Epidermal growth factor stimulates phospholipase D independent of phospholipase C, protein kinase C or phosphatidylinositol-3 kinase activation in immortalized rabbit corneal epithelial cells

Abstract

PURPOSE. Activation of phospholipase D (PLD) is believed to be an important signaling pathway involved in cell growth and differentiation in several tissues, in response to a variety of mitogens. The aim of the present study was to investigate the effect of epidermal growth factor (EGF) on PLD activity in rabbit corneal epithelial cells (RCEC). We have also examined whether the EGF effect is dependent on concurrent activation of phospholipase C (PLC), protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI 3-kinase) in these cells. METHODS. RCEC, immortalized with adenovirus SV-40, were cultured until they became confluent. The cells were labeled with [ 3 H]myristic acid and incubated with or without EGF or other agents for specified time intervals. PLD activity was measured by quantifying [ 3 H]phosphatidylethanol in cells incubated in the presence of ethanol. PLC activity was determined by measuring the radioactivity in inositol trisphosphate in myo[ 3 H]inositol-labeled RCEC. PI 3-kinase activity was assessed by measuring the production of PIP 3 in 32 P-labeled cells. RESULTS. Addition of EGF to RCEC stimulated PLD activity in a time- and dose-dependent manner. The maximal effect was observed with 150 ng/ml EGF and at 10 min of incubation. The PLD activity was also stimulated when phorbol myristate acetate (PMA) was added to the cells. Treatment of the cells with EGF stimulated PLC activity which was inhibited by U73122, a PLC inhibitor. Under the same experimental conditions, the inhibitor had no effect on EGF-stimulated PLD activity. Down-regulation of PKC or treatment of the cells with RO31-8220, a PKC inhibitor, inhibited the PMA- but not EGF-stimulated PLD activity. Incubation of the cells with wortmannin, a PI 3-kinase inhibitor, abolished the EGF-stimulated PI 3-kinase activity, but potentiated the EGF-stimulated PLD activity. The EGF effect was inhibited by treatment of the cells with tyrphostin B 42, a receptor tyrosine kinase inhibitor. CONCLUSIONS. These results indicate that EGF stimulates PLD activity in RCEC by a mechanism that involves tyrosine phosphorylation of a protein(s) in the cascade of biochemical reactions initiated by EGF-receptor interaction, and it is not dependent on concurrent activation of PKC, PLC, or PI 3-kinase in these cells.