ATF4 is necessary and sufficient for ER stress-induced upregulation of REDD1 expression

Abstract

In response to a variety of cell stresses, e.g. endoplasmic reticulum (ER) stress, expression of REDD1 (regulated in development and DNA damage responses) is transcriptionally upregulated. However, the mechanism through which ER stress acts to upregulate REDD1 expression is unknown. In the present study, REDD1 expression was found to be upregulated by ER stress in several cell lines. However, in MEF cells lacking the eIF2alpha kinase PERK, ER stress failed to upregulate REDD1 expression, demonstrating that phosphorylation of eIF2alpha was necessary for the effect. Moreover, ER stress led to upregulated expression of the transcription factor ATF4, but in MEF cells lacking ATF4, REDD1 mRNA expression was not increased by ER stress. In contrast, exogenous expression of ATF4 was sufficient to induce REDD1 expression. Overall, the results suggest that REDD1 expression is upregulated during ER stress through a mechanism involving activation of PERK, phosphorylation of eIF2alpha, and increased ATF4 expression.

Fig. 1

ER stress induces REDD1 expression in a liver-derived cell line. HepG2 cells were exposed to 10 μg/ml tunicamycin, 100 nM thapsigargin, or vehicle control in normal growth medium for 4 h. In separate experiments, RNA or protein was prepared from cell lysates and analyzed by qRT-PCR or Western blot respectively. (A) RNA was analyzed for the REDD1 and GAPDH mRNA expression. The results were normalized to GAPDH mRNA and are expressed as means ± SEM (n=3). *p<0.05 vs. control. (B) Western blot analysis using anti-REDD1 (top panel) and anti-GAPDH (bottom panel) antibodies. The results are representative of 3 studies that were performed.

Fig. 2

Induction of REDD1 expression upon ER stress requires PERK. Wild type (PERK^+/+) and PERK^−/− MEFs were treated with DMSO (vehicle for tunicamycin), 10 μg/ml tunicamycin, ethanol (vehicle for thapsigargin), or 100 nM thapsigargin for 4 h. After treatment, cells were harvested for qRT-PCR analysis as described under Materials and Methods. The results represent mean ± SEM (n=3). *p<0.05 vs. control. (A) PERK^+/+ MEFs. (B) PERK^−/− MEFs. The relative amount of REDD1 mRNA was normalized to GAPDH mRNA. (C) PERK^+/+ and PERK^−/− MEFs were incubated for 0, 3, or 6 h with thapsigargin and then harvested for Western blot analysis or PCR analysis of XBP1 splicing as described under Materials and Methods.

Fig. 3

Induction of REDD1 expression upon ER stress requires ATF4. ATF4^+/+ and ATF4^−/− MEFs were treated with vehicle, 10 μg/ml tunicamycin, or 100 nM thapsigargin for 4 h. The results represent mean ± SEM (n=3). (A) Western blot analysis using anti-ATF4 antibodies. (B) qRT-PCR analysis was performed on REDD1 and GAPDH mRNA extracted from cells. The relative amount of REDD1 mRNA was normalized to GAPDH mRNA. The results represent mean ± SEM (n=3). *p<0.05 vs. control.

Fig. 4

Overexpression of ATF4 leads to induction of REDD1 expression. HEK293T cells were transfected with pMAX-GFP (control) or a plasmid containing ATF4. (A) Western blot analysis was performed on cell homogenates using anti-REDD1, anti-ATF4, and anti-GAPDH antibodies. (B) RNA was extracted from cells and analyzed by qRT-PCR. The relative amount of REDD1 mRNA was normalized to GAPDH mRNA. The results represent mean ± SEM (n=3). *p<0.05 vs. control.