Occludin independently regulates permeability under hydrostatic pressure and cell division in retinal pigment epithelial cells.
OBJECTIVE
The aim of this study was to determine the function of the tight junction protein occludin in the control of permeability, under diffusive and hydrostatic pressures, and its contribution to the control of cell division in retinal pigment epithelium.


METHODS
Occludin expression was inhibited in the human retinal pigment epithelial cell line ARPE-19 by siRNA. Depletion of occludin was confirmed by Western blot, confocal microscopy, and RT-PCR. Paracellular permeability of cell monolayers to fluorescently labeled 70 kDa dextran, 10 kDa dextran, and 467 Da tetramethylrhodamine (TAMRA) was examined under diffusive conditions or after the application of 10 cm H2O transmural pressure. Cell division rates were determined by tritiated thymidine incorporation and Ki67 immunoreactivity. Cell cycle inhibitors were used to determine whether changes in cell division affected permeability.


RESULTS
Occludin depletion increased diffusive paracellular permeability to 467 Da TAMRA by 15%, and permeability under hydrostatic pressure was increased 50% compared with control. Conversely, depletion of occludin protein with siRNA did not alter diffusive permeability to 70 kDa and 10 kDa RITC-dextran, and permeability to 70 kDa dextran was twofold lower in occludin-depleted cells under hydrostatic pressure conditions. Occludin depletion also increased thymidine incorporation by 90% and Ki67-positive cells by 50%. Finally, cell cycle inhibitors did not alter the effect of occludin siRNA on paracellular permeability.


CONCLUSIONS
The data suggest that occludin regulates tight junction permeability in response to changes in hydrostatic pressure. Furthermore, these data suggest that occludin also contributes to the control of cell division, demonstrating a novel function for this tight junction protein.
Phillips
Brett E
BE
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033-0850, USA.
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Limary
L
Tarbell
John M
JM
Antonetti
David A
DA
D001813:Blood-Retinal Barrier / Q000502:physiology*; D015153:Blotting, Western; D002455:Cell Division / Q000502:physiology*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D002470:Cell Survival; D004247:DNA / Q000096:biosynthesis; D003911:Dextrans / Q000378:metabolism; D018274:Electroporation; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006874:Hydrostatic Pressure; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism* / Q000648:ultrastructure; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012235:Rhodamines / Q000378:metabolism; D019108:Tight Junctions / Q000502:physiology*; D014162:Transfection; D062826:Zonula Occludens-1 Protein
D003911:Dextrans; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D012235:Rhodamines; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C005358:tetramethylrhodamine; D004247:DNA