Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

► ATP stimulates PI3K/Akt pathway in osteoblastic cell through P2Y2 receptors. ► PI-PLC, Ca2+, CaM and Src participate in Akt stimulation. ► PKC seems to regulate Akt through Src and the Ca2+ influx/CaM pathway. ► PI3K/Akt activation by ATP is involved in osteoblastic cell proliferation. We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca2+-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca2+ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca2+-containing but not in a Ca2+-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y2 receptors and PI3K/Akt pathway activation involving Ca2+, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca2+ influx/CaM pathway.