Original Article
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World J Gastroenterol. Dec 7, 2014; 20(45): 17049-17064
Published online Dec 7, 2014. doi: 10.3748/wjg.v20.i45.17049
CYLD deletion triggers nuclear factor-κB-signaling and increases cell death resistance in murine hepatocytes
Toni Urbanik, Bruno Christian Koehler, Laura Wolpert, Christin Elßner, Anna-Lena Scherr, Thomas Longerich, Nicole Kautz, Stefan Welte, Nadine Hövelmeyer, Dirk Jäger, Ari Waisman, Henning Schulze-Bergkamen
Toni Urbanik, Bruno Christian Koehler, Laura Wolpert, Christin Elßner, Anna-Lena Scherr, Nicole Kautz, Stefan Welte, Dirk Jäger, Henning Schulze-Bergkamen, National Center for Tumor Diseases, Department of Medical Oncology, University Clinic of Heidelberg, 69120 Heidelberg, Baden-Wuerrtemberg, Germany
Thomas Longerich, Institute of Pathology, University Hospital Heidelberg, 69120 Heidelberg, Baden-Wuerrtemberg, Germany
Nadine Hövelmeyer, Institute for Molecular Medicine, Johannes Gutenberg-University Mainz, 55131 Mainz, Rheinland-Palatinate, Germany
Author contributions: Urbanik T, Wolpert L and Kautz N performed the experiments; Urbanik T designed the study, performed interpretation of the data and statistical analysis; Urbanik T, Koehler BC and Schulze-Bergkamen H wrote the manuscript; Elßner C, Scherr AL, Longerich T, Welte S, Hövelmeyer N, Jäger D and Waisman A made substantial contribution to the discussion of the results; all authors read and approved the final manuscript.
Supported by Research grants from the Dietmar Hopp Stiftung, http://www.dietmar-hopp-stiftung.de and from German Research Foundation (Deutsche Forschungsgemeinschaft, http://www.dfg.de/, DFG SCHU 1443/3-2) to HSB (SFB/TRR 77, associated project)
Correspondence to: Henning Schulze-Bergkamen, MD, National Center for Tumor Diseases, Department of Medical Oncology, University Clinic of Heidelberg, Im Neuenheimer Feld 460, 69120 Heidelberg, Baden-Wuerrtemberg, Germany. henning.schulze@med.uni-heidelberg.de
Telephone: +49-6221560 Fax: +49-6221-5633966
Received: May 29, 2014
Revised: June 30, 2014
Accepted: August 13, 2014
Published online: December 7, 2014
Processing time: 195 Days and 14.8 Hours
Abstract

AIM: To analyze the role of CYLD for receptor-mediated cell death of murine hepatocytes in acute liver injury models.

METHODS: Hepatocyte cell death in CYLD knockout mice (CYLD-/-) was analyzed by application of liver injury models for CD95- (Jo2) and tumor necrosis factor (TNF)-α- [D-GalN/lipopolysaccharide (LPS)] induced apoptosis. Liver injury was assessed by measurement of serum transaminases and histological analysis. Apoptosis induction was quantified by cleaved PARP staining and Western blotting of activated caspases. Nuclear factor (NF)-κB, ERK, Akt and jun amino-terminal kinases signaling were assessed. Primary Hepatocytes were isolated by two step-collagenase perfusion and treated with recombinant TNF-α and with the CD95-ligand Jo2. Cell viability was analyzed by MTT-assay.

RESULTS: Livers of CYLD-/- mice showed increased anti-apoptotic NF-κB signaling. In both applied liver injury models CYLD-/- mice showed a significantly reduced apoptosis sensitivity. After D-GalN/LPS treatment CYLD-/- mice exhibited significantly lower levels of alanine aminotransferase (ALT) (295 U/L vs 859 U/L, P < 0.05) and aspartate aminotransferase (AST) (560 U/L vs 1025 U/L, P < 0.01). After Jo injection CYLD-/- mice showed 2-fold lower ALT (50 U/L vs 110 U/L, P < 0.01) and lower AST (250 U/L vs 435 U/L, P < 0.01) serum-levels compared to WT mice. In addition, isolated CYLD-/- primary murine hepatocytes (PMH) were less sensitive towards death receptor-mediated apoptosis and showed increased levels of Bcl-2, XIAP, cIAP1/2, survivin and c-FLIP expression upon TNF- and CD95-receptor triggering, respectively. Inhibition of NF-κB activation by the inhibitor of NF-κB phosphorylation inhibitor BAY 11-7085 inhibited the expression of anti-apoptotic proteins and re-sensitized CYLD-/- PMH towards TNF- and CD95-receptor mediated cell death.

CONCLUSION: CYLD is a central regulator of apoptotic cell death in murine hepatocytes by controlling NF-κB dependent anti-apoptotic signaling.

Keywords: CYLD; Apoptosis; Nuclear factor-κB; Tumor necrosis factor-α; CD95; Liver

Core tip: Activation of death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor (TNF)-R1, is involved in the pathophysiology of acute and chronic liver diseases. Inactivation of the deubiquitinase CYLD is accompanied by increased survival of different cell types. However, the role of CYLD in death receptor-mediated apoptosis of hepatocytes has not been addressed so far. The study showed for the first time that CYLD negative hepatocytes are less sensitive to CD95 and TNF-R-mediated apoptosis, at least in part via triggering nuclear factor-κB signaling leading to induction of anti-apoptotic proteins. Inhibition of CYLD might represent a therapeutic approach to protect hepatocytes from death receptor-mediated apoptosis.