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 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.

Fig. 1.

Role of MLK3 in APAP-induced JNK activation. A, primary hepatocytes from wild-type and Mlk3(−/−) mice were treated with 5 mM APAP for the indicated times. A representative immunoblot for expression and phosphorylation of JNK, from three independent experiments, is shown. B, wild-type and Mlk3(−/−) mice were treated with APAP (300 mg/kg i.p.) for the indicated times. Livers from three mice per time point and genotype were excised, and total liver lysates were analyzed. A representative immunoblot for expression and phosphorylation of JNK is shown.

Fig. 2.

Role of MLK3 in APAP-induced hepatotoxicity. A, necrosis was determined through Sytox green staining after 20 h of treatment of primary hepatocytes with increasing amounts of APAP. Results are mean ± S.D. from three independent experiments. *, p < 0.05. B and C, hepatotoxicity was analyzed through measurements of serum ALT levels 3 and 6 h (B) and histological analyses of hematoxylin- and eosin-stained samples from WT (left) and MLK3-KO (right) mice 6 h (C) after APAP treatment (300 mg/kg i.p.); top and bottom panels show data for different mice. D, relative interleukin 1β (IL-1) and interleukin 6 (IL-6) mRNA levels 6 h after APAP treatment were determined through quantitative PCR analyses. Results are mean ± S.D. (n = 6 or 7). *, p < 0.05.

Fig. 3.

Role of RIP1 in APAP-induced hepatotoxicity. A, primary hepatocytes from WT and KO mice were treated with 5 mM APAP for 6 h, in the presence or absence of 30 μM necrostatin-1 (Nec-1). Expression and phosphorylation of JNK and expression of RIP1 were examined through immunoblot analyses. RIP1 activity was determined through in vitro kinase assays with ATP and myelin basic protein (MBP) as substrates. Representative data from two independent experiments are shown. B, necrosis was determined through Sytox green staining after 20 h of treatment of WT and KO hepatocytes with increasing amounts of APAP, in the presence or absence of 30 μM necrostatin-1. Results are mean ± S.D. from two independent experiments. *, p < 0.01; **, p < 0.001. NS, not significant.

Fig. 4.

Role of MLK3 in GSH recovery. Wild-type and MLK3-KO mice were treated with APAP (300 mg/kg i.p.) for the indicated times. A, levels of expression of hepatic CYP2E1 and GCLc were examined through immunoblot analyses. B, GSH levels in total liver lysates were determined 1, 3, and 6 h after APAP treatment. Results are mean ± S.D. (n = 6 or 7). *, p < 0.05. C, oxidative stress in total liver lysates was determined through measurements of DCFH-DA fluorescence 0, 3, and 6 h after APAP treatment. Results are mean ± S.D. from three experiments. *, p < 0.05. D, superoxide production was determined through DHE staining after 16 h of treatment of primary hepatocytes with the indicated amounts of APAP. Results are mean ± S.D. from two experiments. *, p < 0.05.

Fig. 5.

Role of MLK3 in Mcl-1 expression. Wild-type and Mlk3(−/−) mice were treated with APAP (300 mg/kg i.p.) for the indicated times, and livers were excised. A, livers from three mice per time point and genotype were excised, and total liver lysates were analyzed. A representative immunoblot for expression of GSK3β and expression and phosphorylation of GS is shown. B, livers from three mice per time point and genotype were excised, and mitochondria were isolated and analyzed. A representative immunoblot for expression of Mcl-1 and cyclooxygenase IV (COXIV) is shown.

Fig. 6.

Model for the role of MLK3 in APAP-induced hepatotoxicity. The metabolism of APAP results in NAPQI formation, GSH depletion, mitochondrial dysfunction, and ROS production. Oxidative stress-induced MLK3 activation amplifies ROS formation by modulating the capacity for GSH biosynthesis through regulation of GCLc expression and by targeting JNK- and GSK3β-dependent Mcl-1 degradation, which ultimately leads to cell death.