Epidermal Growth Factor (EGF)-induced Phospholipase D (PLD) Activation Stimulates Migration and Proliferation in Rabbit Corneal Epithelial Cells (RCEC)

Abstract

Abstract: : Purpose: Phospholipase D (PLD) and its product phosphatidic acid have been implicated in a variety of cellular functions. The purpose of the present work was to identify different isoforms of PLD in RCEC, and to investigate the role of PLD in EGF-induced migration and proliferation in RCEC. The role of small G protein RhoA in PLD activation and cell proliferation was also examined. Methods: Serum-starved, primary RCEC were cultured with or without EGF. At prescribed times, the cultures were terminated, and the cells were trypsinized and counted. The cell migration assay was performed using trans-well chambers. To assay for PLD activity, the cells were labeled with [³H]myristic acid and then cultured with or without EGF in the presence of ethanol. The production of [³H]PEt was taken as a measure for PLD activity. Different isoforms of PLD were identified by Western blotting using specific antibodies. To investigate the role of RhoA in PLD activation and cell proliferation, the RCEC were transiently transfected with constitutively active (CA) and dominant negative (DN) mutant RhoA according to the supplier’s instructions. Results: Addition of EGF to sub-confluent RCEC caused a dose-dependent increase in cell migration and proliferation. The maximal effect of EGF was observed at 5 ng/ml. Western blotting revealed the presence of PLD1 and PLD2 in RCEC. The amount of PLD2 was higher than PLD1. The EGF-treatment also resulted in increased tyrosine phosphorylation of PLD. EGF (5ng/ml) caused a large stimulation of PLD activity in RCEC transfected with constitutively active RhoA. In contrast, the PLD response to EGF was much weaker in RCEC expressing the dominant negative RhoA. The EGF effect on PLD activity in transfected cells was positively correlated with the extent of cell proliferation induced by RCEC. Conclusion: These data demonstrate that activation of PLD is involved in EGF-induced migration and proliferation in RCEC. Further, the effect of EGF is probably mediated by the RhoA-dependent PLD1 isoform of the enzyme.

Keywords: cornea: epithelium • growth factors/growth factor receptors