Phospholipase D and C are essential for bFGF-induced neuronal differentiation of H19-7 cells

Abstract

Phospholipase D (PLD) is widely distributed in mammalian cells and implicated in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. We found at the first time during the bFGF-induced differentiation of immortalized cell line from rat hippocampal neurons (H19-7), PLD activity and PLD1 expression were markedly increased. To confirm the role of PLD1 in neuronal differentiation, we transfected the dominant-negative mutant of PLD1(DN-rPLD1) into H19-7 cells. Transfection of DN-rPLD1 blocked bFGF-induced PLD activation and completely inhibited neuronal differentiation induced by bFGF. In contrast, overexpression of PLD1 in the H19-7 cells could result in neuronal differentiation even in the absence of bFGF. These results suggest that PLD1 activity is important in the neuronal differentiation of H19-7 cells. To elucidate the requirement of Ras and MEK and MAP kinase (ERK1 and ERK2) for the neuronal differentiation, H19-7 cells were treated with the Ras farnesyl transferase inhibitor, manumycin-A and the MEK inhibitor, PD98059. Manumycin-A treatment decreased PLD1 activity and expression induced by bFGF. Also, bFGF-induced neuronal differentiation was completely abolished by treatment of manumycin-A. These results suggest that Ras is also important mediator for the neuronal differentiation of H19-7 cells. In contrast, MEK inhibitor, PD98059, did not block bFGF-induced PLD1 activity and PLD1 expression as well as neuronal differentiation, representing that MAP kinase activation is neither necessary nor sufficient for neuronal differentiation. Treatment of PC-PLC inhibitor, D609 and PI-PLC inhibitor, U73122 partially decreased PLD1 activity and PLD1 expression, respectively. When treated with both of above inhibitors, completely blocked bFGF-induced differentiation as well as activation and expression of PLD1, suggesting that PC-PLC and PI-PLC are required to bFGF-induced neuronal differentiation with PLD1