Activating transcription factor 4 is translationally regulated by hypoxic stress

Abstract

Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2alpha at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2alpha was able to attenuate the phosphorylation of eIF2alpha and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2alpha-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress.

FIG. 1.

Hypoxic stress results in global inhibition of protein translation. (A and B) Polysome profiles (absorbance at 254 nm) in cell lysates fractionated by sucrose density ultracentrifugation. HeLa cells were exposed to hypoxic stress for 16 h (B) or left untreated (A). Cells were treated with CHX at 100 μg/ml (37°C, 3 min) and lysed in a Triton X-100 buffer (4°C). Cell lysates were layered on a 10-ml continuous sucrose gradient (10 to 50%) and ultracentrifuged in an SW41 rotor at 39,000 rpm for 90 min. The positions of the polysomes and ribosomal subunits are indicated. The increase in monosome-bound transcripts and ribosomal subunits combined with the decrease in polysomes apparent in the hypoxia-treated cells is indicative of decreased protein translation. RNA extracted from the polysome fractions was applied to a 1% agarose gel and electrophoresed. The abundant 28S, 18S, and 5S rRNAs were directly visualized by ethidium bromide staining. The polysome fractions isolated for microarray analysis, 0-h poly and 16-h poly, are indicated.

FIG. 2.

Scatter plots of oligonucleotide probes as affected by hypoxic stress. Total and polysomal RNAs were hybridized to the Affymetrix Human Genome U95A (HG-U9Av2) oligonucleotide microarray chip. Relative gene probe intensities (x axis) of total RNA (A) and polysomal mRNA (B and C) of aerobic cells (0 h) were plotted against the corresponding gene probe intensities (y axis) of hypoxia-treated cells (16 h). Gene probes above the top green line represent genes induced >2-fold; gene probes below the bottom green line represent genes repressed >2-fold. (C) Gene probes in green represent genes that were induced >2-fold in the polysomes but whose total mRNA was not induced >2-fold. A short list of these translational candidates is presented in Table 2. Gene probes in yellow represent genes whose expression was induced >2-fold in both the total RNA and polysomal mRNA profiles.

FIG. 3.

ATF4 mRNA is more efficiently translated during hypoxia. (A) Translational candidates. The total mRNA expression does not change during hypoxia. Total RNA was isolated prior to sucrose gradient fractionation from hypoxia treated (16 h) or normoxic (0 h) HeLa cells, reverse transcribed, and quantified by real-time PCR. The quantities of each transcript are described as the number of transcripts isolated from total RNA per cell. Each sample was independently normalized to a spiked internal control. Q-PCR analysis was replicated in triplicate. The results are representative of the average ± the standard error of the mean (SEM) of at least five independent experiments. (B) Translational candidates. Transcripts are enriched in the polysomes during hypoxia. High-molecular-weight polysomes from hypoxia-treated (16 h) or normoxic (0 h) HeLa cells were pooled (fractions 7 to 11), reverse transcribed, and quantified by real-time PCR. The quantities of each transcript are described as the number of transcripts isolated from polysomal RNA per cell. Each sample was independently normalized to a spiked internal control. Q-PCR analysis was repeated in triplicate. The results are representative of the average ± the SEM of at least five independent experiments. (C) Trans-lation efficiency increases for translationally regulated genes. The efficiency of translation during hypoxic treatment (16 h) and normoxia (0 h) was plotted as the percentage of total mRNA associated with polysomes: (quantity of poly mRNA_time x/quantity of total mRNA_time x) × 100%. (D) Hypoxia-induced genes. Total mRNA expression is induced during hypoxia. Total RNA was isolated prior to sucrose gradient fractionation from hypoxia-treated (16 h) or normoxic (0 h) HeLa cells, reverse transcribed, and quantified by real-time PCR. The quantities of each transcript are described as the number of transcripts isolated from total RNA per cell. Each sample was independently normalized to a spiked internal control. Q-PCR analysis was replicated in triplicate. The results are representative of the average ± the SEM of at least three independent experiments. (E) Hypoxia-induced genes. Transcripts are enriched in the polysomes during hypoxia. High-molecular-weight polysomes from hypoxia-treated (16 h) or normoxic (0 h) HeLa cells were pooled (fractions 7 to 11), reverse transcribed, and quantified by real-time PCR. The quantities of each transcript are described as the number of transcripts isolated from polysomal RNA per cell. Each sample was independently normalized to a spiked internal control. Q-PCR analysis was repeated in triplicate. The results are representative of the average ± the SEM of at least three independent experiments. (F) Changes in translation efficiency for hypoxia induced genes. The efficiency of translation during hypoxic treatment (16 h) and normoxia (0 h) was plotted as the percentage of total mRNA associated with polysomes: (quantity of poly mRNA_time x/quantity of total mRNA_time x) × 100%.

FIG. 4.

ATF4 is translationally induced after hypoxic stress. (A) Immunoblot analysis of ATF4 and HIF-1α protein content in HeLa cells exposed to hypoxia or left untreated for the indicated period of time. Actin serves as a loading control. (B) Immunoblot of ATF4 and BiP in HeLa cells treated with hypoxia for the indicated period of time in the presence or absence of the transcriptional inhibitor ActD (100 μM) added 5 min before treatment. Similar results were obtained by using another transcriptional inhibitor, DRB at 100 μM (data not shown). Actin serves as a loading control. (C) Immunoblot of ATF4 and HIF-1α in HeLa cells treated with hypoxia for the indicated period of time in the presence or absence of the translational inhibitor CHX (100 μM). Actin serves as a loading control. (D) Immunoblot of ATF4 and HIF-1α in HeLa cells exposed to hypoxia for the indicated period of time. Cells were first treated with hypoxia, followed by the addition of the translational inhibitor CHX for 15 min, 30 min, or 1 h. Actin served as a loading control.

FIG. 5.

PERK is required for induction of ATF4 in response to hypoxic stress. (A) High-molecular-weight polysomes from normoxic cells (PERK^+/+, PERK^−/−, PKR^−/−, PERK^−/− PKR^−/−, HT29-A1, and HT29-Puro) were pooled (fractions 7 to 11), reverse transcribed, and quantified by real-time PCR. The quantity of ATF4 is described as the number of ATF4 transcripts isolated from polysomal RNA per cell. Each sample was independently normalized to a spiked internal control. Q-PCR analysis was repeated in triplicate. (B) High-molecular-weight polysomes from hypoxia-treated (16 h) or normoxic (0 h) PERK^+/+ and PERK^−/− cells were pooled (fractions 7 to 11), reverse transcribed, and quantified by real-time PCR. The efficiency of ATF4 translation during hypoxic treatment (16 h) and normoxia (0 h) was plotted as the percentage of total mRNA associated with polysomes: (quantity of poly mRNA_time x/quantity of total mRNA_time x) × 100%. Each sample was independently normalized to a spiked internal control. Q-PCR analysis was repeated in triplicate. (C) Immunoblot of ATF4 and eIF2α phosphorylated on Ser-51 [eIF2a(p)] in PERK^+/+ and PERK^−/− MEFs for the indicated period of time after hypoxic stress. Actin served as a loading control.

FIG. 6.

PERK is necessary for ATF4 signal transduction during hypoxic stress. Immunoblot of ATF4, eIF2α(p), total eIF2α, and GADD34 from hypoxia-treated and 100 nM thapsigargin (Tg)-treated wild-type, PKR^−/−, and PKR^−/− PERK^−/− double-knockout MEFs for the indicated periods of time. Actin served as a loading control.

FIG. 7.

Overexpression of GADD34 can antagonize ATF4 signaling during hypoxia. Immunoblot of ATF4, eIF2α(p), total eIF2α, and GADD34 from hypoxia-treated or 100 nM thapsigargin (Tg)-treated HT29-Puro (parental) cells or HT29-A1 cells expressing a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2α (GADD34^trunc) (41). Actin served as a loading control.

FIG. 8.

Model depicting the role of ATF4 during Hypoxic stress. Hypoxia and oxidative stress activate the eIF2α kinase PERK. Phosphorylation of eIF2α results in the global inhibition of protein translation. Some transcripts, such as ATF4, ATF6, and eIF5, are able to escape this general control mechanism. Transcriptional induction of GADD34 results in the activation of PP1 and the dephosphorylation of eIF2α after prolonged exposure to hypoxia. The microarray data also support the notion that numerous ER-resident proteins are induced in response to hypoxic stress, suggesting that cellular adaptation to hypoxic stress may rely on a integrated ER-generated stress signal.