Occludin regulates permeability under hydrostatic pressure and cell division in retinal pigment epithelial cells
Brett Eugene Phillips 1*,
Limary M Cancel 2,
John M Tarbell 3,
and
David A. Antonetti 1
1 Dept. Cellular & Molecular Physiology, Penn State Univ. College of Medicine, Hershey, Pennsylvania, United States
2 Biomedical Engineering Department, The City College of New York, New York, New York, United States
3 Biomedical Engineering, The City College of New York, New York, New York, United States
* To whom correspondence should be addressed. E-mail: bep14{at}pitt.edu.
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Abstract |
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Purpose. The aim of this study was to determine the function of the tight junction protein occludin in control of permeability, under diffusive and hydrostatic pressures, as well as its contribution to control of cell division in retinal pigment epithelia (RPE).
Methods. Occludin expression was inhibited in the human RPE 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 10cm 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 to 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 two-fold 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.
Key Words:
diabetic retinopathy, blood-retinal barrier, retinal pigment epithelium, tight junction
