Abstract
Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1−/− mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis.
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Abbreviations
- Bim:
-
Bcl-2-interacting mediator
- BH3:
-
Bcl-2 homology 3
- BTB:
-
bric-à-brac
- CTR:
-
c-terminal region
- DAPI:
-
4′,6-diamidino-2-phenylindole dihydrochloride
- ER:
-
endoplasmic reticulum
- FFA:
-
free fatty acids
- HKO:
-
hepatocyte specific knockout
- JNK:
-
c-Jun NH2 terminal kinase
- Keap:
-
Kelch-like ECH-associated protein
- Luc:
-
luciferase
- MLK:
-
mixed lineage kinase
- NAFLD:
-
non-alcoholic fatty liver disease
- NASH:
-
non-alcoholic steatohepatitis
- Nrf2:
-
nuclear factor (erythroid-derived 2)-like 2
- OA:
-
oleic acid
- PA:
-
palmitic acid
- SQSTM1:
-
Sequestrosome 1
- PUMA:
-
p53 upregulated modulator of apoptosis
- Sh:
-
short hairpin
- WT:
-
wild-type
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Acknowledgements
We thank Dr. Paul Dent (Massey Cancer Center, VCU, Richmond, VA) for reagents, Dr. Amon Asgharpour and Dr. Robert Vincent (VCU, Richmond, VA, USA) and Dr. Justin Mott (University of Nebraska Medical Center, Omaha, NE, USA) for careful reading of the manuscript, and Kimberly Stratton (VCU Molecular Biology Core Facility) for technical assistance. This work was supported, in whole or in part, by NIH Grants R01 DK081450-03 and T32 07150-37 to AJS, 5R01DK057543 and 1I01BX001390 to HZ, CA93798 and CA167708 to SG and DK081461 to CDK. Support was also provided by VCU Molecular Biology Core Facility, NIH-NCI Cancer Center Support Grant (P30 CA016059).
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Cazanave, S., Wang, X., Zhou, H. et al. Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis. Cell Death Differ 21, 1303–1312 (2014). https://doi.org/10.1038/cdd.2014.49
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DOI: https://doi.org/10.1038/cdd.2014.49
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