Occludin Regulates Permeability and Cell Division in Retinal Pigmented Epithelium Cells
Open Access
- Author:
- Phillips, Brett E
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 19, 2007
- Committee Members:
- David A Antonetti, Committee Chair/Co-Chair
Anthony Edward Pegg, Committee Member
Charles H Lang, Committee Member
Jiyue Zhu, Committee Member
Steven F Abcouwer, Committee Member - Keywords:
- Cell Division
ARPE-19
Tight Junctions
Paracellular Permeability - Abstract:
- Numerous physiological barriers exist in the body to establish compartments with specific substrate concentrations. These compartments are required for various tissue functions such as nutrient absorption in the intestine, waste removal in the kidney, and neural function of the brain and retina. Barriers are established by the adherens and tight junction protein complexes that form junctional protein strands that link adjacent cells. The adherens junction establishes cell adherence and polarization forming uniform cell layers, while the apical tight junction regulates the movement of ions, solutes, and water between cell layers establishing physiologic gradients. The abundance of the tight junction protein occludin inversely correlates with paracellular permeability characteristics but its function remains unclear in physiological settings. Our hypothesis is that a reduction in occludin protein content will result in increased paracellular permeability, specifically with the addition of a physiologic transmural pressure gradient. To explore occludin’s role in the tight junction, occludin content was reduced 60% with transient introduction of short inhibitory RNA in ARPE-19 human retinal pigment epithelium cells. Reduction in occludin content did not alter mRNA or protein abundance of the junctional components zonula occludens-1, claudin-1, claudin-2, or epithelial cadherin, nor inhibit intracellular junction formation. Occludin protein reduction did not affect diffusive permeability to 70 kDa and 10 kDa dextran, but increased permeability to 467 Da tetramethylrhodamine (TAMRA) by 14% compared to control cells. Application of a 10 cmH2O transmural pressure gradient resulted in an initial increase in permeability, followed by an adaptive reduction in permeability over time. This phenomenon has been termed the “sealing effect” and has been previously observed in endothelial cells. Under a 10 cmH2O hydrostatic pressure, permeability to TAMRA increased 50% in occludin depleted cells compared to control. Conversely, permeability to 70 kDa dextran was decreased 50% in occludin-depleted cells compared to control cells. The data suggest that occludin is necessary for the cell’s adaptive response to pressure changes under physiologic conditions and occludin regulates small molecule (467 Da) permeability. Occludin content reduction also caused a 90% increase in DNA synthesis as determined by tritiated thymidine incorporation and a similar increase in cell division was confirmed by Ki67 immuno-staining. The use of cell cycle inhibitors revealed that increased cell division was not responsible for alterations in permeability. These data demonstrate for the first time that reduction of occludin increases cell division rates and reveals occludin’s contribution to small molecule permeability is independent of cell cycle control.