Protein Dephosphorylation and Apoptosis in Osteoblast

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Phosphorylation and dephosphorylation play a central role in the regulation of cellular proliferation and differentiation. It seems likely that the balance between these activities also plays an important parts in apoptosis. Okadaic acid (OA) is a potent inhibitor of protein phosphatases type 1 (PP-1) and type 2A (PP-2A) and increases the phosphorylation of cellular proteins without activation of protein kinase C.We demonstrated that OA induces apoptosis in both Saos-2 cells and MG63 cells in a dose-dependent manner with a maximum effective concentration of 10 nM (Morimoto et al, 1997). Because inhibition of protein phosphatase activity by OA may be a general way of triggering apoptosis in some kinds of cells, it is believed that new protein synthesis is not required for OA-induced apoptosis.To determine if inhibition of the protein synthesis or RNA synthesis could protect against the OA-induced apoptosis in osteoblasts, we cultured the cells in media containing varying concentrations of cycloheximide, actinomycin D, or puromycin in the presence of 10 nM OA.Cycloheximide, actinomycin D, and puromycin significantly protected against OA-induced cytotoxicity and cell death in MG63 cells. The number of apoptotic cells examined by the Hoechst 33342 staining also significantly decreased in MG63 cells treated with these reagents in the presence of OA.Moreover, the intensity of DNA ladder formation was decreased in MG63 cells treated with these reagents in a dose-dependent manner. The maximum effective concentrations of these reagents on the protection of OA-induced apoptosis in MG63 cells are in good agreement with the data concerning thier biological effects in other systems. However, cycloheximide, actinomycin D, and puromycin did not protect against the OA-induced apoptotic cell death in Saos-2 cells determined by the morphological and biochemical techniques (Morimoto et al, in press).