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Abstract

Chronic liver diseases such as viral hepatitis and alcoholic liver disease are a major health burden affecting millions of people worldwide. Chronic liver damage can cause fibrosis, which might ultimately progress to cirrhosis as the major risk factor for the development of hepatocellular carcinoma (HCC). HCC is the most common type of primary liver cancers (accounting for ~ 80 %) and originates from hepatocytes, whereas intrahepatic cholangiocarcinoma (ICC, accounting for ~ 15 %) is a malignant liver tumor arising from biliary epithelial cells. Together, they are among the most frequently diagnosed cancers and a leading cause of cancer-related deaths. NF-kB comprises a family of transcription factors, which regulate the expression of target genes involved in different functions such as inflammation, immunity, cell proliferation, differentiation, and survival. Two major signaling pathways termed as the canonical and non-canonical NF-κB pathway have been described leading to receptor-mediated NF-kB activation. Both pathways are dependent on different signaling components and activate distinct target genes. While activation of canonical NFkB signaling and its key player RELA have been reported to play a crucial role in chronic liver diseases, the role of non-canonical NF-kB signaling with RELB as its key transcriptional factor remains largely elusive. Therefore, the main objective of this work was to investigate the functional importance of RELB for the liver, chronic liver diseases, and hepatocarcinogenesis. For this purpose, an established mouse model with a specific disruption of the cylindromatosis gene (Cyld) in liver parenchymal cells (LPC) was used. CYLD is a deubiquitinase and acts as a negative regulator of NF-kB signaling. Mice lacking liver-specific full-length CYLD (CyldΔLPC) exhibit a severe biliary phenotype consisting of ductular reaction and biliary fibrosis, accompanied by highly upregulated RELB expression. To determine whether RELB overexpression contributes to this biliary phenotype, a double knockout mouse containing an additional deletion of RELB (Cyld/RelbΔLPC mice) was generated. Analysis of Cyld/RelbΔLPC mice revealed that nuclear translocation of RELB is essential for the ductular reaction, activation of the oval cell compartment and development of biliary fibrosis in CyldΔLPC mice, as the spontaneous phenotype of CyldΔLPC mice is completely reverted in Cyld/RelbΔLPC mice. Moreover, sustained RELB activation induced a specific cytokine response without significant increased immune cell infiltration. The finding that cholangiocytes of CyldΔLPC mice acquire a reactive phenotype with the ability to express proinflammatory cytokines such as LTβ points to a perpetuating feedback mechanism of constitutive RELB upregulation ultimately promoting ductular reaction in CyldΔLPC mice. RELB deficiency also affected the protein expression of NF-kB signaling molecules involved in the canonical and non-canonical signaling pathway. Whereas liver lysates of CyldΔLPC mice showed elevated NF-kB activation, protein levels of Cyld/RelbΔLPC livers were similar to control mice. To assess the role of RELB in the injured liver, different chemically induced chronic liver damage models were used. While both RELB and CYLD had no impact on the fibrotic response induced by direct hepatocyte damage, CyldΔLPC mice showed in a cholangiopathy mouse model the most severe biliary phenotype with cholestasis and biliary fibrosis compared to treated control mice. This pronounced biliary injury was RELB driven, as additional RELB deletion was able to revert this phenotype. Interestingly, Cyld/RelbΔLPC mice were protected against cholestatic damage, maintained body weight, and displayed normal bilirubin levels. In the second part of this thesis, the importance of RELB for human chronic liver diseases and hepatocarcinogenesis was investigated. In liver parenchymal cells from patients with chronic infection with hepatitis B or C viruses, autoimmune hepatitis, alcoholic liver disease, early stage primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), an increased nuclear translocation of RELB was observable. Especially in cholangiocytes of early disease stage of PBC and PSC, LTβ acts as a possible upstream trigger leading to sustained RELB activation. In liver tissues from patients with HCC or ICC, nuclear translocation of RELB was significantly increased. Nuclear RELB expression was significantly associated with poor overall survival of HCC patients identifying RELB as a novel prognostic factor to predict the outcome of HCC patients. Taken together, the results of the present study uncover for the first time a crucial role of non-canonical NF-kB signaling for ductular reaction, progenitor cell activation and biliary driven fibrosis in a mouse model. The finding, that RELB is activated in human diseased liver, HCC, and ICC could harbor broad implications for basic research and clinical application.