Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor.
OBJECTIVE
To evaluate the effect of hepatocyte growth factor (HGF) on the integrity and function of tight junctions and adherens junctions in the retinal pigment epithelial (RPE) monolayer.


METHODS
Fresh bovine eyes were dissected to obtain 2- to 3-mm(2) explants of intact RPE with underlying choroid and sclera. Explants were cultured with or without HGF (20 ng/mL) for various periods (20 minutes to 72 hours). Junction integrity was assessed by transmission and scanning electron microscopy; localization, expression, and phosphorylation of junction proteins; and measurement of transepithelial resistance (TER), diffusion of fluorescent labeling in the plasma membrane, and the migration of RPE cells from the monolayer.


RESULTS
Untreated explants consisted of polarized cells with apical microvilli and well-developed tight and adherens junctions. After HGF treatment, the explants showed loss of tight and adherens junctions ultrastructurally, diffusion of fluorescent label from apical to lateral membrane domains, and increased chemotactic migration of RPE cells from the monolayer. Primary cultures of confluent RPE cells showed a progressive decrease in TER. Western blot analysis showed rapid tyrosine phosphorylation of ZO-1, occludin, and beta-catenin within 20 minutes of stimulation. There was a marked loss of ZO-1 protein within 1 hour of HGF treatment. After 6 hours of treatment with HGF, occludin, claudin-1, and beta-catenin were redistributed from the membrane to the cytoplasm.


CONCLUSIONS
Treatment of RPE explants with HGF results in rapid disassembly of tight and adherens junctions associated with loss or redistribution of junctional proteins, decreased TER, and increased migration of RPE cells from the monolayer.
Jin
Manlin
M
Department of Pathology, Keck School of Medicine of the University of Southern California Los Angeles, California 90033, USA.
Barron
Ernesto
E
He
Shikun
S
Ryan
Stephen J
SJ
Hinton
David R
DR
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002465:Cell Movement; D002478:Cells, Cultured; D062445:Claudin-1; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004553:Electric Conductivity; D019084:Fluorescent Antibody Technique, Indirect; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D007365:Intercellular Junctions / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D011233:Precipitin Tests; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin
D062445:Claudin-1; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; D017228:Hepatocyte Growth Factor