Uptake of denatured collagen into hepatic stellate cells: evidence for the involvement of urokinase plasminogen activator receptor-associated protein/Endo180

Biochem J. 2005 Apr 1;387(Pt 1):39-46. doi: 10.1042/BJ20040966.

Abstract

Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase-PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / pharmacology
  • Calcium / metabolism
  • Cattle
  • Collagen / metabolism*
  • Endocytosis / drug effects
  • Endocytosis / physiology
  • Eptifibatide
  • Hepatocytes / metabolism*
  • Hydrogen-Ion Concentration
  • Iodine Radioisotopes / metabolism
  • Ligands
  • Liver / cytology
  • Liver / metabolism
  • Mannose-Binding Lectins / metabolism*
  • Membrane Glycoproteins / metabolism*
  • Ovalbumin / metabolism
  • Peptide Hydrolases / metabolism
  • Peptides / immunology
  • Protein Binding / drug effects
  • Protein Binding / physiology
  • Protein Denaturation
  • Proton Pump Inhibitors
  • Rats
  • Rats, Wistar
  • Receptors, Cell Surface / metabolism*
  • Receptors, Mitogen / metabolism*
  • Urokinase-Type Plasminogen Activator / pharmacology

Substances

  • Antibodies
  • Endo180
  • Iodine Radioisotopes
  • Ligands
  • MRC2 protein, human
  • Mannose-Binding Lectins
  • Membrane Glycoproteins
  • Peptides
  • Proton Pump Inhibitors
  • Receptors, Cell Surface
  • Receptors, Mitogen
  • Ovalbumin
  • Collagen
  • Peptide Hydrolases
  • Urokinase-Type Plasminogen Activator
  • Eptifibatide
  • Calcium