Okadaic acid stimulates the expression of receptor activator of nuclear factor-kappa B ligand (RANKL) in mouse osteoblastic cells

Receptor activator of nuclear factor-κB ligand (RANKL) is a membrane-bound signal transducer responsible for differentiation and maintenance of osteoclasts. To investigate a possible relationship between the status of protein phosphorylation and the regulation of RANKL in osteoblasts, we examined the effects of okadaic acid, a potent inhibitor of protein phosphatases, on cultured mouse osteoblastic MC3T3-E1 cells. Okadaic acid increased the amounts of RANKL protein in a dose-dependent fashion, with the maximum effective concentration being 100 nM. Okadaic acid at 100 nM stimulated the expression of RANKL mRNA and protein in MC3T3-E1 cells in a time-dependent manner up to 3 h. Okadaic acid also stimulated the expression of the Cbfa1 protein, a transcription factor required for osteoblast differentiation, in a dose-dependent manner. Binding of nuclear proteins prepared from MC3T3-E1 cells to Cbfa1 consensus sequence increased in a time-dependent manner when the cells had been treated with 100 nM okadaic acid. A 200-fold excess of unlabelled Cbfa1 oligonucleotide completely inhibited the DNA-protein complex formation. Our study demonstrate that okadaic acid stimulate RANKL production in MC3T3-E1 cells, indicating that the serine/threonine protein phosphatases play an important role in the regulation of bone metabolism.