The transforming growth factor-a and cyclin D1 genes are direct targets of b-catenin signaling in hepatocyte proliferation Cyril Torre   , Samira Benhamouche   , Claudia Mitchell, Cécile Godard, Philippe Veber, Franck Letourneur, Nicolas Cagnard, Sébastien Jacques, Laetitia Finzi, Christine Perret, Sabine Colnot ⇑ Institut Cochin, Université Paris Descartes, CNRS UMR 8104 Paris, France; INSERM, U1016 Paris, France Background & Aims: b-Catenin is an oncogene frequently mutated in hepatocellular carcinoma. In this study, we investi- gated target genes of b-catenin signaling in hepatocyte proliferation. Methods: We studied transgenic mice displaying either inactiva- tion or activation of the b-catenin pathway, focusing on analysis of liver proliferation due to aberrant b-catenin activation, and on the regeneration process during which b-catenin signaling is transiently activated. We localized in situ the various partners involved in proliferation or identified as targets of b-catenin in these transgenic and regenerating livers. We also performed comparative transcriptome analyses, using microarrays. Finally, we extracted, from deep-sequencing data, both the DNA regula- tory elements bound to the b-catenin/Tcf nuclear complex and the expression levels of critical targets identified in microarrays. Results: b-Catenin activation during liver regeneration occurred during G1/S cell cycle progression and allowed zonal extension of the normal territory of active b-catenin and panlobular prolif- eration. We found that b-catenin controlled both cell-autono- mous and non-cell-autonomous hepatocyte proliferation, through direct transcriptional and complex control of cyclin D1 gene expression and of the expression of a new target gene, Tgfa. Conclusions: We propose that b-catenin controls panlobular hepatocyte proliferation partly by controlling, together with its Tcf4 nuclear partner, expression of the pro-proliferation cyclin D1 and Tgfa genes. This study constitutes a first step toward understanding the oncogenic properties of this prominent signal- ing pathway in the liver. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction b-Catenin signaling plays an important role in liver homeostasis, embryonic development, and proliferation ([9,25], for reviews). In particular, inappropriate b-catenin signaling occurs in 30–40% of human hepatocellular carcinomas (HCCs) [13,24]. The b-catenin pathway is activated by Wnt signals in develop- mental and physiological processes, or by mutations affecting its partners during tumor formation. Without such activating sig- nals, a degradation complex consisting of kinases and the tumor suppressors APC (adenomatous polyposis coli) and axins, is responsible for the phosphorylation and degradation of b-catenin. The binding of Wnt to frizzled receptors or the occurrence of oncogenic mutations renders the b-catenin degradation complex inefficient. As a consequence, the unphosphorylated b-catenin accumulates and translocates to the nucleus, where it binds to Tcf/Lef transcription factors to activate target genes highly spe- cific for the tissular and cellular context. The adult hepatocyte is a quiescent, highly differentiated cell, with the unique property to re-enter the cell cycle after injury. After 70% partial hepatectomy (PH) for example, hepatocyte syn- chronous cell cycling regenerates liver mass within a few days in rodents [14,22]. The intensive use of transgenic mice and of the powerful PH model has helped to unravel signaling networks underlying hepatocyte proliferation, but also underlying tumor formation. We have previously shown that the activation of a b-catenin signal in mouse liver is oncogenic [11]. Moreover, the massive activation of b-catenin in more than 70% of hepatocytes in mice, leads to hepatomegaly, partly due to hepatocyte proliferation [7,8,19,28]. Conversely, liver weight has been shown to be 20% lower in adult mice with a liver-specific b-catenin gene inactiva- tion than in wild-type mice, and a role for b-catenin in liver regeneration has also been reported [15,30,31,34]. However, the direct transcriptional targets of b-catenin involved in hepatocyte proliferation remain elusive. We investigated the role of Wnt/b-catenin signaling in hepa- tocellular proliferation in a model of liver regeneration and in a model of liver hyperplasia induced by the aberrant activation of Journal of Hepatology 2011 vol. 55 j 86–95 Received 7 May 2010; received in revised form 11 October 2010; accepted 18 October 2010; available online 30 November 2010 ⇑ Corresponding author. Address: Institut Cochin, INSERM U1016, UMR CNRS 8104, Université René Descartes, Département Endocrinologie, Métabolisme et Cancer, Bâtiment Faculté, 24, rue du Faubourg Saint-Jacques, 75014 Paris, France. Tel.: +33 144412567; fax: +33 144412421. E-mail address: sabine.colnot@inserm.fr (S. Colnot).   These authors contributed equally to this work. Abbreviations: HCC, hepatocellular carcinomas; APC, adenomatous polyposis coli; PH, partial hepatectomy; Tgf-a, transforming growth factor-a; bcat ko , inducible liver-specific inactivation of b-catenin; Apc ko , inducible liver-specific inactivation of Apc; qPCR, real-time quantitative polymerase chain reaction; ISH, in situ hy- bridization; Arg1, arginase 1; Gls2, glutaminase 2; GS or Glul, glutamine Synth- etase; ChIP, chromatin immunoprecipitation; ChIP-Seq, deep-sequencing of ChIP products; RNA-Seq, deep-sequencing of mRNAs; Pck1, Pepck1; PC, pericentral; PP, periportal. Research Article