Abstract
c-Jun NH2-terminal kinase (JNK) activation plays a major role in acetaminophen (APAP)-induced hepatotoxicity. However, the exact mechanism of APAP-induced JNK activation is incompletely understood. It has been established that apoptosis signal-regulating kinase 1 (ASK1) regulates the late phase of APAP-induced JNK activation, but the mitogen-activated protein kinase kinase kinase that mediates the initial phase of APAP-induced JNK activation has not been identified. Oxidative stress produced during APAP metabolism causes JNK activation, which promotes mitochondrial dysfunction and results in the amplification of oxidative stress. Therefore, inhibition of the initial phase of JNK activation may be key to protection against APAP-induced liver injury. The goal of this study was to determine whether mixed-lineage kinase 3 (MLK3) mediates the initial, ASK1-independent phase of APAP-induced JNK activation and thus promotes drug-induced hepatotoxicity. We found that MLK3 was activated by oxidative stress and was required for JNK activation in response to oxidative stress. Loss of MLK3 attenuated APAP-induced JNK activation and hepatocyte death in vitro, independent of receptor-interacting protein 1. Moreover, JNK and glycogen synthase kinase 3β activation was significantly attenuated, and Mcl-1 degradation was inhibited in APAP-treated MLK3-knockout mice. Furthermore, we showed that loss of MLK3 increased expression of glutamate cysteine ligase, accelerated hepatic GSH recovery, and decreased production of reactive oxygen species after APAP treatment. MLK3-deficient mice were significantly protected from APAP-induced liver injury, compared with wild-type mice. Together, these studies establish a novel role for MLK3 in APAP-induced JNK activation and hepatotoxicity, and they suggest MLK3 as a possible target in the treatment of APAP-induced liver injury.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK082583].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- APAP
- acetaminophen
- MLK
- mixed-lineage kinase
- JNK
- c-Jun NH2-terminal kinase
- ASK
- apoptosis-regulating kinase
- MAP3K
- mitogen-activated protein kinase kinase kinase
- ROS
- reactive oxygen species
- NAPQI
- N-acetyl-p-benzoquinoneimine
- GSK
- glycogen synthase kinase
- ALT
- alanine transaminase
- GS
- glycogen synthase
- RIP
- receptor-interacting protein
- DCFH-DA
- 2′,7′-dichlorodihydrofluorescein diacetate
- DCF
- 2′,7′-dichlorofluorescein
- Src I1
- 6,7-dimethoxy-N-(4-phenoxyphenyl)-4-quinazolinamine
- DHE
- dihydroethidium
- WT
- wild-type
- KO
- knockout
- GCLc
- glutamate-cysteine ligase catalytic subunit.
- Received May 7, 2012.
- Accepted August 23, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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