title: Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis.
abstract: BACKGROUND
  In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.
  
  
  METHODS
  Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.
  
  
  RESULTS
  Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated.
  
  
  CONCLUSIONS
  This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.
authors.affiliation: Department of Internal Medicine I, University of Bonn Bonn, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Görtzen
authors.affiliation: Department of Internal Medicine I, University of Bonn Bonn, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Schierwagen
authors.affiliation: Department of General, Visceral, Thoracic, and Vascular Surgery, University of Bonn Bonn, Germany.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Bierwolf
authors.affiliation: Department of Internal Medicine I, University of Bonn Bonn, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Klein
authors.affiliation: Department of Internal Medicine I, University of Bonn Bonn, Germany.
authors.forename: Frank E
authors.initials: FE
authors.lastname: Uschner
authors.affiliation: Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn Bonn, Germany.
authors.forename: Peter F
authors.initials: PF
authors.lastname: van der Ven
authors.affiliation: Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn Bonn, Germany.
authors.forename: Dieter O
authors.initials: DO
authors.lastname: Fürst
authors.affiliation: Department of Internal Medicine I, University of Bonn Bonn, Germany.
authors.forename: Christian P
authors.initials: CP
authors.lastname: Strassburg
authors.affiliation: Department of Internal Medicine, University Hospital Gasthuisberg Leuven, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Laleman
authors.affiliation: Department of General, Visceral, Thoracic, and Vascular Surgery, University of Bonn Bonn, Germany.
authors.forename: Jörg-Matthias
authors.initials: JM
authors.lastname: Pollok
authors.affiliation: Department of Internal Medicine I, University of Bonn Bonn, Germany ; Faculty of Health Sciences, University of Southern Denmark Odense, Denmark.
authors.forename: Jonel
authors.initials: J
authors.lastname: Trebicka
keywords: PP2; RhoA; SRC; hepatic stellate cells; liver fibrosis; matrix stiffness