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The role of TNF and Fas dependent signaling in animal models of inflammatory liver injury and liver cancer
European Journal of Cell Biology 91 (2012) 582–589
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European Journal of Cell Biology journal homepage: www.elsevier.de/ejcb
Review
The role of TNF and Fas dependent signaling in animal models of inflammatory liver injury and liver cancer Christian Liedtke ∗ , Christian Trautwein ∗∗ Department of Medicine III, University Hospital Aachen, RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany
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Article history: Received 21 June 2011 Received in revised form 23 August 2011 Accepted 6 October 2011 Keywords: Liver Inflammation Hepatitis Death ligands NF-B signaling I-kappa B kinase complex Hepatocellular carcinoma Apoptosis Caspase-8
a b s t r a c t Tumor Necrosis Factor (TNF) alpha is a pleiotropic cytokine triggering either pro-inflammatory effects via NF-B related pathways or apoptosis through activation of caspase-8. The related death ligands Fas and TRAIL use homologous receptors and similar signaling cascades but predominantly induce apoptosis. Here, we summarize our experimental approaches to analyze the mechanisms and consequences of TNF and Fas signaling with the ultimate aim to define molecular targets for the treatment of inflammatory liver disease and liver cancer. By using conditional knockout technology in mice we genetically dissected the I-kappa B kinase (IKK) complex consisting of IKK1/IKK␣, IKK2/IKK and IKK␥/NEMO. We demonstrated that IKK2/IKK, but not IKK␥/NEMO might be a promising target for the prevention of liver injury after ischemia and reperfusion or treating steatohepatitis. Genetic inactivation of IKK␥/NEMO defined a new animal model of spontaneous hepatitis and hepatocarcinogenesis involving constitutive activation of caspase-8 and basal apoptosis. We further show that caspase-8 is not only regulated by post-translational modifications as suggested earlier, but also by complex transcriptional regulation. Targeted stimulation of the caspase-8 promoter by interferons alpha and gamma, cytotoxic drugs or p53 can substantially sensitize hepatoma cells for apoptosis, whereas hepatocellular carcinoma frequently present an inactive caspase-8 gene promoter. In conclusion, our work demonstrates that therapeutic intervention in the TNF-NF-B-caspase-8 network is technically feasible and could be of potential benefit in inflammatory liver disease. © 2011 Elsevier GmbH. All rights reserved.
Introduction Tumor Necrosis Factor (TNF) alpha is a pleiotropic cytokine mediating several signals via two different receptors, TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF-R2), respectively. In liver physiology, TNF-R1 plays a predominant role. After ligation of TNF to TNF-R1, the adaptor protein TNF receptor-1 associated protein (TRADD) is recruited to the so called death domain of TNF-R1. TRADD is a central molecule for TNF signaling and regulates the activation of at least three different, and in part even contradictory signaling cascades as illustrated in Fig. 1. Association of TRADD with the receptor interacting protein kinase 1 (RIP1) and the TNF receptor associated protein 2 (TRAF2) triggers the interaction with the I-kappa B kinase (IKK) complex. The IKK complex consists of the three subunits IKK1/IKK␣, IKK2/IKK and NEMO/IKK␥. IKK1 and IKK2 are the kinase components of the
∗ Corresponding author. Tel.: +49 0241 80 89249; fax: +49 0241 80 82455. ∗∗ Corresponding author. Tel.: +49 0241 80 80866; fax: +49 0241 80 82455. E-mail addresses: [email protected] (C. Liedtke), [email protected] (C. Trautwein). 0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.10.001
IKK complex whereas NEMO acts as the regulatory subunit. The IKK complex phosphorylates I-kappa B molecules such as I-B␣ at strongly conserved N-terminal serine residues Ser32 and Ser36, which results in its ubiquitination and proteolytic degradation (Fig. 1). As the function of I-kB␣ is to sequester the transcription factor NF-B in the cytoplasm, I-B␣ degradation eventually results in release of NF-B and subsequent nuclear translocation and activation of pro-inflammatory and anti-apoptotic target genes. In addition, association of TRADD with TRAF2 can activate the cJun N-terminal kinases (JNK) via the N-terminal zinc-finger domain of TRAF2. Although the precise activation mechanism remains elusive, it has been demonstrated that activation of the upstream kinases ASK1 (apoptosis signal-related kinase) and the mitogenactivated kinases MKK4 and MKK7 are essential for TNF-mediated JNK activation. Alternatively, interaction of TRADD with the adaptor protein FADD (Fas-associated death domain) and procaspase-8 results in the formation of a death-inducing signaling complex (DISC). DISC formation depends on internalization of the TNF-R1 complex and results in conversion of the premature procaspase-8 into its activated form via an autoproteolytic process. Activated caspase-8 in
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Fig. 1. TNF signaling in the liver. TNF binds its receptor TNF-R1 and can activate the pro-inflammatory and anti-apoptotic NF-B pathway via activation of the IKK complex consisting of NEMO (=IKK␥), IKK1 (=IKK␣) and IKK2 (=IKK). Active IKK complexes phosphorylate IB␣ resulting in its ubiquitination and thus release and nuclear translocation of NF-B subunits p50 and p65, where this transcription factor activates pro-inflammatory and anti-apoptotic genes. Alternatively TNF can induce the pro-apoptotic caspase–cascade via adaptor proteins TRADD and FADD and proteolytic activation of procaspase-8. Activated caspase-8 in turn cleaves and thus activates effector caspases such as caspase-3. Finally, TNF activates cJun N-terminal kinases JNK1 and JNK2 in the liver and subsequent induction of the transcription factor cJun and its target genes. The network of JNK interactions is complex and incompletely understood but may also induce apoptosis or trigger hepatocyte proliferation. Further details are given in the main text.
turn can trigger apoptosis via two different signaling pathways. In type I cells, caspase-8 directly cleaves target proteins such as caspase-3 and triggers apoptosis. In type II cells including hepatocytes, caspase-8-mediated signals undergo signal amplification through a mitochondrial pathway: The cytosolic protein Bid (BH3interacting domain death agonist) is proteolytically activated by caspase-8 and thereby converted into its active form tBID which translocates into the mitochondrial membrane and contributes to increased mitochondrial permeability (mitochondrial permeability transition, MPT) and subsequent release of cytochrome C from the mitochondria into the cytosol. Cytochrome C release triggers the formation of the apoptosome – a complex consisting of Cytochrom c, Apaf-1 and caspase-9 – which is capable of processing and activating the effector caspase, caspase-3. Via positive feedback loops, caspase-3 may then activate further procaspase-8 molecules, but also directly triggers apoptosis by cleavage of cytoplasmic and nuclear substrates. Caspase-8 activity is also controlled by the antagonist c-FLIP, which shares strong homology with procaspase-8, but lacks protease activity and competes via its death effector domain with procaspase-8 for binding at FADD. In addition, c-FLIP is a target gene of NF-kB. Accordingly, the anti-apoptotic function of NF-B results in part from activation of c-FLIP upon TNF stimulation. In this mini review we summarize our findings on TNF and Fas signaling in the liver in the context of published work from other groups with a strong focus on the IKK complex and caspase-8. We
hypothesized that inhibition of IKK subunits or caspase-8 could be of therapeutic benefit and tested this hypothesis in animal models of acute liver failure, chronic hepatitis, ischemia with cold reperfusion or hepatocarcinogenesis.
General role of the IKK complex for embryonic liver development Earlier studies demonstrated that the components of the canonical IKK complex are essential for general embryonic development whereas the role for liver morphogenesis seems to be different for the three subunits. IKK1−/− embryos complete development but die shortly after birth. Loss of IKK1 is not associated with failure to activate IKK or blunted degradation of I-B by pro-inflammatory stimuli, but most strikingly results in failure to form stratified, well-differentiated epidermis and a defect in proliferation and differentiation of epidermal keratinocytes (Hu et al., 1999). In contrast, mice lacking IKK2 die at approximately 14.5 days of gestation in utero as a result of TNF-induced hepatocyte apoptosis (Li et al., 1999; Tanaka et al., 1999) and embryonic fibroblasts derived from these mice have both reduced basal NF-B activity and impaired cytokine-induced NF-B activation. Accordingly, IKK2 – in contrast to IKK1, is essential for proper liver development. Constitutive deletion of the regulatory subunit NEMO revealed similar results as observed in IKK2 mice. NEMO−/− embryos die at around day 13 p.c. from severe liver damage due to apoptosis and murine
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embryonic fibroblasts from these mice have a defect in phosphorylation and degradation of I-B␣ and cannot activate NF-B after challenging with TNF (Rudolph et al., 2000). The role of IKK2 in hepatocytes and non-parenchymal liver cells Data from constitutive IKK2 and NEMO knockout mice demonstrated that these genes have an important function in the liver at least during embryonic development. To bypass the embryonic lethality of IKK2- and NEMO-deficient mice, a conditional knockout approach using the powerful cre/loxP system (Rajewsky et al., 1996) was applied for the directed inactivation of both genes in hepatocytes in vivo. Unexpectedly, hepatocyte-specific inactivation of IKK2 does not lead to impaired activation of NF-B or increased apoptosis after TNF stimulation (Luedde et al., 2005) suggesting that IKK2 performs two different functions during development and in the adult liver. Although IKK2 is essential for liver development, in the adult liver or in primary hepatocytes, the IKK1/Nemo complex is apparently sufficient to fully activate NF-B (e.g. following TNF signaling) even in the absence of IKK2 suggesting that IKK1 can substitute the function of IKK2 in the adult liver. However, conditional deletion of IKK2 is clearly protective in an experimental model of liver ischemia and reperfusion (I/R) and results in reduced necrotic liver injury and decreased aminotransferase levels in comparison to wildtype control animals. Apparently, NF-B activation after I/R happens in a manner different than after TNF-␣ stimulation (Luedde et al., 2005), although the underlying mechanism remains obscure so far. Similarly, hepatic I/R injury can also be improved in wildtype mice by application of a novel, pharmacological inhibitor of IKK2 (AS602868, Frelin et al., 2003). In agreement with the findings in knockout mice, pharmacological inhibition of IKK2 does not impair NF-B activation after I/R, hinting at an NF-B-independent function of IKK2 for ischemic liver injury. Another benefit of IKK2 inhibition was recently discovered in an animal model of non-alcoholic steatohepatitis (NASH). Mice that are fed a high sucrose diet containing 1% orotic acid (HSD) usually develop obesity, insulin resistance and liver macrosteatosis (Feldstein et al., 2003, 2004) which mimics the NASH pathology in humans. IKK2-dependent NF-B signaling is involved in the pathogenesis of NASH as it plays an important role in the development of insulin resistance during dietary-induced obesity (Kim et al., 2001; Yuan et al., 2001). In this context, we recently showed that application of the IKK2 inhibitor AS602868 in HSD fed mice significantly reduced steatosis progression and NF-B activation especially in non-parenchymal cells (Beraza et al., 2008). This is very likely responsible for the observed reduction of the pro-inflammatory TNF- and IL-6 response in these animals. As a consequence, pharmacological IKK2 inhibition protects from NASH-related apoptosis and subsequent liver fibrosis even under conditions of excessive energy uptake. IKK␥/NEMO: an essential key player for liver homeostasis and tumor suppression In contrast to IKK2, the regulatory subunit IKK␥/NEMO is absolutely essential for NF-B activation in the liver. Accordingly, NF-B activation is completely inhibited in mice with a hepatocyte-specific deletion of NEMO (NEMOhepa ). As NF-B mediates protective and anti-apoptotic effects, NEMO-deficient hepatocytes are hypersensitive for massive TNF-induced apoptosis in vivo and in vitro (Beraza et al., 2007). In the experimental model of ischemia and reperfusion, 100% of NEMOhepa mice die within 24 h of excessive apoptosis whereas wildtype control animals survive with predominant necrotic liver injury. In conclusion,
ablation of NEMO shifts the mode of cell death from necrosis to apoptosis after I/R. Even more interesting, NEMOhepa mice provide a complex spontaneous phenotype with basal caspase-8 activation, subsequent hepatocyte apoptosis and elevated aminotransferase levels. These mice also show spontaneous steatohepatitis and finally develop liver fibrosis and hepatocellular carcinoma at the age of 6–12 months with 100% penetrance as a consequence of chronic liver inflammation. This strong phenotype seems to result from over-activation of death receptor signaling as it can be rescued by simultaneous inactivation of FADD (Luedde et al., 2007). Initially, NF-B was considered to be an important mediator of chronic hepatitis and hepatocarcinogenesis (Karin, 2009). However, this paradigm has to be expanded due to the unexpected findings in NEMOhepa mice. The hypothesis that chronic NF-B activation triggers development of hepatocellular carcinoma was derived from earlier studies in Mdr2 (multidrug resistance protein 2) knockout mice (Pikarsky et al., 2004). These mutant animals develop cholestatic hepatitis and spontaneous HCC which is associated with continuous NF-B activation due to elevated TNF levels. Inhibition of NF-B during hepatitis progression was protective and resulted in decreased tumor progression and increased apoptosis. Therefore, it was suggested that the anti-apoptotic properties of NF-B are responsible for hepatocarcinogenesis in Mdr2−/− mice. In view of the recent data, it is suggested that NF-B can either enhance or suppress hepatocarcinogenesis depending on the inflammatory environment. It is still conceivable that NF-B is rather a tumor promoter in the majority of inflammatory liver diseases. However, the persisting steatohepatitis and the continuous cycles of apoptotic cell death and regeneration which are induced in the absence of NEMO are presumably sufficient to drive genomic instability, and thus hepatocarcinogenesis even in the absence of NF-B. The fact that NEMOhepa FADDhepa double mutants, which are protected from extrinsic apoptosis, do not develop HCC (Luedde et al., 2007), further supports this hypothesis. In conclusion, the initial concept that blocking NF-B by therapeutical inhibition of NEMO might be promising for the treatment of inflammatory liver disease and hepatocellular carcinoma could not be proved. Instead, the present data suggests that any interference with the precisely regulated NF-B activity seems to disturb liver homeostasis.
Identifying the actors: liver damage in NEMO-deficient livers is triggered by NK/NKT cells and TRAIL By inactivating NEMO in parenchymal cells of the liver, a new inflammatory animal model was generated that reproduces important steps of human pathogenesis from chronic hepatitis to HCC via steatohepatitis and liver fibrosis. Due to the complexity of the observed phenotype in NEMOhepa mice this is unlikely to originate from a single pathway but rather from a complete disturbance of several signaling cascades. In fact, NEMOhepa mice displayed de-regulated death-receptor signaling shown by the elevated levels of TNF especially in the non-parenchymal cell compartment and we have previously shown that these animals are hypersensitive to TNF-induced liver damage (Beraza et al., 2007). At a first glance it was therefore surprising that NEMOhepa mice were resistant against Fas-induced acute liver failure (Beraza et al., 2009). However, this unexpected phenomenon is associated with dramatic down-regulation of Fas in NEMO-deficient hepatocytes. Fas is tightly regulated on the transcriptional level and induction of Fas depends on binding of activated NF-B to the Fas promoter (Kuhnel et al., 2000). Therefore, Fas resistance can be explained by insufficient receptor expression due to lack of NF-B activity. In contrast, NEMOhepa mice over-express the TRAIL-specific death receptor DR5 especially on hepatocytes (Beraza et al., 2009),
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Fig. 2. Current model explaining spontaneous liver injury and apoptosis in NEMOhepa mice. NF-B activation is completely inhibited in NEMO-deficient hepatocytes leading to spontaneous inflammation with activation of Kupffer cells (KC) and natural killer (NK) cells, lack of anti-apoptotic response and thus apoptosis-hypersensitivity. Moreover, inactivation of NEMO results in strong up-regulation of the death receptor DR5. The DR5 ligand TRAIL is predominantly expressed by NK cells and is essentially involved in basal liver injury of NEMOhepa mice.
which is very likely the consequence of steatohepatitis in these animals as reported earlier (Malhi et al., 2007). These data would suggest that at least part of the inflammatory liver injury is mediated via TRAIL/DR5 signaling. Indeed, TRAIL-expressing natural killer (NK) cells are activated in NEMOhepa livers and depletion of these cells substantially improved basal liver inflammation. NEMOhepa mice were also shown to be hypersensitive to hepatitis induced by the hepatotoxic lectin Concanavalin A and again, selective NK cell depletion or adoptive transfer of TRAIL-deficient mononuclear cells could protect from increased liver injury. Thus, NK cells are essential mediators of hepatic inflammatory injury after inactivation of the NF-B pathway by hyper-activating TRAILdependent signals. Accordingly, it was suggested that liver disease progression in NEMO-deficient livers is determined by a two step process (see Fig. 2) involving up-regulation of DR5 on hepatocytes (due to excessive accumulation of free fatty acids) and activation of NK cells, which express high amounts of TRAIL thereby triggering DR5-mediated hepatocyte apoptosis. Although blocking TRAIL substantially improves the NEMO-deficient liver histology, it cannot be excluded, that TNF expressed by hepatic Kupffer cells also contributes to liver damage in this mouse model. In conclusion, the NEMO/NF-B axis is essential to maintain liver homeostasis and control the innate immune system thereby preventing spontaneous liver injury. Caspase-8: a tightly regulated mediator of extrinsic apoptosis in liver and hepatoma cells The cysteine-aspartate protease caspase-8 has a central function as the initiator caspase in extrinsic apoptotic signaling pathways. As many other caspases, caspase-8 is expressed as a premature zymogen termed procaspase-8. Binding of two procaspase-8 molecules to the adaptor protein FADD via their death effector domains (DED) results in a two-step proteolytic cleavage of the proenzyme resulting in the activated caspase. Activated caspase-8 consists of a homodimer with two p18 and p20 subunits, respectively (Chang et al., 2003; Medema et al., 1997). Transcriptional control of caspase-8 mediates fine tuning of apoptosis in hepatoma cells Based on the current knowledge on caspase-regulation, it was suggested that caspase-8 is predominantly regulated on a posttranslational level by proteolytic processing. However, ectopic
over-expression of the caspase-8 gene in MCF-7 breast carcinoma cells results in spontaneous induction of apoptosis even without stimulation of death receptors (Muzio et al., 1997) which already indicates that apoptosis could also be controlled by gene regulation of caspase-8. Further support for this hypothesis came from our own work by cloning the human caspase-8 promoter and characterizing promoter regulation in human hepatoma cells (Liedtke et al., 2003). The caspase-8 gene is constitutively expressed and a detailed promoter-deletion analysis identified the core promoter essential for basal caspase-8 transcription. Basal gene expression of caspase-8 basically depends on a single binding site for SP1, a transcription factor which is known to regulate gene expression of TATA-box free housekeeping genes (Wierstra, 2008). Control of basal caspase-8 gene expression by SP1 seems to also have a clinical relevance. A polymorphism in this SP1 binding sequence was found in a cohort of Chinese cancer patients and was shown to inactivate SP1 binding (Sun et al., 2007). More interestingly, this polymorphism affects the apoptosis response in T lymphocytes and results in reduced predisposition for several tumor species (Sun et al., 2007; Yang et al., 2008). However, these findings are controversially discussed as such a correlation was not detected in cancer patients from the United States or the UK (Haiman et al., 2008; Pittman et al., 2008). In addition to basal expression we could demonstrate that the caspase-8 gene could be further activated by adenoviral infection, interferon alpha (IFN␣) and several cytotoxic drugs such as Mitomycin C. Caspase-8 induction by adenoviruses is only observed in hepatoma cells with a functional p53 gene (e.g. HepG2 cells) but not in cell lines with a mutated or deleted p53 (Huh7, Hep3B, respectively), and a p53 responsive promoter element was identified in close proximity to the transcriptional start site of the caspase-8 promoter (Liedtke et al., 2003). Although these findings were clearly confirmed by other studies (Yao et al., 2007), a direct physical interaction of p53 with the caspase-8 promoter could not be detected so far suggesting that p53 activates caspase-8 transcription through a not yet identified mediator. However, this mechanism may contribute to the pro-apoptotic function of p53 in general. Caspase-8 gene induction by IFN␣ is rather moderate and depends on an Interferon-stimulated Response Element (ISRE) closely linked to the start site of transcription (Liedtke et al., 2006) and results in a very slight increase of caspase-8 protein and subsequent apoptosis. The same ISRE element is capable of binding interferon-regulatory factor 1 (IRF-1) after induction with interferon gamma in breast carcinoma cells (Ruiz-Ruiz et al., 2004) and neuroblastoma (De Ambrosis et al., 2007). However, transcriptional
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Fig. 3. Organization and regulation of the caspase-8 promoter. Caspase-8 is a constitutive active gene with basal gene transcription controlled by transcription factor SP1. Of notice, SP1 binding sites usually contain CG dinucleotides which are frequently methylated (Me) in murine hepatocellular carcinoma (HCC) resulting in gene silencing. Caspase-8 gene transcription can be further enhanced by activating Jun/AP1, IFN␣- and IFN␥ dependent genes and p53.
up-regulation of caspase-8 by IFN␣ sensitizes hepatoma cells for stronger apoptosis induced by a second stimulus such as TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing Ligand) which is most likely the general physiological role of caspase-8 regulation on the transcriptional level. This mechanism could be useful for the development of a more efficient chemotherapy in tumor patients with the ultimate goal of triggering apoptosis in tumor cells. We thus screened for cytotoxic drugs, which have the potential to activate the caspase-8 promoter. One of these compounds, Mitomycin C, resulted in strong up-regulation of caspase-8 transcription and subsequent apoptosis of hepatoma cells which could be synergistically enhanced by pre-treatment with IFN␣ (Liedtke et al., 2007). Mechanistically, Mitomycin C activates N-terminal Jun kinases (JNK) resulting in phosphorylation of c-Jun, one of the subunits of the transcription factor AP1. Indeed, the caspase-8 promoter contains a functional AP1 binding site which is essential for the transcriptional caspase-8 response on Mitomycin C and coincidentally caspase-8 expression was essential for Mitomycin C-induced cell death. In conclusion, caspase-8 is not only regulated by posttranslational modifications as expected earlier, but also via complex regulation at the level of gene transcription. Basal caspase8 gene expression is ensured by SP1 and can be further induced via p53, Jun/AP1 and interferon-regulated factors as illustrated in Fig. 3. This expands our knowledge of how p53 and JNKs might contribute to apoptosis induction. We observed that exceeding a threshold level of caspase-8 mRNA expression e.g. after p53 induction or combined treatment with Mitomycin C and INF␣ results in caspase-8 dependent apoptosis even without stimulation of death receptors suggesting that high caspase-8 concentration triggers proteolytic auto-activation in the absence of death ligands (Fig. 4) and thus targeted drug-induced up-regulation of the caspase-8 gene might be of benefit for tumor therapy in patients. Caspase-8: tumor suppressor or tumor promoter? The acquired resistance to cell death was proposed to be a hallmark of cancer (Hanahan and Weinberg, 2011). Healthy hepatocytes are usually very sensitive to Fas- and TNF-mediated apoptosis in vivo and in vitro. However, Hepatocellular Carcinoma (HCC) poorly responds to chemotherapy (Worns et al., 2009) indicating that hepatoma cells have acquired apoptosis resistance
during malignant transformation. As earlier results demonstrated that appropriate caspase-8 gene expression might be relevant for drug-mediated apoptosis e.g. by Mitomycin C treatment, it was hypothesized that caspase-8 is a tumor suppressor during hepatocarcinogenesis. In fact, in several animal models of hepatocarcinogenesis involving over-expression of the oncogenes c-myc or EGF (Dalemans et al., 1990; Sandgren et al., 1993; Tonjes et al., 1995) caspase-8 is frequently silenced or down-regulated (Liedtke et al., 2005) indicating that tumorigenesis is associated with inactivation of caspase-8. In line with these findings, caspase-8 is also inactivated in human hepatocellular carcinoma (Soung et al.,
Fig. 4. The threshold model of apoptosis induction by enhanced caspase-8 gene expression. From our data we conclude that the transcriptional expression level of caspase-8 determines the vitality and apoptosis response of cultured cells. Normal hepatoma cells have some caspase-8 gene expression but do not undergo apoptosis. Moderate induction of caspase-8 transcription e.g. by IFN␣ will not result in spontaneous apoptosis but sensitizes for apoptosis induced by a second stimulus such as TRAIL or Mitomycin C. However, exceeding a threshold level of caspase-8 expression usually results in apoptosis of hepatoma cells even without stimulation of death receptors such as TNF or TRAIL.
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2005), childhood neuroblastoma (Teitz et al., 2000), lung cancer (Shivapurkar et al., 2002) and breast cancer cells (Wu et al., 2010). Interestingly, in tissue derived from murine liver tumors, the murine caspase-8 promoter is strongly methylated at CG dinucleotides. This is especially intriguing as two of these methylated CG dinucleotides are embedded in functional binding sites for SP1 which are essential for basal caspase-8 expression also in mice (Liedtke et al., 2005). We demonstrated in vitro, that CG methylation at the SP1 binding motif within the caspase-8 promoter completely inhibits SP1 binding and thus basal promoter transactivation. In conclusion, at least in murine liver tumors silencing of caspase8 seems to be mediated via blocking the basal SP1 transcription machinery, although it cannot be excluded that other mechanisms such as somatic point mutations may also contribute to caspase-8 inactivation in other tissues. However, to date it is still not clarified, if caspase-8 inactivation is an enhancer or even prerequisite for liver tumor initiation or simply a consequence of tumor cell selection during tumor progression. As mentioned before, conditional knockout mice deficient of NEMO in hepatocytes undergo spontaneous apoptosis associated with basal caspase-8 activation and eventually develop hepatocellular carcinoma, which could be rescued by simultaneous ablation of the adaptor protein FADD (Luedde et al., 2007). This argues that caspase-8 acts as a tumor promoter in an inflammatory environment with dominant apoptosis. As conventional knockout mice for caspase-8 are not viable due to developmental defects (Varfolomeev et al., 1998), future studies with conditional caspase-8 knockout mice in appropriate tumor models will be essential to finally address this important question.
Caspase-8: a suitable target gene for therapeutic intervention? Caspase activation and apoptosis is frequently associated with acute and chronic liver injury e.g. during acute liver failure (Leifeld et al., 2006) or in patients with chronic hepatitis C (Bantel et al., 2001). Especially acute liver failure requires the immediate stabilization of liver homeostasis or alternatively liver transplantation to avoid the death of the patient. Therefore, current approaches including our own studies address the probability that targeted inhibition of caspases might be a practicable approach to treat apoptosis-mediated liver disease. In a pilot study, it was demonstrated that therapeutic application of a small interfering RNA (siRNA) directed against caspase-8 is feasible at least in mice and efficiently targeted the liver after hydrodynamic injection. Moreover, delivery of caspase-8 specific siRNA was protective in a mouse model of acute liver injury induced by the Fas-agonistic antibody (Zender et al., 2003). However, these experiments did not reflect the situation in patients, where therapy is only possible after onset of liver injury. Interestingly, caspase-8 siRNA delivery was even protective if applied after onset of acute liver failure pointing to a high therapeutic potential of caspase8 inhibition at least in Fas-mediated liver disease. However, it remains questionable, if siRNA per se will be of therapeutic value in human patients instead of caspase-specific small molecular inhibitors, which are already available. In this context, application of a pan-caspase inhibitor in a small cohort of hepatitis C patients was shown to decrease aminotransferase activity, which is an important parameter for the level of liver injury (Pockros et al., 2007). These findings suggest that caspase-inhibition indeed could be a promising approach for therapy of liver disease. However, this strategy is now called into question as several independent studies revealed a new and rather unexpected function of caspase-8, FADD and cFLIP which is the inhibition of programmed necrosis (reviewed in
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Vandenabeele et al., 2010). The concept of programmed necrosis (also termed necroptosis) was first postulated in 1998 based on the finding that L929 fibrosarcoma cells undergo necrosis-like cell death after simultaneous caspase-inhibition and TNF stimulation (Vercammen et al., 1998). This alternative mode of cell death is strongly regulated and essentially depends on the receptorinteracting kinases RIP1 and RIP3, respectively (Cho et al., 2009; He et al., 2009). Using an elegant knockout approach very recent work finally demonstrated that embryonic lethality in Casp8−/− , FLIP−/− and FADD−/− embryos is due to programmed necrosis rather than apoptosis deficiency and can be rescued by simultaneous inactivation of RIP3 or RIP1, respectively (Kaiser et al., 2011; Oberst et al., 2011; Zhang et al., 2011). Accordingly, inactivating caspase-8 may protect from apoptosis but also seems to trigger programmed necrosis especially in an inflammatory environment with strong TNF expression. Future work using conditional knockout mice for caspase-8 will further evaluate this pro-necrotic potential. Summary We intensively investigated TNF-, Fas- and TRAIL-related signaling pathways in the murine liver and in hepatoma cells with a strong focus on the two axes leading to activation of NF-B or caspase-8, respectively. The data revealed that therapeutic inhibition of IKK2/IKK could be of substantial benefit in several pathological situations of the liver e.g. after ischemia and reperfusion or in steatohepatitis. By conditional genetic inactivation of IKK␥/NEMO in hepatocytes we defined a new animal model of liver inflammation and tumorigenesis, which reflects several important aspects of chronic hepatitis and hepatocarcinogenesis in human patients. Therefore, NEMOhepa mice will be a valuable tool to study mechanisms of inflammatory liver disease in the future. However, our studies also demonstrated that NEMO is essential for liver homeostasis. Thus, therapeutic inhibition of NEMO – although technically feasible – does not seem to be a promising approach for treatment of hepatitis or prevention of HCC. Instead, the findings in NEMOhepa mice challenge the paradigm that apoptosis is a tumorsuppressive mechanism as chronic apoptosis in these mice is clearly associated with hepatocarcinogenesis. In this line of evidence the postulated role of caspase-8 as a potential tumor suppressor has to be re-defined by future investigations. Some data hint at caspase8 as a tumor suppressor as caspase-8 is silenced in several tumor species including hepatocellular carcinoma but constitutively activated in NEMOhepa mice where it likely acts as a tumor promoter. Our data also modifies the old paradigm that caspase-8 is predominantly regulated on the posttranslational level. In fact, caspase-8 activity and apoptosis can also be modulated by triggering the caspase-8 promoter. Control of basal transcription e.g. by SP1 may explain some phenomenon such as increased tumor predisposition and apoptosis resistance in tumors. Finally, understanding the crosstalk between cytotoxic drugs, the caspase-8 promoter and apoptosis induction may help to define more efficient combination therapies in tumor treatment. Conflict of interest The authors declare that they have no competing interests. Acknowledgement Work reported in this mini review was supported in part by the Deutsche Forschungsgemeinschaft (SFB542, C15) to C. Liedtke and C. Trautwein.
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European Journal of Cell Biology journal homepage: www.elsevier.de/ejcb
Review
The role of TNF and Fas dependent signaling in animal models of inflammatory liver injury and liver cancer Christian Liedtke ∗ , Christian Trautwein ∗∗ Department of Medicine III, University Hospital Aachen, RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany
a r t i c l e
i n f o
Article history: Received 21 June 2011 Received in revised form 23 August 2011 Accepted 6 October 2011 Keywords: Liver Inflammation Hepatitis Death ligands NF-B signaling I-kappa B kinase complex Hepatocellular carcinoma Apoptosis Caspase-8
a b s t r a c t Tumor Necrosis Factor (TNF) alpha is a pleiotropic cytokine triggering either pro-inflammatory effects via NF-B related pathways or apoptosis through activation of caspase-8. The related death ligands Fas and TRAIL use homologous receptors and similar signaling cascades but predominantly induce apoptosis. Here, we summarize our experimental approaches to analyze the mechanisms and consequences of TNF and Fas signaling with the ultimate aim to define molecular targets for the treatment of inflammatory liver disease and liver cancer. By using conditional knockout technology in mice we genetically dissected the I-kappa B kinase (IKK) complex consisting of IKK1/IKK␣, IKK2/IKK and IKK␥/NEMO. We demonstrated that IKK2/IKK, but not IKK␥/NEMO might be a promising target for the prevention of liver injury after ischemia and reperfusion or treating steatohepatitis. Genetic inactivation of IKK␥/NEMO defined a new animal model of spontaneous hepatitis and hepatocarcinogenesis involving constitutive activation of caspase-8 and basal apoptosis. We further show that caspase-8 is not only regulated by post-translational modifications as suggested earlier, but also by complex transcriptional regulation. Targeted stimulation of the caspase-8 promoter by interferons alpha and gamma, cytotoxic drugs or p53 can substantially sensitize hepatoma cells for apoptosis, whereas hepatocellular carcinoma frequently present an inactive caspase-8 gene promoter. In conclusion, our work demonstrates that therapeutic intervention in the TNF-NF-B-caspase-8 network is technically feasible and could be of potential benefit in inflammatory liver disease. © 2011 Elsevier GmbH. All rights reserved.
Introduction Tumor Necrosis Factor (TNF) alpha is a pleiotropic cytokine mediating several signals via two different receptors, TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF-R2), respectively. In liver physiology, TNF-R1 plays a predominant role. After ligation of TNF to TNF-R1, the adaptor protein TNF receptor-1 associated protein (TRADD) is recruited to the so called death domain of TNF-R1. TRADD is a central molecule for TNF signaling and regulates the activation of at least three different, and in part even contradictory signaling cascades as illustrated in Fig. 1. Association of TRADD with the receptor interacting protein kinase 1 (RIP1) and the TNF receptor associated protein 2 (TRAF2) triggers the interaction with the I-kappa B kinase (IKK) complex. The IKK complex consists of the three subunits IKK1/IKK␣, IKK2/IKK and NEMO/IKK␥. IKK1 and IKK2 are the kinase components of the
∗ Corresponding author. Tel.: +49 0241 80 89249; fax: +49 0241 80 82455. ∗∗ Corresponding author. Tel.: +49 0241 80 80866; fax: +49 0241 80 82455. E-mail addresses: [email protected] (C. Liedtke), [email protected] (C. Trautwein). 0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.10.001
IKK complex whereas NEMO acts as the regulatory subunit. The IKK complex phosphorylates I-kappa B molecules such as I-B␣ at strongly conserved N-terminal serine residues Ser32 and Ser36, which results in its ubiquitination and proteolytic degradation (Fig. 1). As the function of I-kB␣ is to sequester the transcription factor NF-B in the cytoplasm, I-B␣ degradation eventually results in release of NF-B and subsequent nuclear translocation and activation of pro-inflammatory and anti-apoptotic target genes. In addition, association of TRADD with TRAF2 can activate the cJun N-terminal kinases (JNK) via the N-terminal zinc-finger domain of TRAF2. Although the precise activation mechanism remains elusive, it has been demonstrated that activation of the upstream kinases ASK1 (apoptosis signal-related kinase) and the mitogenactivated kinases MKK4 and MKK7 are essential for TNF-mediated JNK activation. Alternatively, interaction of TRADD with the adaptor protein FADD (Fas-associated death domain) and procaspase-8 results in the formation of a death-inducing signaling complex (DISC). DISC formation depends on internalization of the TNF-R1 complex and results in conversion of the premature procaspase-8 into its activated form via an autoproteolytic process. Activated caspase-8 in
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Fig. 1. TNF signaling in the liver. TNF binds its receptor TNF-R1 and can activate the pro-inflammatory and anti-apoptotic NF-B pathway via activation of the IKK complex consisting of NEMO (=IKK␥), IKK1 (=IKK␣) and IKK2 (=IKK). Active IKK complexes phosphorylate IB␣ resulting in its ubiquitination and thus release and nuclear translocation of NF-B subunits p50 and p65, where this transcription factor activates pro-inflammatory and anti-apoptotic genes. Alternatively TNF can induce the pro-apoptotic caspase–cascade via adaptor proteins TRADD and FADD and proteolytic activation of procaspase-8. Activated caspase-8 in turn cleaves and thus activates effector caspases such as caspase-3. Finally, TNF activates cJun N-terminal kinases JNK1 and JNK2 in the liver and subsequent induction of the transcription factor cJun and its target genes. The network of JNK interactions is complex and incompletely understood but may also induce apoptosis or trigger hepatocyte proliferation. Further details are given in the main text.
turn can trigger apoptosis via two different signaling pathways. In type I cells, caspase-8 directly cleaves target proteins such as caspase-3 and triggers apoptosis. In type II cells including hepatocytes, caspase-8-mediated signals undergo signal amplification through a mitochondrial pathway: The cytosolic protein Bid (BH3interacting domain death agonist) is proteolytically activated by caspase-8 and thereby converted into its active form tBID which translocates into the mitochondrial membrane and contributes to increased mitochondrial permeability (mitochondrial permeability transition, MPT) and subsequent release of cytochrome C from the mitochondria into the cytosol. Cytochrome C release triggers the formation of the apoptosome – a complex consisting of Cytochrom c, Apaf-1 and caspase-9 – which is capable of processing and activating the effector caspase, caspase-3. Via positive feedback loops, caspase-3 may then activate further procaspase-8 molecules, but also directly triggers apoptosis by cleavage of cytoplasmic and nuclear substrates. Caspase-8 activity is also controlled by the antagonist c-FLIP, which shares strong homology with procaspase-8, but lacks protease activity and competes via its death effector domain with procaspase-8 for binding at FADD. In addition, c-FLIP is a target gene of NF-kB. Accordingly, the anti-apoptotic function of NF-B results in part from activation of c-FLIP upon TNF stimulation. In this mini review we summarize our findings on TNF and Fas signaling in the liver in the context of published work from other groups with a strong focus on the IKK complex and caspase-8. We
hypothesized that inhibition of IKK subunits or caspase-8 could be of therapeutic benefit and tested this hypothesis in animal models of acute liver failure, chronic hepatitis, ischemia with cold reperfusion or hepatocarcinogenesis.
General role of the IKK complex for embryonic liver development Earlier studies demonstrated that the components of the canonical IKK complex are essential for general embryonic development whereas the role for liver morphogenesis seems to be different for the three subunits. IKK1−/− embryos complete development but die shortly after birth. Loss of IKK1 is not associated with failure to activate IKK or blunted degradation of I-B by pro-inflammatory stimuli, but most strikingly results in failure to form stratified, well-differentiated epidermis and a defect in proliferation and differentiation of epidermal keratinocytes (Hu et al., 1999). In contrast, mice lacking IKK2 die at approximately 14.5 days of gestation in utero as a result of TNF-induced hepatocyte apoptosis (Li et al., 1999; Tanaka et al., 1999) and embryonic fibroblasts derived from these mice have both reduced basal NF-B activity and impaired cytokine-induced NF-B activation. Accordingly, IKK2 – in contrast to IKK1, is essential for proper liver development. Constitutive deletion of the regulatory subunit NEMO revealed similar results as observed in IKK2 mice. NEMO−/− embryos die at around day 13 p.c. from severe liver damage due to apoptosis and murine
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embryonic fibroblasts from these mice have a defect in phosphorylation and degradation of I-B␣ and cannot activate NF-B after challenging with TNF (Rudolph et al., 2000). The role of IKK2 in hepatocytes and non-parenchymal liver cells Data from constitutive IKK2 and NEMO knockout mice demonstrated that these genes have an important function in the liver at least during embryonic development. To bypass the embryonic lethality of IKK2- and NEMO-deficient mice, a conditional knockout approach using the powerful cre/loxP system (Rajewsky et al., 1996) was applied for the directed inactivation of both genes in hepatocytes in vivo. Unexpectedly, hepatocyte-specific inactivation of IKK2 does not lead to impaired activation of NF-B or increased apoptosis after TNF stimulation (Luedde et al., 2005) suggesting that IKK2 performs two different functions during development and in the adult liver. Although IKK2 is essential for liver development, in the adult liver or in primary hepatocytes, the IKK1/Nemo complex is apparently sufficient to fully activate NF-B (e.g. following TNF signaling) even in the absence of IKK2 suggesting that IKK1 can substitute the function of IKK2 in the adult liver. However, conditional deletion of IKK2 is clearly protective in an experimental model of liver ischemia and reperfusion (I/R) and results in reduced necrotic liver injury and decreased aminotransferase levels in comparison to wildtype control animals. Apparently, NF-B activation after I/R happens in a manner different than after TNF-␣ stimulation (Luedde et al., 2005), although the underlying mechanism remains obscure so far. Similarly, hepatic I/R injury can also be improved in wildtype mice by application of a novel, pharmacological inhibitor of IKK2 (AS602868, Frelin et al., 2003). In agreement with the findings in knockout mice, pharmacological inhibition of IKK2 does not impair NF-B activation after I/R, hinting at an NF-B-independent function of IKK2 for ischemic liver injury. Another benefit of IKK2 inhibition was recently discovered in an animal model of non-alcoholic steatohepatitis (NASH). Mice that are fed a high sucrose diet containing 1% orotic acid (HSD) usually develop obesity, insulin resistance and liver macrosteatosis (Feldstein et al., 2003, 2004) which mimics the NASH pathology in humans. IKK2-dependent NF-B signaling is involved in the pathogenesis of NASH as it plays an important role in the development of insulin resistance during dietary-induced obesity (Kim et al., 2001; Yuan et al., 2001). In this context, we recently showed that application of the IKK2 inhibitor AS602868 in HSD fed mice significantly reduced steatosis progression and NF-B activation especially in non-parenchymal cells (Beraza et al., 2008). This is very likely responsible for the observed reduction of the pro-inflammatory TNF- and IL-6 response in these animals. As a consequence, pharmacological IKK2 inhibition protects from NASH-related apoptosis and subsequent liver fibrosis even under conditions of excessive energy uptake. IKK␥/NEMO: an essential key player for liver homeostasis and tumor suppression In contrast to IKK2, the regulatory subunit IKK␥/NEMO is absolutely essential for NF-B activation in the liver. Accordingly, NF-B activation is completely inhibited in mice with a hepatocyte-specific deletion of NEMO (NEMOhepa ). As NF-B mediates protective and anti-apoptotic effects, NEMO-deficient hepatocytes are hypersensitive for massive TNF-induced apoptosis in vivo and in vitro (Beraza et al., 2007). In the experimental model of ischemia and reperfusion, 100% of NEMOhepa mice die within 24 h of excessive apoptosis whereas wildtype control animals survive with predominant necrotic liver injury. In conclusion,
ablation of NEMO shifts the mode of cell death from necrosis to apoptosis after I/R. Even more interesting, NEMOhepa mice provide a complex spontaneous phenotype with basal caspase-8 activation, subsequent hepatocyte apoptosis and elevated aminotransferase levels. These mice also show spontaneous steatohepatitis and finally develop liver fibrosis and hepatocellular carcinoma at the age of 6–12 months with 100% penetrance as a consequence of chronic liver inflammation. This strong phenotype seems to result from over-activation of death receptor signaling as it can be rescued by simultaneous inactivation of FADD (Luedde et al., 2007). Initially, NF-B was considered to be an important mediator of chronic hepatitis and hepatocarcinogenesis (Karin, 2009). However, this paradigm has to be expanded due to the unexpected findings in NEMOhepa mice. The hypothesis that chronic NF-B activation triggers development of hepatocellular carcinoma was derived from earlier studies in Mdr2 (multidrug resistance protein 2) knockout mice (Pikarsky et al., 2004). These mutant animals develop cholestatic hepatitis and spontaneous HCC which is associated with continuous NF-B activation due to elevated TNF levels. Inhibition of NF-B during hepatitis progression was protective and resulted in decreased tumor progression and increased apoptosis. Therefore, it was suggested that the anti-apoptotic properties of NF-B are responsible for hepatocarcinogenesis in Mdr2−/− mice. In view of the recent data, it is suggested that NF-B can either enhance or suppress hepatocarcinogenesis depending on the inflammatory environment. It is still conceivable that NF-B is rather a tumor promoter in the majority of inflammatory liver diseases. However, the persisting steatohepatitis and the continuous cycles of apoptotic cell death and regeneration which are induced in the absence of NEMO are presumably sufficient to drive genomic instability, and thus hepatocarcinogenesis even in the absence of NF-B. The fact that NEMOhepa FADDhepa double mutants, which are protected from extrinsic apoptosis, do not develop HCC (Luedde et al., 2007), further supports this hypothesis. In conclusion, the initial concept that blocking NF-B by therapeutical inhibition of NEMO might be promising for the treatment of inflammatory liver disease and hepatocellular carcinoma could not be proved. Instead, the present data suggests that any interference with the precisely regulated NF-B activity seems to disturb liver homeostasis.
Identifying the actors: liver damage in NEMO-deficient livers is triggered by NK/NKT cells and TRAIL By inactivating NEMO in parenchymal cells of the liver, a new inflammatory animal model was generated that reproduces important steps of human pathogenesis from chronic hepatitis to HCC via steatohepatitis and liver fibrosis. Due to the complexity of the observed phenotype in NEMOhepa mice this is unlikely to originate from a single pathway but rather from a complete disturbance of several signaling cascades. In fact, NEMOhepa mice displayed de-regulated death-receptor signaling shown by the elevated levels of TNF especially in the non-parenchymal cell compartment and we have previously shown that these animals are hypersensitive to TNF-induced liver damage (Beraza et al., 2007). At a first glance it was therefore surprising that NEMOhepa mice were resistant against Fas-induced acute liver failure (Beraza et al., 2009). However, this unexpected phenomenon is associated with dramatic down-regulation of Fas in NEMO-deficient hepatocytes. Fas is tightly regulated on the transcriptional level and induction of Fas depends on binding of activated NF-B to the Fas promoter (Kuhnel et al., 2000). Therefore, Fas resistance can be explained by insufficient receptor expression due to lack of NF-B activity. In contrast, NEMOhepa mice over-express the TRAIL-specific death receptor DR5 especially on hepatocytes (Beraza et al., 2009),
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Fig. 2. Current model explaining spontaneous liver injury and apoptosis in NEMOhepa mice. NF-B activation is completely inhibited in NEMO-deficient hepatocytes leading to spontaneous inflammation with activation of Kupffer cells (KC) and natural killer (NK) cells, lack of anti-apoptotic response and thus apoptosis-hypersensitivity. Moreover, inactivation of NEMO results in strong up-regulation of the death receptor DR5. The DR5 ligand TRAIL is predominantly expressed by NK cells and is essentially involved in basal liver injury of NEMOhepa mice.
which is very likely the consequence of steatohepatitis in these animals as reported earlier (Malhi et al., 2007). These data would suggest that at least part of the inflammatory liver injury is mediated via TRAIL/DR5 signaling. Indeed, TRAIL-expressing natural killer (NK) cells are activated in NEMOhepa livers and depletion of these cells substantially improved basal liver inflammation. NEMOhepa mice were also shown to be hypersensitive to hepatitis induced by the hepatotoxic lectin Concanavalin A and again, selective NK cell depletion or adoptive transfer of TRAIL-deficient mononuclear cells could protect from increased liver injury. Thus, NK cells are essential mediators of hepatic inflammatory injury after inactivation of the NF-B pathway by hyper-activating TRAILdependent signals. Accordingly, it was suggested that liver disease progression in NEMO-deficient livers is determined by a two step process (see Fig. 2) involving up-regulation of DR5 on hepatocytes (due to excessive accumulation of free fatty acids) and activation of NK cells, which express high amounts of TRAIL thereby triggering DR5-mediated hepatocyte apoptosis. Although blocking TRAIL substantially improves the NEMO-deficient liver histology, it cannot be excluded, that TNF expressed by hepatic Kupffer cells also contributes to liver damage in this mouse model. In conclusion, the NEMO/NF-B axis is essential to maintain liver homeostasis and control the innate immune system thereby preventing spontaneous liver injury. Caspase-8: a tightly regulated mediator of extrinsic apoptosis in liver and hepatoma cells The cysteine-aspartate protease caspase-8 has a central function as the initiator caspase in extrinsic apoptotic signaling pathways. As many other caspases, caspase-8 is expressed as a premature zymogen termed procaspase-8. Binding of two procaspase-8 molecules to the adaptor protein FADD via their death effector domains (DED) results in a two-step proteolytic cleavage of the proenzyme resulting in the activated caspase. Activated caspase-8 consists of a homodimer with two p18 and p20 subunits, respectively (Chang et al., 2003; Medema et al., 1997). Transcriptional control of caspase-8 mediates fine tuning of apoptosis in hepatoma cells Based on the current knowledge on caspase-regulation, it was suggested that caspase-8 is predominantly regulated on a posttranslational level by proteolytic processing. However, ectopic
over-expression of the caspase-8 gene in MCF-7 breast carcinoma cells results in spontaneous induction of apoptosis even without stimulation of death receptors (Muzio et al., 1997) which already indicates that apoptosis could also be controlled by gene regulation of caspase-8. Further support for this hypothesis came from our own work by cloning the human caspase-8 promoter and characterizing promoter regulation in human hepatoma cells (Liedtke et al., 2003). The caspase-8 gene is constitutively expressed and a detailed promoter-deletion analysis identified the core promoter essential for basal caspase-8 transcription. Basal gene expression of caspase-8 basically depends on a single binding site for SP1, a transcription factor which is known to regulate gene expression of TATA-box free housekeeping genes (Wierstra, 2008). Control of basal caspase-8 gene expression by SP1 seems to also have a clinical relevance. A polymorphism in this SP1 binding sequence was found in a cohort of Chinese cancer patients and was shown to inactivate SP1 binding (Sun et al., 2007). More interestingly, this polymorphism affects the apoptosis response in T lymphocytes and results in reduced predisposition for several tumor species (Sun et al., 2007; Yang et al., 2008). However, these findings are controversially discussed as such a correlation was not detected in cancer patients from the United States or the UK (Haiman et al., 2008; Pittman et al., 2008). In addition to basal expression we could demonstrate that the caspase-8 gene could be further activated by adenoviral infection, interferon alpha (IFN␣) and several cytotoxic drugs such as Mitomycin C. Caspase-8 induction by adenoviruses is only observed in hepatoma cells with a functional p53 gene (e.g. HepG2 cells) but not in cell lines with a mutated or deleted p53 (Huh7, Hep3B, respectively), and a p53 responsive promoter element was identified in close proximity to the transcriptional start site of the caspase-8 promoter (Liedtke et al., 2003). Although these findings were clearly confirmed by other studies (Yao et al., 2007), a direct physical interaction of p53 with the caspase-8 promoter could not be detected so far suggesting that p53 activates caspase-8 transcription through a not yet identified mediator. However, this mechanism may contribute to the pro-apoptotic function of p53 in general. Caspase-8 gene induction by IFN␣ is rather moderate and depends on an Interferon-stimulated Response Element (ISRE) closely linked to the start site of transcription (Liedtke et al., 2006) and results in a very slight increase of caspase-8 protein and subsequent apoptosis. The same ISRE element is capable of binding interferon-regulatory factor 1 (IRF-1) after induction with interferon gamma in breast carcinoma cells (Ruiz-Ruiz et al., 2004) and neuroblastoma (De Ambrosis et al., 2007). However, transcriptional
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Fig. 3. Organization and regulation of the caspase-8 promoter. Caspase-8 is a constitutive active gene with basal gene transcription controlled by transcription factor SP1. Of notice, SP1 binding sites usually contain CG dinucleotides which are frequently methylated (Me) in murine hepatocellular carcinoma (HCC) resulting in gene silencing. Caspase-8 gene transcription can be further enhanced by activating Jun/AP1, IFN␣- and IFN␥ dependent genes and p53.
up-regulation of caspase-8 by IFN␣ sensitizes hepatoma cells for stronger apoptosis induced by a second stimulus such as TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing Ligand) which is most likely the general physiological role of caspase-8 regulation on the transcriptional level. This mechanism could be useful for the development of a more efficient chemotherapy in tumor patients with the ultimate goal of triggering apoptosis in tumor cells. We thus screened for cytotoxic drugs, which have the potential to activate the caspase-8 promoter. One of these compounds, Mitomycin C, resulted in strong up-regulation of caspase-8 transcription and subsequent apoptosis of hepatoma cells which could be synergistically enhanced by pre-treatment with IFN␣ (Liedtke et al., 2007). Mechanistically, Mitomycin C activates N-terminal Jun kinases (JNK) resulting in phosphorylation of c-Jun, one of the subunits of the transcription factor AP1. Indeed, the caspase-8 promoter contains a functional AP1 binding site which is essential for the transcriptional caspase-8 response on Mitomycin C and coincidentally caspase-8 expression was essential for Mitomycin C-induced cell death. In conclusion, caspase-8 is not only regulated by posttranslational modifications as expected earlier, but also via complex regulation at the level of gene transcription. Basal caspase8 gene expression is ensured by SP1 and can be further induced via p53, Jun/AP1 and interferon-regulated factors as illustrated in Fig. 3. This expands our knowledge of how p53 and JNKs might contribute to apoptosis induction. We observed that exceeding a threshold level of caspase-8 mRNA expression e.g. after p53 induction or combined treatment with Mitomycin C and INF␣ results in caspase-8 dependent apoptosis even without stimulation of death receptors suggesting that high caspase-8 concentration triggers proteolytic auto-activation in the absence of death ligands (Fig. 4) and thus targeted drug-induced up-regulation of the caspase-8 gene might be of benefit for tumor therapy in patients. Caspase-8: tumor suppressor or tumor promoter? The acquired resistance to cell death was proposed to be a hallmark of cancer (Hanahan and Weinberg, 2011). Healthy hepatocytes are usually very sensitive to Fas- and TNF-mediated apoptosis in vivo and in vitro. However, Hepatocellular Carcinoma (HCC) poorly responds to chemotherapy (Worns et al., 2009) indicating that hepatoma cells have acquired apoptosis resistance
during malignant transformation. As earlier results demonstrated that appropriate caspase-8 gene expression might be relevant for drug-mediated apoptosis e.g. by Mitomycin C treatment, it was hypothesized that caspase-8 is a tumor suppressor during hepatocarcinogenesis. In fact, in several animal models of hepatocarcinogenesis involving over-expression of the oncogenes c-myc or EGF (Dalemans et al., 1990; Sandgren et al., 1993; Tonjes et al., 1995) caspase-8 is frequently silenced or down-regulated (Liedtke et al., 2005) indicating that tumorigenesis is associated with inactivation of caspase-8. In line with these findings, caspase-8 is also inactivated in human hepatocellular carcinoma (Soung et al.,
Fig. 4. The threshold model of apoptosis induction by enhanced caspase-8 gene expression. From our data we conclude that the transcriptional expression level of caspase-8 determines the vitality and apoptosis response of cultured cells. Normal hepatoma cells have some caspase-8 gene expression but do not undergo apoptosis. Moderate induction of caspase-8 transcription e.g. by IFN␣ will not result in spontaneous apoptosis but sensitizes for apoptosis induced by a second stimulus such as TRAIL or Mitomycin C. However, exceeding a threshold level of caspase-8 expression usually results in apoptosis of hepatoma cells even without stimulation of death receptors such as TNF or TRAIL.
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2005), childhood neuroblastoma (Teitz et al., 2000), lung cancer (Shivapurkar et al., 2002) and breast cancer cells (Wu et al., 2010). Interestingly, in tissue derived from murine liver tumors, the murine caspase-8 promoter is strongly methylated at CG dinucleotides. This is especially intriguing as two of these methylated CG dinucleotides are embedded in functional binding sites for SP1 which are essential for basal caspase-8 expression also in mice (Liedtke et al., 2005). We demonstrated in vitro, that CG methylation at the SP1 binding motif within the caspase-8 promoter completely inhibits SP1 binding and thus basal promoter transactivation. In conclusion, at least in murine liver tumors silencing of caspase8 seems to be mediated via blocking the basal SP1 transcription machinery, although it cannot be excluded that other mechanisms such as somatic point mutations may also contribute to caspase-8 inactivation in other tissues. However, to date it is still not clarified, if caspase-8 inactivation is an enhancer or even prerequisite for liver tumor initiation or simply a consequence of tumor cell selection during tumor progression. As mentioned before, conditional knockout mice deficient of NEMO in hepatocytes undergo spontaneous apoptosis associated with basal caspase-8 activation and eventually develop hepatocellular carcinoma, which could be rescued by simultaneous ablation of the adaptor protein FADD (Luedde et al., 2007). This argues that caspase-8 acts as a tumor promoter in an inflammatory environment with dominant apoptosis. As conventional knockout mice for caspase-8 are not viable due to developmental defects (Varfolomeev et al., 1998), future studies with conditional caspase-8 knockout mice in appropriate tumor models will be essential to finally address this important question.
Caspase-8: a suitable target gene for therapeutic intervention? Caspase activation and apoptosis is frequently associated with acute and chronic liver injury e.g. during acute liver failure (Leifeld et al., 2006) or in patients with chronic hepatitis C (Bantel et al., 2001). Especially acute liver failure requires the immediate stabilization of liver homeostasis or alternatively liver transplantation to avoid the death of the patient. Therefore, current approaches including our own studies address the probability that targeted inhibition of caspases might be a practicable approach to treat apoptosis-mediated liver disease. In a pilot study, it was demonstrated that therapeutic application of a small interfering RNA (siRNA) directed against caspase-8 is feasible at least in mice and efficiently targeted the liver after hydrodynamic injection. Moreover, delivery of caspase-8 specific siRNA was protective in a mouse model of acute liver injury induced by the Fas-agonistic antibody (Zender et al., 2003). However, these experiments did not reflect the situation in patients, where therapy is only possible after onset of liver injury. Interestingly, caspase-8 siRNA delivery was even protective if applied after onset of acute liver failure pointing to a high therapeutic potential of caspase8 inhibition at least in Fas-mediated liver disease. However, it remains questionable, if siRNA per se will be of therapeutic value in human patients instead of caspase-specific small molecular inhibitors, which are already available. In this context, application of a pan-caspase inhibitor in a small cohort of hepatitis C patients was shown to decrease aminotransferase activity, which is an important parameter for the level of liver injury (Pockros et al., 2007). These findings suggest that caspase-inhibition indeed could be a promising approach for therapy of liver disease. However, this strategy is now called into question as several independent studies revealed a new and rather unexpected function of caspase-8, FADD and cFLIP which is the inhibition of programmed necrosis (reviewed in
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Vandenabeele et al., 2010). The concept of programmed necrosis (also termed necroptosis) was first postulated in 1998 based on the finding that L929 fibrosarcoma cells undergo necrosis-like cell death after simultaneous caspase-inhibition and TNF stimulation (Vercammen et al., 1998). This alternative mode of cell death is strongly regulated and essentially depends on the receptorinteracting kinases RIP1 and RIP3, respectively (Cho et al., 2009; He et al., 2009). Using an elegant knockout approach very recent work finally demonstrated that embryonic lethality in Casp8−/− , FLIP−/− and FADD−/− embryos is due to programmed necrosis rather than apoptosis deficiency and can be rescued by simultaneous inactivation of RIP3 or RIP1, respectively (Kaiser et al., 2011; Oberst et al., 2011; Zhang et al., 2011). Accordingly, inactivating caspase-8 may protect from apoptosis but also seems to trigger programmed necrosis especially in an inflammatory environment with strong TNF expression. Future work using conditional knockout mice for caspase-8 will further evaluate this pro-necrotic potential. Summary We intensively investigated TNF-, Fas- and TRAIL-related signaling pathways in the murine liver and in hepatoma cells with a strong focus on the two axes leading to activation of NF-B or caspase-8, respectively. The data revealed that therapeutic inhibition of IKK2/IKK could be of substantial benefit in several pathological situations of the liver e.g. after ischemia and reperfusion or in steatohepatitis. By conditional genetic inactivation of IKK␥/NEMO in hepatocytes we defined a new animal model of liver inflammation and tumorigenesis, which reflects several important aspects of chronic hepatitis and hepatocarcinogenesis in human patients. Therefore, NEMOhepa mice will be a valuable tool to study mechanisms of inflammatory liver disease in the future. However, our studies also demonstrated that NEMO is essential for liver homeostasis. Thus, therapeutic inhibition of NEMO – although technically feasible – does not seem to be a promising approach for treatment of hepatitis or prevention of HCC. Instead, the findings in NEMOhepa mice challenge the paradigm that apoptosis is a tumorsuppressive mechanism as chronic apoptosis in these mice is clearly associated with hepatocarcinogenesis. In this line of evidence the postulated role of caspase-8 as a potential tumor suppressor has to be re-defined by future investigations. Some data hint at caspase8 as a tumor suppressor as caspase-8 is silenced in several tumor species including hepatocellular carcinoma but constitutively activated in NEMOhepa mice where it likely acts as a tumor promoter. Our data also modifies the old paradigm that caspase-8 is predominantly regulated on the posttranslational level. In fact, caspase-8 activity and apoptosis can also be modulated by triggering the caspase-8 promoter. Control of basal transcription e.g. by SP1 may explain some phenomenon such as increased tumor predisposition and apoptosis resistance in tumors. Finally, understanding the crosstalk between cytotoxic drugs, the caspase-8 promoter and apoptosis induction may help to define more efficient combination therapies in tumor treatment. Conflict of interest The authors declare that they have no competing interests. Acknowledgement Work reported in this mini review was supported in part by the Deutsche Forschungsgemeinschaft (SFB542, C15) to C. Liedtke and C. Trautwein.
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