Method: Retroviral vector were constructed to over express dectin-1 in mouse monocyte RAW264.7 cells (d-RAW) and RAW264.7 cells infected with retroviral vector carrying Neo gene were used as a control cells (c-RAW). Cells were cultured in the presence or absence of RANKL and curdlan. In some experiments, mouse bone marrow cells were cultured with RANKL, macrophage-colony stimulating factor (M-CSF) and curdlan. Following tartrate resistant acid phosphatase (TRAP) staining, TRAP-positive multinucleated cells were counted. To estimate bone resorption activity, cells were cultured on Osteo Assay Stripwell PlateⓇ. Phalloidin staining in osteoclasts served to examine actin ring formation. The level of mRNA of osteoclast stimulatory transmembrane protein (OC-STAMP) was determined by real-time RT-PCR.
Result: Curdlan enhanced TRAP positive multinucleated cell formation induced by RANKL in a dose-dependent manner. In addition, we found that curdlan down-regulated pit formation and actin ring formation induced by RANKL, and that curdlan suppressed the expression levels of OC-STAMP stimulated with RANKL.
Conclusion: These results indicate that activation of dectin-1 signaling by curdlan regulates osteoclast differentiation and function.