Soluble EphB4 inhibition of PDGF-induced RPE migration in vitro.
OBJECTIVE
EphB4 receptor (EphB4) and its ligand (EphrinB2) play an important role in the regulation of cell adhesion, growth, and migration. The purpose of this study was to determine the effects of EphB4 blockade by soluble EphB4 (sEphB4) on retinal pigment epithelial (RPE) cell migration and proliferation, induced by platelet-derived growth factor-BB (PDGF), and to establish its relevance to proliferative vitreoretinopathy (PVR).


METHODS
The expression of EphB4 and EphrinB2 in early-passage human RPE cells and in human PVR membranes was evaluated by confocal microscopy. The effect of sEphB4 (0.1-3 microg/mL) on PDGF (20 ng/mL)-induced RPE migration and proliferation was evaluated using a modified Boyden chamber assay and an MTT assay, respectively. Attachment to basement membrane matrix and fibronectin was assayed by MTT. Phosphorylation of FAK and p42/44 mitogen-activated protein kinase (MAPK) in retinal pigment epithelium was determined by Western blot analysis after exposure to sEphB4. The effect of sEphB4 on the phosphorylation of EphB4/EphrinB2 was demonstrated with the use of immunoprecipitation assays.


RESULTS
EphrinB2 and EphB4 were expressed on human RPE cells in vitro and in cells within human PVR membranes. sEphB4 blocked EphB4 and EphrinB2 phosphorylation in RPE cells in vitro. sEphB4 reduced RPE migration in response to PDGF stimulation (P < 0.01). Similarly, sEphB4 inhibited RPE attachment and proliferation in a dose-dependent manner (P < 0.05). PDGF-induced phosphorylation of FAK and MAPK was inhibited by sEphB4.


CONCLUSIONS
EphB4 and EphrinB2 are expressed in RPE cells and PVR membranes. sEphB4 inhibits PDGF-induced RPE cell attachment, proliferation, and migration. This effect may result from the inhibition of FAK and MAPK phosphorylation.
He
Shikun
S
Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California 90089, USA.
Kumar
S Ram
SR
Zhou
Peng
P
Krasnoperov
Valery
V
Ryan
Stephen J
SJ
Gill
Parkash S
PS
Hinton
David R
DR
D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D036388:Ephrin-B2 / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D036224:Receptor, EphB4 / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000196:embryology / Q000378:metabolism*; D018630:Vitreoretinopathy, Proliferative / Q000378:metabolism*
D036388:Ephrin-B2; D005353:Fibronectins; D008024:Ligands; D010982:Platelet-Derived Growth Factor; D036224:Receptor, EphB4; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
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