Activated protein C suppresses osteoclast differentiation via endothelial protein C receptor, protease-activated receptor-1, sphingosine 1-phosphate receptor, and apolipoprotein E receptor 2.

INTRODUCTION

Bone remodeling relies on a delicate balance between formation and resorption of bone tissues, processes in which bone-forming osteoblasts and bone-resorbing osteoclasts play central roles. Recently, we reported that anticoagulant activated protein C (APC) promotes osteoblast proliferation, but the role of the blood coagulation system in bone remodeling remains unclear. In this study, to further elucidate the relationship between bone remodeling and blood coagulation, we investigated the effect of APC on osteoclast differentiation.

MATERIALS AND METHODS

Normal human osteoclast precursor cells were cultured in their growth medium including soluble RANKL, M-CSF, and FBS, and on days 4 and 7, the culture medium was replaced with the same medium containing various concentrations of APC, protein C (PC), sphingosine 1-phosphate (S1P) receptor agonist, FTY720, or APC+various substances without FBS. On day 8, TRAP-positive multinucleated cells (≥3 nuclei) were counted manually using a light microscope. The effects of APC on NF-κB and NFATc1 activation were evaluated using specific ELISA.

RESULTS

APC suppressed RANKL-induced osteoclast differentiation, and this APC-induced suppression of osteoclast differentiation was inhibited by zymogen protein C and aprotinin, a serine protease inhibitor. Immunohistochemistry and RT-PCR analyses suggested that endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) were expressed in osteoclast precursor cells and osteoclasts. Both anti-PAR-1 antibody and anti-EPCR antibody (RCR-252), which blocks APC binding to EPCR, inhibited the APC-induced suppression of osteoclast differentiation. FTY720 had no effect on osteoclast differentiation. However, FTY 720 and S1P receptor antagonist, VP 23019, inhibited the APC-induced suppression of osteoclast differentiation. On the other hand, recombinant soluble human ApoER2 and anti-human ApoER2 inhibited the APC-induced suppression of osteoclast differentiation. Further, APC had no effect on NF-κB and NFATc1 activation.

CONCLUSIONS

APC suppresses human osteoclast differentiation mainly by inhibiting the formation of multinucleated cells via EPCR, PAR-1, S1P receptor, and ApoER2 in a manner that depends on APC protease activity.