Osteoprotegerin exposure at different stages of osteoclastogenesis differentially affects osteoclast formation and function

This study aimed to investigate the effects of osteoprotegerin (OPG), a decoy receptor for receptor activator for nuclear factor kappa B ligand (RANKL), during the various stages of osteoclast differentiation, and additionally investigate its effects on osteoclast adhesion and activity. RAW264.7 murine monocytic cells were incubated with macrophage colony-stimulating factor and RANKL for 1, 3, 5, or 7 days, followed by an additional 24-h incubation in the presence or absence of OPG (80 ng/mL). We examined osteoclast differentiation and adhesion capacity using the tartrate-resistant acid phosphatase (TRAP) assay and immunofluorescence microscopy, and additionally examined cell growth in real time using the xCELLigence system. Furthermore, the expression levels of TRAP, RANK, integrin beta 3, matrix metalloproteinase 9, cathepsin K, carbonic anhydrase II, and vesicular-type H+-ATPase A1 were examined using western blotting. OPG exposure on day 1 enhanced the osteoclast growth curve as well as adhesion, and increased RANK and integrin beta 3 expression. In contrast, exposure to OPG at later time points (days 3-7) inhibited osteoclast differentiation, adhesion structure formation, and protease expression. In conclusion, the biological effects of OPG exposure at the various stages of osteoclast differentiation were varied, and included the enhanced adhesion and survival of preosteoclasts, the block of differentiation from the early to the terminal stages of osteoclastogenesis, and suppression of mature osteoclast activation following OPG exposure during the terminal differentiation stage, suggesting that the effects of OPG exposure differ based on the stage of differentiation.