Regulation of phospholipase D2 by H2O2 in PC12 cells

Abstract

Phospholipase D2 (PLD2) is expressed in brain and inhibited by synuclein, which is involved in Parkinson's and Alzheimer's diseases. However, the activation mechanism of PLD2 in neuronal cells has not been defined clearly. Hydrogen peroxide (H2O2) plays roles in the neurodegenerative diseases and also acts as a second messenger of various molecules such as nerve growth factor. To study regulation mechanisms of PLD2 by H2O2 in neuronal cells, we have made stable PC12 cell lines expressing PLD2 (PLC2-PC12 cells). H2O2 treatment stimulated PLD activity in PLD2-PC12 cells in a dose- and time-dependent manner. This activation was inhibited by the treatment with protein kinase C (PKC) inhibitors or by depletion of PKCα, -δ and -ε. Phorbol ester markedly activated PLC2. Co-treatment with phorbol ester and H2O2 did not show an additive effect. Chelation of extracellular calcium substantially blocked the H2O2-induced activation of PLD2. A calcium ionophore induced PLD2 activation in a PKC-dependent manner. Protein-tyrosine kinase inhibitors inhibited H2O2-induced PLD activation slightly. These data indicate that H2O2 can activate PLD2 in PC12 cells and that this activation is largely dependent on PKC and Ca2+ ions and minimally dependent on tyrosine phosphorylation.