Abstract
Extensive research efforts over the years have provided us with great insights into how bone-resorbing osteoclasts (OCs) develop and function and, based on such work, valuable antiresorptive therapies have been developed to help combat the excessive bone loss that occurs in numerous skeletal disorders. The RAW 264.7 murine cell line has proven to be an important tool for in vitro studies of OC formation and function, having particular advantages over the use of OCs generated from primary bone marrow cell populations or directly isolated from murine bones. These include their ready access and availability, simple culture for this pure macrophage/pre-OC population, sensitive and rapid development into highly bone-resorptive OCs expressing hallmark OC characteristics following their RANKL stimulation, abundance of RAW cell-derived OCs that can be generated to provide large amounts of study material, relative ease of transfection for genetic and regulatory manipulation, and close correlation in characteristics, gene expression, signaling, and developmental or functional processes between RAW cell-derived OCs and OCs either directly isolated from murine bones or formed in vitro from primary bone marrow precursor cells. Here, we describe methods for the culture and RANKL-mediated differentiation of RAW cells into bone-resorptive OCs as well as procedures for their enrichment, characterization, and general use in diverse analytical assays.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Roodman, G. (1996) Advances in bone biology – the osteoclast. Endocrine Rev. 17, 308–332.
Mancini, L., Moradi-Bidhendi, N., Brandi, M., Perretti, M., and McIntyre, I. (2000) Modulation of the effects of osteoprotegerin (OPG) ligand in a human leukemic cell line by OPG and calcitonin. Biochem. Biophys. Res. Commun. 279, 391–397.
Nagai, M., Kyakumoto, S., and Sato, N. (2000) Cancer cells responsible for humoral hypercalcemia express mRNA encoding a secreted form of ODF/TRANCE that induces osteoclast formation. Biochem. Biophys. Res. Commun. 269, 532–536.
Hentunen, T., Reddy, S., Boyce B. et al.,(1998) Immortalization of osteoclast precursors by targeting Bcl-XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice. J. Clin. Invest. 102, 88–97.
Chen, W., and Li Y. (1998) Generation of mouse osteoclastogenic cell lines immortalized with SV40 large T antigen. J. Bone. Miner. Res. 13, 1112–1123.
Takeshita, S., Kaji, K., and Kudo, A. (2000) Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. J. Bone Miner. Res. 15, 1477–1488.
Takahashi, N., Udagawa, N., and Suda, T. (1999) A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem. Biophys. Res. Commun. 256, 449–455.
Chambers, T. (2000) Regulation of the differentiation and function of osteoclasts. J. Pathol. 192, 4–13.
Schoppet, M., Preissner, K., and Hofbauer, L. (2002) RANK ligand and osteoprotegerin. Paracrine regulators of bone metabolism and vascular function. Arterioscler. Thromb. Vasc. Biol. 22, 549–553.
Hsu, H., Lacey, D., Dunstan, C., et al (1999) Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. USA 96, 3540–3545.
Yamamoto, A., Miyazaki, T., Kadono, Y., et al (2002) Possible involvement of IkappaB kinase 2 and MKK7 in osteoclastogenesis induced by receptor activator of nuclear factor kappaB ligand. J. Bone Miner. Res. 17, 612–621.
Mizukami, J., Takaesu, G., Akatsuka, H., et al (2002) Receptor activator of NF-kappaB ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6. Mol. Cell Biol. 22, 992–1000.
Raschke, W., Baird, S., Ralph, P., and Nakoinz, I. (1978) Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 15, 261–267.
Shadduck, R., Waheed, A., Mangan, K., and Rosenfeld, C. (1993) Preparation of a monoclonal antibody directed against the receptor for murine colony-stimulating factor-1. Exp. Hematol. 21, 515–520.
Sells-Galvin, R., Cullison, J., Avioli, L., and Osdoby, P. (1994) Influence of osteoclasts and osteoclast-like cells on osteoblast alkaline phosphatase activity and collagen synthesis. J. Bone Miner. Res. 9, 1167–1178.
Cappellen, D., Luong-Nguyen, N., Bongiovanni, S., Grenet, O., Wanke, C., and Susa, M. (2002) Transcriptional program of mouse osteoclast differentiation governed by the macrophage colony-stimulating factor and the ligand for the receptor activator of NF-kappa B. J. Biol. Chem. 277, 21971–21982.
Cassady, A., Luchin, A., Ostrowski, M., et al (2003) Regulation of the murine TRAP gene promoter. J. Bone Miner. Res. 18, 1901–1904.
Watanabe T, Kukita T, Kukita A, et al. (2004) Direct stimulation of osteoclastogenesis by MIP-1a: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL. J. Endocr. 180, 193–201.
Vincent, C., Kogawa, M., Findlay, D., et al (2009) The generation of osteoclasts from RAW 264.7 precursors in defined, serum-free conditions. J. Bone Miner. Metab. 27, 114–119.
Koseki, T., Gao, Y., Okahashi, N., et al (2002) Role of TGF-beta family in osteoclastogenesis induced by RANKL. Cell Signal 14, 31–36.
Shin, J., Kim, I., Lee, J., Koh, G., Lee, Z., and Kim, H. (2002) A novel zinc finger protein that inhibits osteoclastogenesis and the function of tumor necrosis factor receptor-associated factor 6. J. Biol. Chem. 277, 8346–8353.
Trebec-Reynolds, D., Voronov, I., Heersche, J., et al (2010) IL-1alpha and IL-1beta have different effects on formation and activity of large osteoclasts. J. Cell Biochem. 109, 975–982.
Acknowledgments
We are greatly indebted to the Drs. Xuefeng Yu and Hong Zheng for their advice and many valuable contributions to an earlier version of this chapter. This work was supported by NIH Grants AR32927, AG15435, and AR32087 to P.O.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Collin-Osdoby, P., Osdoby, P. (2012). RANKL-Mediated Osteoclast Formation from Murine RAW 264.7 cells. In: Helfrich, M., Ralston, S. (eds) Bone Research Protocols. Methods in Molecular Biology, vol 816. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-415-5_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-415-5_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-414-8
Online ISBN: 978-1-61779-415-5
eBook Packages: Springer Protocols