Occludin independently regulates permeability under hydrostatic pressure and cell division in retinal pigment epithelial cells

Abstract

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.

Figure 1

Junctional protein and mRNA content after occludin depletion. ARPE-19 cells were transfected with negative control (CN) or occludin-targeted siRNA (siRNA). Cells were harvested 4 days later, and mRNA and protein content were analyzed. (A) RT-PCR quantification, mean, and SE of the mean (SEM) of CN and siRNA-treated cells (n = 6). (B) Western blot analysis of ZO-1, occludin, and claudin-1. (C) Densitometric quantification of Western blot, mean, and SEM (n = 3). Of the junctional components examined, only the tight junctional protein occludin showed significant changes in mRNA and protein content. Two-tailed t-test was performed to determine significance. **P < 0.01; ***P < 0.001.

Figure 2

Occludin siRNA treatment did not inhibit ARPE-19 tight junction formation. ARPE-19 cells were treated with control negative and occludin-targeted siRNA (siRNA). Cells reached confluence 1 day after treatment and were fixed on day 4. (A) Immunocytochemistry was performed on ARPE-19 monolayers for tight junction proteins occludin (green) and ZO-1 (red). Occludin content and localization at the plasma membrane were reduced with occludin siRNA treatment, while ZO-1 was unaffected. (B) Sections were cut perpendicularly to the plane of the fixed cell monolayer and were examined by electron microscopy. These images were of cells exposed to transmural 10 cm H₂O pressure before fixation. Nonpressurized cells were similar in appearance (data not shown).

Figure 3

Diffusive paracellular permeability of ARPE-19 monolayers is modestly affected by occludin siRNA treatment. Graphs display permeability mean and SEM. (A) Paracellular permeability to 70 kDa (n = 20) and 10 kDa (n = 10) RITC-dextran molecules was measured over a 3.5-hour time course and the 467 Da TAMRA (n = 15) over a 2-hour time course. (B) ARPE-19 monolayer TER was measured twice per transwell filter and averaged (n = 32). Two-tailed t-test was performed to determine significance. *P < 0.05; ***P < 0.001.

Figure 4

Occludin dynamically regulates paracellular permeability under hydrostatic pressure. (A). Ten centimers H₂O transmural pressure was applied to ARPE-19 monolayers at 0 minutes, and permeability to 70 kDa was calculated over 5-minute time intervals for 2 hours (CN, n = 8; siRNA, n = 7). Pressure application initially resulted in a fourfold to sevenfold elevation of permeability to 70 kDa dextran, followed by a reduction over the next 60 minutes. Statistical significance was determined from 65 to 115 minutes by two-way ANOVA (P < 0.05) and two-tailed t-test performed on AUC values (P < 0.01). (B) ARPE-19 permeability to 467 Da TAMRA was measured starting 1 hour after pressure application, and statistical significance was determined over 65 to 145 minutes (ANOVA, P < 0.05; AUC, P < 0.05; CN, n = 6; siRNA, n = 8). (C) Comparison of diffusive permeability (Fig. 3A) and average permeability under pressure (over the same timeframe described) for 70 kDa dextran and TAMRA permeability. Statistical significance was determined as stated (*P < 0.05). Occludin siRNA treatment increased permeability to TAMRA in diffusive and pressured conditions but decreased permeability to 70kDa RITC-dextran compared with control-treated cells under pressure, suggesting occludin dynamically regulates permeability.

Figure 5

ARPE-19 cell division rates increase after occludin siRNA treatment. ARPE-19 cells were treated with control (CN) and occludin-targeted siRNA (siRNA). Graphs display mean and SEM values. (A) Immunocytochemistry was performed with Hoechst nuclear stain (gray cells) and Ki67 antibody (white cells) on confluent ARPE-19 monolayers. Ki67 is a nuclear protein expressed only in actively dividing cells during the G1, S, G2, and mitotic cell cycle phases. (B) Cell density was quantified by counting the number of Hoechst-stained cells per field and was unchanged between treatment conditions (n = 3). (C) The percentage of cells that stained positive for Ki67 increased 50% with occludin siRNA treatment compared with the control scrambled treatment (n = 3). (D) ³H-thymidine incorporation over 2 hours increased 90% in occludin siRNA-treated cells compared with control scrambled-treated cells (n = 12). (E) Flow cytometry was performed on resuspended ARPE-19 monolayers (CN, n = 7; siRNA, n = 8). Cells were labeled with Annexin V-PE, which binds to the phosphatidylserine exposed on the cell membrane surface during early apoptosis, and the vital cell dye 7-AAD, which is excluded from living cells. Quadrant analysis was performed, and cells stained positively for Annexin V or 7-AAD were designated as dead, whereas unstained cells were designated as live. ARPE-19 monolayers were incubated with (F) calcein, which is taken up by living cells, and (G) ethidium bromide, which is excluded from living cells (CN, n = 9; siRNA, n = 8; alcohol, n = 4). A positive cell death control was treated with 70% ethanol for 30 minutes. Relative incorporation was measured with a spectrophotometer. No significant difference in cell viability existed between siRNA-treated and control APRE-19 cells. DNA content was measured in APRE-19 (H) cell monolayers and (I) cell culture media on cells from day 3 to day 4 after siRNA treatments (n = 6). DNA content did not change in the cell monolayer but significantly increased twofold in cell media from control scrambled-treated to occludin siRNA-treated cells. These data demonstrate occludin siRNA treatment increased cell division rates without changing cell number or viability within ARPE-19 monolayers compared with control. Excess cells are released into the media. Two-tailed t-test or ANOVA (if more than two conditions) was performed to determine significance (*P < 0.05; ***P < 0.001).

Figure 6

Paracellular permeability is independent of cell division rates. ARPE-19 cells were treated with control (CN) and occludin-targeted siRNA (siRNA). Graphs display mean and SEM values. (A) Tritiated thymidine incorporation was measured in ARPE-19 monolayers (n = 5). Treatment with aphidicolin or roscovitine blocked tritiated thymidine incorporation and eliminated any effect of occludin siRNA on DNA synthesis. ARPE-19 monolayers were assessed for (B) TER (DMSO, n = 29; aphidicolin, n = 2; roscovitine, n = 18) and permeability to (C) 70 kDa dextran (n = 12–14) and (D, E) TAMRA (n = 19 –22) under diffusive conditions. Inhibition of cell division had no significant effect on TER or solute permeability. Statistical significance was determined by ANOVA (*P < 0.05; ***P < 0.001).