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.
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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
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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).
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Dietrich, J., Sommersdorf, C., Gohlke, S. et al. Okadaic acid activates Wnt/β-catenin-signaling in human HepaRG cells. Arch Toxicol 93, 1927–1939 (2019). https://doi.org/10.1007/s00204-019-02489-4
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DOI: https://doi.org/10.1007/s00204-019-02489-4