Activated protein C stimulates osteoblast proliferation via endothelial protein C receptor.
BACKGROUND
Bone is continually remodeled by the action of osteoblasts, osteocytes, and osteoclasts. Resting osteoblasts are able to proliferate and differentiate into mature osteoblasts when physiologically required, as after tissue injury. Activated protein C (APC) is a serine protease that functions in anticoagulation, anti-inflammation, anti-apoptosis, cell proliferation, and wound repair. In this study, we examined the effect of APC on osteoblast proliferation and differentiation.


METHODS
We examined the presence of protein C in human fracture hematoma by immunohistochemical staining. We then evaluated the effect of APC, diisopropyl fluorophosphate-inactivated APC (DIP-APC) or protein C zymogen on normal human osteoblast (NHOst) proliferation using tetrazolium salt assay in the presence or absence of aprotinin, hirudin, protein C, antibody against protein C, endothelial protein C receptor (EPCR) or protease-activated receptor (PAR)-1. Finally, activation of p44/42 MAP kinase was evaluated by Western blot analysis.


RESULTS
Both APC and DIP-APC increased osteoblast proliferation in a dose-dependent manner, while protein C did not. The APC-induced increased proliferation of osteoblast was not affected by aprotinin, hirudin, and anti-protein C antibody which inhibits the protease activity of APC. Treatment with protein C or anti-EPCR antibody which inhibits APC binding to EPCR inhibited APC-mediated osteoblast proliferation, while treatment with anti-PAR-1 antibody did not. APC promoted the phosphorylation of p44/42 MAP kinase within osteoblasts; this effect was inhibited by the anti-EPCR antibody.


CONCLUSIONS
APC stimulates osteoblast proliferation by activating p44/42 MAP kinase through a mechanism that requires EPCR but not PAR-1 or the proteolytic activity of APC. APC generated at fracture sites may contribute to fracture healing by promoting osteoblast proliferation.
Kurata
Tatsuya
T
Department of Molecular Pathobiology, Mie University Graduate School of Medicine, Tsu-city, Mie 514-8507, Japan.
Hayashi
Tatsuya
T
Yoshikawa
Tomoaki
T
Okamoto
Takayuki
T
Yoshida
Kakunoshin
K
Iino
Takahiro
T
Uchida
Atsumasa
A
Suzuki
Koji
K
D015703:Antigens, CD; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D000074283:Endothelial Protein C Receptor; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D011486:Protein C / Q000378:metabolism* / Q000494:pharmacology*; D011956:Receptors, Cell Surface
D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; C478370:PROCR protein, human; D011486:Protein C; D011956:Receptors, Cell Surface