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The aim of the present study was to determine the effects of the Ca2+/calmodulin‑dependent protein kinase pathway inhibitor KN93 on osteoclastogenesis. RAW264.7 cells were incubated with macrophage colony‑stimulating factor (M‑CSF) + receptor activator of nuclear factor kappa‑light‑chain‑enhancer of activated B cells ligand (RANKL) to stimulate osteoclastogenesis and then treated with 10 µM KN93. The methods included tartrate‑resistant acid phosphatase (TRAP) staining, bone resorption activity assays, filamentous (F)‑actin staining, determination of intracellular calcium ([Ca2+]i) levels, monitoring of osteoclast‑specific gene expression levels and measurement of key transcription factors protein levels. The results suggested that KN93 inhibited the formation of TRAP‑positive multinucleated cells, shaping of F‑actin rings and resorption activity of the cells. In addition, KN93 decreased the concentration of [Ca2+]i, expression levels of osteoclast specific genes and protein levels of critical transcription factors in the M‑CSF + RANKL‑induced osteoclast model. In summary, KN93 may directly affect the differentiation and activation of osteoclasts, potentially through the Ca2+/calmodulin‑dependent protein kinase signaling pathway.

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Background: Osteoclasts are large terminal-differentiated cells with multiple nuclei and are the only cells with bone resorption activity in the body. The abnormal migration and differentiation of osteoclasts may accelerate bone absorption, a crucial process in the occurrence and development of osteoporosis. Regulating the differentiation and activation of osteoclasts is a breakthrough point for the prevention and treatment of osteoporosis. Coumarin derivative of 3-(4-methoxyl)-1-(2-(4-coumarin) prop)-2-en-1-one (MCPEO) was selected in this study. We aimed to investigate the effects of (MCPEO) on osteoclast differentiation. Methods: Bone marrow mononuclear cells (BM-MNCs) were collected from 6-week-old ICR mice and inoculated in vitro. BM-MNC and RAW264.7 cells were induced by the receptor activator of nuclear factor κ B ligand (RANKL) to differentiate them into osteoclasts. The cells were then added with 0.01, 0.05, 0.1, 0.5, and 1 μM MCPEO. The cell viability of differentiated osteoclasts was analyzed by methylthiazolyldiphenyl-tetrazolium bromide assay. The differentiated osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. Miuse osteoclast activation was investigated by absorptive activity analysis. Filamentous actin (F-actin) staining was employed to identify the formation of F-actin rings in the differentiated mouse osteoclasts. The change level of critical transcription factors related to osteoclast differentiation was determined by Western blot analysis. Results: Data show that MCPEO affected the cell viability of differentiated osteoclasts, inhibited the formation of TRAP-positive polynuclear cells, and decreased the absorption activity and the formation of F-actin rings in the differentiated osteoclasts. Furthermore, MCPEO influenced the change level of crucial transcription factors related to osteoclast differentiation. Conclusions: MCPEO inhibited the differentiation of RANKL-induced BM-MNC and RAW264.7 cells into osteoclasts.