Oxidative stress affects the junctional integrity of retinal pigment epithelial cells.
OBJECTIVE
Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration. The cell line ARPE-19 was therefore examined for response to oxidative stress and its effect on stress protein induction and junctional integrity.


METHODS
ARPE-19 cell viability after 1 week or 5 weeks in culture was assessed in response to different concentrations of hydrogen peroxide. The response to sublethal doses was assessed by examination of heme oxygenase (HO)-1, Hsp27 and Hsp70 by immunofluorescence and Western blot analysis. Immunofluorescence was used to investigate the localization of the junctional proteins zonula occludens (ZO)-1, occludin, and N-cadherin, and beta-catenin. Subcellular fractionation was used to assess any redistribution of beta-catenin. Monolayer integrity was examined by measurement of flux of rhodamine-conjugated dextrans from the apical to basal aspect of cells.


RESULTS
ARPE-19 cells cultured for 5 weeks were less sensitive to chronic oxidative stress induced by hydrogen peroxide than those cultured for 1 week. The more differentiated ARPE-19 cells had higher steady state levels of Hsp27 and Hsp70. The response to stress also differed with time in culture. The localization of junctional proteins, which became strongly peripheral after 5 weeks in culture, became disrupted after oxidative stress, and cytosolic beta-catenin increased. Chronic oxidative stress also increased paracellular flux across the monolayer.


CONCLUSIONS
Increased resistance to chronic oxidative stress with differentiation in ARPE-19 cells correlated with higher steady state levels of Hsp27 and Hsp70. Oxidative stress disrupted RPE cell junction and barrier integrity, which may contribute to the pathogenesis of diseases related to RPE through disruption of the blood-retinal barrier.
Bailey
Tracey A
TA
Division of Pathology, Institute of Ophthalmology, University College London, London, United Kingdom.
Kanuga
Naheed
N
Romero
Ignacio A
IA
Greenwood
John
J
Luthert
Philip J
PJ
Cheetham
Michael E
ME
D001813:Blood-Retinal Barrier / Q000187:drug effects; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism; D051547:Heme Oxygenase-1; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000378:metabolism; D062793:Occludin; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress; D010750:Phosphoproteins / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000378:metabolism / Q000473:pathology*; D015534:Trans-Activators / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin
C495270:CTNNB1 protein, human; D015820:Cadherins; D003598:Cytoskeletal Proteins; D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; C568985:OCLN protein, human; D062793:Occludin; D016877:Oxidants; D010750:Phosphoproteins; C569042:TJP1 protein, human; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; D006861:Hydrogen Peroxide; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1