The role of TNF and Fas dependent signaling in animal models of inflammatory liver injury and liver cancer

Abstract

Tumor Necrosis Factor (TNF) alpha is a pleiotropic cytokine triggering either pro-inflammatory effects via NF-κB 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/IKKα, IKK2/IKKβ and IKKγ/NEMO. We demonstrated that IKK2/IKKβ, but not IKKγ/NEMO might be a promising target for the prevention of liver injury after ischemia and reperfusion or treating steatohepatitis. Genetic inactivation of IKKγ/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-κB-caspase-8 network is technically feasible and could be of potential benefit in inflammatory liver disease.

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.