Osteoclast Differentiation In Cultures Research Articles

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D3 and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

The immunomodulatory adapter proteins DAP12 and Fc receptor gamma-chain (FcRgamma) regulate development of functional osteoclasts through the Syk tyrosine kinase.

Osteoclasts, the only bone-resorbing cells, are central to the pathogenesis of osteoporosis, yet their development and regulation are incompletely understood. Multiple receptors of the immune system use a common signaling paradigm whereby phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within receptor-associated adapter proteins recruit the Syk tyrosine kinase. Here we demonstrate that a similar mechanism is required for development of functional osteoclasts. Mice lacking two ITAM-bearing adapters, DAP12 and the Fc receptor gamma-chain (FcRgamma), are severely osteopetrotic. DAP12(-/-)FcRgamma(-/-) bone marrow cells fail to differentiate into multinucleated osteoclasts or resorb bone in vitro and show impaired phosphorylation of the Syk tyrosine kinase. syk(-/-) progenitors are similarly defective in osteoclast development and bone resorption. Intact SH2-domains of Syk, introduced by retroviral transduction, are required for functional reconstitution of syk(-/-) osteoclasts, whereas intact ITAM-domains on DAP12 are required for reconstitution of DAP12(-/-) FcRgamma(-/-) cells. These data indicate that recruitment of Syk to phosphorylated ITAMs is critical for osteoclastogenesis. Although DAP12 appears to be primarily responsible for osteoclast differentiation in cultures directly stimulated with macrophage-colony stimulating factor and receptor activator of NF-kappaB ligand cytokines, DAP12 and FcRgamma have overlapping roles in supporting osteoclast development in osteoblast-osteoclast cocultures, which mirrors their overlapping functions in vivo. These results provide new insight into the biology of osteoclasts and suggest novel therapeutic targets in diseases of bony remodeling.