Signaling mechanism for PLD1-induced Bcl-2 expression leading to neurite outgrowth of neural stem cell differentiation

Abstract

We studied the possible role of phospholipase D1 (PLD1) in neuronal differentiation including neurite formation of neural stem cells. Expression level of PLD1 increased during neuronal differentiation in neural stem cells. Bcl-2, which was defined to have a critical role in neurite outgrowing in H19-7 cells from our previous data, also showed increased expression level during neuronal differentiation. To investigate whether PLD1 is involved in neuronal differentiation of rat neural stem cells, the cells were infected with DN-PLD1. Downregulation of PLD1 using DN-PLD1 showed inhibited neurite outgrowth and down-regulation of Bcl-2 expression. PLD activity was dramatically reduced by PLCγ inhibitor (U73122), Ca2+ chelator (Bapta-am), PKCα inhibitor (Ro320432). These results indicate that PLCγ, Ca2+ and PKCα act as upstream activators of PLD in neural stem cells. Furthermore, treatment with arachidonic acid (AA) upregulated phosphorylation of p38 MAPK, CREB, and Bcl-2 expressions during differentiation condition, suggesting that AA is involved in the differentiation process generated by PLD1 activation in neural stem cells. We found that PGE2, which is a cyclooxygenase product of AA, was increased during neuronal differentiation of neural stem cells. Moreover, PGE2-stimulated phosphorylation of P38 MAPK as well as CREB were decreased by PKA inhibitor (Rp-cAMP). As expected, inhibition of P38 MAPK resulted in deactivation of CREB. When CREB activity was blocked with CREB siRNA, Bcl-2 expression was decreased. Taken together, PLD1 acts as a mediator during neuronal differentiation through PLCγ/PKCα/PLD1/PA/AA/PGE2/PKA/P38 MAPK/CREB/Bcl-2 pathway in neural stem cells.