Development of High-throughput Screening System for Identifying Osteoclastogenesis-Related Compounds

Small compounds that potently affect osteoclastogenesis could form the basis for effective therapeutic strategies against alveolar bone resorption. Objectives: To establish a monitoring system for identifying osteoclastogenesis-related compounds using a cell-based sensor. Methods: Murine osteoclast precursor cells (RAW264.7) were transfected with the NFAT response element/luciferase reporter vector (pGL4.30. Promega) and selected using hygromycin. Of the hygromycin-resistant cells, a clone that stably luminesced upon osteoclastogenic stimulation by RANKL was selected (pNFAT/Luc-RAW264.7). An orphan ligand library (Screen-Well^TM, Biomol) containing 84 compounds with defined or putative biological activity but whose protein binding partners have not been identified was applied to the screening. pNFAT/Luc-RAW264.7 cells were cultured in osteoclast induction medium in 96-well plates, and each compound was added at 10 µM. After 24 hours of RANKL stimulation, the luciferase activity of each well was measured using a microplate reader (GloMax-Multi Detection System, Promega). Real-time PCR and western blotting analyses were conducted to confirm the activation of NFATc1 in RAW264.7 or mouse bone marrow-derived osteoclast precursor cells. Results: Luciferase activity of pNFAT/Luc-RAW264.7 cells was induced by RANKL stimulation in a concentration- and time-dependent manner. This activity was abolished in the presence of an NFAT activation inhibitor (FK506). Library screening identified three NFAT activator and two repressor candidates in osteoclasts. One of the activator candidates was the ß-carboline derivative harmine. This compound induced expression of NFATc1 mRNA and protein during osteoclastogenesis either with or without RANKL stimulation. Conclusion: We used a chemical biology strategy to identify novel modulator compounds for NFAT activity in osteoclasts. The pNFAT/Luc-RAW264.7 system could be useful for identifying novel compounds associated with osteoclastogenesis, as in the present study it identified harmine as a novel activator for NFAT activity in osteoclasts.