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

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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 eIF2α kinase PERK, ER stress failed to upregulate REDD1 expression, demonstrating that phosphorylation of eIF2α 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 eIF2α, and increased ATF4 expression.

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Materials and methods

Cell culture, transfections and plasmids. Human HepG2 cells and HEK293T cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with 10% fetal bovine serum (Atlas Biologicals) and 1% penicillin–streptomycin (Invitrogen) at 37 °C. PERK+/+, PERK−/−, ATF4+/+, and ATF4−/− MEFs (kindly provided by Drs. David Ron and Heather Harding, NY University School of Medicine) were similarly maintained with the addition of 1.5 μg/ml puromycin to the medium of the PERK MEFs. ATF4

Results

In two previous reports [4], [18], REDD1 mRNA expression was shown to be induced in response to ER stress. Herein, we confirm the results of the previous reports using HepG2 hepatoma cells subjected to ER stress induced by tunicamycin or thapsigargin. Consistent with the previous reports, ER stress induced by either agent resulted in upregulated REDD1 mRNA expression (Fig. 1A). Moreover, the upregulated mRNA expression corresponded to an elevation in REDD1 protein (Fig. 1B).

We next examined the

Discussion

Previous reports have shown that expression of the mRNA encoding REDD1 is upregulated during ER stress [4], [18]. In the present study, we show that ER stress-induced upregulation of REDD1 expression is dependent on PERK, suggesting that the effect requires eIF2α phosphorylation. This suggestion is supported by the finding that in PERK−/− MEFs, histidine deprivation, which activates the eIF2α kinase GCN2 [27], increased both eIF2α phosphorylation and REDD1 expression (data not shown). In one of

Acknowledgments

The authors would like to thank Holly Lacko for performing the qRT-PCR assays described herein. The studies described in this manuscript were supported by a grant from the National Institutes of Health (DK13499).

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