Abstract
Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK−/− cells display disturbed ER morphology and Ca2+ signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK−/− cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress.
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Abbreviations
- CHOP:
-
C/EBP homologous protein
- eIF2α:
-
eukaryotic initiation factor-2α
- ER:
-
endoplasmic reticulum
- GRP78:
-
glucose regulated protein 78
- IP3R:
-
inositol 1,4,5-trisphosphate receptor
- IRE1:
-
inositol requiring enzyme 1
- JNK:
-
c-Jun N-terminal kinase
- MAM:
-
mitochondria-associated ER membrane
- MEF:
-
murine embryonic fibroblast
- MFN2:
-
mitofusin 2
- PERK:
-
RNA-dependent protein kinase (PKR)-like ER kinase
- Phox:
-
photo-oxidative
- ROS:
-
reactive oxygen species
- SERCA:
-
sarco/endoplasmic reticulum Ca2+ ATPase
- TG:
-
thapsigargin
- UPR:
-
unfolded protein response
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Acknowledgements
This work is supported by GOA grant of the KU Leuven and by F.W.O grant G.0492.05 to PA, G.0788.11N, and OTSRT1/10/44 to GB. This paper presents research results of the IAP6/18 and IAP6/28, funded by the Interuniversity Attraction Poles Programme, initiated by the Belgian State, Science Policy Office. NR is a post-doctoral fellow supported by F.R.S-FNRS (grant F/5/4/5-MCF/KP). J-PD is a recipient of a PhD Fellowship from the Agency for Innovation of Science and Technology (IWT). We thank Dr. R Kaufman (University of Michigan Medical Center, Ann Arbor, MI, USA) for the WT, PERK−/−, CHOP−/− and IRE1−/− fibroblasts, Dr. A Diehl (Abramson Family Cancer Research Institute Philadelphia, USA) for the PERK-shRNA transduced MDA-MB468 cells, Dr. D Krysko (University of Ghent, Belgium) for the PERK-shRNA CT26 cells and Dr. W Martinet (University of Antwerpen, Belgium) for the electron microscopy analysis. We thank Professor M Fransen for the use of the Neon Electroporation equipment. We acknowledge Dr. S Bontems for excellent support in transforming MEFs with KRAS, Tomas Luyten for excellent support in the Ca2+ experiments, Veerle Daniels for help with MACs isolation, and Jan Piessens and Kristine Rillaerts for skillful technical support. Nikon confocal microscope was acquired through a Hercules Type 1 AKUL/09/037 to W Annaert.
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Verfaillie, T., Rubio, N., Garg, A. et al. PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress. Cell Death Differ 19, 1880–1891 (2012). https://doi.org/10.1038/cdd.2012.74
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DOI: https://doi.org/10.1038/cdd.2012.74
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