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M Islam, GI Liou, O Chaudhary, RA Akhta; Epidermal Growth Factor (EGF)-induced Expression of Phospholipase C1 (PLC1) Promoted Rabbit Corneal Epithelial Cell (RCEC) Migration and Proliferation Via Activation of Map Kinase (Erk1/Erk2) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1632.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously, we have reported that EGF stimulates RCEC proliferation with a corresponding increase in PLCγ1 activity. The goal of the current study was to determine whether EGF exerts its effect on PLCγ1 at the mRNA level, and to identify signaling proteins, subsequent to PLCγ1 activation, involved in RCEC migration and proliferation. Methods: Serum-starved, primary RCECs were cultured with or without EGF and either PD-98059 (Erk1/Erk2 inhibitor) or U-73122 (PLC inhibitor). At a prescribed time, the cultures were terminated, and the cells were trypsinized and counted. To determine changes in the level of PLCγ1protein and mRNA, the cells were subjected to Western and Northern analyses. A 32P-UTP-labeled cDNA probe, synthesized by random priming, was used for Northern analysis. Activation of Erk1/Erk2 was assessed by Western analysis using Erk and phospho-Erk antibodies. Epithelial cell migration was monitored by a trans-well chambers method. Results: Addition of EGF (5-50 ng/ml) to primary RCECs resulted in a dose-dependent increase in cell migration and proliferation that correlated with elevation of both PLCγ1protein and mRNA levels. U-73122 (2 µM), when added to the cells, inhibited EGF-induced cell migration and proliferation. Addition of EGF dose-dependently increased the phosphorylation of Erk1/Erk2, which was inhibited by PD-98059 (10 µM). PD-98059 also inhibited the EGF-induced RCEC migration and proliferation. Pretreatment of the cells with U-73122 rendered the cells resistant to EGF-induced activation of Erk1/Erk2. Conclusion: The data suggests that EGF stimulates RCEC migration and proliferation by up-regulating PLCγ1 mRNA. Further, the inhibition of EGF-activated Erk1/Erk2 by U-73122 and PD-98059 suggests that Erk1/Erk2 lies downstream of PLCγ1pathway to regulate RCEC migration and proliferation.
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