8 induces severe biliary damage, fibrosis and increases hepatocarcinogenesis in mice

Research Article

Liver specific deletion of CYLDexon7/8 induces severe biliary damage, fibrosis and increases hepatocarcinogenesis in mice Toni Urbanik1, Regina Johanna Boger1, Thomas Longerich2, Katharina Becker1, Karl Roland Ehrenberg1, Nadine Hövelmeyer3, Matthias Hahn3, Marcus Schuchmann4, Dirk Jäger1, Ari Waisman3, Marcus Alexander Wörns4, , Henning Schulze-Bergkamen1,⇑,  1

National Center for Tumor Diseases, Department of Medical Oncology, University Clinic of Heidelberg, Heidelberg, Germany; 2 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; 3Institute for Molecular Medicine, Johannes Gutenberg-University Mainz, Mainz, Germany; 4First Department of Medicine, Johannes Gutenberg-University Mainz, Mainz, Germany See Editorial, pages 937–939

Background & Aims: CYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. Reduced CYLD expression has been observed in other tumor entities, including hepatocellular carcinoma. In the present study, we analyzed the role of CYLD in liver homeostasis and hepatocarcinogenesis in vivo. Methods: Mice with liver-specific deletion of CYLDexon7/8 (CYLDFFxAlbCre) were generated. Liver tissues were histologically analyzed and oval cell activation was investigated. Hepatocarcinogenesis was induced by diethylnitrosamine/phenobarbital (DEN/PB). Microarray expression profiling of livers was performed in untreated as well as DEN/PB-treated mice. NF-jB signaling was assessed by ELISA, quantitative real-time PCR, and Western blotting.

Keywords: Hepatocellular carcinoma; Cholangiocellular carcinoma; DEN; NF-jB; TNF-a; Oval cells; Fibrosis. Received 11 January 2012; received in revised form 4 June 2012; accepted 14 June 2012; available online 21 June 2012 q DOI of original article: http://dx.doi.org/10.1016/j.jhep.2012.07.039. ⇑ Corresponding author. Address: National Center for Tumor Diseases, Department of Medical Oncology, University Clinic of Heidelberg, Im Neuenheimer Feld 460, 69120 Heidelberg, Germany. Tel.: +49 06221 56 0; fax: +49 06221 56 33966. E-mail address: [email protected] (H. Schulze-Bergkamen).   These authors share last authorship. Abbreviations: NF-jB, Nuclear factor kappa B; IKK, inhibitor of NF-jB kinase complex; NEMO/IKKc, NF-jB essential modulator; IjB, inhibitor of NF-jB; BCL-3, Bcell lymphoma-leukemia-3; RIP, Receptor interacting protein; PMH, primary murine hepatocytes; PMC, primary murine cholangiocytes; DEN, diethylnitrosamine; PB, phenobarbital; TAK1, TGF-beta activated kinase 1; IL-1/6, Interleukin 1/6; MCP-1, monocyte chemoattractant protein-1; TRAF2/3/6, Tumor necrosis factor receptor-associated factor 2/3/6; gadd45ß, Growth arrest and DNA damage 45beta; TGF-b, transforming growth factor-beta; K-7/19, Cytokeratin-7/19; HPRT, Hypoxanthine-phosphoribosyltransferase; NIK, NF-jB inducing kinase; TLR-2, Toll like receptor-2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Mdr2, Multidrug resistance 2.

Results: CYLDFFxAlbCre hepatocytes and cholangiocytes did not express full-length CYLD (FL-CYLD) protein but showed increased expression of the naturally occurring short-CYLD splice variant (s-CYLD). CYLDFFxAlbCre mice exhibited a prominent biliary phenotype with ductular reaction and biliary-type fibrosis. In addition, CYLDFFxAlbCre mice showed a significantly increased sensitivity towards DEN/PB-induced hepatocarcinogenesis. Moreover, we could observe the development of cholangiocellular carcinoma, in line with enhanced oval cell activity. NF-jB-signaling was increased in livers of CYLDFFxAlbCre mice and likely contributed to the inflammatory and fibrotic response. Conclusions: The deletion of exon7/8 of the CYLD gene activates oval cells, leads to a biliary phenotype, and increases the susceptibility towards carcinogenesis in the liver. Thus, our study presents a novel model of biliary damage and liver fibrosis, followed by cancer development. Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Ubiquitination is a fundamental post-translational modification, regulating protein expression, localization, and activity. The functional outcome of ubiquitination processes is determined by the linkage type of single or poly-ubiquitin chains to the targeted substrate. Protein degradation is a well-known event as a result of lysine-48 (K-48) linkage of poly-ubiquitin chains [1]. New functional aspects of ubiquitination were determined by analysis of Lys-63-(K-63-) linked poly-ubiquitin chains, which lead to promotion of signaling processes such as protein kinase activation or protein–protein interactions. Ubiquitination is a reversible process, providing an additional cellular tool to control signaling events [2].

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Research Article The tumor suppressor cylindromatosis gene (CYLD) was identified as a gene mutated in familial cylindromatosis, which is an autosomal-dominant predisposition to tumors of the skin appendages. The product of the CYLD gene contains a ubiquitin C-terminal hydrolase domain allowing it to remove K-63-linked poly-ubiquitin chains from distinct proteins involved in the NF-jB signaling pathway [3]. A central step in NF-jB induction is activation of the IKK complex [3]. In vitro studies have shown that CYLD binds to NEMO, the regulatory subunit of the IKK complex. CYLD removes K-63-linked ubiquitin chains from NEMO, thereby inhibiting proteasomal degradation of the NF-jB inhibitors (IjBs) and nuclear translocation of the NF-jB subunits. It has also been shown that CYLD deubiquitinates the IjB-a homologue BCL-3. In contrast to the NF-jB inhibitory function of IjB-a, BCL3 acts as an NF-jB co-activator by forming transcriptionally active heterodimers with the NF-jB subunits p50 and p52 [4]. CYLD also targets other proteins involved in the NF-jB pathway including RIP, TAK1, TRAF2, and TRAF6 [3]. CYLD knockout mice do not spontaneously develop tumors, but they are more sensitive to chemically-induced skin and colon tumorigenesis [4,5]. Recently, we identified a naturally occurring short splice variant of CYLD (short (s)-CYLD) in CYLDex7/8 mice. The s-CYLD isoform lacks the binding sites for TRAF2 and NEMO, but is still capable of deubiquitinating BCL-3 [6]. CYLD expression was found to be downregulated in hepatocellular carcinoma (HCC) [7,8]. Knockdown of CYLD led to an increase of NF-jB activity in HCC cells in vitro, which was accompanied by a reduced sensitivity of HCC cells towards chemotherapy- and TNF-a-mediated apoptosis [8]. However, the current knowledge about the contribution of CYLD to liver homeostasis and HCC development is limited.

Isolation, culturing, and treatment of primary murine hepatocytes Hepatocytes were isolated and cultured as previously described [9]. After 24 h, cells were incubated with TNF-a (Biomol, Hamburg, Germany) or SN-50 (EnzoLife-Science, Lörrach, Germany). Isolation of primary murine cholangiocytes See Supplementary Materials and methods. Induction of carcinogenesis To induce liver carcinogenesis, a single diethylnitrosamine (DEN) (Sigma–Aldrich, Munich, Germany) i.p. injection (0.05 mg per mouse) was performed at day 7 post partum. Promotion of carcinogenesis was achieved by continuously adding phenobarbital (PB) (0.05% w/v) to the drinking water from the age of 3 weeks. Analysis of livers and immunohistochemistry Livers were assessed visually and the amount of appearing tumor nodules at the liver surface was counted. Additionally, tumor size (diameter) was measured. To investigate liver architecture and tumor histology, 3-lm thick sections were made from formalin-fixed paraffin-embedded liver tissues and stained with hematoxylin and eosin (H&E). Modified Gomori staining was used to assess fibrotic remodeling and architectural distortion. For detection of active caspase 3, CD3, CD68, and Ki67 positive cells, shock frozen liver tissues were sectioned (5 lm) and further processed using the NovoLink™ Min Polymer Detection System (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s instructions. Primary antibodies are listed in Supplementary Materials and methods. Microarray analysis See Supplementary Materials and methods. Western blotting

Materials and methods ex7/8

Generation and genotyping of the liver specific CYLD (CYLDFFxAlbCre)

mutant mice

The generation of CYLDex7/8 mutant mice, harboring two loxP-sites (CYLDFF), had been previously described [6]. To generate liver-specific CYLDex7/8 mutant mice, CYLDFF mice were crossed with mice expressing the Cre recombinase (both C57BL/6 background) under control of an albumin promoter. This leads to hepatocyte specific excision of exon 7 and, subsequently, to alternative splicing from exon 6 to exon 9 [6]. CYLDFFxAlbCre offspring were compared to their control littermates with the genotype CYLDFF (referred to as WT). Animals were bred at the animal facility of the University of Mainz. All experiments were done in accordance with the governmental and institutional guidelines and were performed under written approval of the state animal care commission. PCR-based genotyping was performed using specific primers listed in Supplementary Materials and methods (Supplementary methods Table 1). All experiments were performed with male mice.

Tissue lysis, protein extraction, and preparation of nuclear and cytosolic extracts were performed as previously described [8,9]. SDS–PAGE and Western blotting were performed according to standard procedures. Immunodetection was performed using primary antibodies listed in Supplementary Materials and methods. NF-jB activity ELISA To quantify NF-jB transcription factor activation, the TransAMÒ NF-jB Family Transcription Factor Assay Kit (Active Motiv, La Hulpe, Belgium) was used according to the manufacturer’s instructions. The assay is based on immobilized oligonucleotides containing NF-jB consensus sites. For each well, 3 lg nuclear cell extract was used. Statistical analysis See Supplementary Materials and methods.

Quantitative real-time polymerase chain reaction (q-RT PCR) Isolation of total RNA and cDNA synthesis were performed as previously described [9]. Specific FL- and s-CYLD mRNA transcripts were quantified using LightCyclerÒ FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany) and primers listed in Supplementary Materials and methods, Table 2. Q-RT PCR of collagen I, IL-1, IL-6, TNF-a, MCP-1, TGF-b1/2, K-7, K-19, survivin, gadd45b, BCL3, and GAPDH was performed using primer kits (Qiagen, Hilden, Germany). mRNA expression was normalized to the expression of the housekeeping gene. Alanine aminotransferase levels About 100 ll of blood was collected from the tail vein. Alanine aminotransferase (ALT) was measured at the Institute of Clinical and Laboratory Medicine at the University Hospital Mainz according to standard procedures.

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Results Liver specific CYLDex7/8 mutant mice (CYLDFFxAlbCre) The CYLDFFxAlbCre genotype was confirmed by PCR (Fig. 1A). As shown by Western blot analysis, FL-CYLD expression was not detectable in livers and isolated primary murine hepatocytes (PMH) of 3-month-old CYLDFFxAlbCre mice. In accordance with our previous findings in CYLDex7/8 knockout B cells [6], CYLDFFxAlbCre livers and PMH showed an increased protein expression of s-CYLD (Fig. 1B, upper panel).

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bp 426 325

flox WT

510

actin Cre

310 CYLD exon 7 WT Albumin Cre CYLDFF x AlbCre X

B

LL

f/WT X

f/f X

WT + X

f/WT f/f + + X √

CYLDFF x AlbCre

WT

FL-CYLD mRNA expression was substantially reduced in livers of CYLDFFxAlbCre mice (p <0.001). In contrast, s-CYLD mRNA expression was slightly increased in CYLDFFxAlbCre compared to WT mice (1.2-fold). In CYLDFFxAlbCre PMH, FL-CYLD reduction and s-CYLD overexpression (2-fold compared to WT, p <0.01) were even more apparent (Fig. 1B, lower panel). CYLDFFxAlbCre mice showed a significantly higher liver/body weight ratio at the age of 9 months compared to WT (0.05 vs. 0.035; p <0.05) (Fig. 1C), while their body weight was comparable to WT animals (data not shown). ALT serum levels in CYLDFFxAlbCre mice were not different from WT animals (Supplementary Fig. 1A). CYLDFFxAlbCre mice were fertile and did not spontaneously develop liver tumors during a 12-month observation period.

kDa Unspecific FL-CYLD s-CYLD Tubulin

106 86 55

PMH

CYLDFF x AlbCre kDa

WT

106 86

FL-CYLD s-CYLD Tubulin

55

WT CYLDFF x AlbCre

LL

PMH ***

0.04 **

0.02

**

0.06

YL

YL

D

D C

WT

0.08

D

0.02 0 3

6

age (mo) WT CYLDFF x AlbCre

9

CYLD x AlbCre

0.04

Immunohistological staining of active caspase 3 indicated no apoptotic liver damage in CYLDFFxAlbCre mice (Supplementary Fig. 1B). At the age of 3 and 6 months, livers from CYLDFFxAlbCre mice were macroscopically normal (data not shown), whereas livers from 9-month-old CYLDFFxAlbCre showed indurations on the surface and focal white densities that histologically corresponded to collagenous septa, radiating from subcapsular portal tracts into the liver capsule (Fig. 1D). Histopathological analysis revealed a biliary phenotype in livers of merely 3-month-old CYLDFFxAlbCre mice that was characterized by proliferation of peri-portal biliary ductules associated with fibrosis and sparse inflammatory infiltrates known as ductular reaction (Supplementary Fig. 1C). At later time points, this phenotype had progressed, resulting in a porto-portal bridging fibrosis (Fig. 2A). Additionally, the biliary phenotype was detectable in CYLDex7/8 mice (Supplementary Fig. 2). Fibrogenesis in livers of CYLDFFxAlbCre mice was validated by collagen I mRNA expression analysis. In the liver of 3- and 9-month-old CYLDFFxAlbCre mice, the expression was increased by 4.4- and 8.4-fold compared to WT, respectively (p <0.05, Fig. 2B, upper panel). Livers of CYLDFFxAlbCre mice showed increased peri-portal T cell (CD3+, Fig. 2C) and macrophage infiltrates (CD68+, Supplementary Fig. 3). mRNA expression analysis of inflammatory mediators revealed a significant 2.8-fold increase in TNF-a and a 1.2-fold increase in IL-6 expression in CYLDFFxAlbCre livers at 3 months of age (p <0.05). IL-1 expression was slightly increased compared to WT mice (not significant). MCP-1 mRNA levels were 5.7-fold increased in livers of 6-month-old CYLDFFxAlbCre mice (p <0.01, Fig. 2D). Q-RT PCR analysis of TGF-b1 and TGF-b2 revealed 2.3- and 5-fold increased mRNA levels, respectively, in livers of 3-month-old CYLDFFxAlbCre mice compared to WT, (p <0.01, Fig. 2B, lower panel). CYLDFFxAlbCre mice are highly susceptible towards chemicallyinduced hepatocarcinogenesis

FF

Liver/body weight ratio

n = 7 vs. 8 6 vs. 7 6 vs. 6

s-

FL

s-

C

-C

YL

YL

D

D

0.00

FL

C

n = 6 vs. 6

***

-C

Relative mRNA expression

n = 6 vs. 6

Histopathological analysis of the livers from CYLDFFxAlbCre mice

1 cm

Fig. 1. Liver phenotype of CYLDFFxAlbCre mice. (A) Gene expression of WT or floxed (flox) CYLD and Cre was analyzed. A positive PCR for flox/flox (f/f) and Cre confirmed the CYLDFFxAlbCre genotype (U). (B) Western blot analysis of liver lysates (LL) and PMH for CYLD expression (upper panels). q-RT PCR analysis of FLand s-CYLD expression in livers (LL) and PMH (lower panels). Median values ± SD are shown. (C) Liver/body weight ratio of WT and CYLDFFxAlbCre mice. Values represent the means ± SD. (D) WT and CYLDFFxAlbCre livers (9 months of age). ⁄⁄ p <0.01, ⁄⁄⁄p <0.001.

We applied the DEN/PB model to test tumor susceptibility. Hepatocarcinogenesis was highly increased in CYLDFFxAlbCre compared to WT mice (Fig. 3A) at 3 months of age (0.9 visible tumors at the liver surface compared to 0.5 tumors). After 6 (27.4 vs. 6.5 tumors) and 9 months (72 vs. 29.4 tumors, p <0.001), differences were even higher (Fig. 3B, left panel). The liver tumors of CYLDFFxAlbCre mice also exhibited approximately 2-fold increased sizes (in diameter) (Fig. 3B, right panel). We could not observe any metastasis or tumor development in other organs.

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Research Article CYLDFF x AlbCre

WT

B

WT CYLDFF x AlbCre

Collagen I/GAPDH mRNA expression (10-4) mRNA expression (10-3)

CYLDFF x AlbCre

WT

*

6 3 0

3 mo

9 mo

20

**

15 10 5 0 TGF-β1/ GAPDH

4

**

3 2 1 0 TGF-β2/ GAPDH

10

*

7.5 5

2.5 0

6

3 mo

6 mo

*

4 2 0

3 mo

6 mo

MCP-1/GAPDH IL-1/GAPDH mRNA expression (10-3) mRNA expression (10-4)

WT CYLDFF x AlbCre

IL-6/GAPDH TNF-α/GAPDH -5 mRNA expression (10-4) mRNA expression (10 )

D

9

WT CYLDFF x AlbCre

C

20 15 10 5 0

3 mo

6 mo **

15 10 5 0

3 mo

6 mo

Fig. 2. Histopathological analysis of livers from untreated CYLDFFxAlbCre mice. (A) Histological pictures of WT and CYLDFFxAlbCre livers. CYLDFFxAlbCre showed a biliary phenotype consisting of a ductular reaction and formation of porto-portal bridging fibrosis. (B) q-RT PCR analysis of collagen I, TGF-b1, and -b2. Both single (dots/squares) and median values (bars) are presented; n = 6. (C) Inflammatory T cell (CD3 + cells) infiltrates in CYLDFFxAlbCre livers (scale bar: 40 lm). (D) q-RT PCR analysis of IL-6, IL-1, TNF-a, and MCP-1 expression levels. ⁄ p <0.05, ⁄⁄p <0.01. [This figure appears in colour on the web.]

The liver/body weight ratios of CYLDFFxAlbCre mice were significantly elevated, mainly as a consequence of tumor development and cachexia (Fig. 3C, left panel). The ALT serum concentrations during DEN/PB treatment showed significantly higher levels in 9-month-old CYLDFFxAlbCre (114 U/L) compared to WT mice (27 U/L, p <0.001) (Fig. 3C, right panel). Analysis of CYLD protein expression in tumoral compared to peri-tumoral liver tissue did not reveal any alterations in WT mice. CYLDFFxAlbCre mice showed a downregulation of s-CYLD expression in 2 out of 4 matched paired tumorous and peritumorous samples (Supplementary Fig. 4). 998

Development of cholangiocellular carcinoma after DEN/PB treatment in CYLDFFxAlbCre mice and analysis of cholangiocellular CYLD expression

*

12

mRNA expression (10-3)

A

DEN/PB-treated CYLDFFxAlbCre mice showed a biliary phenotype comparable to untreated mice (Fig. 3D). Interestingly, one CYLDFFxAlbCre mouse developed a cholangiocellular carcinoma (CCC) at the age of 6 months (Fig. 3E), while at the age of 9 months, even 5 out of 8 mice showed at least one CCC lesion in addition to HCC development, eventually fulfilling the diagnostic criteria of HCC-CCC (Fig. 3E). Taking into consideration that experiments utilizing the albumin promoter to regulate genes affect oval cells as well as hepatocytes [10], we assumed that also cholangiocytes were affected by the albumin Cre-mediated deletion of exon 7/8 of the CYLD gene. We isolated primary murine cholangiocytes (PMC) out of CYLDFFxAlbCre and WT mice. K-7 mRNA expression in the PMC fraction was 75-fold increased in comparison to expression levels measured in whole liver lysates, demonstrating the efficiency of PMC isolation (Fig. 3F). CYLDFFxAlbCre PMCs indeed showed FL-CYLD deletion and increased expression of s-CYLD (Fig. 3G). Analysis of biliary/progenitor cell markers and proliferation Damage of hepatocytes and/or cholangiocytes leads to activation of the oval cell compartment (in humans called the progenitor cell compartment) [11]. Expression analysis of the biliary/oval cell markers K-19 and K-7 revealed a significant 8.8- and 8.6-fold upregulation of K-19 in livers of 3- and 6-month-old CYLDFFxAlbCre mice in comparison to WT mice. K-7 expression was 9.6- and 7.4-fold increased, respectively (Fig. 3H). Analysis of tumorous tissues of DEN/PB-treated CYLDFFxAlbCre mice showed increased K-19 and K-7 levels as well (6.5- and 6-fold compared to WT tumors, respectively, Supplementary Fig. 5). Immunohistochemical Ki67 stainings of livers from 6-monthold CYLDFFxAlbCre and WT mice did not show any parenchymal hepatocellular proliferation. However, in line with our histological analysis, we observed an increased number of Ki67 positive cells in the surrounding of peri-portal biliary ductules in CYLDFFxAlbCre livers (Supplementary Fig. 6). Ki67 analysis of livers from 6-month-old DEN/PB-treated mice revealed comparable results (data not shown). Functional analysis of mRNA microarray data Microarray expression profiling of livers from untreated CYLDFFxAlbCre mice revealed a total of 314 genes with significant expression fluctuations in comparison to WT mice. Upload of this dataset to the Ingenuity Pathway Analysis tool (filter: species = mouse, tissue = liver) led to the detection of 36 genes, which were functionally associated with lipid metabolism, molecular transport, and small molecule biochemistry networks. Furthermore, we identified genes like 11b-hydroxysteroid dehydrogenase type 1 (HSD11B1) and apolipoprotein E (APOE) as connected to cancer development and inflammatory response. Interestingly, the upregulated expression of ATP-binding cassette, subfamily B member 11 (ABCB11) is associated with biliary hyperplasia, cholestasis, and inflammation. Profiling of livers from DEN/PB-treated CYLDFFxAlbCre mice revealed a total of 121 genes with significant alterations in expression compared to DEN/PB-treated WT mice. Application

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***

Tumor number

3 mo

100 80 60 40 20 0

6 mo

C

WT CYLDFF x AlbCre

*

15

***

3

*

20

10

6 9 Age (mo)

5 0

***

3

***

0.18

*

0.12 0.06 3

150 100 50

6 9 Age (mo)

3

6 9 Age (mo)

9 mo

9 mo

6 mo

***

200

0

0

6 9 Age (mo)

E

D

WT CYLDFF x AlbCre

ALT (IU/L)

CYLDFF x AlbCre

Liver/body weight ratio

WT

Max. tumor size (mm)

A

0.10

WT

0.05 0

K-7 Tubulin

kDa 54 55

CYLDFF x AlbCre kDa

FL-CYLD s-CYLD K-7 Tubulin

106 86 54 55

H

WT CYLDFF x AlbCre

9

**

**

6 3 0

3 mo

6 mo

K-19/GAPDH mRNA expression (10-3)

G

K-7/GAPDH mRNA expression (10-3)

0.15

L PM L H U C PM F C

K-7/GAPDH mRNA expression

F

**

**

15 10 5 0

3 mo

6 mo

Fig. 3. Increased liver tumor development in CYLDFFxAlbCre mice in DEN/PB-induced carcinogenesis. (A) Pronounced tumor formation in CYLDFFxAlbCre mice. (B) Tumor count and tumor size (diameter), (C) liver weight and ALT serum concentration in DEN/PB-treated mice. Values represent the means ± SD; n = 8. (D) DEN/PB-treated CYLDFFxAlbCre mice showed a biliary phenotype consisting of a ductular reaction and porto-portal bridging fibrosis. (E) Mixed HCC-CCCs were found in CYLDFFxAlbCre livers. Besides hepatocellular differentiation (left panel), cholangiocellular carcinoma was evident (middle panel). In areas of CCC differentiation, formation of ductular structures and desmoplastic stroma were observed (H&E, Gomori’s, 100). The right panel shows a HCC-CCC with a ductular CCC component (left part) intimately intermingled with a classical HCC. Desmoplastic stroma is present in the CCC component (H&E, Gomori’s, 100). (F) K-7 mRNA and protein level indicate enrichment of cholangiocytes during the isolation process (LL, liver lysate; PMH, primary murine hepatocytes; UCF, unlabeled cell fraction; PMC, primary murine cholangiocytes). (G) Western blot analysis of cholangiocytes for FL-, s-CYLD, and K-7 expression. (H) q-RT PCR analysis of K-7 and K-19 expression. Both single (dots/squares) and median values (bars) are shown; n = 6. ⁄ p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001. [This figure appears in colour on the web.]

of the same pathway analysis setting identified 25 genes mainly involved in lipid metabolism, molecular transport, and small molecule biochemistry networks. Genes that were overexpressed in CYLDFFxAlbCre mice like early growth response protein 1 (EGR1), nuclear receptor subfamily 1, group I, member 3 (NR1I3) and Toll-like-receptor-2 (TLR-2) were associated with inflammatory response and hepatic system disease. Heatmaps of all regulated genes and the summary of pathway analysis are presented in Supplementary data (Supplementary Fig. 12, Tables 3 and 4). Increased NF-jB activity in livers of CYLDFFxAlbCre mice Microarray expression profiling did not reveal significant alterations in the expression of NF-jB regulatory genes, some of which represent interaction partners of FL- and s-CYLD. However, CYLDFFxAlbCre mice showed a tendency to higher NF-jB1/p105, TLR-2 and TRAF3 expression. BCL-3 was slightly upregulated in

livers of CYLDFFxAlbCre mice. RelB and the NF-jB-inducing kinase (NIK, MAP3K14) were also slightly increased (Supplementary Fig. 7A). As CYLD exerts its major role in posttranslational modification of NF-jB activity, we subsequently investigated NF-jB signaling on protein level. The analysis of whole liver lysates of CYLDFFxAlbCre mice showed enhanced expression of p50, p52, and its precursor proteins p105 and p100. RelB was also increasingly expressed. RelA and c-Rel were found to be slightly upregulated compared to WT mice (Fig. 4A). Western blot analysis of NF-jB regulatory proteins revealed a slight overexpression of RIP1, TRAF2, BCL-3 and, most profoundly, TRAF3 in CYLDFFxAlbCre livers. In contrast to enhanced NIK mRNA expression (see microarray data), NIK was reduced at protein level in CYLDFFxAlbCre mice. Furthermore, CYLDFFxAlbCre livers showed enhanced IKKa (Chuk) protein levels (Fig. 4B). To explore whether there were any differences in the inducible activity of NF-jB transcription factors, we isolated PMH

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Research Article out of WT and CYLDFFxAlbCre mice. After stimulation of the PMH with TNF-a, we performed an ELISA-based quantification of nuclear NF-jB transcription factors. Our measurements revealed an increased basal NF-jB activity in CYLDFFxAlbCre compared to WT PMH. In CYLDFFxAlbCre PMH, we observed 1.6-fold increase of basal p50 and 1.3-fold increase of TNF-a-stimulated p50 activity level compared to WT (p <0.05; Fig. 4C, upper left panel). Activation analysis of RelA showed a 1.2-fold higher basal activity in CYLDFFxAlbCre compared to WT (p <0.001). Application of TNF-a strongly induced RelA activation in both PMH cultures, but to a higher extent in CYLDFFxAlbCre (1.2-fold compared to WT) (Fig. 4C, upper right panel). Examination of the non-canonical NF-jB pathway revealed higher basal p52 and RelB activity levels in CYLDFFxAlbCre PMH. We found a 1.3-fold increase in basal p52 (p <0.01) and a 7-fold increase (p <0.001) in basal RelB activity in CYLDFFxAlbCre compared to WT PMH, respectively (Fig. 4C, lower panels). Western blot analysis of whole liver lysates revealed unaltered IjB-a and p-IjB-a levels in CYLDFFxAlbCre mice (Fig. 4D). Analysis of cytosolic fractions obtained from TNF-a-stimulated

IKK-α Tubulin

84 55

NIK Tubulin

130 55

at re nt U ed at nt re U

WT

p-IκB-α IκB-α Tubulin

E p-TAK1 TAK1 Tubulin

WT

CYLDFF x AlbCre kDa 37 37 55 CYLD x AlbCre kDa 82 82 55 FF

***

0.2 0.1 0

F c

n

c

n

c

n

c

n kDa 50 65 68 100 55

p50 RelA RelB Nucleolin Tubulin 0

1

5

SN-50 [µM]

10

Relative mRNA expression (compared to GAPDH)

D

0.3

T (3 NF 0 -α m in )

78 55

**

T (3 NF 0 -α m in )

c-Rel Tubulin

0.4 0.3 0.2 0.1 0

ed

60 55

at

BCL-3 Tubulin

re

68 55

nt

RelB Tubulin

0

ed

62 55

0.1

U

TRAF3 Tubulin

0

0.2

at

65 55

0.02

***

nt re

RelA Tubulin

0.04

0.3

U

63 48 55

0.06

*

ed

100 TRAF6 52 NEMO 55 Tubulin

***

*

74 53 55

p100 p52 Tubulin

WT CYLDFF x AlbCre

RelA activity (OD450)

105 RIP1 50 TRAF2 55 Tubulin

C

RelB activity (OD450)

CYLDFF x AlbCre kDa

WT

T (3 NF 0 -α m in )

p105 p50 Tubulin

B

T (3 NF 0 -α m in )

CYLDFF x AlbCre kDa

p50 activity (OD450)

WT

p52 activity (OD450)

A

PMH showed comparable IjB-a degradation kinetics in CYLDFFxAlbCre as observed in WT, whereas the basal cytosolic IjB-a level was slightly reduced (Supplementary Fig. 8). Western blot analysis of p-TAK1 levels revealed an increased TAK1 activity in CYLDFFxAlbCre livers (Fig. 4E). To further investigate NF-jB activity, we performed q-RT PCR experiments to analyze the expression of the NF-jB inducible genes survivin, Gadd45b, and BCL-3. Survivin was significantly increased by 6-fold in CYLDFFxAlbCre compared to WT mice (p <0.05), Gadd45b by 2.7-fold (p <0.01, Supplementary Fig. 9) and BCL-3 showed an increase by 1.6-fold (not significant, data not shown). Expression profiling from livers of 3-month-old, DEN/PB-treated mice revealed a significantly higher expression of TLR-2 and TRAF3 in CYLDFFxAlbCre (p <0.05). Additionally, IKK-a, NEMO (IkBkg), NF-jB1/p105, IKK-b (Ikbkb), RelA, RelB, TNF-R1 (Tnfrsf1a), and BCL-3 mRNAs showed a tendency to elevated expression in CYLDFFxAlbCre compared to WT mice (Supplementary Fig. 7B). Protein expression analysis of the NF-jB subunits in livers of 6month-old DEN/PB-treated mice revealed even more increased

1.2 1.0 0.8 0.6 0.4 0.2 0

TNF-α IL-6 TGF-β1 TGF-β2

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Fig. 4. Enhanced NF-jB activity in livers from CYLDFFxAlbCre mice. (A) Western blot analysis of liver lysates for NF-jB subunits and (B) NF-jB regulating factors. (C) NFjB transcription factor activation. Values represent the means ± SD. Assays were performed in triplicates and are representative of two independent experiments. (D–F) Western blot analysis of liver lysates for (D) p-IjB-a, and IjB-a; (E) p-TAK1 and TAK1 expression and (F) cytosolic (c) and nuclear (n) fractions of 24 h SN-50-treated CYLDFFxAlbCre PMH. Nucleolin and tubulin served as control for n and c fractions, respectively. q-RT PCR analysis of TNF-a, IL-6, TGF-b1, and -b2 after SN-50 treatment of CYLDFFxAlbCre PMH (right panel). ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001.

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JOURNAL OF HEPATOLOGY levels in CYLDFFxAlbCre, especially of RelB, (Supplementary Fig. 10). The expression of BCL-3 was significantly increased after DEN/PB treatment, but no nuclear BCL-3 accumulation was evident (Supplementary Fig. 11). Inhibition of nuclear NF-jB translocation reduces expression of proinflammatory and pro-fibrotic cytokines in CYLDFFxAlbCre PMH To test the relevance of elevated NF-jB activity for the inflammatory and fibrotic response in livers of CYLDFFxAlbCre mice, isolated PMH were treated with the cell permeable peptide SN-50 for 24 h. SN-50 blocks the nuclear translocation of p50:RelA dimer by masking p50 nuclear translocation sequence, but it also exerts minor effects on RelB nuclear import [12]. SN-50 markedly reduced the nuclear levels of p50, RelA, and RelB at a concentration of 10 lM (Fig. 4F, left panel). Q-RT PCR analysis of CYLDFFxAlbCre PMH after SN-50 treatment revealed a dose dependent reduction of the analyzed cytokines TNF-a, IL-6, TGF-b1, and -b2. The strongest effect was observed for TGF-b1 with a significant reduction of the mRNA expression level at 1 (0.6-fold), 5 (0.55-fold), and 10 lM (0.47-fold) SN-50 (compared to the expression level of untreated PMH set to 1, p <0.001). We also observed significantly reduced mRNA levels for TGF-b2 at 5 (0.68-fold, p <0.05) and 10 lM (0.52-fold, p <0.01). IL-6 expression was significantly (0.47-fold) reduced by SN-50 (10 lM, p <0.05). For TNF-a, we also observed reduced levels of mRNA expression (not significant, Fig. 4F, right panel).

Discussion In the present study, we analyzed the role of CYLD for liver homeostasis and hepatocarcinogenesis using CYLDFFxAlbCre mice. Our findings show for the first time that deletion of CYLDexon7/8 results in a severe biliary phenotype including a ductular reaction that progresses to biliary fibrosis and significantly increases susceptibility towards chemically-induced hepatocellular as well as cholangiocellular carcinoma. This phenotype is comparable to the findings in Mdr2 knockout mice [13,14]. Since not only hepatocytes are affected by the albumin Cre-mediated deletion of exon7/8 of the CYLD gene, our study provides evidence for an oval cell origin of combined HCC-CCC development in CYLDFFxAlbCre mice. Furthermore, CYLDFFxAlbCre livers showed an increased NF-jB activation level which correlated with the increased inflammatory and fibrotic response. A ductular reaction including increased proliferation of biliary epithelial cells occurs in a wide variety of liver diseases [15]. Unchanged caspase 3 activity demonstrated a lack of apoptotic liver injury in untreated CYLDFFxAlbCre mice. By contrast, analysis of TGF-b1/2, IL-1, IL-6, TNF-a and MCP-1 expression revealed an inflammatory milieu in livers of CYLDFFxAlbCre mice, which was supported by increased peri-portal T cell and macrophage infiltrates. Several studies have shown that TGF-b activates hepatic stellate cells, which are known to produce extracellular matrix and trigger fibrosis [16]. TGF-b levels correlated with hepatic fibrosis in other models [17] and may contribute to liver injury and fibrogenesis in our model as well. To analyze the involvement of CYLD in HCC development, we used the DEN/PB carcinogenesis model. DEN-induced murine HCCs exhibit high proliferation rates and high ratios of ubiquiti-

nated proteins [18]. This might also involve the loss of function of deubiquitinating enzymes including CYLD. DEN/PB-induced tumors of WT mice did not show altered CYLD expression levels compared to non-tumorous liver tissue. However, CYLDFFxAlbCre developed liver tumors significantly faster compared to WT mice. Notably, additional formation of CCC was detectable. CYLD expression analysis in PMCs isolated from CYLDFFxAlbCre mice showed that cholangiocytes were equally affected by the albumin Cre-mediated deletion of exon7/8 of the CYLD gene. Several studies raised evidence that conditional gene targeting utilizing Cre recombinase driven by albumin promoter sequences will inactivate the gene of interest in hepatocytes and oval cells in parallel [10]. Oval cells can differentiate into both hepatocytes and cholangiocytes [11]. Thus, not only hepatocyte-specific effects of CYLDexon7/8 inactivation may contribute to the phenotype of CYLDFFxAlbCre mice. Ki67 staining of livers from CYLDFFxAlbCre mice revealed increased proliferation of a cellular subset surrounding the portal fields, which can be interpreted as an increased proliferation of the oval cell compartment [11]. Expression analysis of the biliary/oval cell markers K-7 and K-19 showed a dramatic upregulation in livers and liver tumors of CYLDFFxAlbCre mice, possibly indicating the intermediate hepatobiliary cell stage in a dedifferentiation process of mature hepatocytes to pluripotent oval cells [19]. Importantly, hepatic progenitor cells are activated in the majority of chronic liver diseases, forming a target cell population for carcinogenesis [11]. Another possible reason for CCC development might be an increased cholangiocellular differentiation due to increased TGF-b levels [20]. An unequivocal discrimination of a distinct liver cell type as a the origin of CCC and/or HCC tumor development is difficult, as markers for oval cells e.g. OV-1, OV-6, K-7, and K-19 are additionally expressed in cholangiocytes [11]. However, several observations are consistent with a progenitor cell origin of CCCs, HCCs, and combined HCC-CCC with increased K-7 and K-19 expression [21]. NF-jB is a well-known target of CYLD and plays a critical role in cell death, proliferation, and carcinogenesis [3,16]. Depending on the experimental setting, NF-jB is able to act as a promoter or suppressor of carcinogenesis. Inhibition of NF-jB activation by ablation of NEMO caused spontaneous development of steatohepatitis and HCC in mice [22]; while suppression of NF-jB in cholestatic hepatitis inhibited HCC development [14]. It is assumed that NF-jB is essential to suppress spontaneous or carcinogen-induced tumor development [23,24]. By contrast, several studies on human tumor tissues provided evidence that increased NF-jB activity is accompanied by a poor prognosis [24]. Livers of CYLDFFxAlbCre mice showed a persistent basal NF-jB activation. We assume that the underlying severe phenotype favors the tumor promoting effect of an increased NF-jB activation in DEN/PB-induced carcinogenesis in our model. Analysis of CYLD target proteins revealed higher TRAF2 and TRAF3 levels in CYLDFFxAlbCre livers, which has been described to positively affect the formation of the TNF/TNFR1 complex and therewith TNF-induced NF-jB activation [25]. Our findings concerning TRAF2 and TRAF3 contradict the current paradigm of non-canonical NF-jB signaling, which suggests a contribution of TRAF2/3 to the destabilization of NIK in order to inhibit the non-canonical NF-jB pathway [26]. We indeed observed reduced NIK levels in CYLDFFxAlbCre livers. Nevertheless, we detected higher

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Research Article concentrations of IKK-a, which may explain the elevated p52 levels due to enforced processing of NF-jB2/p100 [26]. In addition to the adaptor function, TRAFs act as K-63-specific ubiquitin ligases providing docking sites for molecular adaptors and activating downstream kinases including TAK1 [27]. Livers of CYLDFFxAlbCre mice exhibited increased TAK1 activity indicating again an increased activation of NF-jB transduction cascades. A liver specific TAK1 knockout led to spontaneous HCC development, which contrasts with to our findings [28]. Consequently, the role of impaired NF-jB signaling might depend on the mouse model, the type or degree of liver inflammation, and the use of carcinogens [28]. IngenuityÒ pathway analysis did not identify NF-jB signaling as the key pathway, but confirmed the histopathological phenotype of CYLDFFxAlbCre mice. Since NF-jB binding sequences were found in the regulatory regions of more than 200 target genes, making NF-jB a highly complex regulator of several physiological processes [16], it is not surprising that our analysis revealed a diversity of pathways. However, literature research suggests that most of them are somehow connected to NF-jB signaling. Importantly, the microarray analysis revealed a higher TLR-2 expression in untreated and, even more strikingly, in DEN/PB-treated CYLDFFxAlbCre mice. An increased expression of TLR-2 was observed in hepatitis B, C, and alcoholic liver disease. Moreover, persistent activation of TLR-Myd88-NF-jB/AP-1 signaling is implicated in liver fibrosis due to enhanced growth factor and cytokine production [29]. Inhibition of NF-jB signaling by SN-50 led to a significant reduction of pro-inflammatory and pro-fibrotic cytokine expression in CYLDFFxAlbCre PMH. Thus, our study demonstrates a link between increased NF-jB activation and the inflammatory and fibrotic phenotype of CYLDFFxAlbCre mice. A loss of CYLD expression or its function is accompanied by enhanced anti-cell death signaling which promotes survival of cancer cells. The survival signals emerge from NF-jB-induced anti-apoptotic genes such as survivin, which we found upregulated in livers of CYLDFFxAlbCre mice [8]. Additionally, CYLD was identified as the key substrate of caspase 8 implicated in the conversion of a pro-survival response to necroptotic cell death [30]. NF-jB-induced anti-apoptotic effects have also been reported to be involved in HCC development in Mdr2 knockout mice [14]. In livers of DEN/PB-treated CYLDFFxAlbCre mice, we detected a significantly higher BCL-3 expression, which may be the result of increased NF-jB dependent de novo synthesis. In contrast to our previous findings in CYLDexon7/8 deleted B cells and dendritic cells [6,31], we did not observe increased nuclear BCL-3 levels. Thus, there is no evidence for an accelerated BCL-3 nuclear translocation in CYLDFFxAlbCre livers, confirming the remaining ability of s-CYLD to deubiquitinate BCL-3. In conclusion, the present study describes for the first time an implication of CYLD in biliary injury, fibrosis, and hepatocarcinogenesis in mice. Whether the absence of FL-CYLD or the enforced expression of the s-CYLD splice variant is responsible for the phenotype of CYLDFFxAlbCre mice should be addressed in further studies. Recent studies provide evidence for a positive regulatory function of s-CYLD in NF-jB signaling [6,31], but a possible dominant-negative effect should also be addressed. It will be important to study CYLD dysregulation in chronic liver diseases in the future.

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Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Financial support This study was supported by research grants from the Dietmar Hopp Stiftung, St. Leon-Rot, Germany and Merck Serono GmbH, Darmstadt, Germany, to HSB and from Johannes Gutenberg-University Mainz (MAIFOR) to MAW. Acknowledgements The authors are grateful to Nicole Kautz, Anna Spille, and Petra Adams-Quack for the excellent technical assistance.

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