PKC-dependent ERK phosphorylation is essential for P2X(7) receptor-mediated neuronal differentiation of neural progenitor cells

Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X(7) receptors (P2X(7)Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X(7)R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X(7)R might be expressed in neural progenitor cells (NPCs). P2X(7)R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X(7)R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X(7)Rs induced Ca(2+) influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X(7)R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca(2+) concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X(7)R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca(2+) and treatment with blockers for P2X(7)R and PKC activity. Stimulation of P2X(7)R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X(7)R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.