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

The blood-retinal barrier is composed of epithelial cells of the retinal pigment epithelium and endothelial cells of the retinal vasculature that are under transmural hydrostatic pressure gradients and are composed of cells with a very low mitotic index with well-developed tight junctions. Tight junctions establish physiological barriers that regulate the movement of ions, small solutes, and fluid between tissue compartments.1,2 The retinal pigment epithelium contributes to the outer portion of the blood retinal barrier,3,4 and the retinal pigment epithelial (RPE) dysfunction caused by tight junction opening and increased permeability results in macular edema associated with retinitis pigmentosa.5–8 Transmembrane proteins that contribute to tight junction barrier properties include claudins, tricellulin, and occludin.9–12 Occludin is a tetraspan transmembrane protein located at tight junctions but its precise cellular functions remain unclear. Changes in occludin content correlate with altered permeability, making occludin a likely regulator of overall paracellular permeability of the tight junction.13–16 Similarly, the loss of occludin protein is associated with a number of abnormalities such as diabetic retinopathy14 and inflammatory bowel disease.17 Treatment of RPE cells with hepatocyte growth factor18 or interleukin-1β19 reduced occludin content associated with increased permeability. Furthermore, treatment of ARPE19 cells with matrix metalloproteinases 2 and 9, two proteinases elevated in the retina in diabetes, specifically decreased occludin content and increased permeability.20 These studies demonstrate that cytokines and proteinases that are altered in a number of retinal diseases, including diabetic retinopathy, reduce occludin content associated with increased permeability. The contribution of occludin to barrier properties remains controversial. Blocking the extracellular loops21–25 and reducing the protein content26 of occludin alter paracellular permeability in a number of cell systems. Occludin-deficient mice are viable; tight junction ultrastructure appears unaltered, and isolated intestinal tissues demonstrate normal transepithelial resistance (TER) and permeability to mannitol. These mice do, however, display a number of aberrant phenotypes in tissues containing tight junctions, including testicular atrophy in males, inability to suckle in females, gastric mucus cell hyperplasia, and calcification of the brain.27,28 Additional evidence for occludin in regulating epithelial cell division has been suggested by the ability of exogenous occludin expression to revert the phenotype of raf-transformed rat salivary gland epithelial cells.29 Most occludin permeability studies are performed at or near atmospheric pressure, and the role of occludin has yet to be assessed under a transmural pressure gradient that more closely models physiological conditions. The net movement of water across tissue barriers is governed by differential fluid and oncotic pressure, as expressed in Starling’s equations and subsequent adaptations (for a review, see Salathe30). Application of a transmural hydrostatic pressure gradient across endothelial cell monolayers rapidly increases effective permeability of solutes through convective forces. However, during the 30 to 60 minutes after pressurization, the endothelial cells respond by reducing effective solute and water permeability.31–33 This sealing effect34 is an adaptive biological response to changes in hydrostatic pressure.33 In the present study, the contribution of occludin to regulation of the blood-retinal barrier after application of hydrostatic pressure was examined using the RPE cell line ARPE-19. This cell line has well-developed tight junctions in stable monolayers.3,4 In addition, these studies addressed the novel hypothesis, suggested by occludin knockout mice, that occludin may contribute to growth control, and they examined the relationship of growth control to permeability under diffusive conditions.