FIG. 4.
eIF2α-dependent hypoxic inhibition of NMD stabilizes ATF-4, ATF-3, and CHOP to augment the stress response. (A) U2OS cells were rendered hypoxic or normoxic for 3 h prior to treatment with DRB. RNA was collected for analysis at time zero and then at 2, 4, and 6 h after addition of DRB (because of the short half-life of ATF-4, only time zero and 2 h were assessed), and the messages of NMD targets were determined by reverse transcription-PCR. Normoxic and hypoxic mRNA expression levels at time zero were normalized to 1. Real-time PCR was preformed in triplicate, and the graphs depict the averages of four separate experiments ± standard errors. The hypoxic/normoxic ratios of mRNA levels at each time point are shown in the right panel, with values greater than 1 reflecting stability in hypoxic cells. (B) Wild-type MEFs (eIF2α +/+) or MEFs which cannot phosphorylate eIF2α (eIF2α S51A) were treated as described for panel A, except the stability of ATF-4 mRNA was assessed after 1 h. (C) ATF-4−/− MEFs were transfected with ATF-4 genomic constructs expressing either the coding region of ATF-4 or an ATF-4 cDNA containing the uORFs. The alternative uORFs are depicted by circles and hexagons. The region for ATF-4 coding is shown as a black rectangle, and introns are depicted as lines. Cells were rendered hypoxic for 3 h and then treated with an inhibitor of RNA synthesis. One hour after treatment, RNA was harvested and quantitated by real-time PCR. Normoxic cells were similarly treated. The endogenous ATF-4 half-life was determined with wild-type MEFs. mRNA expression in normoxic and hypoxic cells was normalized to 1 at time zero. Real-time PCR was preformed in triplicate, and the graphs depict the averages of four separate experiments ± standard errors. (D) U2OS cells were either infected with a lentivirus with a scrambled shRNA (scr) or a lentivirus with a Rent1/Upf1 sh RNA (shRent1/Upf1) as in Fig. 1A. Cells were then treated with 2.5 μg/ml of tunicamycin for short (A) or longer (B) times, protein was collected, and immunoblot assays were performed for proteins induced during the unfolded protein response (ATF-4, ATF-3, CHOP, and gadd34). Both actin and total eIF2α served as loading controls.