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Loss of Caspase-8 Protects Mice Against Inflammation-Related Hepatocarcinogenesis but Induces Non-Apoptotic Liver Injury
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BASIC AND TRANSLATIONAL—LIVER Loss of Caspase-8 Protects Mice Against Inflammation-Related Hepatocarcinogenesis but Induces Non-Apoptotic Liver Injury CHRISTIAN LIEDTKE,* JÖRG–MARTIN BANGEN,* JULIA FREIMUTH,* NAIARA BERAZA,* DANIELA LAMBERTZ,* FRANCISCO J. CUBERO,* MAXIMILIAN HATTING,* KARLIN R. KARLMARK,* KONRAD L. STREETZ,* GABRIELE A. KROMBACH,‡ FRANK TACKE,* NIKOLAUS GASSLER,§ DIETER RIETHMACHER,储 and CHRISTIAN TRAUTWEIN* *Department of Medicine III, University Hospital, Aachen, Germany; ‡Department of Radiology, Justus-Liebig University, Giessen, Germany; §Institute of Pathology, University Hospital Aachen, Germany; and 储Human Genetics Division, University of Southampton, Southampton General Hospital, Southampton, United Kingdom
See editorial on page 1969.
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BACKGROUND & AIMS: Disruption of the nuclear factor-B (NF-B) essential modulator (NEMO) in hepatocytes of mice (NEMO⌬hepa mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO⌬hepa mice or after induction of acute liver injury. METHODS: We created mice with conditional deletion of Casp8 in hepatocytes (Casp8⌬hepa) and Casp8⌬hepaNEMO⌬hepa double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS: Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8⌬hepaNEMO⌬hepa mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3—these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS: Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment. Keywords: Fas-associated Protein With Death Domain; FADD; Inflammation; Tumor Necrosis Factor; TNF Signaling.
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wo mechanisms, apoptosis and necrosis, are frequently involved in acute and chronic liver injury. Apoptosis is a precisely regulated and genetically determined process that can be induced via death receptormediated, extrinsic pathways or through intrinsic mechanisms activated by intracellular stress.1 The cystein-aspartate protease caspase-8 (Casp8) is the apical initiator caspase in death receptor-mediated apoptosis and constitutively expressed as a premature zymogene. After death receptor activation by its cognate ligand, the formation of a death-inducing signaling complex consisting of a death receptor, the adaptor protein Fas-associated protein with death domain (FADD), and procaspase-8 is induced leading to activation of Casp8 by an autocatalytic process. Casp8 can in turn activate downstream effector caspases such as caspase-3 or—like in hepatocytes—activate intrinsic apoptosis signalling.2 Casp8 is essential for death receptor-mediated apoptosis. Although constitutive disruption of the murine Casp8 gene is embryonically lethal because of impaired heart muscle development and hyperemia, Casp8-deficient murine embryonic fibroblasts were shown to be protected from cell death induced by Fas, tumor necrosis factor-related apoptosis inducing ligand (TRAIL), or tumor necrosis factor (TNF).3 TNF is a pleiotropic cytokine and does not only induce apoptosis but predominantly triggers antiapoptotic and proinflammatory signals via NF-B or alternatively activates the c-Jun N-terminal protein kinase (JNK) pathway.4 Abbreviations used in this paper: Casp8, caspase-8; ConA, Concanavalin A; FADD, Fas-associated protein with death domain; GalN, D-galactosamine; HCC, hepatocellular carcinoma; IKK, inhibitor of nuclear factor-B kinase; JNK, c-Jun N-terminal protein kinase; LPS, lipopolysaccharides; MRI, magnetic resonance imaging; NEMO, NF-B essential modulator; NF-B, nuclear factor-B; RIP, receptor-interacting protein; TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, tumor necrosis factor receptor type 1-associated death domain protein; TRAF, TNF receptor-associated factor; TRAIL, Tumor Necrosis Factor-Related Apoptosis Inducing Ligand; TUNEL, terminal desoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling; WT, wild type. © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2011.08.037
Following TNF binding, recruitment of the adaptor protein tumor necrosis factor receptor type 1-associated death domain protein (TRADD) to the TNF receptor 1 (TNFR1) is a key event for activation of at least 3 different, in part opposing, signaling cascades: Association of TRADD with the receptor interacting protein (RIP) 1 and TNF receptor-associated factor (TRAF) 2 (complex I) activates the inhibitor of nuclear factor-B kinase (IKK) complex (IKK␣, IKK, and IKK␥/NF-B essential modulator [NEMO]), which initiates NF-B activation.5 The regulatory subunit IKK␥/NEMO is crucial for NF-B activation. Constitutive disruption of the NEMO gene in vivo results in complete NF-B inhibition and embryonic lethality.6 Hepatocyte-specific deletion of NEMO and subsequent NF-B inhibition results in chronic liver apoptosis promoting spontaneous steatohepatitis, fibrosis progression, and development of hepatocellular carcinoma (HCC).7 Association of TRADD and TRAF2 can also activate JNK signaling pathways.8 Alternatively, after TNF binding TNFR1/TRADD can associate with the adaptor protein FADD and pro-caspase-8 (complex II) leading to complex internalization and apoptosis induction.9 The immediate response to TNF stimulation is usually dominated by proinflammatory and antiapoptotic NF-B target genes In contrast, inhibition of NF-B in the liver, eg, by blocking gene transcription with D-galactosamine (GalN) or by genetic ablation, triggers Casp8-mediated apoptosis because of down-regulation of antiapoptotic genes.10 Necrosis has classically been viewed as an accidental and unregulated form of cell death. However, recent findings suggested that TNF is also able to induce tightly regulated cell necrosis if caspase activity is blocked. This phenomenon has been termed programmed necrosis and depends on synergistic action of receptor-interacting serine threonine kinases RIP1 and RIP3, respectively.11,12 Recent data demonstrate that Casp8 is essential for preventing RIP-mediated necrosis.13 However, the physiologic role of programmed necrosis for the liver is poorly understood. In the present study, we investigated potential benefits of hepatocyte-specific Casp8 ablation in mouse models of acute or chronic liver disease. We demonstrate that Casp8 inactivation protects from liver apoptosis but triggers necrotic cell death in the Concanavalin A (ConA) model of T-cell hepatitis and in NEMO-deficient mice.
Materials and Methods Housing and Breeding of Mice All animals were maintained in the animal facility of the University Hospital Aachen in a temperature-controlled room with 12-hour light/dark cycle. Animal husbandry and procedures were approved by the authority for environment conservation and consumer protection of the state North Rhine-Westfalia (LANUV, Germany). We used mice of male sex carrying a hepatocyte-specific deletion (⌬hepa) of Casp8 and/or NEMO genes (Casp8⌬hepa; NEMO⌬hepa; Casp8⌬hepaNEMO⌬hepa) and crenegative littermates (Casp8f/f; NEMOf/f; Casp8f/fNEMOf/f) as controls. Generation of Casp8⌬hepa mice is described in the Supplementary Materials and Methods section. Animals were kept under specific pathogen-free conditions after embryo transfer.
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Animal Models of Liver Injury The Fas-activating antibody Jo2 was obtained from BD Bioscience (San Jose, CA) and administrated intravenously at a concentration of 0.5 g/g of body weight. ConA, lipopolysaccharide (LPS), and GalN were from Sigma-Aldrich (St. Louis, MO). ConA was administered intravenously (25 mg/kg of body weight) alone, or 1 hour after intraperitoneal injection of GalN (1000 mg/kg). Similarly, for induction of septic liver injury, mice were first injected intraperitoneally with 1000 mg/kg GalN 1 hour prior to LPS administration (750 mg/kg, intraperitoneally).
Magnetic Resonance Imaging of Murine HCC To monitor progression of HCC in NEMO⌬hepa and mice, animals were investigated by magnetic resonance imaging (MRI) using an Achieva 1.5-Tesla Scanner MR System (Philips Medical Systems, Best, The Netherlands) as recently described.14 A 47-mm-diameter coil (Philips Medical Systems) was used for signal reception. For contrast enhancement, 0.025-mmol/kg body weight gadoxetic acid disodium (Primovist; Schering, Berlin, Germany) was administered by tail vein injection using a catheter with injection port (Biovalve, 22 gauge; 1.0 ⫻ 25 mm; Vygon, Ecouen, France).
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Statistical Analysis Data are expressed as mean ⫾ standard deviation of the mean. Statistical significance was determined by 2-way analysis of variance followed by a Student t test.
Results Casp8⌬hepa Mice Show Basal Liver Inflammation and Are Protected From Fasand LPS-Mediated Liver Injury Constitutive Casp8 knockout mice die in utero of impaired heart muscle development.3 Therefore, we generated hepatocyte-specific conditional Casp8 knockout mice (Casp8⌬hepa) using the cre/loxP system (Supplementary Figure 1A and B). Casp8⌬hepa mice did not show morphologic differences in comparison with their wildtype (WT) (Casp8f/f) littermates (Supplementary Figure 1C). Efficient, cre-mediated deletion of Casp8 exons III-IV was confirmed on the genomic DNA and protein level using total liver samples or primary hepatocytes (Supplementary Figure 1D). Liver histology of Casp8⌬hepa mice revealed small inflammatory infiltrates of granulocytes and monocytes and increased serum transaminases (Figure 1A), suggesting that ablation of Casp8 in hepatocytes results in moderate basal liver inflammation. As a proof of principle, we investigated whether Casp8⌬hepa mice are protected from liver apoptosis in 2 established models of acute liver injury by administrating either the Fas-activating antibody Jo2 (Figure 1B) or a combination of lipopolysaccharide/D-galactosamine (LPS/GalN, Figure 1C). As expected, in both models, control mice showed strong increase in serum transaminases, strong apoptosis as shown by caspase-3 activation and terminal desoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and approximately 90% mortality within 24 hours. In contrast, 100% of Casp8⌬hepa mice survived Jo2 and LPS/GalN treatment without indications of liver apoptosis.
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Figure 1. Casp8⌬hepa mice show moderate basal liver inflammation and are protected from Fas- and LPS-mediated liver injury. (A) H&E staining of liver sections and basal serum transaminases from untreated controls (Casp8f/f) and Casp8⌬hepa mice. Arrow: focal basal infiltration of inflammatory cells. (B and C) Control animals (Casp8f/f) and Casp8⌬hepa mice were injected with (B) Fas-activating antibody Jo2 or (C) LPS and GalN. Liver injury was determined by measurement of serum transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), caspase-3 activity from murine liver extracts, survival within 24 hours, and TUNEL staining of liver sections. TUNEL-positive apoptotic cells are stained in green. **P ⬍ .01; ***P ⬍ .001.
Casp8 Deletion Triggers Enhanced Nonapoptotic Liver Injury During ConAInduced Hepatitis The lectin ConA is a T-cell mitogen and rapidly induces liver injury involving CD4⫹ T cells, natural killer T cells, and macrophages/Kupffer cells.15 The mode of ConA-mediated cell death is controversial, whereas cotreatment with ConA and GalN was shown to induce a predominant proapoptotic response.16 Unexpectedly, Casp8⌬hepa mice showed impaired survival (Figure 2A) and increased aspartate aminotransferase and alanine aminotransferase levels in comparison with Casp8f/f controls after treatment with sublethal ConA dose. This outcome was completely reverted by concomitant application of GalN resulting in further increase of serum transaminases in the control group in contrast to
remarkable attenuation of injury in Casp8⌬hepa mice (Figure 2B). Opposing effects of ConA vs ConA/GalN were confirmed in liver histology and TUNEL analysis (Figure 2C). Following ConA treatment, Casp8⌬hepa livers revealed increased hepatocyte injury and a striking staining pattern of abundant TUNEL-positive hepatocytes with focal concentration, which was not evident in the control group. However, in the ConA/GalN model, we observed only moderate cell death of single hepatocytes and few TUNEL-positive cells in Casp8⌬hepa livers, whereas the control group revealed strong hepatocellular damage of grouped hepatocytes with morphologic aspects of apoptosis-related cell death and high number of TUNEL-positive cells. To determine further the impact of apoptosis in both injury models, caspase-3 activity was measured in
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Figure 2. Casp8 deletion does not protect from ConA mediated hepatitis. Casp8f/f control mice and Casp8⌬hepa animals were injected with ConA or ConA and GalN where indicated. (A) Kaplan–Maier survival curves for ConA-treated animals. (B) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum transaminase levels. (C) H&E staining (original magnification, ⫻400) and TUNEL assay of liver sections 7 hours after ConA or ConA/GalN treatment; dying cells in the TUNEL assay are stained in green. Dying hepatocytes with morphologic signs of apoptosis are highlighted by arrows. (D) Comparison of caspase-3 activity in ConA-treated vs ConA/GalN-treated mice. Activity is shown as fold induction compared with untreated controls. (E) Reactive oxygen species were assayed by measuring malondialdehyde in liver homogenates. Untreated Casp8f/f livers served as reference (ctrl). *P ⬍ .05; ***P ⬍ .001.
Casp8⌬hepa mice and controls after treatment with ConA or ConA/GalN (Figure 2D). ConA treatment alone triggered only minor caspase-3 activation in both Casp8f/f and Casp8⌬hepa mice, suggesting that apoptosis in the ConA model is Casp8-independent and contributes only little to liver injury. However, combined ConA/GalN treatment remarkable activated caspase-3 in control mice, but not in Casp8⌬hepa animals. Instead, Casp8⌬hepa livers produce a higher amount of reactive oxygen species after ConA
treatment as evidenced by increased levels of malondialdehyde (Figure 2E). Thus, ablation of Casp8 may trigger enhanced nonapoptotic injury via increased oxidative stress in the ConA model. Additionally, we detected slightly increased TNF and interferon-␥ expression in Casp8⌬hepa mice after ConA treatment (Supplementary Figure 2A). In line with this finding, already 2 hours after ConA treatment Casp8⌬hepa livers showed increased infiltration of CD11b⫹F4/80⫹
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macrophages (Supplementary Figure 2B and C), suggesting that Casp8⌬hepa mice have an accelerated immune response after ConA, which might affect downstream cytokine signalling.
Increased ConA-Mediated Liver Injury in Casp8⌬Hepa Mice Is Associated With Aberrant TNF Signaling TNF activates at least 3 different pathways via TNFR1 triggering apoptosis through Casp8 or inflammation via NF-B or JNK/p38 mitogen-activated protein kinases. In addition, TNF may also induce necrosis via RIP1/RIP3-dependent mechanisms (Supplementary Figure 3). We thus systematically analyzed TNF signaling in ConA-treated Casp8⌬hepa mice. We detected aberrant high basal RIP1 expression and further up-regulation in Casp8⌬hepa livers upon ConA stimulation, which was less evident in controls (Figure 3A). RIP3 expres-
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sion was barely regulated by ConA in either group, whereas overall RIP3 expression in Casp8⌬hepa mice was slightly reduced. However, FADD expression was regulated during ConA-induced hepatitis showing accelerated induction in Casp8⌬hepa mice correlating with earlier onset of injury. Immunoprecipitation experiments revealed that in ConA-treated Casp8⌬hepa but not in control livers, a FADD-RIP1-RIP3 complex is formed. RIP3 was constitutively associated with FADD in both untreated and ConA-treated Casp8⌬hepa livers (Figure 3A) demonstrating that expression and localization of RIP1—in contrast to RIP3—is differentially controlled in Casp8⌬hepa mice. FADD-RIP1-RIP3 complexes precipitated from Casp8⌬hepa livers revealed kinase activity as they efficiently phosphorylated the substrate myelin basic protein (Figure 3B). Interestingly, RIP1 and RIP3 were colocalized in the cytoplasm of ConA-treated
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Figure 3. ConA treatment induces aberrant TNF signaling and the formation of RIP1-RIP3-FADD complexes in Casp8⌬hepa mice. ConAtreated Casp8f/f and Casp8⌬hepa mice were analyzed at time points indicated. (A) Upper part: Western blot analysis of total protein levels for RIP1, RIP3, and the adaptor protein FADD after ConA treatment. GAPDH was detected as loading control. Lower part: Immunoprecipitation (IP) of FADD complexes; recruitment of RIP1 and RIP3 to FADD was examined by Western blot. (B) Four hours after ConA treatment, FADD complexes were immunoprecipitated (IP:FADD) and subjected to in vitro kinase assay using myelin basic protein (MBP) as artificial substrate. (C) Coimmunofluorescence staining for RIP1 and RIP3 in liver sections from Casp8f/f and Casp8⌬hepa mice 7 hours after ConA treatment. Green: RIP1-positive cells; Red: RIP3-positive cells. Signals in orange indicate colocalization of RIP1 and RIP3. Sections are counterstained with DAPI (blue). (D) Western blot analysis of phosphorylated JNK1/JNK2 and p-cJun. Pro-Casp8 expression was determined to monitor knockout efficiency. (E) Analysis of NF-B activation. Upper part: EMSA analysis with NF-B consensus sequence and nuclear extracts as indicated. FP, free probe. Signals for active p50 homodimers and p50/p65 heterodimers are indicated by arrows. Supershift: WT sample (Casp8f/f) 2 hours after ConA treatment was incubated with antibodies against NF-B subunits p50 or p65, respectively. Lower part: Cellular localization of NF-B p65 was investigated by Western blot analysis of cytoplasmic (p65 cyt) and nuclear (p65 nuc) liver protein extracts. Loading controls: GAPDH (cytoplasmic extracts [GAPDH cyt]); TBP (nuclear extracts [TBP nuc]). (F) Quantitative real-time PCR for IB-␣.
Casp8⌬hepa liver and showed strong focal expression along with injured tissue, which was not evident in control livers (Figure 3C). Ablation of Casp8 also resulted in prolonged JNK activation after ConA delivery, whereas phosphorylation of its target c-Jun was even diminished in Casp8⌬hepa mice (Figure 3D). In parallel, ConA also induced prolonged NF-B activation and nuclear localization in Casp8⌬hepa mice (Figure 3E), resulting in increased up-regulation of the NF-B target gene IB-␣ (Figure 3F).
Simultaneous Ablation of Casp8 and NEMO in Liver Parenchymal Cells Results in Massive Necrosis and Cholestasis The contrasting functions of Casp8 upon acute liver injury prompted us to investigate its role in the NEMO⌬hepa model, which recapitulates human chronic liver disease progression in a unique manner. NEMO⌬hepa mice show strong basal inflammation, spontaneous apoptosis, steatosis, liver fibrosis, and, finally, develop HCC.7,17,18 We thus investigated whether inactivation of Casp8 in NEMO⌬hepa mice attenuates progression of liver disease. Unexpectedly, 6- to 8-week-old Casp8⌬hepaNEMO⌬hepa mice revealed a complex liver phenotype with bile inclusions and infarcts as well as hepatomegaly. The penetrance of this phenotype showed variations reflecting differences in disease severity. Thus, Casp8⌬hepaNEMO⌬hepa livers were grouped dependent on the strength of the phenotype (types I, II, and III). From Casp8⌬hepaNEMO⌬hepa mice initially investigated (n ⫽ 43), 34% were classified as type I and revealed few white liver lesions (Figure 4A) that were identified as necrotic areas (Figure 4B and Supplementary Figure 4B). The majority (40%) of mice was designated type II defined by multilocular, macroscopically visible white liver lesions displaying histomorphologic features of parenchymal necroses or bile infarcts (Figure 4A and B and Supplementary Figure 4C). These animals had normal body size (Figure 4C) but significantly increased liver-to-body weight ratio compared with WT controls (Figure 4D). Type III livers (26%) displayed yellow appearance, increased organ size, and multilocular yellow lesions, resulting in massive necrotic destruction of the liver associated with multiple bile infarcts (Figure 4A and B, Supplementary Figure 4D). In addition, the animals were characterized by dwarfism in combination with hepatomegaly leading to an abnormal liver-to-body weight ratio of 9.3% (Figure 4C and D). Of notice, the severity of liver necrosis in Casp8⌬hepaNEMO⌬hepa livers correlated with increasing numbers of CD11b-positive cells specifically localized at necrotic foci (Supplementary Figure 5A and B). Types II and III Casp8⌬hepaNEMO⌬hepa mice showed increased serum transaminase levels, and no significant difference was found compared with NEMO⌬hepa mice (Figure 4E). Alkaline phosphatase levels best differentiated among the 3 phenotypes and were elevated in close correlation to the severity of liver injury (Figure 4E). Accordingly, type III mice had strongly and significantly increased bile acid and bilirubin levels in comparison with
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both Casp8⌬hepa and NEMO⌬hepa mice. Thus, combined hepatocyte-specific Casp8 and NEMO ablation triggers massive liver necrosis and cholestasis, which was not observed in either single knockout strain. Similar to our observations in Fas-induced acute liver injury, ablation of Casp8 rescued spontaneous liver apoptosis of NEMO⌬hepa livers because no TUNEL-positive cells were found in type I livers (Supplementary Figure 5(C). In contrast, in necrotic foci of type II-III livers, TUNEL-positive signals were found reflecting apoptosis of nonparenchymal cells. Additionally, increased cell proliferation was evident in all Casp8⌬hepaNEMO⌬hepa livers as evidenced by measuring BrdU incorporation (Supplementary Figure 5D). Costainings of proliferation- (Ki67) and hepatocyte-specific markers (HNF4␣) excluded hepatocyte proliferation and suggest that bile duct cells (Supplementary Figure 5E) proliferate in Casp8⌬hepaNEMO⌬hepa livers.
FADD-RIP1-RIP3 Kinase Complexes Are Formed Spontaneously in Casp8⌬HepaNEMO⌬Hepa Mice and Localize at Foci of Necrotic Liver Injury Disease severity in Casp8⌬hepaNEMO⌬hepa livers correlated with increasing basal RIP1 and FADD expression showing highest levels in type III mice. Interestingly, basal RIP1 expression in NEMO⌬hepa mice was significantly lower compared with WT controls (Figure 5A). In Casp8⌬hepaNEMO⌬hepa livers, but not in Casp8⌬hepa or NEMO⌬hepa mice, FADD constitutively formed complexes with both RIP1 and RIP3 as evidenced by immunoprecipitation experiments (Figure 5A). These complexes had kinase activity that was proportional to the strength of liver injury and thus strongest in type III and barely detectable in type I livers (Figure 5B). RIP1 can be proteolytically degraded by Casp8 resulting in a cleavage product of approximately 38 kilodaltons.19 Accordingly, we detected cleaved RIP1 in untreated NEMO⌬hepa mice, but not in Casp8⌬hepaNEMO⌬hepa mice (Figure 5C). RIP1 immunhistochemistry revealed that RIP1 is not equally distributed in Casp8⌬hepaNEMO⌬hepa liver tissue but concentrated in foci of intact liver tissue of type I and type II mice or in close proximity to necrotic areas in type III animals (Figure 5D). Importantly, RIP1 colocalized with RIP3 in Casp8⌬hepaNEMO⌬hepa tissue, which was not observed in the control group (Figure 5D), further proving the existence of pronecrotic RIP-FADD complexes at loci of liver injury. The severity of liver injury in Casp8⌬hepaNEMO⌬hepa mice further correlated with the level of JNK activation and subsequent c-Jun phosphorylation (Supplementary Figure 6A and B) suggesting that JNK signaling is also associated with necrosis induction in these animals.
Aging Casp8⌬hepaNEMO⌬hepa Mice Recover From Necrotic Liver Injury and Are Protected From Steatosis but Develop Liver Fibrosis Analysis of age-dependent disease progression of Casp8⌬hepaNEMO⌬hepa mice revealed normal serum lev-
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Figure 4. Simultaneous ablation of Casp8 and NEMO in liver parenchyma results in massive liver necrosis and cholestasis. (A) Macroscopic appearance of male Casp8⌬hepaNEMO⌬hepa livers at the age of 6 – 8 weeks. Organs were classified according to their phenotype as I (macroscopically normal), II (white lesions), or III (yellow lesions, hepatomegaly, dwarfism). Frequency of each phenotype is given in parentheses. Representative lesions are highlighted by arrows. (B) Histologic analysis (H&E) of types I–III livers in comparison with samples from either single knockout (Casp8⌬hepa, NEMO⌬hepa) and WT controls (Casp8f/fNEMOf/f). Original magnification, ⫻200. (C) Macroscopic appearance of type III Casp8⌬hepaNEMO⌬hepa mice (right) showing dwarfism in comparison with type II (middle) and cre-negative (left) littermate controls. (D) Liver mass index of 6to 8-week-old Casp8⌬hepaNEMO⌬hepa mice classified as types I–III. (E) Analysis of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), bile acids, and total bilirubin levels in 6- to 8-week-old Casp8⌬hepaNEMO⌬hepa mice and controls. Serum parameters are given both for total Casp8⌬hepaNEMO⌬hepa mice and subtypes I–III, respectively. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001; n.s., not significant.
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Figure 5. Spontaneous formation of FADD-RIP1-RIP3 kinase complexes in Casp8⌬hepaNEMO⌬hepa mice and localization at foci of necrotic liver injury. Six- to 8-week-old Casp8⌬hepaNEMO⌬hepa mice and controls were investigated for hepatic RIP1 expression, RIP1/RIP3 interaction, and in situ localization. WT, Casp8f/fNEMOf/f controls. (A) Top: Total expression of RIP1 and FADD. Bottom: Protein complexes were isolated by immunoprecipitation with antibodies for FADD (IP:FADD) and subjected to Western blot analysis for RIP1 and RIP3 expression. (B) FADD complexes from total liver extracts were isolated by immunoprecipitation and subjected to radioactive in vitro kinase assay using myelin basic protein (MBP) as substrate. (C) Total RIP1 was enriched by immunoprecipitation (IP:RIP1) and subjected to RIP1 immunoblot. IgG, heavy chain immunoglobulin. (D) Localization of RIP1 and RIP3 in situ. Top: Liver tissue sections from Casp8⌬hepaNEMO⌬hepa mice with different grade of liver injury (types I–III) and WT controls were probed with RIP1 (RIP1 IHC). Encircled areas define foci with strong RIP1 expression. Bottom: Costaining for RIP1 and RIP3 analyzed by immunofluorescence microscopy. Green: RIP1; red: RIP3; yellow: RIP1-RIP3 colocalization.
els for aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and bile acids in juvenile mice at the age of 3 weeks (Figure 6A). Liver injury was maximal at 6 – 8 weeks but continuously ameliorated with aging, indicating that generation of necrosis and cholestasis is restricted to juvenile liver development. Beginning at the age of 25 weeks, serum parameters in Casp8⌬hepaNEMO⌬hepa mice reached almost normal values compared with WT controls, and classification of type I-III mice was no longer possible because of substantially improvement of liver histology (Supplementary Figure 7A). Of notice, despite extensive liver injury of young Casp8⌬hepaNEMO⌬hepa mice, we did not observe increased mortality of these animals compared with controls within 1 year (Figure 6B and Supplementary Figure 7B). We determined the age-dependent efficiency of simultaneous Casp8 and NEMO inactivation. In total liver from 6to 8-week-old Casp8⌬hepaNEMO⌬hepa mice, we unexpectedly
detected substantial levels of full-length Casp8 and NEMO messenger RNA. However, primary hepatocytes from these samples showed almost complete inactivation of Casp8 and NEMO (Figure 6C, top), suggesting that the residual Casp8 and NEMO expression derives from excessive infiltrating and ductular cells. Protein analysis confirmed efficient ablation of Casp8 and NEMO in type I livers but substantial expression of processed Casp8 (44-kilodalton cleavage product) in type II and type III livers (Figure 6C, bottom) potentially because of excessive infiltrating cells. Of notice, in NEMO⌬hepa mice, Casp8 was highly expressed as a preactivated 44-kilodalton protein further highlighting their predisposition to extrinsic apoptosis. In contrast, at the age of 26 weeks, expression of Casp8 and NEMO was completely absent in Casp8⌬hepaNEMO⌬hepa livers. NEMO⌬hepa mice develop spontaneous steatosis and steatohepatitis.7 Deletion of Casp8 in NEMO⌬hepa mice resulted in complete recovery from liver steatosis because they lack extensive fat accumulation and show normal
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Figure 6. Aging Casp8⌬hepaNEMO⌬hepa mice recover from necrotic liver injury and are protected from steatosis but develop liver fibrosis. (A) Age-dependent analysis of serum AST, ALT, alkaline phosphatase (AP), and bile acid levels in Casp8⌬hepaNEMO⌬hepa mice and WT (Casp8f/fNEMOf/f) controls. All animals were included irrespective of their classification in types I–III. Parameters were measured at the age of 3, 8, 12, 26, and 52 weeks. (B) Kaplan–Meier survival curve for Casp8⌬hepaNEMO⌬hepa mice (n ⫽ 474) and WT (Casp8f/fNEMOf/f; n ⫽ 259) controls. (C) Age-dependent efficiency of Casp8 and NEMO inactivation in liver samples and primary hepatocytes from 6- to 8-week-old Casp8⌬hepaNEMO⌬hepa mice was determined by rtPCR (top) or analyzed by Western blot analysis (bottom). Subgroups I–III (6 – 8 weeks) or 3 independent samples (26 weeks) were loaded. Localization of full-length pro-Casp8 (55 kilodaltons) and the preactivated cleavage product (44 kilodaltons) is indicated. (D) Evaluation of steatosis markers in 6-month-old NEMO⌬hepa and Casp8⌬hepaNEMO⌬hepa mice. Top: Oil-red staining of paraffin liver sections. Red staining indicates fat deposition. Original magnification, ⫻200. Bottom: Measurement of triglycerides and cholesterol in liver homogenates. (E) H&E and Sirius red staining of liver paraffin sections from 1-year-old mice. Sirius red staining was documented using polarized light showing collagen fibers in red. Original magnification, ⫻200. (F) Quantitative real-time PCR for collagen 1 of 1-year-old mice. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001; n.s., not significant.
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levels for triglycerides and cholesterol (Figure 6D). Accordingly Casp8 may, directly or indirectly, interfere with hepatic fat metabolism and assist steatosis. However, Casp8⌬hepaNEMO⌬hepa mice developed liver fibrosis similar to NEMO⌬hepa mice as evidenced by Sirius red staining (Figure 6E) and collagen 1 gene expression (Figure 6F).
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lowing 10 weeks. Interestingly, heterozygous deletion of Casp8 in Casp8⫹/⫺NEMO⌬hepa mice clearly attenuated carcinogenesis. First neoplastic lesions were only found at 40 weeks of age, and, macroscopically, these livers were still cirrhotic but free of HCC. In contrast, none of the Casp8⌬hepaNEMO⌬hepa mice investigated showed any abnormality in MRI analysis within 40 weeks, and all of these animals (n ⫽ 23) remained tumor free up to 60 weeks of age. Histologically, Casp8⌬hepaNEMO⌬hepa tissues were free of dysplasia, whereas NEMO⌬hepa mice showed dysplastic nodules (Figure 7B).
Discussion Caspase-8 is the apical initiator caspase in death receptor-mediated apoptosis and thereby involved in different forms of acute and chronic liver diseases.20 In our present study, we investigated the consequences of Casp8 inactivation in models of acute and chronic liver injury. Genetic inactivation of Casp8 protected from Fas- and LPS/GalN induced liver failure. These findings are in agreement with earlier studies3,21,22 and thus confirmed
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NEMO⌬hepa mice undergo continuous spontaneous liver apoptosis and develop HCC between 9 and 12 months of age.7 We next investigated whether Casp8 acts procarcinogenicaly in NEMO⌬hepa mice by promoting death receptor-mediated apoptosis. Casp8⌬hepaNEMO⌬hepa mice were periodically monitored by MRI for liver tumor development and compared with NEMO⌬hepa mice. Of note, we did not observe spontaneous hepatocarcinogenesis in Casp8⌬hepa mice (n ⫽ 17) within 1 year (Figure 7A and data not shown). MRI analysis (Figure 7A) revealed first neoplastic lesions in NEMO⌬hepa mice at the age of 30 weeks, which developed into large HCCs within the fol-
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Figure 7. Ablation of Casp8 rescues spontaneous hepatocarcinogenesis in NEMO-deficient mice. (A) Casp8⌬hepaNEMO⌬hepa mice and controls were subjected to MRI at the age of 30 and 40 weeks, respectively. T1-weighted, contrast-enhanced images are shown. Hypointense areas represent tumorigenic liver modifications and are highlighted by arrows. After 40 weeks, animals were killed, and the macroscopic appearance of the livers was documented. (B) H&E staining of paraffin liver sections from 1-year-old Casp8⌬hepaNEMO⌬hepa mice and corresponding controls. Liver dysplasia in NEMO⌬hepa liver is encircled with dotted line. Original magnification, ⫻100 (upper part) and ⫻400 (lower part).
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the relevance of this pathway for hepatocyte apoptosis and also demonstrated the functionality of our strategy. Hepatic ablation of Casp8 revealed 2 unexpected effects: First, Casp8 deficiency triggered increased nonapoptotic liver injury and oxidative stress in ConA-induced hepatitis; and, second, Casp8 deletion induced massive liver necrosis and cholestasis but completely rescued spontaneous apoptosis, steatosis, and hepatocarcinogenesis in NEMO-deficient mice. In both models, we could experimentally link these findings to a general mechanism. RIP1 was proteolytically degraded by Casp8 during inflammation, eg, in NEMO⌬hepa mice and thus accumulated in Casp8-deficient livers. In addition, lack of Casp8 promoted the formation of a pronecrotic kinase complex consisting of FADD, RIP1, and RIP3 in the inflamed liver and also in other experimental settings as described recently.11,12 This kinase is functionally related to TNFinduced over-production of reactive oxygen species and cell necrosis.23 In line with these findings, we detected increased markers of oxidative stress in ConA-treated Casp8⌬hepa liver. In situ, we proved colocalization of RIP1 and RIP3 exclusively in the prenecrotic tissue, which was most evident in Casp8⌬hepaNEMO⌬hepa liver. Our data therefore strongly suggest that nonapoptotic liver injury in Casp8-deficient mice with hepatitis (ConA mediated or NEMO dependent) is a direct consequence of RIP1-RIP3 kinase formation and programmed necrosis. The mechanisms leading to ConA-mediated liver cell injury are still incompletely understood. Different studies suggested a predominance of apoptosis involving JNK1mediated activation of Casp824 or caspase-independent necrosis.16 Using Casp8⌬hepa mice, we aimed to clarify the role of death receptor-mediated apoptosis in the ConA model. In fact, apoptosis contributed only little to ConAmediated injury in our experimental setting and was completely Casp8 independent, hinting at a moderate activation of intrinsic apoptosis by ConA. Our findings show differences compared with recent results published by Kaufmann et al,22 who showed a protective effect of Casp8 deletion in the ConA model, which would suggest that ConA-mediated injury is predominantly mediated by Casp8-dependent apoptosis. We could not clarify these discrepancies so far and speculate that functional differences in the knockout alleles used and dosage effects (sublethal vs lethal ConA dosage) may contribute to this contradiction. In our experiments, ablation of Casp8 was only protective if ConA was administered together with the liver-specific transcription inhibitor GalN, which induces apoptosis in addition to necrosis.16 This suggests that protective effects in Casp8⌬hepa mice strictly depend on receptor-mediated apoptosis, whereas increased ConAinduced liver damage in Casp8⌬hepa mice relies on general transcriptional activity. Elevated liver injury in ConA-treated Casp8⌬hepa mice was associated with prolonged activation of JNK and NF-B. NF-B is activated by TNF via formation of a TNFR1-RIP1-TRAF2 complex.25 Additionally, the RIP1TRAF2 axis has been shown to activate JNK and p38.26
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Thus, prolonged JNK and NF-B activation could be a direct consequence of higher RIP1 expression in Casp8⌬hepa mice. Although JNK1 and JNK2 both significantly contribute to ConA damage,27 the impact for increased necrotic liver injury in our experimental model is unclear so far. NEMO⌬hepa mice develop a complex liver phenotype with chronic inflammation, basal apoptosis, steatosis, and signs of end-stage liver disease (fibrosis and HCC)7,17 reflecting the progression of chronic liver disease in humans. The complex phenotype observed by simultaneous ablation of Casp8 and NEMO is especially intriguing because inhibition of FADD completely rescued disease progression of NEMO⌬hepa livers.7 These results suggested that FADD is absolutely essential for spontaneous necrosis and cholestasis seen in Casp8⌬hepaNEMO⌬hepa livers. The mechanisms leading to hepatocarcinogenesis by NEMO inactivation are complex and still incompletely understood. Ablation of Casp8 completely protected from hepatocyte apoptosis and HCC development. Thus, our data suggest that Casp8-dependent apoptosis and compensatory proliferation are key events for tumour development in this model. However, Casp8⌬hepaNEMO⌬hepa mice also showed hepatitis along with excessive necrotic liver injury and several studies suggested that tissue necrosis is also a strong inducer of compensatory proliferation and HCC.28 This discrepancy could be explained by the observed reversion of necrotic injury with aging, resulting in almost normal liver tissue at the age of 6 months. We could exclude that this is a selection process rather than a reversion because Casp8⌬hepaNEMO⌬hepa mice did not show increased mortality at any age. Thus, transient liver necrosis triggered liver fibrosis but was not sufficient to induce HCC if Casp8 was deleted. At present, we cannot exclude that the tumor-promoting effect of Casp8 is specific for NEMO⌬hepa mice and thus has to be investigated in future studies. Additionally, NEMO⌬hepa-dependent steatosis and steatohepatitis were completely rescued by Casp8 inhibition. This suggests an unknown function of Casp8 in hepatic fat metabolism. Steatohepatitis may trigger growth of HCC, and lack of steatosis in Casp8⌬hepaNEMO⌬hepa mice is most likely another important factor contributing to tumor-free survival in these animals. In summary, we demonstrate that Casp8 has a dual function for liver homeostasis because it triggers apoptosis and HCC but also prevents from necrotic injury in NEMO⌬hepa livers. Our findings may also explain recent results showing elevated transaminases in hepatitis C virus patients treated with caspase inhibitors.29 We conclude that Casp8 inhibition in hepatitis patients likely is of limited benefit because of the higher risk of programmed necrosis. However, combined inhibition of Casp8 and RIP kinases might be a promising approach for future therapy.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2011.08.037. References 1. Malhi H, Gores GJ, Lemasters JJ. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology 2006;43:S31–S44. 2. Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998;17:1675–1687. 3. Varfolomeev EE, Schuchmann M, Luria V, et al. Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 1998;9:267–276. 4. Ding WX, Yin XM. Dissection of the multiple mechanisms of TNF␣-induced apoptosis in liver injury. J Cell Mol Med 2004;8:445– 454. 5. Ermolaeva MA, Michallet MC, Papadopoulou N, et al. Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIFdependent inflammatory responses. Nat Immunol 2008;9:1037– 1046. 6. Schmidt-Supprian M, Bloch W, Courtois G, et al. NEMO/IKK ␥-deficient mice model incontinentia pigmenti. Mol Cell 2000;5:981– 992. 7. Luedde T, Beraza N, Kotsikoris V, et al. Deletion of NEMO/IKK␥ in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell 2007;11:119 –132. 8. Dempsey PW, Doyle SE, He JQ, et al. The signaling adaptors and pathways activated by TNF superfamily. Cytokine Growth Factor Rev 2003;14:193–209. 9. Schneider-Brachert W, Tchikov V, Neumeyer J, et al. Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 2004;21:415– 428. 10. Dong QG, Sclabas GM, Fujioka S, et al. The function of multiple IB:NF-B complexes in the resistance of cancer cells to Taxolinduced apoptosis. Oncogene 2002;21:6510 – 6519. 11. Cho YS, Challa S, Moquin D, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009;137:1112–1123. 12. He S, Wang L, Miao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-␣. Cell 2009;137: 1100 –1111. 13. Kaiser WJ, Upton JW, Long AB, et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 2011;471:368 –72. 14. Freimuth J, Gassler N, Moro N, et al. Application of magnetic resonance imaging in transgenic and chemical mouse models of hepatocellular carcinoma. Mol Cancer 2010;9:94. 15. Tiegs G, Hentschel J, Wendel A. A T cell-dependent experimental liver injury in mice inducible by concanavalin A. J Clin Invest 1992;90:196 –203. 16. Ni HM, Chen X, Ding WX, et al. Differential roles of JNK in ConA/ GalN and ConA-induced liver injury in mice. Am J Pathol 2008; 173:962–972. 17. Beraza N, Ludde T, Assmus U, et al. Hepatocyte-specific IKK␥/ NEMO expression determines the degree of liver injury. Gastroenterology 2007;132:2504 –2517. 18. Beraza N, Malato Y, Sander LE, et al. Hepatocyte-specific NEMO deletion promotes NK/NKT cell- and TRAIL-dependent liver damage. J Exp Med 2009;206:1727–1737.
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19. Rajput A, Kovalenko A, Bogdanov K, et al. RIG-I RNA helicase activation of IRF3 transcription factor is negatively regulated by caspase-8-mediated cleavage of the RIP1 protein. Immunity;34: 340 –351. 20. Canbay A, Friedman S, Gores GJ. Apoptosis: the nexus of liver injury and fibrosis. Hepatology 2004;39:273–278. 21. Kang TB, Ben-Moshe T, Varfolomeev EE, et al. Caspase-8 serves both apoptotic and nonapoptotic roles. J Immunol 2004;173: 2976 –2984. 22. Kaufmann T, Jost PJ, Pellegrini M, et al. Fatal hepatitis mediated by tumor necrosis factor TNF␣ requires caspase-8 and involves the BH3-only proteins Bid and Bim. Immunity 2009;30:56 – 66. 23. Zhang DW, Shao J, Lin J, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009;325:332–336. 24. Chang L, Kamata H, Solinas G, et al. The E3 ubiquitin ligase itch couples JNK activation to TNF␣-induced cell death by inducing c-FLIP(L) turnover. Cell 2006;124:601– 613. 25. Ting AT, Pimentel-Muinos FX, Seed B. RIP mediates tumor necrosis factor receptor 1 activation of NF-B but not Fas/APO-1-initiated apoptosis. EMBO J 1996;15:6189 – 6196. 26. Lee TH, Huang Q, Oikemus S, et al. The death domain kinase RIP1 is essential for tumor necrosis factor ␣ signaling to p38 mitogenactivated protein kinase. Mol Cell Biol 2003;23:8377– 8385. 27. Maeda S, Chang L, Li ZW, et al. IKK is required for prevention of apoptosis mediated by cell-bound but not by circulating TNF␣. Immunity 2003;19:725–737. 28. Naugler WE, Sakurai T, Kim S, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 2007;317:121–124. 29. Manns M, Palmer M, Flisiak R, et al. A Phase-2B trial to evaluate the safety, tolerability and efficacy of a caspase inhibitor GS9450, in adults failing PEG/RBV therapy for chronic HCV infection J Hepatol 2011;54(Suppl1):S55–S56. Received Febrary 7, 2011. Accepted August 11, 2011. Reprint requests Address requests for reprints to: Christian Liedtke, PhD, and Christian Trautwein, MD, Department of Medicine III, University Hospital Aachen, RWTH Aachen University, Pauwelsstrasse 30, D52074 Aachen, Germany. e-mail: [email protected]; and [email protected]; fax: (49) 0241-80 82455. Acknowledgments J.F.’s current address is UCSF Helen Diller Family Comprehensive Cancer Center 1450, San Francisco, California. N.B.’s current address is CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberedh), Bizkaia, Spain. J-M.B. and J.F. contributed equally to this work. This manuscript is dedicated to Michael P. Manns in honor of his 60th birthday. Conflicts of Interest The authors disclose no conflicts. Funding Supported by a grant of the Deutsche Krebshilfe (grant No. 107682) and the Deutsche Forschungsgemeinschaft (DFG), SFB TRR57; by the Instituto de Salud Carlos III (Ministry of Health, Spain; FIS09/02010; to N.B.); and by the program Ramón y Cajal (Ministry of Science and Innovation, Spain).
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BASIC AND TRANSLATIONAL—LIVER Loss of Caspase-8 Protects Mice Against Inflammation-Related Hepatocarcinogenesis but Induces Non-Apoptotic Liver Injury CHRISTIAN LIEDTKE,* JÖRG–MARTIN BANGEN,* JULIA FREIMUTH,* NAIARA BERAZA,* DANIELA LAMBERTZ,* FRANCISCO J. CUBERO,* MAXIMILIAN HATTING,* KARLIN R. KARLMARK,* KONRAD L. STREETZ,* GABRIELE A. KROMBACH,‡ FRANK TACKE,* NIKOLAUS GASSLER,§ DIETER RIETHMACHER,储 and CHRISTIAN TRAUTWEIN* *Department of Medicine III, University Hospital, Aachen, Germany; ‡Department of Radiology, Justus-Liebig University, Giessen, Germany; §Institute of Pathology, University Hospital Aachen, Germany; and 储Human Genetics Division, University of Southampton, Southampton General Hospital, Southampton, United Kingdom
See editorial on page 1969.
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BACKGROUND & AIMS: Disruption of the nuclear factor-B (NF-B) essential modulator (NEMO) in hepatocytes of mice (NEMO⌬hepa mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO⌬hepa mice or after induction of acute liver injury. METHODS: We created mice with conditional deletion of Casp8 in hepatocytes (Casp8⌬hepa) and Casp8⌬hepaNEMO⌬hepa double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS: Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8⌬hepaNEMO⌬hepa mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3—these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS: Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment. Keywords: Fas-associated Protein With Death Domain; FADD; Inflammation; Tumor Necrosis Factor; TNF Signaling.
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wo mechanisms, apoptosis and necrosis, are frequently involved in acute and chronic liver injury. Apoptosis is a precisely regulated and genetically determined process that can be induced via death receptormediated, extrinsic pathways or through intrinsic mechanisms activated by intracellular stress.1 The cystein-aspartate protease caspase-8 (Casp8) is the apical initiator caspase in death receptor-mediated apoptosis and constitutively expressed as a premature zymogene. After death receptor activation by its cognate ligand, the formation of a death-inducing signaling complex consisting of a death receptor, the adaptor protein Fas-associated protein with death domain (FADD), and procaspase-8 is induced leading to activation of Casp8 by an autocatalytic process. Casp8 can in turn activate downstream effector caspases such as caspase-3 or—like in hepatocytes—activate intrinsic apoptosis signalling.2 Casp8 is essential for death receptor-mediated apoptosis. Although constitutive disruption of the murine Casp8 gene is embryonically lethal because of impaired heart muscle development and hyperemia, Casp8-deficient murine embryonic fibroblasts were shown to be protected from cell death induced by Fas, tumor necrosis factor-related apoptosis inducing ligand (TRAIL), or tumor necrosis factor (TNF).3 TNF is a pleiotropic cytokine and does not only induce apoptosis but predominantly triggers antiapoptotic and proinflammatory signals via NF-B or alternatively activates the c-Jun N-terminal protein kinase (JNK) pathway.4 Abbreviations used in this paper: Casp8, caspase-8; ConA, Concanavalin A; FADD, Fas-associated protein with death domain; GalN, D-galactosamine; HCC, hepatocellular carcinoma; IKK, inhibitor of nuclear factor-B kinase; JNK, c-Jun N-terminal protein kinase; LPS, lipopolysaccharides; MRI, magnetic resonance imaging; NEMO, NF-B essential modulator; NF-B, nuclear factor-B; RIP, receptor-interacting protein; TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, tumor necrosis factor receptor type 1-associated death domain protein; TRAF, TNF receptor-associated factor; TRAIL, Tumor Necrosis Factor-Related Apoptosis Inducing Ligand; TUNEL, terminal desoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling; WT, wild type. © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2011.08.037
Following TNF binding, recruitment of the adaptor protein tumor necrosis factor receptor type 1-associated death domain protein (TRADD) to the TNF receptor 1 (TNFR1) is a key event for activation of at least 3 different, in part opposing, signaling cascades: Association of TRADD with the receptor interacting protein (RIP) 1 and TNF receptor-associated factor (TRAF) 2 (complex I) activates the inhibitor of nuclear factor-B kinase (IKK) complex (IKK␣, IKK, and IKK␥/NF-B essential modulator [NEMO]), which initiates NF-B activation.5 The regulatory subunit IKK␥/NEMO is crucial for NF-B activation. Constitutive disruption of the NEMO gene in vivo results in complete NF-B inhibition and embryonic lethality.6 Hepatocyte-specific deletion of NEMO and subsequent NF-B inhibition results in chronic liver apoptosis promoting spontaneous steatohepatitis, fibrosis progression, and development of hepatocellular carcinoma (HCC).7 Association of TRADD and TRAF2 can also activate JNK signaling pathways.8 Alternatively, after TNF binding TNFR1/TRADD can associate with the adaptor protein FADD and pro-caspase-8 (complex II) leading to complex internalization and apoptosis induction.9 The immediate response to TNF stimulation is usually dominated by proinflammatory and antiapoptotic NF-B target genes In contrast, inhibition of NF-B in the liver, eg, by blocking gene transcription with D-galactosamine (GalN) or by genetic ablation, triggers Casp8-mediated apoptosis because of down-regulation of antiapoptotic genes.10 Necrosis has classically been viewed as an accidental and unregulated form of cell death. However, recent findings suggested that TNF is also able to induce tightly regulated cell necrosis if caspase activity is blocked. This phenomenon has been termed programmed necrosis and depends on synergistic action of receptor-interacting serine threonine kinases RIP1 and RIP3, respectively.11,12 Recent data demonstrate that Casp8 is essential for preventing RIP-mediated necrosis.13 However, the physiologic role of programmed necrosis for the liver is poorly understood. In the present study, we investigated potential benefits of hepatocyte-specific Casp8 ablation in mouse models of acute or chronic liver disease. We demonstrate that Casp8 inactivation protects from liver apoptosis but triggers necrotic cell death in the Concanavalin A (ConA) model of T-cell hepatitis and in NEMO-deficient mice.
Materials and Methods Housing and Breeding of Mice All animals were maintained in the animal facility of the University Hospital Aachen in a temperature-controlled room with 12-hour light/dark cycle. Animal husbandry and procedures were approved by the authority for environment conservation and consumer protection of the state North Rhine-Westfalia (LANUV, Germany). We used mice of male sex carrying a hepatocyte-specific deletion (⌬hepa) of Casp8 and/or NEMO genes (Casp8⌬hepa; NEMO⌬hepa; Casp8⌬hepaNEMO⌬hepa) and crenegative littermates (Casp8f/f; NEMOf/f; Casp8f/fNEMOf/f) as controls. Generation of Casp8⌬hepa mice is described in the Supplementary Materials and Methods section. Animals were kept under specific pathogen-free conditions after embryo transfer.
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Animal Models of Liver Injury The Fas-activating antibody Jo2 was obtained from BD Bioscience (San Jose, CA) and administrated intravenously at a concentration of 0.5 g/g of body weight. ConA, lipopolysaccharide (LPS), and GalN were from Sigma-Aldrich (St. Louis, MO). ConA was administered intravenously (25 mg/kg of body weight) alone, or 1 hour after intraperitoneal injection of GalN (1000 mg/kg). Similarly, for induction of septic liver injury, mice were first injected intraperitoneally with 1000 mg/kg GalN 1 hour prior to LPS administration (750 mg/kg, intraperitoneally).
Magnetic Resonance Imaging of Murine HCC To monitor progression of HCC in NEMO⌬hepa and mice, animals were investigated by magnetic resonance imaging (MRI) using an Achieva 1.5-Tesla Scanner MR System (Philips Medical Systems, Best, The Netherlands) as recently described.14 A 47-mm-diameter coil (Philips Medical Systems) was used for signal reception. For contrast enhancement, 0.025-mmol/kg body weight gadoxetic acid disodium (Primovist; Schering, Berlin, Germany) was administered by tail vein injection using a catheter with injection port (Biovalve, 22 gauge; 1.0 ⫻ 25 mm; Vygon, Ecouen, France).
Casp8⌬hepaNEMO⌬hepa
Statistical Analysis Data are expressed as mean ⫾ standard deviation of the mean. Statistical significance was determined by 2-way analysis of variance followed by a Student t test.
Results Casp8⌬hepa Mice Show Basal Liver Inflammation and Are Protected From Fasand LPS-Mediated Liver Injury Constitutive Casp8 knockout mice die in utero of impaired heart muscle development.3 Therefore, we generated hepatocyte-specific conditional Casp8 knockout mice (Casp8⌬hepa) using the cre/loxP system (Supplementary Figure 1A and B). Casp8⌬hepa mice did not show morphologic differences in comparison with their wildtype (WT) (Casp8f/f) littermates (Supplementary Figure 1C). Efficient, cre-mediated deletion of Casp8 exons III-IV was confirmed on the genomic DNA and protein level using total liver samples or primary hepatocytes (Supplementary Figure 1D). Liver histology of Casp8⌬hepa mice revealed small inflammatory infiltrates of granulocytes and monocytes and increased serum transaminases (Figure 1A), suggesting that ablation of Casp8 in hepatocytes results in moderate basal liver inflammation. As a proof of principle, we investigated whether Casp8⌬hepa mice are protected from liver apoptosis in 2 established models of acute liver injury by administrating either the Fas-activating antibody Jo2 (Figure 1B) or a combination of lipopolysaccharide/D-galactosamine (LPS/GalN, Figure 1C). As expected, in both models, control mice showed strong increase in serum transaminases, strong apoptosis as shown by caspase-3 activation and terminal desoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and approximately 90% mortality within 24 hours. In contrast, 100% of Casp8⌬hepa mice survived Jo2 and LPS/GalN treatment without indications of liver apoptosis.
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Figure 1. Casp8⌬hepa mice show moderate basal liver inflammation and are protected from Fas- and LPS-mediated liver injury. (A) H&E staining of liver sections and basal serum transaminases from untreated controls (Casp8f/f) and Casp8⌬hepa mice. Arrow: focal basal infiltration of inflammatory cells. (B and C) Control animals (Casp8f/f) and Casp8⌬hepa mice were injected with (B) Fas-activating antibody Jo2 or (C) LPS and GalN. Liver injury was determined by measurement of serum transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), caspase-3 activity from murine liver extracts, survival within 24 hours, and TUNEL staining of liver sections. TUNEL-positive apoptotic cells are stained in green. **P ⬍ .01; ***P ⬍ .001.
Casp8 Deletion Triggers Enhanced Nonapoptotic Liver Injury During ConAInduced Hepatitis The lectin ConA is a T-cell mitogen and rapidly induces liver injury involving CD4⫹ T cells, natural killer T cells, and macrophages/Kupffer cells.15 The mode of ConA-mediated cell death is controversial, whereas cotreatment with ConA and GalN was shown to induce a predominant proapoptotic response.16 Unexpectedly, Casp8⌬hepa mice showed impaired survival (Figure 2A) and increased aspartate aminotransferase and alanine aminotransferase levels in comparison with Casp8f/f controls after treatment with sublethal ConA dose. This outcome was completely reverted by concomitant application of GalN resulting in further increase of serum transaminases in the control group in contrast to
remarkable attenuation of injury in Casp8⌬hepa mice (Figure 2B). Opposing effects of ConA vs ConA/GalN were confirmed in liver histology and TUNEL analysis (Figure 2C). Following ConA treatment, Casp8⌬hepa livers revealed increased hepatocyte injury and a striking staining pattern of abundant TUNEL-positive hepatocytes with focal concentration, which was not evident in the control group. However, in the ConA/GalN model, we observed only moderate cell death of single hepatocytes and few TUNEL-positive cells in Casp8⌬hepa livers, whereas the control group revealed strong hepatocellular damage of grouped hepatocytes with morphologic aspects of apoptosis-related cell death and high number of TUNEL-positive cells. To determine further the impact of apoptosis in both injury models, caspase-3 activity was measured in
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Figure 2. Casp8 deletion does not protect from ConA mediated hepatitis. Casp8f/f control mice and Casp8⌬hepa animals were injected with ConA or ConA and GalN where indicated. (A) Kaplan–Maier survival curves for ConA-treated animals. (B) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum transaminase levels. (C) H&E staining (original magnification, ⫻400) and TUNEL assay of liver sections 7 hours after ConA or ConA/GalN treatment; dying cells in the TUNEL assay are stained in green. Dying hepatocytes with morphologic signs of apoptosis are highlighted by arrows. (D) Comparison of caspase-3 activity in ConA-treated vs ConA/GalN-treated mice. Activity is shown as fold induction compared with untreated controls. (E) Reactive oxygen species were assayed by measuring malondialdehyde in liver homogenates. Untreated Casp8f/f livers served as reference (ctrl). *P ⬍ .05; ***P ⬍ .001.
Casp8⌬hepa mice and controls after treatment with ConA or ConA/GalN (Figure 2D). ConA treatment alone triggered only minor caspase-3 activation in both Casp8f/f and Casp8⌬hepa mice, suggesting that apoptosis in the ConA model is Casp8-independent and contributes only little to liver injury. However, combined ConA/GalN treatment remarkable activated caspase-3 in control mice, but not in Casp8⌬hepa animals. Instead, Casp8⌬hepa livers produce a higher amount of reactive oxygen species after ConA
treatment as evidenced by increased levels of malondialdehyde (Figure 2E). Thus, ablation of Casp8 may trigger enhanced nonapoptotic injury via increased oxidative stress in the ConA model. Additionally, we detected slightly increased TNF and interferon-␥ expression in Casp8⌬hepa mice after ConA treatment (Supplementary Figure 2A). In line with this finding, already 2 hours after ConA treatment Casp8⌬hepa livers showed increased infiltration of CD11b⫹F4/80⫹
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macrophages (Supplementary Figure 2B and C), suggesting that Casp8⌬hepa mice have an accelerated immune response after ConA, which might affect downstream cytokine signalling.
Increased ConA-Mediated Liver Injury in Casp8⌬Hepa Mice Is Associated With Aberrant TNF Signaling TNF activates at least 3 different pathways via TNFR1 triggering apoptosis through Casp8 or inflammation via NF-B or JNK/p38 mitogen-activated protein kinases. In addition, TNF may also induce necrosis via RIP1/RIP3-dependent mechanisms (Supplementary Figure 3). We thus systematically analyzed TNF signaling in ConA-treated Casp8⌬hepa mice. We detected aberrant high basal RIP1 expression and further up-regulation in Casp8⌬hepa livers upon ConA stimulation, which was less evident in controls (Figure 3A). RIP3 expres-
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sion was barely regulated by ConA in either group, whereas overall RIP3 expression in Casp8⌬hepa mice was slightly reduced. However, FADD expression was regulated during ConA-induced hepatitis showing accelerated induction in Casp8⌬hepa mice correlating with earlier onset of injury. Immunoprecipitation experiments revealed that in ConA-treated Casp8⌬hepa but not in control livers, a FADD-RIP1-RIP3 complex is formed. RIP3 was constitutively associated with FADD in both untreated and ConA-treated Casp8⌬hepa livers (Figure 3A) demonstrating that expression and localization of RIP1—in contrast to RIP3—is differentially controlled in Casp8⌬hepa mice. FADD-RIP1-RIP3 complexes precipitated from Casp8⌬hepa livers revealed kinase activity as they efficiently phosphorylated the substrate myelin basic protein (Figure 3B). Interestingly, RIP1 and RIP3 were colocalized in the cytoplasm of ConA-treated
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Figure 3. ConA treatment induces aberrant TNF signaling and the formation of RIP1-RIP3-FADD complexes in Casp8⌬hepa mice. ConAtreated Casp8f/f and Casp8⌬hepa mice were analyzed at time points indicated. (A) Upper part: Western blot analysis of total protein levels for RIP1, RIP3, and the adaptor protein FADD after ConA treatment. GAPDH was detected as loading control. Lower part: Immunoprecipitation (IP) of FADD complexes; recruitment of RIP1 and RIP3 to FADD was examined by Western blot. (B) Four hours after ConA treatment, FADD complexes were immunoprecipitated (IP:FADD) and subjected to in vitro kinase assay using myelin basic protein (MBP) as artificial substrate. (C) Coimmunofluorescence staining for RIP1 and RIP3 in liver sections from Casp8f/f and Casp8⌬hepa mice 7 hours after ConA treatment. Green: RIP1-positive cells; Red: RIP3-positive cells. Signals in orange indicate colocalization of RIP1 and RIP3. Sections are counterstained with DAPI (blue). (D) Western blot analysis of phosphorylated JNK1/JNK2 and p-cJun. Pro-Casp8 expression was determined to monitor knockout efficiency. (E) Analysis of NF-B activation. Upper part: EMSA analysis with NF-B consensus sequence and nuclear extracts as indicated. FP, free probe. Signals for active p50 homodimers and p50/p65 heterodimers are indicated by arrows. Supershift: WT sample (Casp8f/f) 2 hours after ConA treatment was incubated with antibodies against NF-B subunits p50 or p65, respectively. Lower part: Cellular localization of NF-B p65 was investigated by Western blot analysis of cytoplasmic (p65 cyt) and nuclear (p65 nuc) liver protein extracts. Loading controls: GAPDH (cytoplasmic extracts [GAPDH cyt]); TBP (nuclear extracts [TBP nuc]). (F) Quantitative real-time PCR for IB-␣.
Casp8⌬hepa liver and showed strong focal expression along with injured tissue, which was not evident in control livers (Figure 3C). Ablation of Casp8 also resulted in prolonged JNK activation after ConA delivery, whereas phosphorylation of its target c-Jun was even diminished in Casp8⌬hepa mice (Figure 3D). In parallel, ConA also induced prolonged NF-B activation and nuclear localization in Casp8⌬hepa mice (Figure 3E), resulting in increased up-regulation of the NF-B target gene IB-␣ (Figure 3F).
Simultaneous Ablation of Casp8 and NEMO in Liver Parenchymal Cells Results in Massive Necrosis and Cholestasis The contrasting functions of Casp8 upon acute liver injury prompted us to investigate its role in the NEMO⌬hepa model, which recapitulates human chronic liver disease progression in a unique manner. NEMO⌬hepa mice show strong basal inflammation, spontaneous apoptosis, steatosis, liver fibrosis, and, finally, develop HCC.7,17,18 We thus investigated whether inactivation of Casp8 in NEMO⌬hepa mice attenuates progression of liver disease. Unexpectedly, 6- to 8-week-old Casp8⌬hepaNEMO⌬hepa mice revealed a complex liver phenotype with bile inclusions and infarcts as well as hepatomegaly. The penetrance of this phenotype showed variations reflecting differences in disease severity. Thus, Casp8⌬hepaNEMO⌬hepa livers were grouped dependent on the strength of the phenotype (types I, II, and III). From Casp8⌬hepaNEMO⌬hepa mice initially investigated (n ⫽ 43), 34% were classified as type I and revealed few white liver lesions (Figure 4A) that were identified as necrotic areas (Figure 4B and Supplementary Figure 4B). The majority (40%) of mice was designated type II defined by multilocular, macroscopically visible white liver lesions displaying histomorphologic features of parenchymal necroses or bile infarcts (Figure 4A and B and Supplementary Figure 4C). These animals had normal body size (Figure 4C) but significantly increased liver-to-body weight ratio compared with WT controls (Figure 4D). Type III livers (26%) displayed yellow appearance, increased organ size, and multilocular yellow lesions, resulting in massive necrotic destruction of the liver associated with multiple bile infarcts (Figure 4A and B, Supplementary Figure 4D). In addition, the animals were characterized by dwarfism in combination with hepatomegaly leading to an abnormal liver-to-body weight ratio of 9.3% (Figure 4C and D). Of notice, the severity of liver necrosis in Casp8⌬hepaNEMO⌬hepa livers correlated with increasing numbers of CD11b-positive cells specifically localized at necrotic foci (Supplementary Figure 5A and B). Types II and III Casp8⌬hepaNEMO⌬hepa mice showed increased serum transaminase levels, and no significant difference was found compared with NEMO⌬hepa mice (Figure 4E). Alkaline phosphatase levels best differentiated among the 3 phenotypes and were elevated in close correlation to the severity of liver injury (Figure 4E). Accordingly, type III mice had strongly and significantly increased bile acid and bilirubin levels in comparison with
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both Casp8⌬hepa and NEMO⌬hepa mice. Thus, combined hepatocyte-specific Casp8 and NEMO ablation triggers massive liver necrosis and cholestasis, which was not observed in either single knockout strain. Similar to our observations in Fas-induced acute liver injury, ablation of Casp8 rescued spontaneous liver apoptosis of NEMO⌬hepa livers because no TUNEL-positive cells were found in type I livers (Supplementary Figure 5(C). In contrast, in necrotic foci of type II-III livers, TUNEL-positive signals were found reflecting apoptosis of nonparenchymal cells. Additionally, increased cell proliferation was evident in all Casp8⌬hepaNEMO⌬hepa livers as evidenced by measuring BrdU incorporation (Supplementary Figure 5D). Costainings of proliferation- (Ki67) and hepatocyte-specific markers (HNF4␣) excluded hepatocyte proliferation and suggest that bile duct cells (Supplementary Figure 5E) proliferate in Casp8⌬hepaNEMO⌬hepa livers.
FADD-RIP1-RIP3 Kinase Complexes Are Formed Spontaneously in Casp8⌬HepaNEMO⌬Hepa Mice and Localize at Foci of Necrotic Liver Injury Disease severity in Casp8⌬hepaNEMO⌬hepa livers correlated with increasing basal RIP1 and FADD expression showing highest levels in type III mice. Interestingly, basal RIP1 expression in NEMO⌬hepa mice was significantly lower compared with WT controls (Figure 5A). In Casp8⌬hepaNEMO⌬hepa livers, but not in Casp8⌬hepa or NEMO⌬hepa mice, FADD constitutively formed complexes with both RIP1 and RIP3 as evidenced by immunoprecipitation experiments (Figure 5A). These complexes had kinase activity that was proportional to the strength of liver injury and thus strongest in type III and barely detectable in type I livers (Figure 5B). RIP1 can be proteolytically degraded by Casp8 resulting in a cleavage product of approximately 38 kilodaltons.19 Accordingly, we detected cleaved RIP1 in untreated NEMO⌬hepa mice, but not in Casp8⌬hepaNEMO⌬hepa mice (Figure 5C). RIP1 immunhistochemistry revealed that RIP1 is not equally distributed in Casp8⌬hepaNEMO⌬hepa liver tissue but concentrated in foci of intact liver tissue of type I and type II mice or in close proximity to necrotic areas in type III animals (Figure 5D). Importantly, RIP1 colocalized with RIP3 in Casp8⌬hepaNEMO⌬hepa tissue, which was not observed in the control group (Figure 5D), further proving the existence of pronecrotic RIP-FADD complexes at loci of liver injury. The severity of liver injury in Casp8⌬hepaNEMO⌬hepa mice further correlated with the level of JNK activation and subsequent c-Jun phosphorylation (Supplementary Figure 6A and B) suggesting that JNK signaling is also associated with necrosis induction in these animals.
Aging Casp8⌬hepaNEMO⌬hepa Mice Recover From Necrotic Liver Injury and Are Protected From Steatosis but Develop Liver Fibrosis Analysis of age-dependent disease progression of Casp8⌬hepaNEMO⌬hepa mice revealed normal serum lev-
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Figure 4. Simultaneous ablation of Casp8 and NEMO in liver parenchyma results in massive liver necrosis and cholestasis. (A) Macroscopic appearance of male Casp8⌬hepaNEMO⌬hepa livers at the age of 6 – 8 weeks. Organs were classified according to their phenotype as I (macroscopically normal), II (white lesions), or III (yellow lesions, hepatomegaly, dwarfism). Frequency of each phenotype is given in parentheses. Representative lesions are highlighted by arrows. (B) Histologic analysis (H&E) of types I–III livers in comparison with samples from either single knockout (Casp8⌬hepa, NEMO⌬hepa) and WT controls (Casp8f/fNEMOf/f). Original magnification, ⫻200. (C) Macroscopic appearance of type III Casp8⌬hepaNEMO⌬hepa mice (right) showing dwarfism in comparison with type II (middle) and cre-negative (left) littermate controls. (D) Liver mass index of 6to 8-week-old Casp8⌬hepaNEMO⌬hepa mice classified as types I–III. (E) Analysis of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), bile acids, and total bilirubin levels in 6- to 8-week-old Casp8⌬hepaNEMO⌬hepa mice and controls. Serum parameters are given both for total Casp8⌬hepaNEMO⌬hepa mice and subtypes I–III, respectively. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001; n.s., not significant.
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Figure 5. Spontaneous formation of FADD-RIP1-RIP3 kinase complexes in Casp8⌬hepaNEMO⌬hepa mice and localization at foci of necrotic liver injury. Six- to 8-week-old Casp8⌬hepaNEMO⌬hepa mice and controls were investigated for hepatic RIP1 expression, RIP1/RIP3 interaction, and in situ localization. WT, Casp8f/fNEMOf/f controls. (A) Top: Total expression of RIP1 and FADD. Bottom: Protein complexes were isolated by immunoprecipitation with antibodies for FADD (IP:FADD) and subjected to Western blot analysis for RIP1 and RIP3 expression. (B) FADD complexes from total liver extracts were isolated by immunoprecipitation and subjected to radioactive in vitro kinase assay using myelin basic protein (MBP) as substrate. (C) Total RIP1 was enriched by immunoprecipitation (IP:RIP1) and subjected to RIP1 immunoblot. IgG, heavy chain immunoglobulin. (D) Localization of RIP1 and RIP3 in situ. Top: Liver tissue sections from Casp8⌬hepaNEMO⌬hepa mice with different grade of liver injury (types I–III) and WT controls were probed with RIP1 (RIP1 IHC). Encircled areas define foci with strong RIP1 expression. Bottom: Costaining for RIP1 and RIP3 analyzed by immunofluorescence microscopy. Green: RIP1; red: RIP3; yellow: RIP1-RIP3 colocalization.
els for aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and bile acids in juvenile mice at the age of 3 weeks (Figure 6A). Liver injury was maximal at 6 – 8 weeks but continuously ameliorated with aging, indicating that generation of necrosis and cholestasis is restricted to juvenile liver development. Beginning at the age of 25 weeks, serum parameters in Casp8⌬hepaNEMO⌬hepa mice reached almost normal values compared with WT controls, and classification of type I-III mice was no longer possible because of substantially improvement of liver histology (Supplementary Figure 7A). Of notice, despite extensive liver injury of young Casp8⌬hepaNEMO⌬hepa mice, we did not observe increased mortality of these animals compared with controls within 1 year (Figure 6B and Supplementary Figure 7B). We determined the age-dependent efficiency of simultaneous Casp8 and NEMO inactivation. In total liver from 6to 8-week-old Casp8⌬hepaNEMO⌬hepa mice, we unexpectedly
detected substantial levels of full-length Casp8 and NEMO messenger RNA. However, primary hepatocytes from these samples showed almost complete inactivation of Casp8 and NEMO (Figure 6C, top), suggesting that the residual Casp8 and NEMO expression derives from excessive infiltrating and ductular cells. Protein analysis confirmed efficient ablation of Casp8 and NEMO in type I livers but substantial expression of processed Casp8 (44-kilodalton cleavage product) in type II and type III livers (Figure 6C, bottom) potentially because of excessive infiltrating cells. Of notice, in NEMO⌬hepa mice, Casp8 was highly expressed as a preactivated 44-kilodalton protein further highlighting their predisposition to extrinsic apoptosis. In contrast, at the age of 26 weeks, expression of Casp8 and NEMO was completely absent in Casp8⌬hepaNEMO⌬hepa livers. NEMO⌬hepa mice develop spontaneous steatosis and steatohepatitis.7 Deletion of Casp8 in NEMO⌬hepa mice resulted in complete recovery from liver steatosis because they lack extensive fat accumulation and show normal
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Figure 6. Aging Casp8⌬hepaNEMO⌬hepa mice recover from necrotic liver injury and are protected from steatosis but develop liver fibrosis. (A) Age-dependent analysis of serum AST, ALT, alkaline phosphatase (AP), and bile acid levels in Casp8⌬hepaNEMO⌬hepa mice and WT (Casp8f/fNEMOf/f) controls. All animals were included irrespective of their classification in types I–III. Parameters were measured at the age of 3, 8, 12, 26, and 52 weeks. (B) Kaplan–Meier survival curve for Casp8⌬hepaNEMO⌬hepa mice (n ⫽ 474) and WT (Casp8f/fNEMOf/f; n ⫽ 259) controls. (C) Age-dependent efficiency of Casp8 and NEMO inactivation in liver samples and primary hepatocytes from 6- to 8-week-old Casp8⌬hepaNEMO⌬hepa mice was determined by rtPCR (top) or analyzed by Western blot analysis (bottom). Subgroups I–III (6 – 8 weeks) or 3 independent samples (26 weeks) were loaded. Localization of full-length pro-Casp8 (55 kilodaltons) and the preactivated cleavage product (44 kilodaltons) is indicated. (D) Evaluation of steatosis markers in 6-month-old NEMO⌬hepa and Casp8⌬hepaNEMO⌬hepa mice. Top: Oil-red staining of paraffin liver sections. Red staining indicates fat deposition. Original magnification, ⫻200. Bottom: Measurement of triglycerides and cholesterol in liver homogenates. (E) H&E and Sirius red staining of liver paraffin sections from 1-year-old mice. Sirius red staining was documented using polarized light showing collagen fibers in red. Original magnification, ⫻200. (F) Quantitative real-time PCR for collagen 1 of 1-year-old mice. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001; n.s., not significant.
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levels for triglycerides and cholesterol (Figure 6D). Accordingly Casp8 may, directly or indirectly, interfere with hepatic fat metabolism and assist steatosis. However, Casp8⌬hepaNEMO⌬hepa mice developed liver fibrosis similar to NEMO⌬hepa mice as evidenced by Sirius red staining (Figure 6E) and collagen 1 gene expression (Figure 6F).
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lowing 10 weeks. Interestingly, heterozygous deletion of Casp8 in Casp8⫹/⫺NEMO⌬hepa mice clearly attenuated carcinogenesis. First neoplastic lesions were only found at 40 weeks of age, and, macroscopically, these livers were still cirrhotic but free of HCC. In contrast, none of the Casp8⌬hepaNEMO⌬hepa mice investigated showed any abnormality in MRI analysis within 40 weeks, and all of these animals (n ⫽ 23) remained tumor free up to 60 weeks of age. Histologically, Casp8⌬hepaNEMO⌬hepa tissues were free of dysplasia, whereas NEMO⌬hepa mice showed dysplastic nodules (Figure 7B).
Discussion Caspase-8 is the apical initiator caspase in death receptor-mediated apoptosis and thereby involved in different forms of acute and chronic liver diseases.20 In our present study, we investigated the consequences of Casp8 inactivation in models of acute and chronic liver injury. Genetic inactivation of Casp8 protected from Fas- and LPS/GalN induced liver failure. These findings are in agreement with earlier studies3,21,22 and thus confirmed
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NEMO⌬hepa mice undergo continuous spontaneous liver apoptosis and develop HCC between 9 and 12 months of age.7 We next investigated whether Casp8 acts procarcinogenicaly in NEMO⌬hepa mice by promoting death receptor-mediated apoptosis. Casp8⌬hepaNEMO⌬hepa mice were periodically monitored by MRI for liver tumor development and compared with NEMO⌬hepa mice. Of note, we did not observe spontaneous hepatocarcinogenesis in Casp8⌬hepa mice (n ⫽ 17) within 1 year (Figure 7A and data not shown). MRI analysis (Figure 7A) revealed first neoplastic lesions in NEMO⌬hepa mice at the age of 30 weeks, which developed into large HCCs within the fol-
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Figure 7. Ablation of Casp8 rescues spontaneous hepatocarcinogenesis in NEMO-deficient mice. (A) Casp8⌬hepaNEMO⌬hepa mice and controls were subjected to MRI at the age of 30 and 40 weeks, respectively. T1-weighted, contrast-enhanced images are shown. Hypointense areas represent tumorigenic liver modifications and are highlighted by arrows. After 40 weeks, animals were killed, and the macroscopic appearance of the livers was documented. (B) H&E staining of paraffin liver sections from 1-year-old Casp8⌬hepaNEMO⌬hepa mice and corresponding controls. Liver dysplasia in NEMO⌬hepa liver is encircled with dotted line. Original magnification, ⫻100 (upper part) and ⫻400 (lower part).
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the relevance of this pathway for hepatocyte apoptosis and also demonstrated the functionality of our strategy. Hepatic ablation of Casp8 revealed 2 unexpected effects: First, Casp8 deficiency triggered increased nonapoptotic liver injury and oxidative stress in ConA-induced hepatitis; and, second, Casp8 deletion induced massive liver necrosis and cholestasis but completely rescued spontaneous apoptosis, steatosis, and hepatocarcinogenesis in NEMO-deficient mice. In both models, we could experimentally link these findings to a general mechanism. RIP1 was proteolytically degraded by Casp8 during inflammation, eg, in NEMO⌬hepa mice and thus accumulated in Casp8-deficient livers. In addition, lack of Casp8 promoted the formation of a pronecrotic kinase complex consisting of FADD, RIP1, and RIP3 in the inflamed liver and also in other experimental settings as described recently.11,12 This kinase is functionally related to TNFinduced over-production of reactive oxygen species and cell necrosis.23 In line with these findings, we detected increased markers of oxidative stress in ConA-treated Casp8⌬hepa liver. In situ, we proved colocalization of RIP1 and RIP3 exclusively in the prenecrotic tissue, which was most evident in Casp8⌬hepaNEMO⌬hepa liver. Our data therefore strongly suggest that nonapoptotic liver injury in Casp8-deficient mice with hepatitis (ConA mediated or NEMO dependent) is a direct consequence of RIP1-RIP3 kinase formation and programmed necrosis. The mechanisms leading to ConA-mediated liver cell injury are still incompletely understood. Different studies suggested a predominance of apoptosis involving JNK1mediated activation of Casp824 or caspase-independent necrosis.16 Using Casp8⌬hepa mice, we aimed to clarify the role of death receptor-mediated apoptosis in the ConA model. In fact, apoptosis contributed only little to ConAmediated injury in our experimental setting and was completely Casp8 independent, hinting at a moderate activation of intrinsic apoptosis by ConA. Our findings show differences compared with recent results published by Kaufmann et al,22 who showed a protective effect of Casp8 deletion in the ConA model, which would suggest that ConA-mediated injury is predominantly mediated by Casp8-dependent apoptosis. We could not clarify these discrepancies so far and speculate that functional differences in the knockout alleles used and dosage effects (sublethal vs lethal ConA dosage) may contribute to this contradiction. In our experiments, ablation of Casp8 was only protective if ConA was administered together with the liver-specific transcription inhibitor GalN, which induces apoptosis in addition to necrosis.16 This suggests that protective effects in Casp8⌬hepa mice strictly depend on receptor-mediated apoptosis, whereas increased ConAinduced liver damage in Casp8⌬hepa mice relies on general transcriptional activity. Elevated liver injury in ConA-treated Casp8⌬hepa mice was associated with prolonged activation of JNK and NF-B. NF-B is activated by TNF via formation of a TNFR1-RIP1-TRAF2 complex.25 Additionally, the RIP1TRAF2 axis has been shown to activate JNK and p38.26
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Thus, prolonged JNK and NF-B activation could be a direct consequence of higher RIP1 expression in Casp8⌬hepa mice. Although JNK1 and JNK2 both significantly contribute to ConA damage,27 the impact for increased necrotic liver injury in our experimental model is unclear so far. NEMO⌬hepa mice develop a complex liver phenotype with chronic inflammation, basal apoptosis, steatosis, and signs of end-stage liver disease (fibrosis and HCC)7,17 reflecting the progression of chronic liver disease in humans. The complex phenotype observed by simultaneous ablation of Casp8 and NEMO is especially intriguing because inhibition of FADD completely rescued disease progression of NEMO⌬hepa livers.7 These results suggested that FADD is absolutely essential for spontaneous necrosis and cholestasis seen in Casp8⌬hepaNEMO⌬hepa livers. The mechanisms leading to hepatocarcinogenesis by NEMO inactivation are complex and still incompletely understood. Ablation of Casp8 completely protected from hepatocyte apoptosis and HCC development. Thus, our data suggest that Casp8-dependent apoptosis and compensatory proliferation are key events for tumour development in this model. However, Casp8⌬hepaNEMO⌬hepa mice also showed hepatitis along with excessive necrotic liver injury and several studies suggested that tissue necrosis is also a strong inducer of compensatory proliferation and HCC.28 This discrepancy could be explained by the observed reversion of necrotic injury with aging, resulting in almost normal liver tissue at the age of 6 months. We could exclude that this is a selection process rather than a reversion because Casp8⌬hepaNEMO⌬hepa mice did not show increased mortality at any age. Thus, transient liver necrosis triggered liver fibrosis but was not sufficient to induce HCC if Casp8 was deleted. At present, we cannot exclude that the tumor-promoting effect of Casp8 is specific for NEMO⌬hepa mice and thus has to be investigated in future studies. Additionally, NEMO⌬hepa-dependent steatosis and steatohepatitis were completely rescued by Casp8 inhibition. This suggests an unknown function of Casp8 in hepatic fat metabolism. Steatohepatitis may trigger growth of HCC, and lack of steatosis in Casp8⌬hepaNEMO⌬hepa mice is most likely another important factor contributing to tumor-free survival in these animals. In summary, we demonstrate that Casp8 has a dual function for liver homeostasis because it triggers apoptosis and HCC but also prevents from necrotic injury in NEMO⌬hepa livers. Our findings may also explain recent results showing elevated transaminases in hepatitis C virus patients treated with caspase inhibitors.29 We conclude that Casp8 inhibition in hepatitis patients likely is of limited benefit because of the higher risk of programmed necrosis. However, combined inhibition of Casp8 and RIP kinases might be a promising approach for future therapy.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2011.08.037. References 1. Malhi H, Gores GJ, Lemasters JJ. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology 2006;43:S31–S44. 2. Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998;17:1675–1687. 3. Varfolomeev EE, Schuchmann M, Luria V, et al. Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 1998;9:267–276. 4. Ding WX, Yin XM. Dissection of the multiple mechanisms of TNF␣-induced apoptosis in liver injury. J Cell Mol Med 2004;8:445– 454. 5. Ermolaeva MA, Michallet MC, Papadopoulou N, et al. Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIFdependent inflammatory responses. Nat Immunol 2008;9:1037– 1046. 6. Schmidt-Supprian M, Bloch W, Courtois G, et al. NEMO/IKK ␥-deficient mice model incontinentia pigmenti. Mol Cell 2000;5:981– 992. 7. Luedde T, Beraza N, Kotsikoris V, et al. Deletion of NEMO/IKK␥ in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell 2007;11:119 –132. 8. Dempsey PW, Doyle SE, He JQ, et al. The signaling adaptors and pathways activated by TNF superfamily. Cytokine Growth Factor Rev 2003;14:193–209. 9. Schneider-Brachert W, Tchikov V, Neumeyer J, et al. Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 2004;21:415– 428. 10. Dong QG, Sclabas GM, Fujioka S, et al. The function of multiple IB:NF-B complexes in the resistance of cancer cells to Taxolinduced apoptosis. Oncogene 2002;21:6510 – 6519. 11. Cho YS, Challa S, Moquin D, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009;137:1112–1123. 12. He S, Wang L, Miao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-␣. Cell 2009;137: 1100 –1111. 13. Kaiser WJ, Upton JW, Long AB, et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 2011;471:368 –72. 14. Freimuth J, Gassler N, Moro N, et al. Application of magnetic resonance imaging in transgenic and chemical mouse models of hepatocellular carcinoma. Mol Cancer 2010;9:94. 15. Tiegs G, Hentschel J, Wendel A. A T cell-dependent experimental liver injury in mice inducible by concanavalin A. J Clin Invest 1992;90:196 –203. 16. Ni HM, Chen X, Ding WX, et al. Differential roles of JNK in ConA/ GalN and ConA-induced liver injury in mice. Am J Pathol 2008; 173:962–972. 17. Beraza N, Ludde T, Assmus U, et al. Hepatocyte-specific IKK␥/ NEMO expression determines the degree of liver injury. Gastroenterology 2007;132:2504 –2517. 18. Beraza N, Malato Y, Sander LE, et al. Hepatocyte-specific NEMO deletion promotes NK/NKT cell- and TRAIL-dependent liver damage. J Exp Med 2009;206:1727–1737.
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19. Rajput A, Kovalenko A, Bogdanov K, et al. RIG-I RNA helicase activation of IRF3 transcription factor is negatively regulated by caspase-8-mediated cleavage of the RIP1 protein. Immunity;34: 340 –351. 20. Canbay A, Friedman S, Gores GJ. Apoptosis: the nexus of liver injury and fibrosis. Hepatology 2004;39:273–278. 21. Kang TB, Ben-Moshe T, Varfolomeev EE, et al. Caspase-8 serves both apoptotic and nonapoptotic roles. J Immunol 2004;173: 2976 –2984. 22. Kaufmann T, Jost PJ, Pellegrini M, et al. Fatal hepatitis mediated by tumor necrosis factor TNF␣ requires caspase-8 and involves the BH3-only proteins Bid and Bim. Immunity 2009;30:56 – 66. 23. Zhang DW, Shao J, Lin J, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009;325:332–336. 24. Chang L, Kamata H, Solinas G, et al. The E3 ubiquitin ligase itch couples JNK activation to TNF␣-induced cell death by inducing c-FLIP(L) turnover. Cell 2006;124:601– 613. 25. Ting AT, Pimentel-Muinos FX, Seed B. RIP mediates tumor necrosis factor receptor 1 activation of NF-B but not Fas/APO-1-initiated apoptosis. EMBO J 1996;15:6189 – 6196. 26. Lee TH, Huang Q, Oikemus S, et al. The death domain kinase RIP1 is essential for tumor necrosis factor ␣ signaling to p38 mitogenactivated protein kinase. Mol Cell Biol 2003;23:8377– 8385. 27. Maeda S, Chang L, Li ZW, et al. IKK is required for prevention of apoptosis mediated by cell-bound but not by circulating TNF␣. Immunity 2003;19:725–737. 28. Naugler WE, Sakurai T, Kim S, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 2007;317:121–124. 29. Manns M, Palmer M, Flisiak R, et al. A Phase-2B trial to evaluate the safety, tolerability and efficacy of a caspase inhibitor GS9450, in adults failing PEG/RBV therapy for chronic HCV infection J Hepatol 2011;54(Suppl1):S55–S56. Received Febrary 7, 2011. Accepted August 11, 2011. Reprint requests Address requests for reprints to: Christian Liedtke, PhD, and Christian Trautwein, MD, Department of Medicine III, University Hospital Aachen, RWTH Aachen University, Pauwelsstrasse 30, D52074 Aachen, Germany. e-mail: [email protected]; and [email protected]; fax: (49) 0241-80 82455. Acknowledgments J.F.’s current address is UCSF Helen Diller Family Comprehensive Cancer Center 1450, San Francisco, California. N.B.’s current address is CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberedh), Bizkaia, Spain. J-M.B. and J.F. contributed equally to this work. This manuscript is dedicated to Michael P. Manns in honor of his 60th birthday. Conflicts of Interest The authors disclose no conflicts. Funding Supported by a grant of the Deutsche Krebshilfe (grant No. 107682) and the Deutsche Forschungsgemeinschaft (DFG), SFB TRR57; by the Instituto de Salud Carlos III (Ministry of Health, Spain; FIS09/02010; to N.B.); and by the program Ramón y Cajal (Ministry of Science and Innovation, Spain).
BASIC AND TRANSLATIONAL LIVER
December 2011