RANKL induces heterogeneous DC-STAMP(lo) and DC-STAMP(hi) osteoclast precursors of which the DC-STAMP(lo) precursors are the master fusogens

Abstract

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.

Keywords: Cell Fusion; Dendritic Cell-Specific Transmembrane Protein (DC-STAMP); Osteoclast Precursors (OCP).

Fig.1

1A2 is a unique anti-DC-STAMP mAb that recognizes the dimeric receptor on OCP and inhibits OC formation. (A) IP-western blotting for DC-STAMP was performed on total protein extracts from RAW 264.7 cells after 2 days of RANKL treatment using 1A2 mAb or commercially available polyclonal anti-sera (KR104) as both the capture and primary antibody, under non-reducing (nr) and reducing (r) conditions. A non-specific mouse IgG mAb was run as a negative control with molecular weight markers (M). The blot is representative of 4 independent experiments. (B) Representative flow cytometric histogram showing surface DC-STAMP expression on unstimulated RAW 264.7 cells. Solid outlined histogram indicates unstained control, while the shaded grey histograms represent the DC-STAMP level measured by either the FITC conjugated 1A2 (left), or KR104 with FITC conjugated anti-rabbit IgG (right) antibodies. For the KR104 antibody, the dotted outlined histogram indicates the background signal attributed to the goat anti-rabbit secondary antibody. Numbers indicate percentage of cells in the indicated region and the mean fluorescence intensity for DC-STAMP of cells in that region. (C) RAW cells were cultured with 100ng/mg of RANKL in the presence of 0, 5, 10, or 20 μg of 1A2 or irrelevant IgG control antibody for 4 days (n=4). Afterwards the cultures were fixed and stained for TRAP to quantify OC numbers. Data are presented as mean +/− SEM (*p<0.05). (D) Primary murine bone marrow monocytes were cultured with 50nl/ml RANKL and 10ng/ml of M-CSF +/− 20 μg of 1A2 for 5 days (n=4). Afterwards the cultures were fixed and stained for TRAP to quantify OC numbers. Data are presented as mean +/− SEM (*p<0.05).

Fig. 2

RANKL stimulation of homogeneous OCP induces a heterogeneous population of DC-STAMP^hi and DC-STAMP^lo cell assessed by 1A2 surface staining. Representative (n=4) flow cytometric histograms showing surface DC-STAMP expression via FITC-1A2 staining of M-CSF enriched CD11b+ adherent bone marrow cells cultured with 100 ng/mL RANKL for 1 (A), 2 (B), or 3 (C) days. Dotted line indicates DC-STAMP level on cells cultured without RANKL, while shaded grey histograms represent DC-STAMP level on RANKL treated cells for the indicated time. Numbers indicate percentage of cells in the indicated region (99% of untreated control). RAW cells were cultured with 100 ng/mL RANKL for 1 (D), 2 (E), or 3 (F) days, and then permeablized prior to FITC-1A2 staining and flow cytometry with selective gating to assess cytoplasmic fluorescence. Representative (n=4) histograms showing the percentage of cells with positive staining for intracellular DC-STAMP. (G) Representative flow cytometric histogram of surface DC-STAMP on M-CSF enriched CD11b+ adherent bone marrow cells cultured with 10 ng/mL RANKL for 3 days.

Fig. 3

RANKL induces translocation and down-regulation of DC-STAM during osteoclastogeneis. (A) RAW cells were cultured with RANKL for 2 days, fixed and stained with PE-conjugated 1A2 (red DC-STAMP), FITC-phalloidin (green actin ring inside the plasma membrane) and DAPI (blue nucleus). The high power 60X fluorescent micrograph highlights the internal DC-STAMP. (B) RAW cells were cultured with RANKL for 3 days to generate heterogeneous populations of mononuclear OCP and large multinucleated OC, and were stained with PE-conjugated 1A2 and FITC-phalloidin, and fluorescent micrographs were taken 40X magnification. Note the bright DC-STAMP signal (red) in mononuclear OCP vs. the dim signal in the large OC. (C) RAW cells were culture with RANKL for 3 days, and surface DC-STAMP^hi and DC-STAMP^lo cells were separated by FACS, permeablized, restained with 1A2 and analyzed by flow cytometry. A representative (n=4) histogram of the mean fluorescence intensity (MFI) levels of intracellular DC-STAMP in the two populations is shown.

Fig. 4

RANKL-induced DC-STAMP^lo cells are necessary for the formation of large TRAP+ multinucleated cells. (A.) RANKL-induced DC-STAMP^lo and DC-STAMP^hi RAW 264.7 cells were sorted based on surface DC-STAMP expression as indicated. The sorted cells were recultured with RANKL for 3 more days either as homogeneous DC-STAMP^lo (B.) or DC-STAMP^hi (C.) populations, or mixed DC-STAMP^lo-to-DC-STAMP^hi population ratios of 10:1 (D.), 1:1 (E.), or 1:10 (F.). Representative photographs are shown of the TRAP-stained cultures to demonstrate the relative osteoclastogenic potential of the different culture conditions. Numbers indicate the average area of the TRAP+ multinucleated cells and are reported in mm².

Fig. 5

RANKL-induced DC-STAMP^lo OCP express higher levels of OC markers and fusagenic genes vs. DC-STAMP^hi OCP. (A.) Relative mRNA fold change for OC marker genes or fusion-related genes in RANKL-induced DC-STAMP^lo (black bars) and DC-STAMP^hi (white bars) cells relative to levels in unsorted cells treated with RANKL. Graphs are representative of experiments done in triplicate and data are normalized to β-actin. * P < 0.05 vs. DC-STAMP^lo gene expression levels.