Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Modeling Osteoclast Precursor Master Fusogens and Mononuclear OCP Donors with Raw Cell Line Clones.
Ju, Yawen, Takahata, Masahiko, Mensah, Kofi, Chiu, Grace, T. Ritchlin, Christopher, Xing, Lianping, M. Schwarz, Edward
Osteoclasts (OC) are multinucleated bone-resorbing cells derived from mononuclear osteoclast precursors (OCP) following stimulation with RANKL and M-CSF. Raw264.7 cells are a heterogeneous murine monocyte/macrophage cell line, of which some can form OCs and others cannot. DC-STAMP is a receptor on OCP, and is required for cell fusion to form multinucleated OC. Recently we demonstrated that the surface expression of DC-STAMP by RANK+ OCPs defines OCP fusion potential. RANKL induces DC-STAMPlo OCPs to function as master fusogens, and DC-STAMPhi OCPs to serve as mononuclear donors that cannot fuse on their own to form mature OCs. In order to investigate the molecular mechanisms by which DC-STAMP contributes to OCP fusion and OC formation, we aimed to isolate and characterize Raw cell clones with OCP master fusogen vs. mononuclear donor phenotypes based on their DC-STAMP expression before and after RANKL stimulation.
Raw 264.7 cells were cloned by limiting dilution: diluted 50 cells in 10ml alpha-MEM media, seeded 100ul/well in 96 well-plate and selected single cell clones. The clones were treated with 100ng/ml RANKL, and their osteoclastogenic potential was assess by TRAP staining. The surface expression of DC-STAMP was determined by flow cytometry following 7AAD staining to gate the live cells, and FITC-conjugated 1A2 monoclonal anti-DC-STAMP antibody.
Seven Raw cell clones were obtained and their OC formation and DC-STAMP surface expression were examined. Clone 5 formed large multinucleated TRAP+ OCs three days after RANKL treatment, while it took five days for the parental raw cell line to form OCs under a same culture condition. Clone 5 also expressed a low basal level of surface DC-STAMP (MFI= 32.0), which was similar to DC-STAMPlo (MFI=16.5) identified in the parental cells following three days of RANKL treatment. In contrast, the Clone 1 could not form OCs after five days of RANKL treatment. Clone 1 also had a high basal level of surface DC-STAMP expression (MFI= 60.8), which was similar to RANKL-induced DC-STAMPhi (MFI=55.4) of the parental cells.
Two Raw 246.7 cell sub-clones were isolated to investigate DC-STAMP-mediated OCP fusion in response to RANKL treatment. Clone 5 forms OCs faster and has low basal DS-STAMP expression, while Clone 1 forms OCs much slower, and expresses high basal DC-STAMP. These clones mimic the heterogeneous DC-STAMP expression levels and OC forming potential of the parental Raw cells, and can be used as cell models of master fusogens (Clone 5) or mononuclear OCP donors (Clone 1). Our ongoing studies to clone DC-STAMP ligand using these clones via differential microarray analysis will be discussed.
To cite this abstract, please use the following information:
Ju, Yawen, Takahata, Masahiko, Mensah, Kofi, Chiu, Grace, T. Ritchlin, Christopher, Xing, Lianping, et al; Modeling Osteoclast Precursor Master Fusogens and Mononuclear OCP Donors with Raw Cell Line Clones. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1492