Proteinase-activated receptor 2 (PAR2) in hepatic stellate cells - evidence for a role in hepatocellular carcinoma growth in vivo

Background: Previous studies have established that proteinase-activated receptor 2 (PAR(2)) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR(2) in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model. Methods: PAR(2) 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 PAR(2) 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 PAR(2) activation in LX-2 cells, various signalling pathways were analysed by immunoblotting and proteome profiler arrays. Results: Following verification of functional PAR(2) 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 PAR(2)) was downregulated by RNA interference (RNAi). In vitro studies confirmed these results demonstrating RNAi mediated inhibition of PAR(2) attenuated Smad2/3 activation in response to TGF-beta 1 stimulation in LX-2 cells and blocked the pro-mitotic effect of LX-2 derived conditioned medium on Hep3B cells. Furthermore, PAR(2) stimulation with trypsin or a PAR(2)-selective activating peptide (PAR(2)-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 PAR(2)-AP-induced migration. Conclusion: PAR(2) in HSCs plays a crucial role in promoting HCC growth presumably by mediating migration and secretion of pro-angiogenic and pro-mitotic factors. Therefore, PAR(2) in stromal HSCs may have relevance as a therapeutic target of HCC.