Liver myofibroblasts activate protein C and respond to activated protein C.
OBJECTIVE
To study the protein C activation system in human liver myofibroblasts, and the effects of activated protein C (APC) on these cells.


METHODS
Human liver myofibroblasts were obtained by outgrowth. Expression of protease activated receptor 1 (PAR-1), endothelial protein C receptor (EPCR) and thrombomodulin (TM) was analyzed by flow cytometry. Extracellular signal-regulated kinase (ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies. Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction (RT-PCR). Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate.


RESULTS
Primary cultures of human liver myofibroblasts expressed EPCR on their surface, together with PAR-1 and TM. This receptor system was functional since exposure of myofibroblasts to APC induced ERK1/2 phosphorylation in a dose- and time-dependent manner. Furthermore, APC significantly upregulated the expression of collagen mRNA, as shown by real-time RT-PCR. Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059. Finally, using a cell-based colorimetric assay, we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin.


CONCLUSIONS
These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.
Gillibert-Duplantier
Jennifer
J
INSERM U889, Victor Segalen Bordeaux 2 University, F-33076 Bordeaux, France.
Rullier
Anne
A
Neaud
Véronique
V
Kisiel
Walter
W
Rosenbaum
Jean
J
D015703:Antigens, CD / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D000074283:Endothelial Protein C Receptor; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D011486:Protein C / Q000378:metabolism* / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D018180:Thrombomodulin / Q000378:metabolism
D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; C478370:PROCR protein, human; D011486:Protein C; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; D018180:Thrombomodulin; D003094:Collagen