Soluble Ephb4 Inhibits Pdgf-Induced Rpe Migration in vitro: Mechanism of Action

Abstract

Purpose:: In a previous study, we found that soluble EphB4 (sEphB4) inhibits platelet derived growth factor (PDGF) induced retinal pigment epithelial (RPE) cell migration in vitro. Here we show that sEphB4 regulates RPE adhesion and migration by inhibition of focal adhesion kinase phosphorylation.

Methods:: Cultured early passage human fetal RPE cells were used in the study. Expression and phosphorylation of focal adhesion kinase (FAK), and mitogen activated protein kinase (MAPK) in response to exposure of RPE cells to sEphB4 (0.1, 0.5,1,3 ug/ml) was analyzed by western blot. A modified MTT assay and immunoblot were used to measure RPE cell attachment and total FAK expression in the presence of sEphB4 with or without pretreatment with a calpain inhibitor. The effect of sEphB4 on F-actin was evaluated using Phalloidin-FITC staining after 1ug/ml of sEphB4 treatment at different time points.

Results:: The expression of total FAK, phospho-FAK, phospho-MAPK and RPE cell attachment were significantly inhibited by sEphB4 treatment (0.5 ug -3 ug/ ml) at 24 hours. The reduction in RPE cell attachment and expression of total FAK were reversed by pretreatment with the calpain specific inhibitor. Treatment with sEphB4 resulted in F-actin redistribution starting at 4hours without PDGF addition; however the effect was delayed to 24 hours in the presence of PDGF.

Conclusions:: The inhibition of RPE migration by sEphB4 may be mediated through the suppression of phosphorylation of FAK and MAPK.

Keywords: choroid: neovascularization • retinal pigment epithelium • pathobiology