Cell Death in the Liver—All Roads Lead to JNK

Abstract

See article on page 165. See article on page 165. The liver performs essential functions related to glucose, protein, and lipid metabolism and is responsible for the detoxification and excretion of various endogenous and exogenous substances. Due to its involvement in a large number of biological processes and its inherent exposure to food-derived toxins and pathogens, liver parenchyma has a high likelihood to sustain irreversible damage leading to cell death. The liver is well equipped to face most of these challenges and possesses an amazing ability to regenerate after loss of functional mass. Moreover, hepatocytes contain high levels of the antioxidant glutathione (GSH) and are thus protected against damage from a variety of toxins. The ingestion of hepatotoxins through food sources has become a relatively rare reason for acute liver failure in modern society, but drug-induced liver toxicity is a common problem. Overdoses of acetaminophen (AAP), an effective analgesic and antipyretic drug, are the most common causes of drug-induced liver failure in the United States.1Lee W.M. Acute liver failure in the United States.Semin Liver Dis. 2003; 23: 217-226Crossref PubMed Scopus (323) Google Scholar While AAP is a very safe drug at therapeutic doses, overdoses of AAP lead to the generation of high amounts of the toxic metabolite N-acetyl-quinoneimine (NAPQI) by CYP2E1. NAPQI binds to and depletes GSH and, after GSH depletion, forms adducts with thiol-containing proteins.2Jaeschke H. Bajt M.L. Intracellular signaling mechanisms of acetaminophen-induced liver cell death.Toxicol Sci. 2006; 89: 31-41Crossref PubMed Scopus (436) Google Scholar GSH depletion and formation of protein adducts are key mechanisms of AAP-induced cell death that lead to increased generation of mitochondrial reactive oxygen species (ROS), mitochondrial permeability transition (MPT), pore formation in the outer mitochondrial membrane by proapoptotic Bcl-2 family members, and mitochondrial release of the proapototic factors cytochrome c and Smac.2Jaeschke H. Bajt M.L. Intracellular signaling mechanisms of acetaminophen-induced liver cell death.Toxicol Sci. 2006; 89: 31-41Crossref PubMed Scopus (436) Google Scholar Cell death induced by AAP is predominantly necrotic and localized in zone III due to high expression of CYYP2E1 in hepatocytes of this zone. In later phases, there is additional immune-mediated liver damage that is primarily mediated by LPS-dependent Kupffer cell activation.3Su G.L. Gong K.Q. Fan M.H. Kelley W.M. Hsieh J. Sun J.M. Hemmila M.R. Arbabi S. Remick D.G. Wang S.C. Lipopolysaccharide-binding protein modulates acetaminophen-induced liver injury in mice.Hepatology. 2005; 41: 187-195Crossref PubMed Scopus (45) Google Scholar In this issue, Gunawan et al4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar demonstrate that pharmacological inhibition or genetic ablation of JNK protects mice from AAP-induced liver failure. Mice treated with the JNK inhibitor SP600125 not only displayed strikingly reduced ALT levels, but were also completely protected from AAP-induced mortality, whereas vehicle-treated mice suffered 60% mortality after 48 hours. The authors convincingly show that SP600125-mediated protection was not caused by inhibition of AAP metabolism, TNF production, or immune responses.4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar JNK has emerged as a key proapoptotic mediator in cell death induced by a variety of stimuli. The precise mechanism by which JNK promotes cell death is still being investigated and probably depends on stimulus and cell type, but a prolonged activation of JNK seems to be a common requirement.5Schwabe R.F. Brenner D.A. Mechanisms of liver injury. I. TNF-alpha-induced liver injury: role of IKK, JNK, and ROS pathways.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G583-G589Crossref PubMed Scopus (590) Google Scholar Although JNK plays a major role in the regulation of AP-1 dependent gene transcription through its target c-Jun, it has been shown that JNK-mediated proapoptotic effects in hepatocytes are independent of c-Jun and transcription.6Schwabe R.F. Uchinami H. Qian T. Bennett B.L. Lemasters J.J. Brenner D.A. Differential requirement for c-Jun NH2-terminal kinase in TNFalpha- and Fas-mediated apoptosis in hepatocytes.FASEB J. 2004; 18: 720-722Crossref PubMed Scopus (135) Google Scholar Recent evidence points toward the ubiquitin E3 ligase Itch as JNK target during cell death in hepatocytes.7Chang L. Kamata H. Solinas G. Luo J.L. Maeda S. Venuprasad K. Liu Y.C. Karin M. 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 (594) Google Scholar Prolonged activation JNK induces Itch-dependent c-Flip(L) ubiquitination and degradation. Decreased levels of the caspase inhibitor c-Flip(L) then allow for the activation of upstream caspases and initiation of the mitochondrial death program.7Chang L. Kamata H. Solinas G. Luo J.L. Maeda S. Venuprasad K. Liu Y.C. Karin M. 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 (594) Google Scholar Alternative mechanisms by which JNK induces cell death include the phosphorylation and inactivation of protective Bcl-2 family members,8Fan M. Goodwin M. Vu T. Brantley-Finley C. Gaarde W.A. Chambers T.C. Vinblastine-induced phosphorylation of Bcl-2 and Bcl-XL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade.J Biol Chem. 2000; 275: 29980-29985Crossref PubMed Scopus (275) Google Scholar, 9Uehara T. Bennett B. Sakata S.T. Satoh Y. Bilter G.K. Westwick J.K. Brenner D.A. JNK mediates hepatic ischemia reperfusion injury.J Hepatol. 2005; 42: 850-859Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar as well as the activation of the proapoptotic Bcl2-family member Bid.10Deng Y. Ren X. Yang L. Lin Y. Wu X. A JNK-dependent pathway is required or TNFalpha-induced apoptosis.Cell. 2003; 115: 61-70Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar The question arises as to where JNK acts in AAP-induced hepatocyte death and whether this is a pathway shared with other forms of JNK-dependent cell death. The role of apoptosis in AAP-mediated cell death is highly controversial and most experimental evidence suggests that although AAP-induced apoptosis can be readily observed in hepatocytes in vitro, it only occurs in a very small percentage of hepatocytes in vivo. However, there is evidence that AAP-induced cell death either utilizes the same signaling components as apoptosis or starts as apoptosis that is later aborted due to ATP depletion and changes into necrosis.11Kon K. Kim J.S. Jaeschke H. Lemasters J.J. Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes.Hepatology. 2004; 40: 1170-1179Crossref PubMed Scopus (392) Google Scholar The fact that JNK inhibition is effective even at later phases of AAP toxicity appears to be evidence against JNK being an upstream mediator of early proapoptotic signals that trigger necrotic cell death. While JNK activation was only observed 6 hours after AAP, severe GSH depletion was already observed 3 hours after AAP.4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar However, it is unlikely that cells were massively depleted of ATP 6 hours after AAP, because JNK activation and activity both require ATP, and SP600125 functions as an ATP-competitive inhibitor. Gunawan et al4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar show that JNK inhibition prevents AAP-induced mitochondrial translocation of Bax. These findings are in line with the current concept that mitochondrial signals are key mechanisms in AAP-mediated cell death as demonstrated by the ability of MPT inhibitors to prevent AAP-mediated cell death in vitro and in vivo.11Kon K. Kim J.S. Jaeschke H. Lemasters J.J. Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes.Hepatology. 2004; 40: 1170-1179Crossref PubMed Scopus (392) Google Scholar, 12Beales D. McLean A.E. Protection in the late stages of paracetamol-induced liver cell injury with fructose, cyslosporin A and trifluoperazine.Toxicology. 1996; 107: 201-208Crossref PubMed Scopus (24) Google Scholar Thus, it is likely that JNK exerts its effects in AAP-induced apoptosis upstream of mitochondria and that these effects are independent of the classical JNK target c-Jun. It is possible that cell death promoting factors upstream of the mitochondria need to reach a threshold, which is only achieved after several hours of AAP exposure and that JNK shifts the balance toward cell death during this interval. While such a scenario is likely, further experimental evidence is required to define the targets of JNK in AAP-induced liver failure and to demonstrate the inability of JNK inhibitors to block AAP-induced liver failure in the absence of these targets, eg, in Bax-deficient mice. Gunawan et al4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar also have not investigated the mechanism by which AAP activates JNK. Recent evidence points to ROS-mediated inhibition of JNK phosphatases as a main mechanism of ROS-induced MAP kinase activation.13Kamata H. Honda S. Maeda S. Chang L. Hirata H. Karin M. Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases.Cell. 2005; 120: 649-661Abstract Full Text Full Text PDF PubMed Scopus (1504) Google Scholar However, it is possible that AAP activates JNK through other or additional mechanisms, eg, direct inactivation of JNK phosphatases by NAPQI. The regulation of cell death in the hepatocyte is a common theme in many areas of liver research. While massive acute hepatocyte death, eg, after acetaminophen intoxication is life-threatening, chronic hepatocyte death occurs in a large number of patients, eg, those with viral hepatitis and nonalcoholic and alcoholic fatty liver disease and activates signals that promote the development of hepatic fibrosis and hepatocellular carcinoma. Among the many pathways that are activated in dying hepatocytes, JNK stands out not only because it is activated by the majority of cell death-inducing stimuli including TNFα, bile-acids, ischemia-reperfusion, acetaminophen, and concanavalin A,6Schwabe R.F. Uchinami H. Qian T. Bennett B.L. Lemasters J.J. Brenner D.A. Differential requirement for c-Jun NH2-terminal kinase in TNFalpha- and Fas-mediated apoptosis in hepatocytes.FASEB J. 2004; 18: 720-722Crossref PubMed Scopus (135) Google Scholar, 14Trautwein C. Rakemann T. Brenner D.A. Streetz K. Licato L. Manns M.P. Tiegs G. Concanavalin A-induced liver cell damage activation of intracellular pathways triggered by tumor necrosis factor in mice.Gastroenterology. 1998; 114: 1035-1045Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 15Matsumaru K. Ji C. Kaplowitz N. Mechanisms for sensitization to TNF-induced apoptosis by acute glutathione depletion in murine hepatocytes.Hepatology. 2003; 37: 1425-1434Crossref PubMed Scopus (136) Google Scholar, 16Bradham C.A. Stachlewitz R.F. Gao W. Qian T. Jayadev S. Jenkins G. Hannun Y. Lemasters J.J. Thurman R.G. Brenner D.A. Reperfusion after liver transplantation in rats differentially activates the mitogen-activated protein kinases.Hepatology. 1997; 25: 1128-1135Crossref PubMed Scopus (188) Google Scholar, 17Graf D. Kurz A.K. Fischer R. Reinehr R. Haussinger D. Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner.Gastroenterology. 2002; 122: 1411-1427Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar but also because it is required for the induction of cell death by these stimuli. Pharmacological or genetic JNK inhibition has been shown to be effective in animal studies of TNFα-mediated cell death, concanavalin A-mediated hepatitis, ischemia-reperfusion injury, and liver transplantation.7Chang L. Kamata H. Solinas G. Luo J.L. Maeda S. Venuprasad K. Liu Y.C. Karin M. 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 (594) Google Scholar, 9Uehara T. Bennett B. Sakata S.T. Satoh Y. Bilter G.K. Westwick J.K. Brenner D.A. JNK mediates hepatic ischemia reperfusion injury.J Hepatol. 2005; 42: 850-859Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 18Maeda S. Chang L. Li Z.W. Luo J.L. Leffert H. Karin M. 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 (265) Google Scholar, 19Uehara T. Xi Peng X. Bennett B. Satoh Y. Friedman G. Currin R. Brenner D.A. Lemasters J. c-Jun N-terminal kinase mediates hepatic injury after rat liver transplantation.Transplantation. 2004; 78: 324-332Crossref PubMed Scopus (81) Google Scholar, 20Wang Y. Singh R. Lefkowitch J.H. Rigoli R.M. Czaja M.J. TNF-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway.J Biol Chem. 2006; 281: 15258-15267Crossref PubMed Scopus (188) Google Scholar Based on these studies, inhibition of JNK appears to be a feasible therapeutic strategy to reduce hepatic cell death in clinical settings in the future. There is still considerable uncertainty regarding the relative role of JNK1 and JNK2 in the regulation of cell death. It has been shown that JNK1 mediates the majority of c-Jun phosphorylation, whereas JNK2 mainly regulates c-Jun stability.21Sabapathy K. Hochedlinger K. Nam S.Y. Bauer A. Karin M. Wagner E.F. Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation.Mol Cell. 2004; 15: 713-725Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar Although several studies showed that JNK1, but not JNK2 is the main mediator of TNF-induced cell death,7Chang L. Kamata H. Solinas G. Luo J.L. Maeda S. Venuprasad K. Liu Y.C. Karin M. 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 (594) Google Scholar, 22Liu J. Minemoto Y. Lin A. c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, is essential for tumor necrosis factor alpha-induced c-Jun kinase activation and apoptosis.Mol Cell Biol. 2004; 24: 10844-10856Crossref PubMed Scopus (172) Google Scholar a recent study concluded that JNK2 plays a more prominent role than JNK1 in TNF-dependent liver injury.20Wang Y. Singh R. Lefkowitch J.H. Rigoli R.M. Czaja M.J. TNF-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway.J Biol Chem. 2006; 281: 15258-15267Crossref PubMed Scopus (188) Google Scholar Gunawan et al4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar found JNK2 to be more important than JNK1 in AAP-mediated hepatocyte cell death, but that JNK1 may to some degree substitute for JNK2.4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar It is reasonable to assume that there may be some redundancy between JNK1 and JNK2 in regard to induction of cell death, and that pan-JNK inhibitors will most likely be more efficient than specific JNK1 or JNK2 inhibitors for the treatment of liver disease. The fact that liver injury was reduced by SP600125 6 hours after AAP at a time point when GSH depletion already occurred4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar suggests that JNK inhibition may have applications in patients with AAP overdose, eg, as treatment in addition to N-acetylcysteine. An important issue to be determined is potential downsides of JNK inhibition. JNK activity is required for hepatocyte proliferation and liver regeneration after partial hepatectomy.23Schwabe R.F. Bradham C.A. Uehara T. Hatano E. Bennett B.L. Schoonhoven R. Brenner D.A. c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration.Hepatology. 2003; 37: 824-832Crossref PubMed Scopus (211) Google Scholar Thus, inhibition of JNK during hepatic injury might hamper the ability of the liver to regenerate. At least during the first 24 to 48 hours, inhibition of JNK-mediated hepatocyte proliferation does not appear to be an issue in AAP-induced liver injury, since survival was markedly increased after pharmacological or genetic JNK inhibition.4Gunawan B.K. Lu Z.-X. Han D. Hanawa N. Gaarde W.A. Kaplowitz N. 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 (360) Google Scholar However, later time points were not investigated in this study. In other forms of cell death, eg, chronic mild liver injury induced by alcohol or viral hepatitis, inhibiting JNK may turn out to be shooting 2 birds with one stone by (1) reducing hepatocyte cell death and (2) blocking compensatory hepatocyte proliferation that leads to hepatocarcinogenesis. In conclusion, inhibition of JNK activity may be a novel option to block cell death in the liver, and further investigations of AAP-induced liver failure may help us to understand the precise role of JNK in the regulation of hepatocyte cell death and to determine whether JNK inhibition may have a role in patients with liver disease. c-Jun N-Terminal Kinase Plays a Major Role in Murine Acetaminophen HepatotoxicityGastroenterologyVol. 131Issue 1PreviewBackground & Aims: In searching for effects of acetaminophen (APAP) on hepatocytes downstream of its metabolism that may participate in hepatotoxicity, we examined the role of stress kinases. Methods: Mouse hepatocytes and C57BL/6 mice were administered a toxic dose of APAP with or without SP600125, a chemical c-jun N-terminal kinase (JNK) inhibitor. JNK activity as reflected in phospho-c-jun levels, serum alanine transaminase (ALT), and liver histology were assessed. Similar experiments were repeated in JNK1 and JNK2 knockout mice and by using antisense oligonucleotide (ASO) to knockdown JNK. Full-Text PDF