Roland Kaufmann1, Franziska Mussbach, Petra Henklein, Utz Settmacher. 1. Department of General, Visceral and Vascular Surgery, Medical Faculty at the Friedrich Schiller University Jena, Erlanger Allee 101, 07747 Jena, Germany. roland.kaufmann@med.uni-jena.de
Abstract
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.
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.
Authors: Patrick J Klein; C Max Schmidt; Chad A Wiesenauer; Jennifer N Choi; Earl A Gage; Michele T Yip-Schneider; Eric A Wiebke; Yufang Wang; Charles Omer; Judith S Sebolt-Leopold Journal: Neoplasia Date: 2006-01 Impact factor: 5.715
Authors: Dennis J Grab; Jose C Garcia-Garcia; Olga V Nikolskaia; Yuri V Kim; Amanda Brown; Carlos A Pardo; Yongqing Zhang; Kevin G Becker; Brenda A Wilson; Ana Paula C de A Lima; Julio Scharfstein; J Stephen Dumler Journal: PLoS Negl Trop Dis Date: 2009-07-21
Authors: Kathryn H Driesbaugh; Marguerite S Buzza; Erik W Martin; Gregory D Conway; Joseph P Y Kao; Toni M Antalis Journal: J Biol Chem Date: 2014-12-17 Impact factor: 5.157
Authors: Franziska Mußbach; Hendrik Ungefroren; Bernd Günther; Kathrin Katenkamp; Petra Henklein; Martin Westermann; Utz Settmacher; Lennart Lenk; Susanne Sebens; Jörg P Müller; Frank-Dietmar Böhmer; Roland Kaufmann Journal: Mol Cancer Date: 2016-07-29 Impact factor: 27.401
Authors: Sonja Stahn; Lisa Thelen; Ina-Maria Albrecht; Jens Bitzer; Thomas Henkel; Nicole Elisabeth Teusch Journal: Pharmacol Res Perspect Date: 2016-06-10