c-Met responsiveness of isolated hepatocytes evaluated in an in vitro de-differentiation model

Int J Mol Med. 2006 Mar;17(3):475-82.

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.

MeSH terms

  • Animals
  • Asialoglycoprotein Receptor / metabolism
  • Cell Differentiation*
  • Cell Shape
  • Cells, Cultured*
  • Collagen
  • Culture Media, Serum-Free
  • Dose-Response Relationship, Drug
  • Hepatocyte Growth Factor / pharmacology
  • Hepatocytes / cytology*
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism*
  • Liver Cirrhosis, Experimental
  • Male
  • Models, Biological
  • Proto-Oncogene Proteins c-met / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Taurochenodeoxycholic Acid / pharmacology
  • Up-Regulation / genetics
  • Ursodeoxycholic Acid / pharmacology

Substances

  • Asialoglycoprotein Receptor
  • Culture Media, Serum-Free
  • Taurochenodeoxycholic Acid
  • ursodoxicoltaurine
  • Hepatocyte Growth Factor
  • Ursodeoxycholic Acid
  • Collagen
  • Proto-Oncogene Proteins c-met