RANKL-induced DC-STAMP is essential for osteoclastogenesis

Abstract

Osteoclasts are bone-resorbing, multinucleated giant cells that are essential for bone remodeling and are formed through cell fusion of mononuclear precursor cells. Although receptor activator of nuclear factor-kappaB ligand (RANKL) has been demonstrated to be an important osteoclastogenic cytokine, the cell surface molecules involved in osteoclastogenesis are mostly unknown. Here, we report that the seven-transmembrane receptor-like molecule, dendritic cell-specific transmembrane protein (DC-STAMP) is involved in osteoclastogenesis. Expression of DC-STAMP is rapidly induced in osteoclast precursor cells by RANKL and other osteoclastogenic stimulations. Targeted inhibition of DC-STAMP by small interfering RNAs and specific antibody markedly suppressed the formation of multinucleated osteoclast-like cells. Overexpression of DC-STAMP enhanced osteoclastogenesis in the presence of RANKL. Furthermore, DC-STAMP directly induced the expression of the osteoclast marker tartrate-resistant acid phosphatase. These data demonstrate for the first time that DC-STAMP has an essential role in osteoclastogenesis.

Figure 1.

Induction of DC-STAMP and its splicing variant DC-STAMPΔT7 in osteoclastogenesis. (A) Structures of DC-STAMP and DC-STAMPΔT7. Noncoding and coding sequences are shown in unshaded and shaded boxes, respectively. PCR primers used in C and D are indicated by arrows. Schematic protein structures are shown below the gene. Shaded regions indicate the putative transmembrane domains. (B) Northern blot analysis for induction of DC-STAMP and DC-STAMPΔT7 in RAW-D cells. RAW-D and RAW-N cells were stimulated for 72 h as indicated. c, control; N, RAW-N; D, RAW-D. (C) RT-PCR analysis on the time course of DC-STAMP and DC-STAMPΔT7 induction in RAW-D cells. RAW-D cells were stimulated with RANKL + TNF-α for indicated periods of time. Expression of DC-STAMP, DC-STAMPΔT7, and osteoclast marker genes (cathepsin K and TRAP) was analyzed. (D) RT-PCR analysis on the time course of DC-STAMP and DC-STAMPΔT7 induction in mouse BM cells. Mouse BM cells were stimulated with 1α,25(OH)₂D₃. Expression of DC-STAMP and DC-STAMPΔT7 was analyzed. The GenBank/EMBL/DDBJ accession nos. of mouse DC-STAMP and DC-STAMPΔT7 are AB109560 and AB109561, respectively.

Figure 2.

Immunological staining of DC-STAMP in osteoclasts. Cells and tissues were stained with anti–DC-STAMP (left) or control preimmune IgG (right). (A) RAW-D cells stimulated with RANKL + TNF-α for 3 d. Nuclei were visualized by staining with hematoxylin. DC-STAMP positive osteoclast-like MNCs and mononuclear cells are indicated by arrows and arrowheads, respectively. (B) Osteoclasts present in the mandibular tissue of newborn mice. (C) Osteoclasts isolated from the tibia of newborn mice. OC, osteoclast. Bars: 25 μm (A), 10 μm (B), and 20 μm (C).

Figure 3.

Inhibition of osteoclastogenesis by DC-STAMP siRNAs and by anti–DC-STAMP. (A) Effects of the siRNAs on the formation of osteoclast-like TRAP positive MNCs in RAW-D cells stimulated with RANKL and TNF-α for 3 d. Specific reduction of DC-STAMP mRNA and DC-STAMPΔT7mRNA by #6 siRNA was evaluated by RT-PCR (right). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Inhibition of osteoclast-like TRAP positive MNC formation in RAW-D cells by anti–DC-STAMP. RAW-D cells were treated with RANKL and TNF-α for 3 d without or with indicated concentrations of anti–DC-STAMP. **, P < 0.01; ***, P < 0.001. IgG, control IgG (20 μg/ml). (C) Inhibition of osteoclastogenesis in mouse BM cells by anti–DC-STAMP. BM cells were cultured in the presence of 1α,25(OH)₂D₃ for 6 d without or with indicated concentrations of anti–DC-STAMP. *, P < 0.05; **, P < 0.01. (D) Anti–DC-STAMP inhibits osteoclast function. Rat osteoclast-like cells formed in BM cultures were seeded on dentin slices and cultured for 3 d in the presence of 10 μg/ml of control IgG or anti–DC-STAMP. *, P < 0.05.

Figure 4.

Promotion of osteoclastogenesis in RAW-D cells by DC-STAMP and DC-STAMPΔT7. (A) Transfection of RAW-D cells with control, DC-STAMP, or DC-STAMPΔT7 expression vectors. Cells were treated without or with RANKL + TNF-α for 3 d. Osteoclast-like TRAP positive MNCs were counted. **, P < 0.01; ***, P < 0.001. (B) Induction of TRAP in RAW-D cells by coculture with mouse pre–B L1.2 cells stably expressing DC-STAMP or DC-STAMPΔT7. RAW-D cells were cocultured with live (left) or fixed (right) L1.2 cells or L1.2 cells stably expressing DC-STAMP or DC-STAMPΔT7 in the absence of osteoclastogenic factors for 3 d. Osteoclast-like TRAP positive MNCs were counted. **, P < 0.01.