Advertisement

Archives of Toxicology

, Volume 93, Issue 7, pp 1927–1939 | Cite as

Okadaic acid activates Wnt/β-catenin-signaling in human HepaRG cells

  • Jessica Dietrich
  • Cornelia Sommersdorf
  • Svenja Gohlke
  • Oliver Poetz
  • Bjoern Traenkle
  • Ulrich Rothbauer
  • Stefanie Hessel-PrasEmail author
  • Alfonso Lampen
  • Albert Braeuning
Molecular Toxicology
  • 351 Downloads

Abstract

The lipophilic phycotoxin okadaic acid (OA) occurs in the fatty tissue and hepatopancreas of filter-feeding shellfish. The compound provokes the diarrhetic shellfish poisoning (DSP) syndrome after intake of seafood contaminated with high levels of the DSP toxin. In animal experiments, long-term exposure to OA is associated with an elevated risk for tumor formation in different organs including the liver. Although OA is a known inhibitor of the serine/threonine protein phosphatase 2A, the mechanisms behind OA-induced carcinogenesis are not fully understood. Here, we investigated the influence of OA on the β-catenin-dependent Wnt-signaling pathway, addressing a major oncogenic pathway relevant for tumor development. We analyzed OA-mediated effects on β-catenin and its biological function, cellular localization, post-translational modifications, and target gene expression in human HepaRG hepatocarcinoma cells treated with non-cytotoxic concentrations up to 50 nM. We detected concentration- and time-dependent effects of OA on the phosphorylation state, cellular redistribution as well as on the amount of transcriptionally active β-catenin. These findings were confirmed by quantitative live-cell imaging of U2OS cells stably expressing a green fluorescent chromobody which specifically recognize hypophosphorylated β-catenin. Finally, we demonstrated that nuclear translocation of β-catenin mediated by non-cytotoxic OA concentrations results in an upregulation of Wnt-target genes. In conclusion, our results show a significant induction of the canonical Wnt/β-catenin-signaling pathway by OA in human liver cells. Our data contribute to a better understanding of the molecular mechanisms underlying OA-induced carcinogenesis.

Keywords

liver toxicity Okadaic acid TCF/LEF-responsive genes Tumor promotion Wnt-signaling pathway 

Abbreviations

APC

Adenomatous polyposis coli

CB

Chromobody

CK1α

Casein kinase 1α

CYP

Cytochrome P450

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

Dimethyl sulfoxide

DSP

Diarrhetic shellfish poisoning

ECT

E-cadherin cytosolic tail

FBS

Fetal bovine serum

GSK3β

Glycogen synthase kinase 3β

GUSB

β-Glucuronidase

ICAT

Inhibitor of β-catenin

LEF

Lymphoid enhancer factor

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

OA

Okadaic acid

PBS

Phosphate-buffered saline

PC

Positive control

PP

Serine/threonine protein phosphatase

qPCR

Quantitative real-time reverse transcriptase polymerase chain reaction

SC

Solvent control

SD

Standard deviation

SFM

Serum-free assay medium

STF

SuperTopFlash

TBST

Tris-buffered saline with Tween 20

TCF

T-cell factor

Notes

Acknowledgements

This work was supported by the German Research Foundation (Grant no LA 1177/11-1) and by the German Federal Institute for Risk Assessment (Grant no. 1322-662).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

204_2019_2489_MOESM1_ESM.docx (85 kb)
Supplementary material 1 (DOCX 85 kb)

References

  1. Amit S et al (2002) Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 16:1066–1076.  https://doi.org/10.1101/gad.230302 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Barker N et al (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003–1007.  https://doi.org/10.1038/nature06196 CrossRefPubMedGoogle Scholar
  3. Bialojan C, Takai A (1988) Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J 256:283–290CrossRefGoogle Scholar
  4. Boe R, Gjertsen BT, Vintermyr OK, Houge G, Lanotte M, Doskeland SO (1991) The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells. Exp Cell Res 195:237–246CrossRefGoogle Scholar
  5. Braeuning A et al (2007) Serum components and activated Ha-ras antagonize expression of perivenous marker genes stimulated by beta-catenin signaling in mouse hepatocytes. FEBS J 274:4766–4777.  https://doi.org/10.1111/j.1742-4658.2007.06002.x CrossRefPubMedGoogle Scholar
  6. Cordier S, Monfort C, Miossec L, Richardson S, Belin C (2000) Ecological analysis of digestive cancer mortality related to contamination by diarrhetic shellfish poisoning toxins along the coasts of France. Environ Res 84:145–150.  https://doi.org/10.1006/enrs.2000.4103 CrossRefPubMedGoogle Scholar
  7. EFSA (2008) Scientific Opinion of the Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfish—okadaic acid and analogue. EFSA J 589:1–62Google Scholar
  8. Ehlers A, Stempin S, Al-Hamwi R, Lampen A (2010) Embryotoxic effects of the marine biotoxin okadaic acid on murine embryonic stem cells. Toxicon 55:855–863.  https://doi.org/10.1016/j.toxicon.2009.12.008 CrossRefPubMedGoogle Scholar
  9. Fang D et al (2007) Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J Biol Chem 282:11221–11229.  https://doi.org/10.1074/jbc.M611871200 CrossRefPubMedPubMedCentralGoogle Scholar
  10. FAO (2004) Marine biotoxins. FAO Food Nutr Pap 80:53–92Google Scholar
  11. Ferron PJ, Hogeveen K, De Sousa G, Rahmani R, Dubreil E, Fessard V, Le Hegarat L (2016) Modulation of CYP3A4 activity alters the cytotoxicity of lipophilic phycotoxins in human hepatic HepaRG cells. Toxicol In Vitro 33:136–146.  https://doi.org/10.1016/j.tiv.2016.02.021 CrossRefPubMedGoogle Scholar
  12. Fessard V, Grosse Y, Pfohl-Leszkowicz A, Puiseux-Dao S (1996) Okadaic acid treatment induces DNA adduct formation in BHK21 C13 fibroblasts and HESV keratinocytes. Mutat Res 361:133–141CrossRefGoogle Scholar
  13. Giles RH, van Es JH, Clevers H (2003) Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta 1653:1–24PubMedGoogle Scholar
  14. Groll N, Sommersdorf C, Joos TO, Poetz O (2015) A bead-based multiplex sandwich immunoassay to assess the abundance and posttranslational modification state of beta-catenin. Methods Mol Biol 1295:441–453.  https://doi.org/10.1007/978-1-4939-2550-6_31 CrossRefPubMedGoogle Scholar
  15. Guo FJ, An TY, Rein KS (2010) The algal hepatoxoxin okadaic acid is a substrate for human cytochromes CYP3A4 and CYP3A5. Toxicon 55:325–332CrossRefGoogle Scholar
  16. Guzman M, Castro J (1991) Okadaic acid stimulates carnitine palmitoyltransferase I activity and palmitate oxidation in isolated rat hepatocytes. FEBS Lett 291:105–108CrossRefGoogle Scholar
  17. Ha NC, Tonozuka T, Stamos JL, Choi HJ, Weis WI (2004) Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation. Mol Cell 15:511–521.  https://doi.org/10.1016/j.molcel.2004.08.010 CrossRefPubMedGoogle Scholar
  18. Hampf M, Gossen M (2006) A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays. Anal Biochem 356:94–99.  https://doi.org/10.1016/j.ab.2006.04.046 CrossRefPubMedGoogle Scholar
  19. He TC et al (1998) Identification of c-MYC as a target of the APC pathway. Science 281:1509–1512CrossRefGoogle Scholar
  20. He TC, Chan TA, Vogelstein B, Kinzler KW (1999) PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99:335–345CrossRefGoogle Scholar
  21. Howe LR, Subbaramaiah K, Chung WJ, Dannenberg AJ, Brown AM (1999) Transcriptional activation of cyclooxygenase-2 in Wnt-1-transformed mouse mammary epithelial cells. Cancer Res 59:1572–1577PubMedGoogle Scholar
  22. Ikeda S, Kishida M, Matsuura Y, Usui H, Kikuchi A (2000) GSK-3beta-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by beta-catenin and protein phosphatase 2A complexed with Axin. Oncogene 19:537–545.  https://doi.org/10.1038/sj.onc.1203359 CrossRefPubMedGoogle Scholar
  23. Jamora C, DasGupta R, Kocieniewski P, Fuchs E (2003) Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 422:317–322.  https://doi.org/10.1038/nature01458 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kasa A, Czikora I, Verin AD, Gergely P, Csortos C (2013) Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells. Microvasc Res 89:86–94.  https://doi.org/10.1016/j.mvr.2013.05.003 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Keller BM, Maier J, Secker KA, Egetemaier SM, Parfyonova Y, Rothbauer U, Traenkle B (2018) Chromobodies to quantify changes of endogenous protein concentration in living cells. Mol Cell Proteomics 17:2518–2533.  https://doi.org/10.1074/mcp.TIR118.000914 CrossRefPubMedGoogle Scholar
  26. Keller BM, Maier J, Weldle M, Segan S, Traenkle B, Rothbauer U (2019) A strategy to optimize the generation of stable chromobody cell lines for visualization and quantification of endogenous proteins in living cells. Antibodies 8:10.  https://doi.org/10.3390/antib8010010 CrossRefPubMedCentralGoogle Scholar
  27. Klein S, Mueller D, Schevchenko V, Noor F (2014) Long-term maintenance of HepaRG cells in serum-free conditions and application in a repeated dose study. J Appl Toxicol 34:1078–1086.  https://doi.org/10.1002/jat.2929 CrossRefPubMedGoogle Scholar
  28. Kolrep F, Hessel S, These A, Ehlers A, Rein K, Lampen A (2016) Differences in metabolism of the marine biotoxin okadaic acid by human and rat cytochrome P450 monooxygenases. Arch Toxicol 90:2025–2036.  https://doi.org/10.1007/s00204-015-1591-9 CrossRefPubMedGoogle Scholar
  29. Le Hegarat L, Puech L, Fessard V, Poul JM, Dragacci S (2003) Aneugenic potential of okadaic acid revealed by the micronucleus assay combined with the FISH technique in CHO-K1 cells. Mutagenesis 18:293–298CrossRefGoogle Scholar
  30. Le Hegarat L, Fessard V, Poul JM, Dragacci S, Sanders P (2004) Marine toxin okadaic acid induces aneuploidy in CHO-K1 cells in presence of rat liver postmitochondrial fraction, revealed by cytokinesis-block micronucleus assay coupled to FISH. Environ Toxicol 19:123–128.  https://doi.org/10.1002/tox.20004 CrossRefPubMedGoogle Scholar
  31. Le Hegarat L, Jacquin AG, Bazin E, Fessard V (2006) Genotoxicity of the marine toxin okadaic acid, in human Caco-2 cells and in mice gut cells. Environ Toxicol 21:55–64.  https://doi.org/10.1002/tox.20154 CrossRefPubMedGoogle Scholar
  32. Liu C et al (2002) Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108:837–847CrossRefGoogle Scholar
  33. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408.  https://doi.org/10.1006/meth.2001.1262 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lopez-Terrada D et al (2009) Histologic subtypes of hepatoblastoma are characterized by differential canonical Wnt and Notch pathway activation in DLK + precursors. Hum Pathol 40:783–794.  https://doi.org/10.1016/j.humpath.2008.07.022 CrossRefPubMedGoogle Scholar
  35. Luckert K, Gotschel F, Sorger PK, Hecht A, Joos TO, Potz O (2011) Snapshots of protein dynamics and post-translational modifications in one experiment–beta-catenin and its functions. Mol Cell Proteomics 10(M110):007377.  https://doi.org/10.1074/mcp.M110.007377 CrossRefPubMedGoogle Scholar
  36. MacDonald BT, Tamai K, He X (2009) Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9–26.  https://doi.org/10.1016/j.devcel.2009.06.016 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Manerio E, Rodas VL, Costas E, Hernandez JM (2008) Shellfish consumption: a major risk factor for colorectal cancer. Med Hypotheses 70:409–412.  https://doi.org/10.1016/j.mehy.2007.03.041 CrossRefPubMedGoogle Scholar
  38. Mann B et al (1999) Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci USA 96:1603–1608CrossRefGoogle Scholar
  39. Marion MJ, Hantz O, Durantel D (2010) The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies. Methods Mol Biol 640:261–272.  https://doi.org/10.1007/978-1-60761-688-7_13 CrossRefPubMedGoogle Scholar
  40. Matias WG, Traore A, Bonini M, Sanni A, Creppy EE (1999) Oxygen reactive radicals production in cell culture by okadaic acid and their implication in protein synthesis inhibition. Hum Exp Toxicol 18:634–639.  https://doi.org/10.1191/096032799678839473 CrossRefPubMedGoogle Scholar
  41. Mitra A, Menezes ME, Pannell LK, Mulekar MS, Honkanen RE, Shevde LA, Samant RS (2012) DNAJB6 chaperones PP2A mediated dephosphorylation of GSK3beta to downregulate beta-catenin transcription target, osteopontin. Oncogene 31:4472–4483.  https://doi.org/10.1038/onc.2011.623 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Morimoto Y, Ohba T, Kobayashi S, Haneji T (1997) The protein phosphatase inhibitors okadaic acid and calyculin A induce apoptosis in human osteoblastic cells. Exp Cell Res 230:181–186.  https://doi.org/10.1006/excr.1996.3404 CrossRefPubMedGoogle Scholar
  43. Nakashima N, Huang CL, Liu D, Ueno M, Yokomise H (2010) Intratumoral Wnt1 expression affects survivin gene expression in non-small cell lung cancer. Int J Oncol 37:687–694PubMedGoogle Scholar
  44. Nunbhakdi-Craig V, Machleidt T, Ogris E, Bellotto D, White CL 3rd, Sontag E (2002) Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol 158:967–978.  https://doi.org/10.1083/jcb.200206114 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Pasdar M, Li Z, Chan H (1995) Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells. Cell Motil Cytoskeleton 30:108–121.  https://doi.org/10.1002/cm.970300203 CrossRefPubMedGoogle Scholar
  46. Peifer M, Pai LM, Casey M (1994) Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase. Dev Biol 166:543–556.  https://doi.org/10.1006/dbio.1994.1336 CrossRefPubMedGoogle Scholar
  47. Peng J, Bowden GT, Domann FE (1997) Activation of AP-1 by okadaic acid in mouse keratinocytes associated with hyperphosphorylation of c-jun. Mol Carcinog 18:37–43CrossRefGoogle Scholar
  48. Ranganathan S, Tan X, Monga SP (2005) beta-Catenin and met deregulation in childhood hepatoblastomas. Pediatr Dev Pathol 8:435–447.  https://doi.org/10.1007/s10024-005-0028-5 CrossRefPubMedGoogle Scholar
  49. Ravindran J, Gupta N, Agrawal M, Bala Bhaskar AS, Lakshmana Rao PV (2011) Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via, mitochondrial mediated caspase-dependent mechanism. Apoptosis 16:145–161.  https://doi.org/10.1007/s10495-010-0554-0 CrossRefPubMedGoogle Scholar
  50. Schonthal AH (1998) Role of PP2A in intracellular signal transduction pathways. Front Biosci 3:D1262–D1273CrossRefGoogle Scholar
  51. Suganuma M et al (1988) Okadaic acid: an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter. Proc Natl Acad Sci USA 85:1768–1771CrossRefGoogle Scholar
  52. Suganuma M, Tatematsu M, Yatsunami J, Yoshizawa S, Okabe S, Uemura D, Fujiki H (1992) An alternative theory of tissue specificity by tumor promotion of okadaic acid in glandular stomach of SD rats. Carcinogenesis 13:1841–1845CrossRefGoogle Scholar
  53. Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO (2006) Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase. J Biol Chem 281:9971–9976.  https://doi.org/10.1074/jbc.M508778200 CrossRefPubMedGoogle Scholar
  54. ten Berge D, Koole W, Fuerer C, Fish M, Eroglu E, Nusse R (2008) Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3:508–518.  https://doi.org/10.1016/j.stem.2008.09.013 CrossRefPubMedPubMedCentralGoogle Scholar
  55. Thompson JJ, Williams CS (2018) Protein phosphatase 2A in the regulation of Wnt signaling, stem cells, and cancer. Genes (Basel) 9:121.  https://doi.org/10.3390/genes9030121 CrossRefGoogle Scholar
  56. Thompson EJ, MacGowan J, Young MR, Colburn N, Bowden GT (2002) A dominant negative c-jun specifically blocks okadaic acid-induced skin tumor promotion. Cancer Res 62:3044–3047PubMedGoogle Scholar
  57. Traenkle B et al (2015) Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells. Mol Cell Proteomics 14:707–723.  https://doi.org/10.1074/mcp.M114.044016 CrossRefPubMedPubMedCentralGoogle Scholar
  58. Wielenga VJ et al (1999) Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway. Am J Pathol 154:515–523.  https://doi.org/10.1016/S0002-9440(10)65297-2 CrossRefPubMedPubMedCentralGoogle Scholar
  59. Yan D et al (2001) Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta -catenin signaling is activated in human colon tumors. Proc Natl Acad Sci USA 98:14973–14978.  https://doi.org/10.1073/pnas.261574498 CrossRefPubMedGoogle Scholar
  60. Yokoyama N, Malbon CC (2007) Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/beta-catenin signaling. J Mol Signal 2:12.  https://doi.org/10.1186/1750-2187-2-12 CrossRefPubMedPubMedCentralGoogle Scholar
  61. Zhang T, Otevrel T, Gao Z, Gao Z, Ehrlich SM, Fields JZ, Boman BM (2001) Evidence that APC regulates survivin expression: a possible mechanism contributing to the stem cell origin of colon cancer. Cancer Res 61:8664–8667PubMedGoogle Scholar
  62. Zhang W, Yang J, Liu Y, Chen X, Yu T, Jia J, Liu C (2009) PR55 alpha, a regulatory subunit of PP2A, specifically regulates PP2A-mediated beta-catenin dephosphorylation. J Biol Chem 284:22649–22656.  https://doi.org/10.1074/jbc.M109.013698 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jessica Dietrich
    • 1
  • Cornelia Sommersdorf
    • 2
  • Svenja Gohlke
    • 1
  • Oliver Poetz
    • 2
  • Bjoern Traenkle
    • 3
    • 4
  • Ulrich Rothbauer
    • 3
    • 4
  • Stefanie Hessel-Pras
    • 1
    Email author
  • Alfonso Lampen
    • 1
  • Albert Braeuning
    • 1
  1. 1.Department of Food SafetyGerman Federal Institute for Risk AssessmentBerlinGermany
  2. 2.SIGNATOPE GmbHReutlingenGermany
  3. 3.NMI Natural and Medical Sciences Institute at the University of TuebingenReutlingenGermany
  4. 4.Eberhard Karls University Tuebingen, Pharmaceutical BiotechnologyTuebingenGermany

Personalised recommendations