Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout.

H Ehlken,M Pasparakis,P Schirmacher,S Krishna-Subramanian,L Ochoa-Callejero,V Kondylis,N E Nadi,H Walczak,B K Straub,G Kollias

doi:10.1038/cdd.2014.83

Abstract

Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO(LPC-KO) mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO(LPC-KO) mice. To address potential functional redundancies between death receptors we generated and analysed NEMO(LPC-KO) mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO(LPC-KO) mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO(LPC-KO) mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO(LPC-KO) mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.

Highlights

Responses in Aging-Associated Diseases (CECAD), University of Cologne, Joseph-Stelzmann-Strasse 26, Cologne D-50931, Germany
The liver of 8-week-old NEMOLPC-KO/FADDLPC-KO mice displayed some focal necrotic lesions that were surrounded by granulocytes (Figure 1b), indicating that Fas-associated with death domain (FADD) ablation protected NF-kappa B essential modulator (NEMO)-deficient hepatocytes from apoptosis but triggered focal necrotic hepatocyte death resulting in mildly elevated serum alanine aminotransferase (ALT) levels
Macroscopic and microscopic quantifications did not reveal significant differences in the number or size of tumours found in the livers of NEMOLPC-KO/TNFR1LPC-KO, NEMOLPC-KO/ TRAILRLPC-KO, NEMOLPC-KO/FASLPC-KO and NEMOLPC-KO/ 3DRLPC-KO mice compared with NEMOLPC-KO mice (Figures 6c–e and Supplementary Figure S1). These results demonstrate that signalling by the three main death receptors, namely TNFR1, Fas and TRAIL-R, in liver parenchymal cells (LPC) is not required for spontaneous hepatocyte apoptosis, chronic hepatitis and hepatocellular carcinoma (HCC) development in NEMOLPC-KO mice

Summary

Introduction

Responses in Aging-Associated Diseases (CECAD), University of Cologne, Joseph-Stelzmann-Strasse 26, Cologne D-50931, Germany. Tel: +49 221 478 84349; Abbreviations: NEMO, NF-kappa B essential modulator; TNF, tumour necrosis factor; FADD, Fas-associated with death domain; Trail, TNF-related apoptosis-inducing ligand; LPS, Lipopolysaccharide; LPC, liver parenchymal cells; DR5, death receptor 5; ALT, alanine aminotransferase; RIP, receptor interacting protein; Ifng, interferon gamma; Ccnd[1], cyclin D1; CK-19, cytokeratin 19; TBP, TATA-binding protein; EBSS, Eagle’s balanced salt solution. In the Mdr[2] À / À mouse model of inflammation-driven liver carcinogenesis, NF-kB inhibition caused by transgenic IkBa super–repressor expression in hepatocytes inhibited HCC progression.[12] hepatocyte-restricted ablation of IKK2 prevented hepatitis and liver tumorigenesis induced by overexpression of lymphotoxins a and b in hepatocytes.[13] mice with hepatocyte-specific IKK2 ablation developed more tumours induced by a single injection of the chemical carcinogen diethylnitrosamine,[14] revealing a tumour suppressor role of NF-kB in this context. Studies in mice lacking NEMO in liver parenchymal cells (LPCs) further supported a tumour suppressor function of IKK/NF-kB signalling in liver cancer. Death Domain (FADD or MORT1), an adapter protein essential for the recruitment of caspase-8 to the Death

Methods

Results

Conclusion