Involvement of GSK3/β-catenin in the action of extracellular ATP on differentiation of primary cultures from rat calvaria into osteoblasts.

Modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the involvement of the GSK3/βcatenin signaling in the action of ATPγ-S on osteogenic differentiation of primary cell cultures from rat calvaria. Our results indicate that the cell treatment with 10 or 100 µM ATPγ-S for 96 h increase the cytoplasmic levels of β-catenin and its translocation to nucleus respect to control. A similar effect was observed after cell treatment with the GSK3 inhibitor LiCl (10 mM). Cell treatments with 4-10 mM LiCl significantly stimulated ALP activity respect to control at 4 and 7 days, suggesting that inhibition of GSK-3 mediates osteoblastic differentiation of rat calvarial cells. Effects comparison between ATP and LiCl shown that ALP activity was significantly increased by 10 µM ATPγ-S and decreased by 10 mM LiCl at 10 day of treatment, respect to control, suggesting that the effect of ATPγ-S was less potent but more persistent than of LiCl in stimulating this osteogenic marker in calvarial cells. Cell culture mineralization was significantly increased by treatment with 10 µM ATPγ-S and decreased by 10 mM LiCl, respect to control. In together, these results suggest that GSK3 inhibition is involved in ATPγ-S action on rat calvarial cell differentiation into osteoblasts at early steadies. In addition such inhibition by LiCl appear promote osteoblasts differentiation at beginning but has a deleterious effect on its function at later steadies as the extracellular matrix mineralization.