ReviewThe role of TNF and Fas dependent signaling in animal models of inflammatory liver injury and liver cancer
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/IKKα, IKK2/IKKβ and NEMO/IKKγ. IKK1 and IKK2 are the kinase components of the IKK complex whereas NEMO acts as the regulatory subunit. The IKK complex phosphorylates I-kappa B molecules such as I-κBα 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-kBα is to sequester the transcription factor NF-κB in the cytoplasm, I-κBα degradation eventually results in release of NF-κB and subsequent nuclear translocation and activation of pro-inflammatory and anti-apoptotic target genes.
In addition, association of TRADD with TRAF2 can activate the c-Jun 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 mitogen-activated 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 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 (BH3-interacting 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-κB 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.
Section snippets
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-κB by pro-inflammatory stimuli, but most strikingly results in failure to form stratified, well-differentiated epidermis and a
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-κB
IKKγ/NEMO: an essential key player for liver homeostasis and tumor suppression
In contrast to IKK2, the regulatory subunit IKKγ/NEMO is absolutely essential for NF-κB activation in the liver. Accordingly, NF-κB activation is completely inhibited in mice with a hepatocyte-specific deletion of NEMO (NEMOΔhepa). As NF-κB 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 NEMOΔhepa mice die
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 NEMOΔhepa mice this is unlikely to originate from a single pathway but rather from a complete disturbance of several signaling cascades. In fact, NEMOΔhepa mice displayed de-regulated death-receptor signaling shown by the
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,
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 post-translational 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
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
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
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-κB or caspase-8, respectively. The data revealed that therapeutic inhibition of IKK2/IKKβ 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 IKKγ/NEMO in hepatocytes we defined a new animal
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.
References (55)
- et al.
Caspase activation correlates with the degree of inflammatory liver injury in chronic hepatitis C virus infection
Hepatology
(2001) - et al.
Hepatocyte-specific IKK gamma/NEMO expression determines the degree of liver injury
Gastroenterology
(2007) - et al.
Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation
Cell
(2009) - et al.
Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse)
Biologicals
(1990) - et al.
Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice
J. Hepatol.
(2003) - et al.
Hallmarks of cancer: the next generation
Cell
(2011) - et al.
Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha
Cell
(2009) - et al.
NFkappaB mediates apoptosis through transcriptional activation of Fas (CD95) in adenoviral hepatitis
J. Biol. Chem.
(2000) - et al.
The human caspase-8 promoter sustains basal activity through SP1 and ETS-like transcription factors and can be up-regulated by a p53-dependent mechanism
J. Biol. Chem.
(2003) - et al.
Interferon-alpha enhances TRAIL-mediated apoptosis by up-regulating caspase-8 transcription in human hepatoma cells
J. Hepatol.
(2006)
Silencing of caspase-8 in murine hepatocellular carcinomas is mediated via methylation of an essential promoter element
Gastroenterology
Deletion of NEMO/IKK gamma in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma
Cancer Cell
FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens
J. Biol. Chem.
The up-regulation of human caspase-8 by interferon-gamma in breast tumor cells requires the induction and action of the transcription factor interferon regulatory factor-1
J. Biol. Chem.
Embryonic lethality, liver degeneration, and impaired NF-kappa B activation in IKK-beta-deficient mice
Immunity
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
Sp1: emerging roles – beyond constitutive activation of TATA-less housekeeping genes
Biochem. Biophys. Res. Commun.
Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation
BMC Cancer
Hepatocyte-specific NEMO deletion promotes NK/NKT cell- and TRAIL-dependent liver damage
J. Exp. Med.
Pharmacological IKK2 inhibition blocks liver steatosis and initiation of non-alcoholic steatohepatitis
Gut
Interdimer processing mechanism of procaspase-8 activation
EMBO J.
An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cells
Int. J. Cancer
Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway
Hepatology
AS602868, a pharmacological inhibitor of IKK2, reveals the apoptotic potential of TNF-alpha in Jurkat leukemic cells
Oncogene
A promoter polymorphism in the CASP8 gene is not associated with cancer risk
Nat. Genet.
Abnormal morphogenesis but intact IKK activation in mice lacking the IKKalpha subunit of IkappaB kinase
Science
RIP3 mediates the embryonic lethality of caspase-8-deficient mice
Nature
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