Research Article
The transforming growth factor-α and cyclin D1 genes are direct targets of β-catenin signaling in hepatocyte proliferation

https://doi.org/10.1016/j.jhep.2010.10.021Get rights and content

Background & Aims

β-Catenin is an oncogene frequently mutated in hepatocellular carcinoma. In this study, we investigated target genes of β-catenin signaling in hepatocyte proliferation.

Methods

We studied transgenic mice displaying either inactivation or activation of the β-catenin pathway, focusing on analysis of liver proliferation due to aberrant β-catenin activation, and on the regeneration process during which β-catenin signaling is transiently activated. We localized in situ the various partners involved in proliferation or identified as targets of β-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 regulatory elements bound to the β-catenin/Tcf nuclear complex and the expression levels of critical targets identified in microarrays.

Results

β-Catenin activation during liver regeneration occurred during G1/S cell cycle progression and allowed zonal extension of the normal territory of active β-catenin and panlobular proliferation. We found that β-catenin controlled both cell-autonomous 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, Tgfα.

Conclusions

We propose that β-catenin controls panlobular hepatocyte proliferation partly by controlling, together with its Tcf4 nuclear partner, expression of the pro-proliferation cyclin D1 and Tgfα genes. This study constitutes a first step toward understanding the oncogenic properties of this prominent signaling pathway in the liver.

Introduction

β-Catenin signaling plays an important role in liver homeostasis, embryonic development, and proliferation ([9], [25], for reviews). In particular, inappropriate β-catenin signaling occurs in 30–40% of human hepatocellular carcinomas (HCCs) [13], [24]. The β-catenin pathway is activated by Wnt signals in developmental and physiological processes, or by mutations affecting its partners during tumor formation. Without such activating signals, a degradation complex consisting of kinases and the tumor suppressors APC (adenomatous polyposis coli) and axins, is responsible for the phosphorylation and degradation of β-catenin. The binding of Wnt to frizzled receptors or the occurrence of oncogenic mutations renders the β-catenin degradation complex inefficient. As a consequence, the unphosphorylated β-catenin accumulates and translocates to the nucleus, where it binds to Tcf/Lef transcription factors to activate target genes highly specific 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 synchronous 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 β-catenin signal in mouse liver is oncogenic [11]. Moreover, the massive activation of β-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 β-catenin gene inactivation than in wild-type mice, and a role for β-catenin in liver regeneration has also been reported [15], [30], [31], [34]. However, the direct transcriptional targets of β-catenin involved in hepatocyte proliferation remain elusive.

We investigated the role of Wnt/β-catenin signaling in hepatocellular proliferation in a model of liver regeneration and in a model of liver hyperplasia induced by the aberrant activation of β-catenin signaling after Apc loss. Microarray and functional analyses demonstrate that the cyclin D1 and Tgfα genes are direct targets of β-catenin involved in β-catenin-dependent hepatocyte proliferation.

Section snippets

Animals and surgical procedures

Two-thirds PH was performed on two-month-old wild-type C57BL/6 (Charles River, France) or transgenic male mice, as previously described [23]. All animal experiments were carried out in accordance with French government regulations.

Mice with a liver-specific knockout of the β-catenin gene were obtained by crossing TTR-CreTam mice [35] with mice carrying a biallelic floxed β-catenin gene (loxP sites located between the 2nd and 6th exon) [5]. Analyses were carried out on male β−cateninlox2−6/lox2–6

Results

We developed two models in which hepatocyte proliferation is at least partly dependent on β-catenin signaling: the PH and the liver-specific tamoxifen-inducible invalidation of Apc [2], [30], [34] (Fig. 1A). We also generated a new model of conditional invalidation of the β-catenin gene in the adult hepatocytes that is tamoxifen-inducible [5], (Fig. 1A and Supplementary Fig. 1A). Here, as in βcatko livers generated in the postnatal period using the alb-Cre transgene [30], [34], β-catenin

Discussion

This study provides new insight into the paradox linking β-catenin to proliferation in the liver [37]: the β-catenin pathway is physiologically activated in quiescent PC hepatocytes, whereas it promotes hepatocyte proliferation and liver carcinogenesis when aberrantly activated and it also controls the G1/S progression of hepatocytes during liver regeneration.

We have clearly defined the liver compartments in which β-catenin is activated following PH. Using different Lef-Tcf/β-catenin reporter

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 work was supported by the Ligue Nationale Contre Le Cancer (2008–10), the Agence Nationale de la Recherche (2008–2009), and Cancersys EU FP7 programme (2008–2011). SB and CT held fellowships from the Association de Recherche contre le Cancer, and from the Ministere de la Recherche et de la Technologie respectively.

Acknowledgments

We would like to thank Dr. Rolf Kemler for providing us with β-catenin floxed mice, Drs. Costantini, Camonis, Ben-Ze’ev for plasmids and Drs. Romagnolo, Couty, Cavard, Denechaud and Gilgenkrantz for critical reading of the manuscript. We warmly thank Isabelle Lagoutte and the staff of the animal facilities of Institut Cochin for skillful care of transgenic mice.

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    These authors contributed equally to this work.

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