Deletion of Apoptosis Signal-Regulating Kinase 1 Attenuates Acetaminophen-Induced Liver Injury by Inhibiting c-Jun N-Terminal Kinase Activation
2008; Elsevier BV; Volume: 135; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2008.07.006
ISSN1528-0012
AutoresHayato Nakagawa, Shin Maeda, Yohko Hikiba, Tomoya Ohmae, Wataru Shibata, Ayako Yanai, Kei Sakamoto, Keiji Ogura, Takuya Noguchi, Michael Karin, Hidenori Ichijo, Masao Omata,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoBackground & Aims: Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure. C-jun N-terminal kinase (JNK) is thought to play a central role in APAP-induced liver injury, although its upstream activator has not yet been identified. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase family and is important for stress-induced JNK activation. We tested the hypothesis that ASK1 was involved in APAP-induced JNK activation and liver injury. Methods: ASK1-deficient (ASK1−/−) mice and wild-type (WT) mice were given 300 mg/kg of APAP. Serum alanine aminotransferase levels and liver histology were assessed. To investigate the involvement of ASK1 in direct hepatocellular damage and the subsequent inflammatory response, we used primary hepatocytes and splenocytes from WT and ASK1−/− mice. Results: In ASK1−/− mouse liver, APAP toxicity was attenuated significantly and the prolonged activation of JNK was inhibited. In addition, thioredoxin, a direct ASK1 inhibitor, dissociated from ASK1 after APAP overdose with concomitant ASK1 activation. Although the prolonged activation of p38 also was attenuated in ASK1−/− mice, the p38 signaling pathway was not likely to be involved in APAP-induced liver injury. Primary hepatocyte culture also revealed that ASK1 and JNK, but not p38, contributed to direct APAP-induced cellular damage. Conclusions: Our data suggest that ASK1 is activated by APAP overdose, most likely via a mechanism involving thioredoxin–ASK1 dissociation, and that it plays a role in APAP-induced liver injury through JNK activation. Background & Aims: Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure. C-jun N-terminal kinase (JNK) is thought to play a central role in APAP-induced liver injury, although its upstream activator has not yet been identified. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase family and is important for stress-induced JNK activation. We tested the hypothesis that ASK1 was involved in APAP-induced JNK activation and liver injury. Methods: ASK1-deficient (ASK1−/−) mice and wild-type (WT) mice were given 300 mg/kg of APAP. Serum alanine aminotransferase levels and liver histology were assessed. To investigate the involvement of ASK1 in direct hepatocellular damage and the subsequent inflammatory response, we used primary hepatocytes and splenocytes from WT and ASK1−/− mice. Results: In ASK1−/− mouse liver, APAP toxicity was attenuated significantly and the prolonged activation of JNK was inhibited. In addition, thioredoxin, a direct ASK1 inhibitor, dissociated from ASK1 after APAP overdose with concomitant ASK1 activation. Although the prolonged activation of p38 also was attenuated in ASK1−/− mice, the p38 signaling pathway was not likely to be involved in APAP-induced liver injury. Primary hepatocyte culture also revealed that ASK1 and JNK, but not p38, contributed to direct APAP-induced cellular damage. Conclusions: Our data suggest that ASK1 is activated by APAP overdose, most likely via a mechanism involving thioredoxin–ASK1 dissociation, and that it plays a role in APAP-induced liver injury through JNK activation. See editorial on page 1047. See editorial on page 1047. Acetaminophen (APAP) is a widely used analgesic and antipyretic agent that is usually safe at therapeutic doses. However, overdose can lead to acute, fatal liver failure. In fact, APAP overdose is the most frequent cause of drug-induced liver failure in the United States.1Lee W.M. Acetaminophen toxicity: changing perceptions on a social/medical issue.Hepatology. 2007; 46: 966-970Crossref PubMed Scopus (112) Google Scholar Because APAP hepatotoxicity is dose-dependent and reproducible in animal models, the underlying mechanism has been well characterized. At high doses, cytochrome P-450 enzymes convert APAP into a reactive quinone form, N-acetyl-p-benzoquinone imine.2Dahlin D.C. Miwa G.T. Lu A.Y. et al.N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen.Proc Natl Acad Sci U S A. 1984; 81: 1327-1331Crossref PubMed Google Scholar, 3Snawder J.E. Roe A.L. 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Signal transduction by the JNK group of MAP kinases.Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3688) Google Scholar and prolonged JNK activation plays an important role in cell death.12Chen Y.R. Meyer C.F. Tan T.H. Persistent activation of c-Jun N-terminal kinase 1 (JNK1) in gamma radiation-induced apoptosis.J Biol Chem. 1996; 271: 631-634Crossref PubMed Scopus (462) Google Scholar, 13Guo Y.L. Baysal K. Kang B. et al.Correlation between sustained c-Jun N-terminal protein kinase activation and apoptosis induced by tumor necrosis factor-alpha in rat mesangial cells.J Biol Chem. 1998; 273: 4027-4034Crossref PubMed Scopus (245) Google Scholar Several MAPK kinase kinases (MAPKKKs) have been identified in the JNK cascade, including apoptosis signal-regulating kinase 1 (ASK1), MAP/extracellular signal-regulating kinase (ERK) kinase kinases, and TGFβ activated kinase 1 (TAK1). These MAPKKKs activate MAP kinase kinase (MKK) 4 and/or MKK7, which in turn activate JNK.11Davis R.J. Signal transduction by the JNK group of MAP kinases.Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3688) Google Scholar Although the physiologic relevance of MAPKKKs in the control of JNK activation is not completely clear, genetic deletion studies have suggested that different stimuli may act through different MAPKKKs to activate the JNK signaling pathway.14Xia Y. Makris C. Su B. et al.MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration.Proc Natl Acad Sci U S A. 2000; 97: 5243-5248Crossref PubMed Scopus (235) Google Scholar, 15Nagai H. Noguchi T. Takeda K. et al.Pathophysiological roles of ASK1-MAP kinase signaling pathways.J Biochem Mol Biol. 2007; 40: 1-6Crossref PubMed Google Scholar In APAP-induced liver injury, hepatic JNK activation is thought to be a consequence of oxidative stress produced during APAP metabolism; however, its upstream activator has not yet been identified. Although JNK inhibition is a promising therapy for the acute effects of APAP-induced liver injury, such a therapy may inhibit recovery from severe APAP-induced liver damage because JNK activity is required for hepatocyte proliferation and liver regeneration.16Schwabe R.F. Bradham C.A. Uehara T. et al.c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration.Hepatology. 2003; 37: 824-832Crossref PubMed Scopus (212) Google Scholar Therefore, identifying the specific upstream activator in APAP-induced JNK activation is very important. ASK1 is a ubiquitously expressed MAPKKK that is activated by various types of stress, including reactive oxygen species, tumor necrosis factor-α, lipopolysaccharide, endoplasmic reticulum stress, and calcium influx.17Ichijo H. Nishida E. Irie K. et al.Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.Science. 1997; 275: 90-94Crossref PubMed Scopus (2049) Google Scholar, 18Nishitoh H. Saitoh M. Mochida Y. et al.ASK1 is essential for JNK/SAPK activation by TRAF2.Mol Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 19Matsuzawa A. Saegusa K. Noguchi T. et al.ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity.Nat Immunol. 2005; 6: 587-592Crossref PubMed Scopus (578) Google Scholar, 20Sekine Y. Takeda K. Ichijo H. The ASK1-MAP kinase signaling in ER stress and neurodegenerative diseases.Curr Mol Med. 2006; 6: 87-97Crossref PubMed Scopus (129) Google Scholar, 21Takeda K. Matsuzawa A. Nishitoh H. et al.Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation.EMBO Rep. 2004; 5: 161-166Crossref PubMed Scopus (161) Google Scholar ASK1 activates the JNK and p38 signaling pathways and is required for both oxidative stress– and cytokine-induced apoptosis.17Ichijo H. Nishida E. Irie K. et al.Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.Science. 1997; 275: 90-94Crossref PubMed Scopus (2049) Google Scholar ASK1 is particularly important for the oxidative stress–mediated activation of JNK and p38. Furthermore, ASK1 has been implicated in a variety of cellular functions, including the inflammatory response.19Matsuzawa A. Saegusa K. Noguchi T. et al.ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity.Nat Immunol. 2005; 6: 587-592Crossref PubMed Scopus (578) Google Scholar We examined whether ASK1 plays a role in APAP-induced liver injury using ASK1-deficient (ASK1−/−) mice. We found that ASK1−/− mice showed significant attenuation of APAP-induced liver injury, and that the APAP-induced prolonged activation of JNK was attenuated in ASK1−/− mice. These results show the important role of the ASK1–JNK pathway in APAP-induced liver injury, and they identify ASK1 as a potential therapeutic target. The generation of ASK1−/−, JNK1−/−, JNK2−/−, and p38α+/– mice has been described previously.22Tobiume K. Matsuzawa A. Takahashi T. et al.ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1024) Google Scholar, 23Chang L. Kamata H. Solinas G. et al.The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover.Cell. 2006; 124: 601-613Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 24Tamura K. Sudo T. Senftleben U. et al.Requirement for p38alpha in erythropoietin expression: a role for stress kinases in erythropoiesis.Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar All mice were back-crossed into the C57BL/6 strain at least 6 times. Male ASK1−/−, JNK1−/−, JNK2−/−, and p38α+/− mice, 8–10 weeks old, and appropriate age-matched male C57BL/6 wild-type mice (WT; Clea Japan, Tokyo, Japan) were used in all experiments. The study design and protocols were approved by our institutional ethics committee for animal experimentation. All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Department of Medicine, the University of Tokyo. All mice were maintained under pathogen-free conditions under a light/dark cycle. After an overnight fast, the mice were injected intraperitoneally with APAP (Sigma–Aldrich, St Louis, MO) dissolved in warmed saline or saline alone. Some mice were pretreated with the JNK inhibitor SP600125 (BIOMOL, Plymouth Meeting, PA) dissolved in 40% polyethylene glycol in phosphate-buffered saline, or the p38 inhibitor SB203580 (Wako, Osaka, Japan) dissolved in 4% dimethyl sulfoxide. Each inhibitor was administered intraperitoneally 1 hour before APAP treatment. Mice were killed at various time points. Whole-liver protein homogenates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene membrane (Amersham Biosciences, Piscataway, NJ). The membrane was probed with primary antibodies, and then incubated with the secondary antibody. Immunocomplexes were detected using the enhanced chemiluminescence system (Amersham Biosciences). Anti-JNK1/2 (1:1000 dilution), anti-JNK2 (1:1000), anti–phospho JNK1/2 (1:1000), anti-MKK4 (1:1000), anti–phospho MKK4 (1:1000), anti-p38 (1:1000), and anti–phospho p38 antibodies (1:1000) were purchased from Cell Signaling (Boston, MA), and anti-ASK1 (1:1000) and anti-JNK1 antibodies (1:1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antithioredoxin antibody (1:3000) was purchased from Redox Bio Science (Kyoto, Japan), and the anti-mouse actin (1:3000) antibody was from Sigma–Aldrich. The anti–phospho-ASK1 antibody (1:2000) was described previously.25Tobiume K. Saitoh M. Ichijo H. Activation of apoptosis signal-regulating kinase 1 by the stress-induced activating phosphorylation of pre-formed oligomer.J Cell Physiol. 2002; 191: 95-104Crossref PubMed Scopus (301) Google Scholar Hepatocytes were isolated and cultured as described previously.26Maeda S. Chang L. Li Z.W. et al.IKKbeta is required for prevention of apoptosis mediated by cell-bound but not by circulating TNFalpha.Immunity. 2003; 19: 725-737Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar APAP was dissolved in warm saline and added to the medium. SP600125 and SB203580 were dissolved in dimethyl sulfoxide and added to the medium. Cytotoxicity was assessed using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using the Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan). Spleen samples were disaggregated through 70-μm cell strainers. Splenocytes were distributed in plates and stimulated. Necrotic hepatocytes were prepared by isolating hepatocytes as described earlier, and then subjecting them to 3 freeze–thaw cycles at −80°C and room temperature. We performed this experiment according to the description of Naugler et al.27Naugler W.E. Sakurai T. Kim S. et al.Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production.Science. 2007; 317: 121-124Crossref PubMed Scopus (1524) Google Scholar Total GSH was measured in liver homogenates using a Total Glutathione Quantification Kit (Dojindo Laboratory). Livers were fixed in 10% formalin, embedded in paraffin, and sectioned. Sections were stained with H&E, and necrosis was graded using a scale previously described: 0, no injury; 1, minimal injury; 2, mild injury; 3, moderate injury; 4, marked injury; and 5, severe injury.28Latchoumycandane C. Goh C.W. Ong M.M. et al.Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury.Hepatology. 2007; 45: 412-421Crossref PubMed Scopus (140) Google Scholar The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed, according to a standard protocol. The number of TUNEL-positive hepatocytes was determined by manually counting cells in 5 low-power fields per liver section. We used the Agilent Mouse Whole Genome Array (Agilent Technologies, Palo Alto, CA) containing 60-mer DNA probes synthesized in situ in a 44-k format. Chips were scanned according to the manufacturer's hybridization protocol (Takara Bio Inc, Mie, Japan). Agilent data were preprocessed using an Agilent GeneSpring GX9. RNA was extracted from liver tissue using the Isogen reagents (Nippon Gene, Tokyo, Japan). The first-strand complementary DNA was synthesized using SuperScript 2 (Invitrogen Life Technologies, Carlsbad, CA), and the relative amount of each messenger RNA (mRNA) was quantified via real-time polymerase chain reaction and normalized against glyceraldehyde-3-phosphate dehydrogenase mRNA expression. Primer sequences are available on request. An interleukin (IL)-6 enzyme-linked immunosorbent assay kit was purchased from R&D Systems (Minneapolis, MN). The data were expressed as the mean ± SD. Statistical analyses were performed using the Student t test or analysis of variance, followed by the Dunnett test when appropriate. A P value of less than .05 was considered statistically significant. A single intraperitoneal injection of APAP (300 mg/kg) induced significant liver injury and marked increases in serum alanine aminotransferase (ALT) levels at 6 hours, peaking at 24 hours, in WT mice. To determine the role of ASK1 in APAP-induced liver injury, ASK1−/− and WT mice were given 300 mg/kg APAP and killed at 6 and 24 hours posttreatment. The increase of serum ALT levels at 6 and 24 hours posttreatment were reduced significantly in ASK1−/− mice compared with WT mice (Figure 1A) The time course and dose-response experiments are shown in supplementary Figure 1 (see supplementary material online at www.gastrojournal.org). Although no significant reduction was observed in ALT level at 6 hours after administration of 400 mg/kg APAP in ASK1−/− mice, the survival rate at 60 hours later improved significantly (44.4% in WT vs 78.5% in ASK1−/−; WT, n = 18; ASK1−/−, n = 14). To confirm the important role of JNK in APAP-induced liver injury,9Gunawan B.K. Liu Z.X. Han D. et al.c-Jun N-terminal kinase plays a major role in murine acetaminophen hepatotoxicity.Gastroenterology. 2006; 131: 165-178Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 10Henderson N.C. Pollock K.J. Frew J. et al.Critical role of c-jun (NH2) terminal kinase in paracetamol-induced acute liver failure.Gut. 2007; 56: 982-990Crossref PubMed Scopus (152) Google Scholar APAP also was administered to JNK1−/− mice, JNK2−/− mice, and WT mice pretreated with the JNK inhibitor SP600125. We confirmed in advance that polyethylene glycol solution, the vehicle for the JNK inhibitor, had no protective effect on APAP-induced liver injury. Consistent with previous reports, serum ALT levels were mildly reduced in JNK2−/− mice and drastically reduced in SP600125-treated mice. In JNK1−/− mice, serum ALT level was reduced significantly at 6 hours, but not at 24 hours, compared with WT mice (Figure 1A). Liver histology was examined to determine the extent of liver necrosis. APAP caused moderate to marked centrilobular necrosis at 6 hours and severe necrosis with hemorrhagic congestion at 24 hours posttreatment in WT mice. In contrast, these histologic changes were attenuated significantly in ASK1−/− mice (Figure 1B and C). Although necrosis is the dominant feature in APAP-induced cell death,29Gujral J.S. Knight T.R. Farhood A. et al.Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis?.Toxicol Sci. 2002; 67: 322-328Crossref PubMed Scopus (348) Google Scholar the involvement of apoptotic pathways also has been reported.30Ferret P.J. Hammoud R. Tulliez M. et al.Detoxification of reactive oxygen species by a nonpeptidyl mimic of superoxide dismutase cures acetaminophen-induced acute liver failure in the mouse.Hepatology. 2001; 33: 1173-1180Crossref PubMed Scopus (138) Google Scholar, 31Vaquero J. Belanger M. James L. et al.Mild hypothermia attenuates liver injury and improves survival in mice with acetaminophen toxicity.Gastroenterology. 2007; 132: 372-383Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Thus, we next analyzed the extent of apoptotic cell death using the TUNEL assay. Mice administered vehicle only revealed almost no TUNEL-positive cells in any group (data not shown). In WT mice, liver sections prepared at 6 hours posttreatment revealed a high frequency of TUNEL-positive cells in centrilobular lesions, whereas very few TUNEL-positive cells were observed in liver sections from ASK1−/− mice (Figure 1D and E). Pretreatment with SP600125 almost completely inhibited centrilobular necrosis and apoptosis in the histologic examination. JNK1−/− and JNK2−/− mice showed mild attenuation of centrilobular necrosis and a significant reduction in apoptotic cells (Figure 1B–E). To rule out the possibility that attenuated liver injury was the result of altered APAP metabolism, we examined the time course of GSH consumption. The decline in hepatic GSH levels is a widely accepted indicator of the generation of the reactive intermediate.28Latchoumycandane C. Goh C.W. Ong M.M. et al.Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury.Hepatology. 2007; 45: 412-421Crossref PubMed Scopus (140) Google Scholar No differences in basal GSH levels or depletion at 1.5 or 6 hours were observed in any group, with the exception of SP6000125-pretreated mice at 6 hours (Figure 1F). Higher GSH recovery levels in mice pretreated with the JNK inhibitor were likely the result of relatively weak liver damage. These results suggest that both JNK and ASK1 play a role in APAP-induced liver injury. Furthermore, to assess the degree of oxidative stress in WT and ASK1−/− mice, we measured lipid peroxidation using thiobarbituric acid reactive substances assay. The thiobarbituric acid reactive substances assay was increased transiently at 3 hours after APAP administration; a similar degree of increase was seen in WT and ASK1−/− mice livers (supplementary Figure 2; see supplementary material online at www.gastrojournal.org). To identify genes involved in ASK1-dependent liver injury, liver samples from 4 groups (untreated and injured livers 6 hours after APAP administration from WT or ASK1−/− mice) were collected and gene expression in each group was monitored using Agilent whole-mouse microarray. We identified 959 annotated genes of the 41,252 represented on the array, which were similar in WT and ASK1−/− untreated livers, and were at least 3-fold up-regulated or 3-fold down-regulated in WT injured liver (Figure 2A). Among these genes, we characterized 140 genes as ASK1-dependent genes, which were at least 2.5-fold up-regulated in WT injured livers compared with ASK1−/− injured livers (Figure 2B). Among these genes, those with known functions are shown in supplementary Table 1 (see supplementary material online at www.gastrojournal.org). The ASK1-dependent genes included Jun and Fos (Figure 2C), which are known to be JNK-dependent genes and reported to be associated with the degree of liver injury caused by APAP.32Beyer R.P. Fry R.C. Lasarev M.R. et al.Multicenter study of acetaminophen hepatotoxicity reveals the importance of biological endpoints in genomic analyses.Toxicol Sci. 2007; 99: 326-337Crossref PubMed Scopus (72) Google Scholar We examined APAP-induced ASK1 activation in WT mouse liver. ASK1 activation began at 3 hours after APAP administration and showed strong activation at 6 hours (Figure 3A). Recent studies have shown that thioredoxin (Trx), a reduction/oxidation regulatory protein, inhibits ASK1 activity via direct binding. Intracellular oxidative stress dissociates Trx from ASK1 and induces ASK1 phosphorylation.33Saitoh M. Nishitoh H. Fujii M. et al.Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1.EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2107) Google Scholar, 34Fujino G. Noguchi T. Takeda K. et al.Thioredoxin and protein kinases in redox signaling.Semin Cancer Biol. 2006; 16: 427-435Crossref PubMed Scopus (131) Google Scholar To determine whether ASK1 is activated through this mechanism in APAP-treated liver, ASK1 was immunoprecipitated, and the interaction of ASK1 with Trx was examined via immunoblotting using a Trx antibody. In normal liver, Trx is associated physically with ASK1; however, Trx–ASK1 interaction decreased at 3 hours post-treatment and then increased again slightly at 6 hours (Figure 3B). To determine whether ASK1 deficiency influences JNK signaling, we used Western blotting to examine the activation of MKK4 and JNK at 6 hours after APAP treatment in liver sections from WT, ASK1−/−, JNK inhibitor–pretreated, JNK1−/−, and JNK2−/− mice. In ASK1−/− mice, JNK phosphorylation decreased compared with WT mice. In addition, JNK phosphorylation was nearly abolished in mice pretreated with the JNK inhibitor and reduced in the corresponding deleted isoform in JNK1−/− and JNK2−/− mice. In contrast, MKK4 phosphorylation decreased only in ASK1−/− mice (Figure 3C). We then examined the time course of JNK activation in WT and ASK1−/− mouse liver. Although JNK activation occurred to a similar degree in both WT and ASK1−/− mice at 1.5 hours, it was attenuated significantly at 3 and 6 hours in ASK1−/− mice compared with WT mice (Figure 3D). This finding suggests that ASK1 is required for prolonged JNK activation in APAP-induced liver injury. Because ASK1 also activates p38 pathways, we examined the effect of ASK1 deletion on APAP-induced p38 activation. In WT mice, p38 activation was observed at 1.5 and 6 hours after APAP administration. Similar to JNK, p38 activation was attenuated at 3 and 6 hours in ASK1−/− mice compared with WT mice (Figure 4A). Thus, we used p38α+/– mice to examine whether the p38 pathway is involved in APAP-induced liver injury. To ascertain whether the p38 signaling pathway was attenuated in p38α+/– mouse liver, we examined total and phosphorylated p38 protein levels in APAP-treated WT and p38α+/– mouse liver. As expected, both total and phosphorylated p38 levels were reduced significantly in p38α+/– mice compared with WT mice at 6 hours post-treatment (Figure 4B). However, no differences in serum ALT level were observed between WT and p38α+/– mice (Figure 4C). We then examined the effect of the p38 inhibitor SB203580 on APAP-induced liver injury. Consistent with the results from p38α+/– mice, no difference in serum ALT levels was observed between p38 inhibitor–pretreated mice and untreated mice at 6 or 24 hours post-treatment (Figure 4D). The serum ALT levels observed in this experiment were lower than in other experiments, probably because of the protective effect of dimethyl sulfoxide on APAP-induced liver injury. These results suggest that the p38 pathway is not likely to be involved in APAP-induced liver injury. We used cultured primary hepatocytes to assess the effect of ASK1 deletion on direct APAP-induced hepatocellular damage. In primary hepatocytes obtained from WT mice, we confirmed that ASK1 was activated by APAP (Figure 5A). Next, we examined APAP-induced JNK and p38 activation in primary hepatocytes from WT and ASK1−/− mice. In ASK1−/− hepatocytes, both JNK and p38 activation were attenuated significantly compared with WT hepatocytes (Figure 5B). Furthermore, primary hepatocytes obtained from WT or ASK1−/− mice were incubated in 10 mmol/L APAP for 24 hours, and cytotoxicity was assessed using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. APAP-induced cell death was suppressed significantly in ASK1−/− hepatocytes compared with WT hepatocytes (Figure 5C). To investigate the involvement of JNK and p38 in direct APAP-induced hepatocellular damage, WT hepatocytes were pretreated with JNK or p38 inhibitor at 1 hour before APAP administration. JNK inhibitor significantly inhibited APAP-induced cell death, whereas the same concentration of p38 inhibitor, which is thought to be sufficient to inhibit p38 kinase activity in mouse primary hepatocytes,35Liu H.Y. Collins Q.F. Xiong Y. et al.Prolonged treatment of primary hepatocytes with oleate induces insulin resistance through p38 mitogen-activated protein kinase.J Biol Chem. 2007; 282: 14205-14212Crossref PubMed Scopus (67) Google Scholar showed no protective effect (Figure 5D). These results suggest that the ASK1–JNK pathway, but not p38, contributes to direct APAP-induced hepatocellular damage. APAP-induced liver injury is thought to consist of direct hepatocellular damage by APAP and a subsequent inflammatory response.6Blazka M.E. Wilmer J.L. Holladay S.D. et al.Role of proinflammatory cytokines in acetaminophen hepatotoxicity.Toxicol Appl Pharmacol. 1995; 133: 43-52Crossref PubMed Scopus (300) Google Scholar, 7Liu Z.X. Govindarajan S. Kaplowitz N. Innate immune system plays a critical role in determining the progression and severity
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