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, 283 (45), 31153-62

Hypoxic Reactive Oxygen Species Regulate the Integrated Stress Response and Cell Survival

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Hypoxic Reactive Oxygen Species Regulate the Integrated Stress Response and Cell Survival

Liping Liu et al. J Biol Chem.

Abstract

Under hypoxic conditions, cells suppress energy-intensive mRNA translation by modulating the mammalian target of rapamycin (mTOR) and pancreatic eIF2alpha kinase (PERK) pathways. Much is known about hypoxic inhibition of mTOR activity; however, the cellular processes activating PERK remain unclear. Since hypoxia is known to increase intracellular reactive oxygen species (ROS), we hypothesized that hypoxic ROS regulate mTOR and PERK to control mRNA translation and cell survival. Our data indicate that although exogenous ROS inhibit mTOR, eIF2alpha, and eEF2, mTOR and eEF2 were largely refractory to ROS generated under moderate hypoxia (0.5% O(2)). In direct contrast, the PERK/eIF2alpha/ATF4 integrated stress response (ISR) was activated by hypoxic ROS and contributed to global protein synthesis inhibition and adaptive ATF4-mediated gene expression. The ISR as well as exogenous growth factors were critical for cell viability during extended hypoxia, since ISR inhibition decreased the viability of cells deprived of O(2) and growth factors. Collectively, our data support an important role for ROS in hypoxic cell survival. Under conditions of moderate hypoxia, ROS induce the ISR, thereby promoting energy and redox homeostasis and enhancing cellular survival.

Figures

FIGURE 1.
FIGURE 1.
Hypoxia inhibits multiple pathways regulating mRNA translation. A and B, schematic diagram of signaling pathways regulating mRNA translation during hypoxia. These include the regulation of translation elongation and availability of eIF4E protein by modulating 4EBP1 phosphorylation (A) and eIF2α phosphorylation (B). A, hypoxia inhibits cap-dependent translation via activating the AMPK/TSC2 and REDD1/TSC2 pathways and promyelocytic leukemia-mediated mTOR nuclear translocation. eIF4E is also regulated by 4E-T sequestration in hypoxic cells. Furthermore, AMPK phosphorylates eEF2 kinase (eEF2K), leading to eEF2 phosphorylation and the inhibition of translation elongation and global protein synthesis. B, hypoxia also inhibits global protein synthesis by PERK-mediated eIF2α phosphorylation. Other stresses (e.g. increased protein load or disruption of protein glycosylation in the ER) and perturbations in Ca2+ homeostasis also result in PERK activation. The ATF4/GADD34/eIF2α negative feedback loop relieves translational inhibition.
FIGURE 2.
FIGURE 2.
PERK is required for induction of the integrated stress response and protein synthesis inhibition during hypoxia. A, eIF2α phosphorylation in PERK+/+ and PERK-/- MEFs exposed to 0-20 h 0.5% O2. Quantitative changes in eIF2α phosphorylation, compared with 0 h hypoxia based on Image J analysis, are indicated. Total eIF2α protein serves as a loading control. Also shown is the accumulation of ATF4 and CHOP proteins in PERK+/+ and PERK-/- MEFs. B, protein synthesis in MEFs after 48 h 0.5% O2 measured by [35S]methionine incorporation. See “Experimental Procedures” for statistical analyses. **, p < 0.01.
FIGURE 3.
FIGURE 3.
Effects of H2O2 on mRNA translation and protein synthesis. A, HEK293 cells and immortalized wild-type mouse embryonic fibroblasts (MEFs) were exposed to 0-500 μm H2O2 for 1 h. Whole cell extracts were blotted for 4EBP1, phospho-rpS6, phospho-eIF2α, total eIF2α, phospho-eEF2, and total eEF2. Hypophosphorylated (α) and phosphorylated (β and γ) forms of 4EBP1 are indicated. Levels of total eIF2α and eEF2 proteins were examined for sample loading and protein stability using the same lysates run on separate gels. Changes in eIF2α phosphorylation (based on Image J analysis) compared with 0 μm H2O2 are shown. B, protein synthesis in H2O2-treated (1 h) MEFs with or without 2 h BME (100 μm) preconditioning. BME (100 μm) was present during the 1-h protein synthesis. **, p < 0.01. C, Western blotting for total 4EBP1 protein and rpS6 phospho-Ser235/236 in H2O2-treated (1 h) TSC2+/+ and TSC2-/- MEFs. Changes in rpS6 phosphorylation compared with 0 μm H2O2 are indicated. D, protein synthesis in TSC2+/+ and TSC2-/- MEFs treated with 100 μm H2O2 (1 h). [35 S]Methionine labeling was carried out in the presence of 100 μm H2O2. Base-line protein synthesis was similar in TSC2+/+ and TSC2-/- cells.
FIGURE 4.
FIGURE 4.
Role of eIF2α kinases in H2O2-induced eIF2α phosphorylation. A, HEK293 cells were exposed to varying concentrations of H2O2 (1 h) or 0.8 μm thapsigargin (4 h). Cell lysates were probed for phospho-eIF2α, total eIF2α, and PERK. The arrows indicate mobility changes for PERK proteins. B, eIF2α phosphorylation in HEK293 cells exposed to 20 μm H2O2 for 0-60 min. C, protein synthesis in MEFs treated with 0-100 μm H2O2 (1 h) (n = 9-10). **, p < 0.01; wild-type (WT) versus PERK-/- or DKO (PERK-/-, GCN2-/-) MEFs subjected to 100 μm H2O2. ##, p < 0.01; TKO (PERK-/-, GCN2-/-, PKR-/-) MEFs showed significantly higher protein synthesis in comparison with PERK-/- and DKO MEFs upon exposure to 100 μm H2O2.
FIGURE 5.
FIGURE 5.
Role of ROS during hypoxic regulation of mRNA translation. A, HEK293 cells were exposed to H2O (vehicle control), H2O2 (20 or 100 μm, 1 h), hypoxia (Hyp; 0.5% O2, 2 or 20 h), or TNFα (10 ng/ml, 6 or 16 h). Protein carbonylation in whole cell extracts was examined. Equivalent sample loading was based on Ponceau staining. B, HEK293 cells were exposed to hypoxia (0.5% O2, 8 or 24 h), H2O2 (20 or 100 μm, 1 h), or thapsigargin (T) (0.8 μm, 4 h). Mobility of total PERK proteins and phosphorylation of eIF2α were determined. C, MEFs infected with adenoviral GFP or catalase were exposed to hypoxia (0.5% O2, 0.5 or 6 h) or H2O2 (50 or 200 μm, 1 h). Phosphorylation of p70S6K and 4EBP1 was examined using anti-phospho-p70S6K (Thr389) and total 4EBP1 antibodies. *, the p80S6K isoform, which did not change appreciably during any treatments. The status of 4EBP1 is indicated by mobility shift from phosphorylated β form to hypophosphorylatedα form. D, HEK293 cells transfected with catalase or empty vector were exposed to 21% (N) or 0.5% O2 (H) for 20 h, or 50 μm H2O2 for 1 h (R). Phosphorylation of rpS6, eIF2α, and eEF2 proteins was determined. Quantitative changes in eIF2α phosphorylation are shown. E, expression of mRNA for GADD34, BiP, CHOP, and phosphoglycerate kinase in HEK293 cells transfected with catalase (CAT) or vector (VEC) following 20 h of 21% or 0.5% O2. F, protein synthesis in adenoviral GFP- or catalase-expressing MEFs after 48 h 0.5% O2.*, p < 0.05; **, p < 0.01.
FIGURE 6.
FIGURE 6.
Mitochondrial ROS activate the ISR during hypoxia. A and B, cytochrome c wild type (EC-WT) or null (EC-Null) embryonic cells were exposed to 0.5% O2 for 0-12 h. Phospho-rpS6, -eEF2, and HIF-1α proteins (A) and phospho-eIF2α and ATF4 proteins (B) were examined by Western blot. Levels of total rpS6 and eEF2 proteins (A) and eIF2α proteins (B) were analyzed for protein stability. N.S., nonspecific protein band for sample loading. Increases in eIF2α phosphorylation and ATF4 protein levels compared with 0 h hypoxia are indicated. C, effects of cytochrome c mutation on hypoxic induction of catalase and ISR genes GADD34, BiP, CHOP, and catalase in EC cells. Cytochrome c wild type and null cells were exposed to 0.5% O2 for 16 h in the presence or absence of BME (100 μm). Cells were then harvested for mRNA analysis. **, p < 0.01; cytochrome c WT versus null EC in the absence of BME. ##, p < 0.01; WT EC in the presence or absence of BME.
FIGURE 7.
FIGURE 7.
The PERK/eIF2α pathway is critical for adaptation to low O2 and growth factor conditions. A, eIF2α S51S and S51A MEFs were exposed to 20 h of 0.5% O2 (H), 1 h 20 μm H2O2 (R), or 4 h of 0.8 μm thapsigargin (T). eIF2α phosphorylation in total lysates was determined. B, eIF2α S51S or S51A MEFs were exposed to 21 or 0.5% O2 for 48 h in medium containing full (10%) or reduced (0.5%) FBS and full (4.5 g/liter) or reduced glucose (Gluc) (0.2 g/liter). Cell survival was examined by colony formation in regular medium (10% FBS/4.5 g/liter glucose) under normoxia for an additional 7 days. Colonies were stained using 4% crystal violet. C, eIF2α phosphorylation in S51S MEFs after growing for 48 h in normoxia in regular medium (10/4.5), or medium containing 0.2 g/liter glucose (10/0.2) or 0.5% FBS (0.5/4.5). D and E, survival for S51S or S51A MEFs exposed to 21 or 0.5% O2 for 24 h in serum-reduced medium containing 0.5% FBS. Shown are representative assays (D) and quantification of colonies (E) (n = 4). **, p < 0.01. F, intracellular ATP levels in S51S and S51A MEFs after 48 h of 21 or 0.5% O2 in medium containing 0.5% FBS, 4.5 g/liter glucose. The numbers were corrected with normoxic ATP levels. *, p < 0.05. G, schematic diagram for hypoxic activation of the ISR and biological significance of this regulation. ROS are the signaling molecules that induce ISR during hypoxia.

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