title: Proteinase-activated receptor 2 (PAR2) in hepatic stellate cells - evidence for a role in hepatocellular carcinoma growth in vivo.
abstract: 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.
  
  
  
  PAR2 expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca(2+)]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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of General, Visceral and Vascular Surgery, Jena University Hospital, Erlanger Allee 101, D-07747, Jena, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Mußbach
authors.affiliation: First Department of Medicine, UKSH and University of Lübeck, Lübeck, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Ungefroren
authors.affiliation: Service Unit Small Animal, Research Center Lobeda (FZL), Jena University Hospital, Jena, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Günther
authors.affiliation: Institute of Pathology, Jena University Hospital, Jena, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Katenkamp
authors.affiliation: Institute of Biochemistry, Charité, Berlin, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Henklein
authors.affiliation: Electron Microscopy Center, Jena University Hospital, Jena, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Westermann
authors.affiliation: Department of General, Visceral and Vascular Surgery, Jena University Hospital, Erlanger Allee 101, D-07747, Jena, Germany.
authors.forename: Utz
authors.initials: U
authors.lastname: Settmacher
authors.affiliation: Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein (UKSH), Campus Kiel, Kiel, Germany.
authors.forename: Lennart
authors.initials: L
authors.lastname: Lenk
authors.affiliation: Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein (UKSH), Campus Kiel, Kiel, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Sebens
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
authors.forename: Jörg P
authors.initials: JP
authors.lastname: Müller
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
authors.forename: Frank-Dietmar
authors.initials: FD
authors.lastname: Böhmer
authors.affiliation: Department of General, Visceral and Vascular Surgery, Jena University Hospital, Erlanger Allee 101, D-07747, Jena, Germany. roland.kaufmann@med.uni-jena.de.
authors.forename: Roland
authors.initials: R
authors.lastname: Kaufmann
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000378:metabolism*; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D055166:Hepatic Stellate Cells / Q000166:cytology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D044464:Receptor, PAR-2 / Q000235:genetics* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D043925:Angiogenesis Inducing Agents; C000621508:F2RL1 protein, human; D044464:Receptor, PAR-2; D043562:Receptors, G-Protein-Coupled
keywords: Angiogenesis; Cell migration; HCC; HCC xenograft; HSCs; Hepatic stellate cells; Hepatocellular carcinoma; LX-2; Met; PAR2; Proteinase-activated receptor 2; Receptor tyrosine kinase; Src