Abstract
In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3′-kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3–Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.
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Abbreviations
- ATF:
-
activating transcription factor
- BFA:
-
brefeldin A
- CHOP:
-
C/EBP homologous protein
- eIF2α:
-
eukaryotic translation initiation factor-2α subunit
- ER:
-
endoplasmic reticulum
- GFP:
-
green fluorescence protein
- JNK:
-
c-Jun N-terminal kinase
- LC3:
-
microtubule-associated protein-1 light-chain 3
- NS:
-
nutrient starvation
- PI3KC3:
-
class III phosphatidylinositol-3′-kinase
- TG:
-
thapsigargin
- Tun:
-
tunicamycin
- UPR:
-
unfolded protein response
- Xbp-1:
-
X-box-binding protein 1
- 3-MA:
-
3-methyladenine
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Acknowledgements
We thank Drs. N Mizushima for the gift of the pEGFP-LC3 expression vector, R Kopito for antibody against LC3 and members of the Lee lab, in particular, Dezheng Dong, for assistance and helpful discussions. This work is supported in part by NIH grants CA027607 and CA111700 (to ASL) and a grant from the Arnold and Mabel Beckman Foundation (to DH). Electron microscopy was performed in the Cell and Tissue Imaging Core Facility supported by Cancer Center Support Grant 5P30 CA14089.
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Li, J., Ni, M., Lee, B. et al. The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells. Cell Death Differ 15, 1460–1471 (2008). https://doi.org/10.1038/cdd.2008.81
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DOI: https://doi.org/10.1038/cdd.2008.81
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