Upregulation of phospholipase Cγ1 activity during EGF-induced proliferation of corneal epithelial cells:: Effect of phosphoinositide-3 kinase

PURPOSE. Previously, the authors showed that epidermal growth factor (EGF) stimulates phospholipase C gamma1 (PLC gamma1) and phosphoinositide-3 kinase (PI3K) activities in confluent rabbit corneal epithelial cells (RCECs). The purpose of this study was to investigate whether PLC gamma1 activity is upregulated during EGF-induced proliferation of RCECs and to determine whether there is any cross-talk between PLC gamma1 and PI3K in these cells. METHODS. Simian virus (SV)40-immortalized RCECs were cultured in the presence and absence of EGF and other agents. At prescribed time intervals, the cultures were terminated and the cells counted. PLC gamma1 activity in intact cells was assessed by measuring the production of [H-3]IP, in [H-3]myoinositol-labeled cells. The in vitro enzyme activity was assayed using immunoprecipitated PLC gamma1 and [H-3]PI(4,5)P-2 as substrate. [H-3]IP3 the product of PLC gamma1, was analyzed by anion-exchange chromatography. The changes in protein content and level of phosphorylation of PLC gamma1 were determined by Western immunoblot analysis, with the appropriate antibodies. RESULTS. Addition of EGF (50 ng/ml) caused a time-dependent increase in proliferation of RCECs. The effect of EGF peaked at approximately 36 hours. Under the same experimental conditions, EGF stimulated PLC gamma1 activity with a time course similar to that of cell proliferation. Data from Western immunoblot analysis revealed that the EGP-stimulated PLC gamma1 activity was due to increased synthesis of the enzyme. Furthermore, during cell proliferation, tyrosine phosphorylation of PLC gamma1 increased in a time-dependent manner that corresponded closely with the expression of PLC gamma1. EGF exerted its effects both on cell proliferation and PLC gamma1 activation in a dose-dependent manner. Treatment of the cells with U-73122, a PLC inhibitor, or myr-GLYRKAMRLRY, a myristoylated PLC gamma1 inhibitor peptide, caused attenuation of both the EGF-stimulated cell proliferation and PLC gamma1 activity. Treatment of the cells with the PI3K inhibitors, wortmannin or LY294002, caused inhibition of both EGF-stimulated cell proliferation and PLC gamma1 activation. Addition of PI(3,4,5)P-3 to the in vitro PLC gamma1 assay mixture stimulated the enzyme activity in a dose-dependent manner. CONCLUSIONS. The data suggest a positive correlation between EGF-stimulated PLC gamma1 activation and cell proliferation in RCECs. The EGF-stimulated PLC gamma1 activity was mirrored by increased synthesis and tyrosine phosphorylation of the enzyme. The data also show that PLC gamma1 activation and cell proliferation were inhibited by PI3K inhibitors, suggesting a role for PI3K in EGF-stimulated proliferation of corneal epithelial cells.