Involvement of the PI3K/AKT pathway in ATP-induced proliferation of developing retinal cells in culture

Abstract

ATP induces the proliferation of chick retinal cells in culture through the activation of P2Y1 receptors, PKC and MAP kinases. Together with MAP kinases, the PI3K/AKT pathway has also been implicated as an important mediator in proliferative events during development. Here we investigated the participation of the PI3K/AKT signal pathway on ATP-induced proliferation of chick embryo retinal cells in culture. When retinal cultures obtained from 7-day-old embryos were cultivated for 1 day and treated with ATP, a transient and dose-dependent phosphorylation of both ERK and AKT was observed, an effect that could be mimicked by 500μM ADP and blocked by 100μM PPADS, a P2 receptor antagonist. Maximal stimulation of both enzymes was obtained with 100μM ATP in 5min, decreasing thereafter. Activation of these pathways by ATP seemed to be independent, since LY294002 and U0126, inhibitors of PI3K and MEK, did not block the activation of ERK and AKT, respectively, although each compound blocked its respective target. Moreover, when the cultures were incubated with ATP in the presence of LY294002, a decreased incorporation of [3H]-thymidine was observed, as compared to cultures treated only with ATP, a decline that was also obtained by incubating the cells with ATP plus 0.5μM API-59CJ-Ome, an inhibitor of AKT. No decrease in cell viability was observed with this concentration of API-59CJ-Ome. An increase in cyclin D1 expression, that could be inhibited by 10μM LY 294002 or 20μM U0126, was observed when cells were incubated with 500μM ADP. No effect of PI3K and MEK inhibitors was observed in the expression of p27kip1 in the cultures. These results suggest that, besides the involvement of the MAP kinases pathway, ATP-induced cell cycling of late developing retinal progenitors in culture also involves the activation of the PI3K/AKT pathway.