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Proteinase-activated receptor 2-mediated calcium signaling in hepatocellular carcinoma cells

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Abstract

Purpose

The proteinase-activated receptor-2 (PAR2), a member of a newly discovered G protein–coupled receptor subfamily has recently been shown to promote hepatocellular carcinoma (HCC) cell invasion, suggesting a function in HCC progression. In this study, the effect of PAR2 on intracellular calcium and its involvement in p42/p44 MAPKinase activation in HEP-3B cells and in two primary HCC cultures established from surgically resected HCC specimens has been investigated.

Methods

[Ca2+]i was measured in single HCC cells with fluo-4 using confocal laser scanning microscopy. For PAR2 gene silencing, a specific PAR2 siRNA was used. P42/p44 MAPK activation was assessed by Western blot employing a phospho-p42/p44 MAPKinase–specific antibody.

Results

Both PAR2-selective-activating peptide (PAR2-AP), 2-furoyl-LIGRLO-NH2, and the PAR2 activator trypsin increased Ca2+ in HCC cells. These effects were reduced by pretreatment of the cells with thapsigargin and by EGTA buffering. In addition, the effect of trypsin and PAR2-AP on [Ca2+]i in HCC cells could be blocked by a PAR2-selective antagonist (Pal-PAR2) and by PAR2 silencing with specific siRNA. Furthermore, PAR2-AP-induced p42/p44 MAPKinase activation could be inhibited by depletion of intracellular calcium stores by thapsigargin and removing extracellular calcium.

Conclusions

Our results imply that PAR2 evokes calcium signals in liver carcinoma cells both by calcium entry and calcium liberation from internal pools. In addition, PAR2-dependent calcium signaling was shown to be critical for p42/p44 MAPKinase activation in HCC cells. Since MAPKinases are key elements in HCC cell invasion, calcium mobilization appears to critically contribute to this crucial intracellular pathway for hepatocellular carcinoma progression.

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Acknowledgments

The authors would like to thank Beate Schulze and Elke Oswald, Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Medical Faculty of the Friedrich Schiller University Jena for the excellent technical assistance. This work was supported in part by German Cancer Aid (R.K., Project 108809).

Conflict of interest

We declare that we have no conflict of interest.

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Authors and Affiliations

  1. Department of General, Visceral and Vascular Surgery, Medical Faculty at the Friedrich Schiller University Jena, Erlanger Allee 101, 07747, Jena, Germany

    Roland Kaufmann, Franziska Mußbach & Utz Settmacher

  2. Institute of Biochemistry, Charité, Humboldt University of Berlin, Oudenarderstr. 16, 13347, Berlin, Germany

    Petra Henklein

  3. Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Medical Faculty at the Friedrich Schiller University Jena, Drackendorfer Str. 1, 07747, Jena, Germany

    Roland Kaufmann

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  1. Roland Kaufmann
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Correspondence to Roland Kaufmann.

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Kaufmann, R., Mußbach, F., Henklein, P. et al. Proteinase-activated receptor 2-mediated calcium signaling in hepatocellular carcinoma cells. J Cancer Res Clin Oncol 137, 965–973 (2011). https://doi.org/10.1007/s00432-010-0961-1

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  • DOI: https://doi.org/10.1007/s00432-010-0961-1

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