Osteoblastic NF-κB pathway is involved in 1α, 25(OH)2D3-induced osteoclast-like cells formation in vitro

1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25(OH)(2)D-3) acts on the osteoblasts to enhance the expressions of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) and induce the formation of osteoclasts. However, the mechanism in osteoblasts by which 1 alpha, 25(OH)(2)D-3 promotes osteoclastogenesis has not yet been completely understood. This study aimed to select the first generation of murine osteoblasts to explore the underlying mechanism of 1 alpha, 25(OH)(2)D-3-induced osteoclastic formation from bone marrow mononuclear cells (BMMNCs). We discovered the activation of osteoblastic NF-kappa B pathway under 10-8 mol/L 1 alpha, 25(OH)(2)D-3 treatment, as evidenced by the transfer of NF-kappa B p65 from cytoplasm to nuclei. Then, the NF-kappa B p65-siRNA was designed, constructed, and transfected into osteoblastic cells. Immunofluorescence assay confirmed the successfully silenced NF-kappa B p65 gene in osteoblasts. In the co-culture system of osteoblasts and BMMNCs with 1 alpha, 25(OH)(2)D-3 added, the multinucleated osteoclast-like cells containing 2-3 nuclei were observed in BMMNCs co-cultured with non-transfection osteoblasts, conversely, silencing osteoblastic NF-kappa B p65 resulted in failed differentiation of BMMNCs along with substantial vacuolar degeneration in cytoplasm. In addition, the expressions of RANKL and M-CSF were notably decreased in NF-kappa B p65-silenced osteoblasts. Taken together, our data indicated that osteoblastic NF-kappa B pathway was involved in 1 alpha, 25(OH)(2)D-3-induced osteoclast-like cells formation from BMMNCs through regulating the expression of RANKL and M-CSF. Therefore, our findings further identified the mechanism of 1 alpha, 25(OH)(2)D-3-induced osteoclastogenesis on the basis of prior studies.