Proteinase-activated receptor 2 (PAR2) in hepatic stellate cells – evidence for a role in hepatocellular carcinoma growth in vivo
Creators: name: Mussbach, Franziska
name: Ungefroren, Hendrik
name: Günther, Bernd
name: Katenkamp, Kathrin
name: Henklein, Petra
name: Westermann, Martin
name: Settmacher, Utz
name: Lenk, Lennart
name: Sebens, Susanne
name: Müller, Jörg P.
name: Böhmer, Frank-Dietmar
name: Kaufmann, Roland
Publisher: name: Charité - Universitätsmedizin Berlin
Year: 2016
Subjects: subject: Proteinase-activated receptor 2
subject: PAR2
subject: Hepatocellular carcinoma
subject: HCC
subject: Hepatic stellate cells
subject: HSCs
subject: LX-2
subject: Cell migration
subject: Receptor tyrosine kinase
subject: Met
subject: Src
subject: HCC xenograft
subject: Angiogenesis
subject: 500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::572 Biochemie
subject: 600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit

description: Background Previous studies have established that proteinase-activated receptor 2 (PAR2) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR2 in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model. Methods PAR2 expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca2+]i measurements, respectively. The impact of LX-2-expressed PAR2 on tumour growth in vivo was monitored using HCC xenotransplantation experiments in SCID mice, in which HCC-like tumours were induced by coinjection of LX-2 cells and Hep3B cells. To characterise the effects of PAR2 activation in LX-2 cells, various signalling pathways were analysed by immunoblotting and proteome profiler arrays. Results Following verification of functional PAR2 expression in LX-2 cells, in vivo studies showed that these cells promoted tumour growth and angiogenesis of HCC xenografts in mice. These effects were significantly reduced when F2RL1 (encoding PAR2) was downregulated by RNA interference (RNAi). In vitro studies confirmed these results demonstrating RNAi mediated inhibition of PAR2 attenuated Smad2/3 activation in response to TGF-β1 stimulation in LX-2 cells and blocked the pro-mitotic effect of LX-2 derived conditioned medium on Hep3B cells. Furthermore, PAR2 stimulation with trypsin or a PAR2-selective activating peptide (PAR2-AP) led to activation of different intracellular signalling pathways, an increased secretion of pro-angiogenic and pro-mitotic factors and proteinases, and an enhanced migration rate across a collagen- coated membrane barrier. Silencing F2RL1 by RNAi or pharmacological inhibition of Src, hepatocyte growth factor receptor (Met), platelet-derived growth factor receptor (PDGFR), p42/p44 mitogen activated protein kinase (MAPK) or matrix-metalloproteinases (MMPs) blocked PAR2-AP-induced migration. Conclusion PAR2 in HSCs plays a crucial role in promoting HCC growth presumably by mediating migration and secretion of pro-angiogenic and pro-mitotic factors. Therefore, PAR2 in stromal HSCs may have relevance as a therapeutic target of HCC.