Critical Role for Mixed-Lineage Kinase 3 in Acetaminophen-Induced Hepatotoxicity

Abstract

c-Jun NH 2 -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 3␤ 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 wildtype 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.

Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein

“…Computational analysis of the amino acid sequences surrounding Thr-166 by GPS (Group-base Prediction System) version 2.1 (41), NetPhosK 1.0 (42), and PhosphoMotif Finder (43) databases predict Thr-166 phosphorylation by MAP3K11 (MLK3), PKC, and casein kinase 1, respectively. Oxidative stress is known to activate PKC and MLK3 activity (44,45). Moreover, apoptosis can be stimulated by MLK3-mediated JNK and p38 signaling (46).…”

Chronic Deletion and Acute Knockdown of Parkin Have Differential Responses to Acetaminophen-induced Mitophagy and Liver Injury in Mice

“…Mcl-1 is an anti-apoptotic Bcl-2 protein that is mostly localized to the mitochondrial membrane (53), and its stabilization has been shown to play a protective role against APAP-induced hepatocellular necrosis (28). Parkin KO mice had increased liver Mcl-1 expression compared with WT mice before and after APAP treatment, which may be another mechanism for their protection against APAP-induced liver injury.…”

“…Mcl-1 is an anti-apoptotic protein that has been shown to be protective against APAP-induced liver injury (28,29). We measured protein levels of Mcl-1 using total lysate from WT and Parkin KO mouse livers 6 h after APAP treatment.…”

Oxidative Stress in Acute Liver Failure

“…Glycogen synthase kinase 3β (GSK3β), another redox-responsive kinase, has also been shown to activate JNK in APAP hepatotoxicity and it was suggested that this protein acts at earlier time points after APAP overdose [ 82 ]. Mixed lineage kinase 3 (MLK3) can also be activated by oxidative stress, and MLK3 knockout mice are protected against APAP and have reduced activation of both JNK and GSK3β [ 81 ]. Once JNK has been activated, it translocates into mitochondria where it is thought to augment the mitochondrial oxidative stress in a feed-forward loop [ 33 , 76 ].…”