Nuclear Translocation of RELB Is Increased in Diseased Human Liver and Promotes Ductular Reaction and Biliary Fibrosis in Mice

Background & Aims Cholangiocyteproliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELBproto-oncogene NF-κB subunit in human cholangiocytesand in mouse models of liver disease characterized by a ductular reaction. Methods We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bankin Germany. Tissues were analyzed by immunohistochemistry for levels of RELBand lymphotoxinβ (LTB). We studied mice with liver parenchymal cell (LPC)-specific disruption of the cylindromatosis (CYLD) lysine 63 deubiquitinase gene ( Cyld ), with or without disruption of Relb( Cyld ΔLPC mice and Cyld/ RelbΔLPC mice) and compared them with C57BL/6 mice (controls). Mice were fed 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or standard chow diets to induce biliary injuryor were given injections of CCl 4 to induce non-cholestatic liver fibrosis. Liver tissues were analyzed by histology, immunohistochemistry, immunoblots, in situ hybridization, and quantitative real-time polymerase chain reaction. Cholangiocyteswere isolated from normal human liver, incubated with LTB receptor agonist, and transfected with small interfering RNAs to knock down RELB. Results In liver tissues from patients with primary sclerosing cholangitis, primary biliary cholangitis, chronic infection with hepatitis B or C virus, autoimmune hepatitis, or alcoholic liver disease, we detected increased nuclear translocation of RELBand increased levels of LTB in cholangiocytesthat formed reactive bile ducts compared with control liver tissues. Human cholangiocytes, but not those with RELBknockdown, proliferated with exposure to LTB. The phenotype of Cyld ΔLPC mice, which included ductular reaction, oval cell activation, and biliary fibrosis, was completely lost from Cyld/ RelbΔLPC mice. Compared with livers from control mice, livers from Cyld ΔLPC mice (but not Cyld/ RelbΔLPC mice) had increased levels of mRNAs encoding cytokines (LTB; CD40; and tumor necrosis factor superfamily [TNFSF] members TNFSF11 [RANKL], TNFSF13B [BAFF], and TNFSF14 [LIGHT]) produced by reactive cholangiocytes. However, these strains of mice developed similar levels of liver fibrosis in response to CCl 4 exposure. Cyld ΔLPC mice and Cyld/ RelbΔLPC mice had improved liver function on the DDC diet compared with control mice fed the DDC diet. Conclusion Reactive bile ducts in patients with chronic liver diseases have increased levels of LTB and nuclear translocation of RELB. RELBis required for the ductular reaction and development of biliary fibrosis in Cyld ΔLPC mice. Deletion of RELBand CYLD from LPCs protects mice from DDC-induced cholestatic liver fibrosis.