RANKL induces heterogeneous DC-STAMP(lo) and DC-STAMP(hi) osteoclast precursors of which the DC-STAMP(lo) precursors are the master fusogens.
Osteoclasts (OC) are multinucleated bone resorbing cells that form via RANKL-induced fusion of heterogeneous mononuclear OC precursors (OCP). Currently, there are no unique surface markers to distinguish these OCP populations, which are diagnostic for erosive and metabolic bone diseases using culture assays. Thus, we investigated expression of DC-STAMP, a surface receptor required for OCP fusion, during osteoclastogenesis in vitro using a novel monoclonal antibody (1A2). Immunoprecipitation-Western blot analysis of OCP membrane proteins detected 106 kDa dimeric and 53 kDa monomeric DC-STAMP in non-denaturing and denaturing conditions, respectively, with greater sensitivity versus rabbit anti-sera (KR104). 1A2 also detected 99.9% of undifferentiated monocytes as a single population by flow cytometry with a MFI 100-fold over background, while KR104 was not useful in this assay. Functionally, 1A2 inhibited OCP fusion in vitro. RANKL stimulation of OCP induced DC-STAMP(lo) and DC-STAMP(hi) cells, which mature into OC and mononuclear cells respectively as determined by fluorescent microscopy and TRAP assays. Addition of DC-STAMP(hi) cells to purified DC-STAMP(lo) cultures produced larger, more nucleated OC vs. pure DC-STAMP(lo) cultures. RT-qPCR analysis of these two populations showed that OC markers (Trap and Oc-stamp) and fusogenic gene expression (Cd9 and Cd47), were significantly increased in DC-STAMP(lo) vs. DC-STAMP(hi) cells. Collectively, these results demonstrate that DC-STAMP is expressed on OCP as a dimer, which is efficiently detected by 1A2 via flow cytometry. RANKL induces osteoclastogenesis by stimulating DC-STAMP internalization in some OCP, and these DC-STAMP(lo) cells display the "master fusogen" phenotype. In contrast, DC-STAMP(hi) OCP can only act as mononuclear donors.
Mensah
Kofi A
KA
Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York 14642, USA.
Ritchlin
Christopher T
CT
Schwarz
Edward M
EM
D000135:Acid Phosphatase / Q000235:genetics; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism*; D051928:CD47 Antigen / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D002459:Cell Fusion; D002478:Cells, Cultured; D005434:Flow Cytometry; D007160:Immunoproliferative Disorders; D007527:Isoenzymes / Q000235:genetics; D008264:Macrophages / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism*; D010641:Phenotype; D055503:Protein Multimerization; D053245:RANK Ligand / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000378:metabolism*; D000071681:Tartrate-Resistant Acid Phosphatase; D060245:Tetraspanin 29; D013997:Time Factors
D015703:Antigens, CD; D015415:Biomarkers; D051928:CD47 Antigen; C497895:Cd47 protein, mouse; C554870:Cd9 protein, mouse; C499636:DC-STAMP protein, mouse; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C527519:OC-STAMP protein, mouse; D053245:RANK Ligand; D012333:RNA, Messenger; D060245:Tetraspanin 29; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase