Phospholipase D-mediated signaling in HEK-293 cells: A balance between pro and anti-proliferative effects

Phospholipase D (PLD) is a ubiquitously expressed enzyme responsible for the production of the lipid second messenger, phosphatidic acid (PA). Two isoforms of PLD exist, PLD1 and PLD2. There is no specific pharmacological inhibitor for PLD, thus it has been difficult to differentiate between their activities within the cell. In this study, the effects of PLD1-mediated signaling was investigated. The tumor promoter 12-O-tetradeconyl-13-phorbol acetate (TPA) stimulates PLD1 in HEK-293 cells. We used overexpression studies to investigate the effects of TPA-stimulation on downstream targets of PLD1. We found PLD1 activation enhanced the phosphorylation of the MAPK ERK and p38 both constitutively and in response to TPA stimulation. In addition, we observed PLD1-dependent expression of cyclooxygenase-2 (Cox-2) and interleukin-8 (IL-8) in response to TPA stimulation. PLD1 transfectant cells exhibited a reduced proliferation rate compared to parental HEK-293 cells. The reduction in proliferation rate was due to PLD1-dependent increases in the expression of the lipid phosphatase PTEN. We found that PLD1 transfectant cells produced the bioactive mediator, lyso phosphatidic acid (LPA). The LPA produced by PLD1 transfectants rescued HEK-293 cells from serum withdrawal-induced apoptosis. Additionally, we observed TPA-stimulated increases in arachidonic acid release and cPLA₂ phosphorylation in PLD1 transfectant cells. These studies suggest PLD1 activity provides a balance between pro and anti-proliferative responses in HEK-293 cells.