Phospholipase D in Corneal Epithelial Cell Motility

Abstract

Abstract: : Purpose: Stimulation of cells by numerous hormones and growth factors results in activation of phospholipase D, an enzyme that hydrolyzes phosphatidyl choline to generate the secondary messenger phosphatidic acid. The purpose of this study was to examine the role of phospholipase D in induction of a motile phenotype in corneal epithelial cells. Methods: The motile state of epithelial cells was induced by acute presentation of permissive culture area. Phospholipase D activities were assayed by labeling cells with [3H]myristic acid, adding 1–BuOH and measuring formation of phosphatidylinositol butanol. Directional migration was measured in Transwell^® migration chambers. Results: Wounding of sheets of corneal epithelial and MDCK cells causes rapid activation of phospholipase D. Short–chain analogues of phosphatidic acid were found to induce a migratory phenotype robustly in several epithelial cell types, including corneal epithelial cells. Addition of exogenous dioctanoyl phosphatidic acid induces marked activation of endogenous phospholipase D suggesting the existence of a feed–forward regulatory loop. When corneal epithelial cells were presented with phosphatidic acid as a gradient in modified Boyden chambers, a strong chemotaxic response was observed. Epidermal growth factor activates PLD and acts as a chemoattractant in corneal epithelial cells. When saturating amounts of epidermal growth factor was present in both compartments of the migration chambers chemotactic movement of corneal epithelial cells towards phosphatidic acid was abolished. This suggests that phospholipase D and epidermal growth factor signal through shared signaling pathways. Differential sensitivity to inhibitors and other observations strongly suggested that the added phosphatidic acid did not act through activation of protein kinase C. Conclusions: These data support an important role for phospholipase D in induction of motility in corneal and other epithelial cells, and suggest that localized concentrations of phosphatidic acid may determine direction of movement.

Keywords: cornea: epithelium • second messengers • wound healing