Abstract
Signaling through the Wnt/β-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining β-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the β-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated β-catenin. Surprisingly, the expression pattern was observed independent of the cell’s position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in β-catenin-deficient liver. Clusters of β-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, β-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of β-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte’s gene expression profile is dependent on the presence of β-catenin, and that β-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.
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References
Aydinlik H, Nguyen TD, Moennikes O et al (2001) Selective pressure during tumor promotion by phenobarbital leads to clonal outgrowth of beta-catenin-mutated mouse liver tumors. Oncogene 20:7812–7816
Benhamouche S, Decaens T, Godard C et al (2006) Apc tumor suppressor gene is the “zonation-keeper” of mouse liver. Dev Cell 10:759–770
Braeuning A (2008) Beta-catenin and Ha-ras–master regulators of zonal gene expression in mouse liver? Logos Verlag, Berlin
Braeuning A, Schwarz M (2010) Beta-catenin as a multilayer modulator of zonal cytochrome P450 expression in mouse liver. Biol Chem 391:139–148
Braeuning A, Ittrich C, Kohle C et al (2007) Zonal gene expression in mouse liver resembles expression patterns of Ha-ras and beta-catenin mutated hepatomas. Drug Metab Dispos 35:503–507
Braeuning A, Sanna R, Huelsken J et al (2009) Inducibility of drug-metabolizing enzymes by xenobiotics in mice with liver-specific knockout of Ctnnb1. Drug Metab Dispos 37:1138–1145
Bruix J, Boix L, Sala M et al (2004) Focus on hepatocellular carcinoma. Cancer Cell 5:215–219
Brulport M, Schormann W, Bauer A et al (2007) Fate of extrahepatic human stem and precursor cells after transplantation into mouse livers. Hepatology 46:861–870
Cadoret A, Ovejero C, Terris B et al (2002) New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 21:8293–8301
Cavard C, Colnot S, Audard V et al (2008) Wnt/beta-catenin pathway in hepatocellular carcinoma pathogenesis and liver physiology. Future Oncol 4:647–660
Colnot S, Decaens T, Niwa-Kawakita M et al (2004) Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas. Proc Natl Acad Sci USA 101:17216–17221
Dagli ML, Hernandez-Blazquez FJ (2007) Roles of gap junctions and connexins in non-neoplastic pathological processes in which cell proliferation is involved. J Membr Biol 218:79–91
Farazi PA, DePinho RA (2006) Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 6:674–687
Gebhardt R (1992) Metabolic zonation of the liver: regulation and implications for liver function. Pharmacol Ther 53:275–354
Gebhardt R, Gaunitz F (1997) Cell–cell interactions in the regulation of the expression of hepatic enzymes. Cell Biol Toxicol 13:263–273
Gebhardt R, Mecke D (1983) Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture. EMBO J 2:567–570
Gebhardt R, Williams GM (1995) Glutamine synthetase and hepatocarcinogenesis. Carcinogenesis 16:1673–1681
Giera S, Braeuning A, Kohle C et al (2010) Wnt/beta-catenin signaling activates and determines hepatic zonal expression of glutathione S-transferases in mouse liver. Toxicol Sci 115:22–33
Godoy P, Hengstler JG, Ilkavets I et al (2009) Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology 49:2031–2043
Gupta S, Rajvanshi P, Sokhi RP et al (1999) Position-specific gene expression in the liver lobule is directed by the microenvironment and not by the previous cell differentiation state. J Biol Chem 274:2157–2165
Hailfinger S, Jaworski M, Braeuning A et al (2006) Zonal gene expression in murine liver: lessons from tumors. Hepatology 43:407–414
Harada N, Miyoshi H, Murai N et al (2002) Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin. Cancer Res 62:1971–1977
Harada N, Oshima H, Katoh M et al (2004) Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations. Cancer Res 64:48–54
Hoehme S, Brulport M, Bauer A et al (2010) Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration. Proc Natl Acad Sci USA 107:10371–10376
Huelsken J, Vogel R, Erdmann B et al (2001) Beta-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 105:533–545
Jungermann K (1995) Zonation of metabolism and gene expression in liver. Histochem Cell Biol 103:81–91
Jungermann K, Katz N (1989) Functional specialization of different hepatocyte populations. Physiol Rev 69:708–764
Jungermann K, Kietzmann T (1996) Zonation of parenchymal and nonparenchymal metabolism in liver. Annu Rev Nutr 16:179–203
Koenig S, Aurich H, Schneider C et al (2007) Zonal expression of hepatocytic marker enzymes during liver repopulation. Histochem Cell Biol 128:105–114
Kruithof-de Julio M, Labruyere WT, Ruijter JM et al (2005) The RL-ET-14 cell line mediates expression of glutamine synthetase through the upstream enhancer/promoter region. J Hepatol 43:126–131
Kuo FC, Darnell JE (1991) Evidence that interaction of hepatocytes with the collecting (hepatic) veins triggers position-specific transcription of the glutamine synthetase and ornithine aminotransferase genes in the mouse liver. Mol Cell Biol 11:6050–6058
Lindros KO (1997) Zonation of cytochrome P450 expression, drug metabolism and toxicity in liver. Gen Pharmacol 28:191–196
Loeppen S, Schneider D, Gaunitz D et al (2002) Overexpression of glutamine synthetase is associated with beta-catenin-mutations in mouse liver tumors during promotion of hepatocarcinogenesis by phenobarbital. Cancer Res 62:5685–5688
Loeppen S, Koehle C, Buchmann A et al (2005) A beta-catenin-dependent pathway regulates expression of cytochrome P450 isoforms in mouse liver tumors. Carcinogenesis 26:239–248
Marx-Stoelting P, Mahr J, Knorpp T et al (2008) Tumor promotion in liver of mice with a conditional Cx26 knockout. Toxicol Sci 103:260–267
Moennikes O, Buchmann A, Romualdi A et al (2000) Lack of phenobarbital-mediated promotion of hepatocarcinogenesis in connexin32-null mice. Cancer Res 60:5087–5091
Nakashima Y, Ono T, Yamanoi A et al (2004) Expression of gap junction protein connexin32 in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. J Gastroenterol 39:763–768
Oinonen T, Lindros KO (1998) Zonation of hepatic cytochrome P-450 expression and regulation. Biochem J 329:17–35
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Quistorff B, Dich J, Grunnet N (1986) Periportal and perivenous hepatocytes retain their zonal characteristics in primary culture. Biochem Biophys Res Commun 139:1055–1061
Schols L, Mecke D, Gebhardt R (1990) Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication. Histochemistry 94:49–54
Schrode W, Mecke D, Gebhardt R (1990) Induction of glutamine synthetase in periportal hepatocytes by cocultivation with a liver epithelial cell line. Eur J Cell Biol 53:35–41
Sekine S, Lan BY, Bedolli M et al (2006) Liver-specific loss of β-catenin blocks glutamine synthesis pathway activity and cytochrome P450 expression in mice. Hepatology 43:817–825
Sekine S, Gutierrez PJ, Lan BY et al (2007) Liver-specific loss of beta-catenin results in delayed hepatocyte proliferation after partial hepatectomy. Hepatology 45:361–368
Sekine S, Ogawa R, Ito R et al (2009) Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis. Gastroenterology 136:2304–2315
Strathmann J, Schwarz M, Tharappel JC et al (2006) PCB 153, a non-dioxin-like tumor promoter, selects for beta-catenin (Catnb) mutated mouse liver tumors. Toxicol Sci 93:34–40
Tan X, Behari J, Cieply B et al (2006) Conditional deletion of beta-catenin reveals its role in liver growth and regeneration. Gastroenterology 131:1561–1572
Thompson MD, Monga SP (2007) WNT/beta-catenin signaling in liver health and disease. Hepatology 45:1298–1305
Willert K, Nusse R (1998) Beta-catenin: a key mediator of Wnt signaling. Curr Opin Genet Dev 8:95–102
Zhang XF, Tan X, Zeng G, et al (2010) Conditional beta-catenin loss in mice promotes chemical hepatocarcinogenesis: role of oxidative stress and platelet-derived growth factor receptor alpha/phosphoinositide 3-kinase signaling. Hepatology 52:954–965
Acknowledgments
The authors acknowledge the expert technical assistance by Johanna Mahr and Elke Zabinsky. We also thank Dr. J. Huelsken (Lausanne, Switzerland) for the kind gift of Ctnnb1 loxP/loxP mice, Dr. R. Wolf (Dundee, UK) for the gift of CYP and GST antisera, and Dr. S. Yamagoe (Tokyo, Japan) for providing the Lect2 antibody. We also thank Dr. Leticia Quintanilla-Fendt for her consultation on histopathological findings. This study was supported by the Deutsche Forschungsgemeinschaft (grant SFB 773), by the European Commission through CancerSys (HEALTH-F4-2008-223188), and by the Bundesministerium für Bildung und Forschung-funded network ‘Virtual Liver’.
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Braeuning, A., Singh, Y., Rignall, B. et al. Phenotype and growth behavior of residual β-catenin-positive hepatocytes in livers of β-catenin-deficient mice. Histochem Cell Biol 134, 469–481 (2010). https://doi.org/10.1007/s00418-010-0747-1
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DOI: https://doi.org/10.1007/s00418-010-0747-1