M Islam1, R A Akhtar. 1. Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Abstract
PURPOSE: Previously, the authors showed that epidermal growth factor (EGF) stimulates phospholipase Cgamma1 (PLCgamma1) and phosphoinositide-3 kinase (PI3K) activities in confluent rabbit corneal epithelial cells (RCECs). The purpose of this study was to investigate whether PLCgamma1 activity is upregulated during EGF-induced proliferation of RCECs and to determine whether there is any cross-talk between PLCgamma1 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. PLCgamma1 activity in intact cells was assessed by measuring the production of [(3)H]IP(3) in [(3)H]myoinositol-labeled cells. The in vitro enzyme activity was assayed using immunoprecipitated PLCgamma1 and [(3)H]PI(4,5)P(2) as substrate. [(3)H]IP(3), the product of PLCgamma1, was analyzed by anion-exchange chromatography. The changes in protein content and level of phosphorylation of PLCgamma1 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 PLCgamma1 activity with a time course similar to that of cell proliferation. Data from Western immunoblot analysis revealed that the EGF-stimulated PLCgamma1 activity was due to increased synthesis of the enzyme. Furthermore, during cell proliferation, tyrosine phosphorylation of PLCgamma1 increased in a time-dependent manner that corresponded closely with the expression of PLCgamma1. EGF exerted its effects both on cell proliferation and PLCgamma1 activation in a dose-dependent manner. Treatment of the cells with U-73122, a PLC inhibitor, or myr-GLYRKAMRLRY, a myristoylated PLCgamma1 inhibitor peptide, caused attenuation of both the EGF-stimulated cell proliferation and PLCgamma1 activity. Treatment of the cells with the PI3K inhibitors, wortmannin or LY294002, caused inhibition of both EGF-stimulated cell proliferation and PLCgamma1 activation. Addition of PI(3,4,5)P(3) to the in vitro PLCgamma1 assay mixture stimulated the enzyme activity in a dose-dependent manner. CONCLUSIONS: The data suggest a positive correlation between EGF-stimulated PLCgamma1 activation and cell proliferation in RCECS. The EGF-stimulated PLCgamma1 activity was mirrored by increased synthesis and tyrosine phosphorylation of the enzyme. The data also show that PLCgamma1 activation and cell proliferation were inhibited by PI3K inhibitors, suggesting a role for PI3K in EGF-stimulated proliferation of corneal epithelial cells.
PURPOSE: Previously, the authors showed that epidermal growth factor (EGF) stimulates phospholipase Cgamma1 (PLCgamma1) and phosphoinositide-3 kinase (PI3K) activities in confluent rabbit corneal epithelial cells (RCECs). The purpose of this study was to investigate whether PLCgamma1 activity is upregulated during EGF-induced proliferation of RCECs and to determine whether there is any cross-talk between PLCgamma1 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. PLCgamma1 activity in intact cells was assessed by measuring the production of [(3)H]IP(3) in [(3)H]myoinositol-labeled cells. The in vitro enzyme activity was assayed using immunoprecipitated PLCgamma1 and [(3)H]PI(4,5)P(2) as substrate. [(3)H]IP(3), the product of PLCgamma1, was analyzed by anion-exchange chromatography. The changes in protein content and level of phosphorylation of PLCgamma1 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 PLCgamma1 activity with a time course similar to that of cell proliferation. Data from Western immunoblot analysis revealed that the EGF-stimulated PLCgamma1 activity was due to increased synthesis of the enzyme. Furthermore, during cell proliferation, tyrosine phosphorylation of PLCgamma1 increased in a time-dependent manner that corresponded closely with the expression of PLCgamma1. EGF exerted its effects both on cell proliferation and PLCgamma1 activation in a dose-dependent manner. Treatment of the cells with U-73122, a PLC inhibitor, or myr-GLYRKAMRLRY, a myristoylated PLCgamma1 inhibitor peptide, caused attenuation of both the EGF-stimulated cell proliferation and PLCgamma1 activity. Treatment of the cells with the PI3K inhibitors, wortmannin or LY294002, caused inhibition of both EGF-stimulated cell proliferation and PLCgamma1 activation. Addition of PI(3,4,5)P(3) to the in vitro PLCgamma1 assay mixture stimulated the enzyme activity in a dose-dependent manner. CONCLUSIONS: The data suggest a positive correlation between EGF-stimulated PLCgamma1 activation and cell proliferation in RCECS. The EGF-stimulated PLCgamma1 activity was mirrored by increased synthesis and tyrosine phosphorylation of the enzyme. The data also show that PLCgamma1 activation and cell proliferation were inhibited by PI3K inhibitors, suggesting a role for PI3K in EGF-stimulated proliferation of corneal epithelial cells.
Authors: Solange Landreville; Stéphanie Coulombe; Patrick Carrier; Michael H Gelb; Sylvain L Guérin; Christian Salesse Journal: Invest Ophthalmol Vis Sci Date: 2004-11 Impact factor: 4.799