| Abstract |
Osteoclast fusion is an essential step in the acquisition of bone-resorbing activity; however, the molecular mechanisms underlying this process remain incompletely understood. In this study, we developed a novel quantitative monitoring system for osteoclast fusion using NanoBiT, a split-luciferase technology. This system enabled sensitive detection of receptor activator of NF-κB ligand (RANKL)-dependent osteoclast fusion in RAW264 cells and bone marrow-derived macrophages, as well as interleukin-4 (IL-4)-induced foreign body giant cell fusion. The time-course analysis of luciferase activity during osteoclast differentiation was closely correlated with osteoclast fusion observed by conventional microscopic analysis. In addition, luciferase activity was inhibited by cyclosporin A in a dose-dependent manner and completely abolished by Dcstamp knockout, demonstrating its validity for chemical screening and for evaluating genes involved in osteoclast fusion. Using this platform, we screened approximately 2300 known bioactive compounds and identified THZ-P1-2, a phosphatidylinositol-5-phosphate 4-kinase type 2 inhibitor, and Brequinar, a dihydroorotate dehydrogenase inhibitor, as novel modulators of osteoclast fusion. Compared with fluorescence-based assays, this luminescence-based system minimizes false-positive signals caused by cellular or compound autofluorescence and provides superior sensitivity and quantifiability. Furthermore, the assay is rapid, simple, and highly compatible with high-throughput screening, making it a useful platform for studying osteoclast fusion and for screening compounds that modulate this process.
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