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 κB 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 β3, 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 β3 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.
KeywordsOsteoclast Osteoprotegerin Differentiation Adhesion Activation
This work was supported by grants from the National Natural Science Foundation of China (Grant Numbers 31172373, 31302154, 31372495), the Specialized Research Fund for the Doctoral Program of Higher Education (Grant Number 20113250110003), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Graduate Innovation Project of Jiangsu Province (Grant Number CXZZ12_0917).
- Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, Henriksen K, Lenhard T, Foged NT, Werb Z, Delaisse JM (2000) Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151:879–889CrossRefGoogle Scholar
- Fu YX, Gu JH, Zhang YR, Tong XS, Zhao HY, Yuan Y, Liu XZ, Bian JC, Liu ZP (2013b) Osteoprotegerin influences the bone resorption activity of osteoclasts. Int J Mol Med 31:1411–1417Google Scholar
- Okada Y, Naka K, Kawamura K, Matsumoto T, Nakanishi I, Fujimoto N, Sato H, Seiki M (1995) Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclasts: implications for bone resorption. Lab Invest 72:311–322Google Scholar
- Price CP, Kirwan A, Vader C (1995) Tartrate-resistant acid phosphatase as a marker of bone resorption. Clin Chem 41:641–643Google Scholar
- Shipman CM, Croucher PI (2003) Osteoprotegerin is a soluble decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand and can function as a paracrine survival factor for human myeloma cells. Cancer Res 63:912–916Google Scholar
- Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Boyle WJ (1997) Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309–319CrossRefGoogle Scholar
- Udagawa N, Takahashi N, Akatsu T, Tanaka H, Sasaki T, Nishihara T, Koga T, Martin TJ, Suda T (1990) Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci USA 87:7260–7264CrossRefGoogle Scholar