title: c-Met responsiveness of isolated hepatocytes evaluated in an in vitro de-differentiation model.
abstract: Hepatocyte growth factor (HGF) accelerates tissue regeneration and ameliorates tissue fibrosis through its ligand c-Met receptor tyrosine kinase. Hence, HGF is currently discussed as an attractive therapeutic candidate for fatal liver diseases. However, it remains unclear whether c-Met of de-differentiated hepatocytes adequately responds to HGF in an impaired liver. Therefore, we investigated c-Met expression and c-Met responsiveness to HGF in an experimental de-differentiation cell culture system. Primary rat hepatocytes were seeded on a two-dimensional collagen matrix or embedded within a three dimensional collagen gel to guarantee intact cell geometry. Cells were cultivated in a growth factor enriched extracellular milieu (de-differentiation medium), or in a chemically defined differentiation medium, representing physiologically intact hepatocytes. c-Met surface expression was determined by flow cytometry. Receptor localisation was examined by confocal microscopy, c-Met and phosphorylated c-Met protein were determined by western blotting. Hepatocyte-specific asialoglycoprotein receptor (ASGPr) was examined to control the differentiation status of the cells. Growth factor enriched milieu induced a rapid loss of ASGPr with a significant increase of c-Met surface level and a decrease in c-Met protein level. Surprisingly, the increased amount of c-Met surface expression was associated with its loss of responsiveness to HGF. The addition of bile acids into the culture medium had significantly delayed the process of de-differentiation and restrained the drastic elevation of c-Met (tauroursodeoxycholic acid > ursodeoxycholic acid). Application of the three-dimensional hepatocellular architecture stabilized the c-Met surface receptor level and rendered c-Met activation. We have demonstrated that growth factor enriched extracellular milieu and loss of intact liver architecture seems to be accompanied by an up-regulation of c-Met surface level. Our findings suggest that irresponsiveness of c-Met to soluble HGF was possibly caused by an excessive HGF production and receptor over-stimulation. Both events should be considered when establishing an HGF-based therapy for fibrosis/cirrhosis.
authors.affiliation: Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Zentrum der Chirurgie, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Boost
authors.forename: He-Jin
authors.initials: HJ
authors.lastname: Kim
authors.forename: Tobias
authors.initials: T
authors.lastname: Engl
authors.forename: Elsie
authors.initials: E
authors.lastname: Oppermann
authors.forename: Dietger
authors.initials: D
authors.lastname: Jonas
authors.forename: Anton
authors.initials: A
authors.lastname: Oertl
authors.forename: Roman A
authors.initials: RA
authors.lastname: Blaheta
mesh_terms: D000818:Animals; D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D002454:Cell Differentiation; D048430:Cell Shape; D002478:Cells, Cultured; D003094:Collagen; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008106:Liver Cirrhosis, Experimental; D008297:Male; D008954:Models, Biological; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013655:Taurochenodeoxycholic Acid / Q000494:pharmacology; D015854:Up-Regulation / Q000235:genetics; D014580:Ursodeoxycholic Acid / Q000494:pharmacology
chemical_list: D037263:Asialoglycoprotein Receptor; D016895:Culture Media, Serum-Free; D013655:Taurochenodeoxycholic Acid; C031655:ursodoxicoltaurine; D017228:Hepatocyte Growth Factor; D014580:Ursodeoxycholic Acid; D003094:Collagen; D019859:Proto-Oncogene Proteins c-met