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This Article Full Text Full Text (PDF) All Versions of this Article: iovs.07-1204v1 49/6/2568    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Phillips, B. E. Articles by Antonetti, D. A. PubMed PubMed Citation Articles by Phillips, B. E. Articles by Antonetti, D. A.

Occludin Independently Regulates Permeability under Hydrostatic Pressure and Cell Division in Retinal Pigment Epithelial Cells

¹From the Departments of Cellular and Molecular Physiology and ³Ophthalmology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania; and the ²Biomedical Engineering Department, City College of New York, New York, New York.

PURPOSE. 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.