Purpose: The proteinase-activated receptor-2 (PAR(2)), 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 PAR(2) 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: [Ca(2+)](i) was measured in single HCC cells with fluo-4 using confocal laser scanning microscopy. For PAR(2) gene silencing, a specific PAR(2) siRNA was used. P42/p44 MAPK activation was assessed by Western blot employing a phospho-p42/p44 MAPKinase-specific antibody.
Results: Both PAR(2)-selective-activating peptide (PAR(2)-AP), 2-furoyl-LIGRLO-NH(2), and the PAR(2) activator trypsin increased Ca(2+) 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 PAR(2)-AP on [Ca(2+)](i) in HCC cells could be blocked by a PAR(2)-selective antagonist (Pal-PAR(2)) and by PAR(2) silencing with specific siRNA. Furthermore, PAR(2)-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 PAR(2) evokes calcium signals in liver carcinoma cells both by calcium entry and calcium liberation from internal pools. In addition, PAR(2)-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.