Role of phospholipases D1 and 2 in astroglial proliferation: effects of specific inhibitors and genetic deletion

Phospholipase D (PLD) activity has been linked to proliferation in many cell types including tumor cells. In the present study, we investigated the effects of genetic deletion of PLD1 and PLD2 and of specific PLD1 and PLD2 inhibitors on PLD activity and cell proliferation in primary mouse astrocytes. Basal and stimulated PLD activity was negligible in PLD1/2 double knockouts. PLD activity was significantly reduced in PLD1-deficient cells when fetal calf serum (FCS), insulin-like growth factor 1 (IGF-1) or phorbol ester was used as a stimulant. The specificity of PLD inhibitors VU0359595 and VU0285655-1 at 500 nM was confirmed in phorbol ester-stimulated cells. Significant reductions of cell proliferation were observed in PLD-deficient cell lines under basal and stimulated conditions. At 500 nM, the PLD1 inhibitor VU0359595 reduced proliferation in PLD2-deficient cells, but also in PLD1-deficient cells stimulated by IGF-1 or phorbol ester. Vice versa, at 500 nM, the PLD2 inhibitor VU0285655-1 reduced proliferation in PLD1-deficient cells, but also in PLD2-deficient cells exposed to IGF-1. At 5 µM, both inhibitors showed non-specific effects because they inhibited cell proliferation even in PLD1/2 double knockouts. Summarizing, inhibition of PLD occurs in parallel with reduced cell proliferation in astrocytes which are deficient in PLD1 or PLD2. Synthetic PLD inhibitors show high specificity for PLD in low (nanomolar) concentrations, but have additional, non-specific effects on cell proliferation when used at high (micromolar) concentrations.