1, BCNU, ethacrynic acid (ECA), and tert-butylhydroperoxide (tBOOH)(D) at the indicated concentrations. Survival was measured after 24 h using the MTT assay. E, 2.5 × 10⁵ A/A and S/S cells per well were grown for 24 h in 96-well plates and treated with Aβ-(1–42) peptide (Aβ_1–42) for 48 h. The effect of Aβ-(1–42) was measured by the MTT assay. All single experiments were performed in quadruplicate, and the MTT value without treatment was normalized to 100%. Each data point represents the means of at least three experiments ± S.E. Statistical analysis was performed by two-way ANOVA. Column factor for A/A versus S/S was highly significant (p < 0.0001) for all insults tested except buthionine sulfoximine, which showed no statistical difference.

FIGURE 2.

Glutathione metabolism in A/A cells is compromised by decreased ATF4 translation and subsequent down-regulation of system X ^-_c. A, GSH is decreased in A/A cells. Exponentially dividing A/A and S/S cells in 60-mm dishes were harvested, and GSH was measured enzymatically. For each experiment, the GSH content of S/S cells was normalized to 100%. B, system X^-_c activity and xCT expression are down-regulated in A/A cells. A/A and S/S cells were plated in 24-well plates, and glutamate-sensitive uptake of [³⁵S]cystine in sodium-free HBSS was measured for 20 min and normalized to total protein (upper panel). Uptake of S/S cells was normalized to 100% for every individual experiment. Similar amounts of purified membranes from A/A and S/S cells were separated by SDS-PAGE, transferred to nitrocellulose, and hybridized with an anti-xCT antibody. Comparable loading was verified by staining of the same membrane with an anti-actin antibody (lower panel). C, amino acid uptake systems distinct from system X^-_c are not compromised in A/A cells. A/A and S/S cells were plated as in B, upper panel. Uptake of 25 μm [³⁵S]cystine co-incubated with 10 μm cysteamine (Cys+Cyst) or 1 μm [³H]leucine was measured for 20 min. Uptake in S/S cells was normalized to 100% for each experiment. D, proximal xCT promoter activity is decreased in A/A cells. 5 × 10⁴ A/A or 2 × 10⁵ S/S cells were grown in 60-mm dishes for 48 h and then transfected with either control plasmid (pGL3) or pGL3 containing the proximal 93, 94, or 4700 bp of the xCT promoter together with the pSV-β-GAL vector. After 24 h, cells were harvested, and luciferase and β-galactosidase activity were measured. Relative luciferase induction by xCT promoter fragments was calculated by normalizing individual luciferase/β-galactosidase ratios of cells transfected with pGL3 to one. E, ATF4 translation is defective in A/A cells. Nuclear extracts of S/S and A/A cells either treated with vehicle or 5 μg/ml tunicamycin (Tuni) for 2 h were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with anti-ATF4. Membranes were hybridized with anti-actin as a loading control (upper panel). S/S and A/A cells were co-transfected as in D with the ATF4 5′UTR luciferase or empty pGL3 vector and the pSV-β-GAL vector. Cells were treated with vehicle or 5 μg/ml tunicamycin (Tuni) for 2 h. The relative increase in luciferase activity by the ATF4 5′UTR compared with pGL3 in control S/S cells was normalized to 100% for every individual experiment (lower panel). F, Nrf2 is similarly induced in A/A and S/S cells. Nrf2 expression in S/S and A/A cells with or without treatment with 25 μm tBHQ for 4 h was analyzed using nuclear extracts and Western blotting (upper panel). System X^-_c activity with and without tBHQ treatment was measured using glutamate-sensitive uptake of [³⁵S]cystine (lower panel). G, ATF4 mediates differential xCT promoter activity in S/S and A/A cells. S/S and A/A cells were co-transfected with the 4700-bp xCT promoter-luciferase construct, pSV-β-GAL, and either ATF4 or control siRNA for S/S cells or pRK7 (Ctrl) or ATF4-pRK7 (ATF4) for A/A cells, respectively. Luciferase/β-galactosidase ratios of S/S cells transfected with control siRNA were normalized to 100% for every individual experiment. All graphs show mean ± S.E. of at least three experiments. Statistical analysis was performed using one-sample t test compared with 100% (A and B) or two-way ANOVA with Bonferroni posttests (D–G) to compare A/A with S/S cells or the effect of tunicamycin or tBHQ versus control A/A and S/S cells (E and F, indicated next to % increase). Relative increase of S/S and A/A in F was compared by Student's unpaired t test. ^***, p < 0.0001; ^**, p < 0.001; n.s., not significant.

FIGURE 3.

eIF2α phosphorylation increases system X^-_c activity and protects against oxidative glutamate toxicity via ATF4. A, 3.3 × 10⁵ HT22 cells were plated in 60-mm dishes treated with 30 μm salubrinal (Salu) or vehicle (Ctrl) for 24 h. Similar amounts of whole cell lysates were analyzed by Western blotting for eIF2α phosphorylation (peIF2α) and ATF4 expression (ATF4). Antibodies recognizing total eIF2α (eIF2α) or actin served as loading controls. System X^-_c activity (X ^-_c) was measured using Na⁺-independent, HCA-sensitive [³H]glutamate uptake in HT22 cells plated at a density of 2.5 × 10⁴ cells/well in 24-well plates and treated with 30 μm salubrinal (Salu) or vehicle (Ctrl) for 24 h. For oxidative glutamate toxicity (right panel), glutamate at the indicated concentrations was added to 2.5 × 10³ HT22 cells grown for 24 h with (Salu) or without (Ctrl) 30 μm salubrinal. Survival was measured by the MTT assay. B, 4.4 × 10⁵ HT22 cells grown for 24 h were transfected with either empty vector (pRK7) or vector expressing ATF4 (ATF4). After another 24 h, cells were replated into 100-mm dishes (8.8 × 10⁵ cells/dish) for Western blot analysis of ATF4 expression (ATF4, left panel), into 24-well plates (3 × 10⁴/well) for system X^-_c activity (X ^-_c) measured as glutamate-sensitive [³⁵S]cystine uptake, into 60-mm dishes (1.65 × 10⁵/dish) for GSH measurement (GSH) or into 96-well plates (2.5 × 10³/well) for oxidative glutamate toxicity assays (Glutamate). Assays and treatment with glutamate were performed after 24 h. Survival was measured by the MTT assay after 24 h of glutamate treatment. All graphs represent the data from three independent experiments. For protein expression or phosphorylation, relative eIF2α phosphorylation or ATF4 expression of control cells was normalized to 1. System X^-_c activity and GSH were normalized to 100% in control cells. Statistical analysis of these experiments was performed by one-sample t test compared with 1 and 100, respectively; ^*, p < 0.05. Relative glutamate sensitivity was analyzed by two-way ANOVA. Cells treated with salubrinal or transfected with ATF4 were significantly more resistant to glutamate (column factor, p < 0.0001).

FIGURE 4.

Salubrinal is a nontoxic mediator of protection against oxidative glutamate toxicity in immature cortical neurons. 1 × 10⁵ primary cortical neurons in 96-well plates were grown for 24 h with (Salu) or without (Ctrl) 30 μm salubrinal and then treated with glutamate at the indicated concentrations or without glutamate for another 24 h followed by an MTT assay. A, viability in the absence of glutamate and salubrinal was normalized to 100%. B, viability without glutamate with or without salubrinal was normalized to 100%. Graphs represent means of four independent experiments each done in triplicate. Statistical analysis was performed by one-sample t test compared with 100 (A) or two-way ANOVA (B). Viability of cultures treated with salubrinal alone was not different from control cells, whereas cells treated with salubrinal were significantly more resistant to glutamate (column factor, p < 0.0001).

FIGURE 5.

Up-regulation of system X^-_c activity, ATF4 protein, and eIF2α phosphorylation but not Nrf2 protein in Aβ-resistant and salubrinal-treated PC12 cells. A and B, for protein, 1.2 × 10⁶ wild-type PC12 cells (PC12) and 6 × 10⁵ clonal Aβ-resistant PC12 cells (PC12r7) were plated in 100-mm dishes and grown for 48 h. 30 μm salubrinal (Salu) or vehicle (Ctrl) was added at the time of plating in B. Western blots of whole cell extracts were analyzed for eIF2α phosphorylation using a phospho-specific anti-eIF2α antibody and a total eIF2α antibody as a loading control. For ATF4, Nrf2, and xCT expression, cells were fractionated. Similar amounts of nuclear extracts for ATF4 and Nrf2 or membrane protein for xCT were analyzed by Western blotting using specific antisera. Actin served as loading control with the exception of the nuclear extracts that were normalized to histone (Hist.). System X^-_c activity (X ^-_c) was measured as glutamate-sensitive [³⁵S]cystine uptake in cells plated at a density of 3 × 10⁵ cells/well in 24-well dishes and grown for 48 h. Graphs represent quantified blots of three (A) and four (B) independent protein preparations and four (A) and three (B) independent uptake assays normalized to control conditions and shown as mean ± S.E. For statistical analysis, values for PC12r7 or salubrinal-treated PC12 cells were compared with 1 by one-sample t test, ^**, p < 0.01; ^*, p < 0.05; n.s., not significant.

FIGURE 6.

Salubrinal treatment of PC12 cells increases cellular GSH and protects against Aβ toxicity. A, 4.5 × 10⁵ PC12 cells were plated in 60-mm dishes and grown for 48 h with 30 μm salubrinal (Salu) or vehicle (Ctrl). Cellular GSH was measured enzymatically and normalized to protein. The graph represents the mean of four experiments with the GSH levels of controls normalized to 1. Statistical analysis of values with salubrinal was performed by one-sample t test compared with 1. ^*, p < 0.05. B, 2 × 10⁵ PC12 cells per well were plated into 96-well plates with either 30 μm salubrinal (Salu) or vehicle (Ctrl). After 24 h, Aβ-(1–42) was added at the indicated concentrations. MTT reduction was measured after 48 h of Aβ exposure. Each experiment was performed in triplicate, and the mean MTT value of cells without Aβ was normalized to 100%. Each data point represents the mean ± S.E. of three independent experiments. Statistical analysis was performed by nonlinear regression and F test. EC₅₀ values of Aβ for MTT reduction are 3.3 × 10^-8 and 3.3 × 10^-7 m for control and salubrinal treated PC12 cells, respectively, and significantly different, p < 0.001.

FIGURE 7.

The eIF2α phosphorylation and ATF4 translation signaling pathway in AD brains. 2% SDS extracts of midfrontal cortex of eight brains of individuals with histopathologically confirmed Alzheimer dementia (A1–A8) and eight age-matched control brains (C1–C8) were analyzed by Western blotting with antibodies against phosphorylated eIF2α (peIF2α) and an antibody to total eIF2α (A) and antibodies against ATF4 and actin (B). A and B show a representative blot of three independently performed Western blots. Each membrane was re-probed with antibodies against actin. C, for quantitative analysis of peIF2α and ATF4 expression, mean band density of controls was normalized to 1. For eIF2α and actin, mean band density of all samples was normalized to 1. For relative eIF2α phosphorylation, mean normalized peIF2α and eIF2α band density for each sample was normalized by mean normalized actin band density of the same sample. Each individual data point in C represents the relative phosphorylation as the ratio of the mean actin-normalized peIF2α band density and the mean actin-normalized eIF2α band density (left panel) or the relative ATF4 expression as the ratio of mean normalized ATF4 band density and mean normalized actin band density of three independent Western blots. Normality was tested by D'Agostino and Pearson omnibus normality test. Mean relative eIF2α phosphorylation (peIF2α) in AD brains (AD) was slightly (1.4-fold) higher than mean eIF2α phosphorylation in control brains (Ctrl). The difference was not significantly different (p = 0.083, Mann-Whitney test) unless C8, which fulfilled the criteria of an extreme outlier (3.04, Q3 + 3xIQR, 1/62), was excluded (p < 0.05, Mann-Whitney test). Mean relative ATF4 expression was higher in AD brains as compared with controls (1.9-fold, p < 0.05, Mann-Whitney test). There was a highly significant correlation of relative eIF2α phosphorylation and ATF4 expression in the whole group of samples (Spearman r 0.79, p < 0.001).