The Tight Junction Protein Occludin Contributes to Centrosome Separation, Cell Cycle Progression, and Barrier Properties

Open Access
- Author:
- Runkle, Edwin Aaron
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 14, 2010
- Committee Members:
- David Antonetti, Dissertation Advisor/Co-Advisor
David A Antonetti, Committee Chair/Co-Chair
Leonard Shelton Jefferson Jr., Committee Chair/Co-Chair
Scot R Kimball, Committee Member
Christopher J Lynch, Committee Member
Lisa M Shantz, Committee Member
Christopher Charles Norbury, Committee Member - Keywords:
- Mitosis
Centrosomes
Tight Junctions
Occludin
Barrier Function - Abstract:
- The transmembrane tight junction (TJ) protein occludin has been implicated in a variety of cell processes including barrier properties particularly towards small cations, regulation of permeability through endocytosis, and growth control. A number of studies have identified phosphorylation sites on occludin that regulate its interaction with other junctional proteins and may regulate its function. The overall goal of this dissertation was to test the hypothesis that phosphorylation of the tight junction protein occludin on Ser490 contributes to growth control and barrier function. In this study, the Madin-Darby canine kidney (MDCK) cell line was used to study the function of a previously identified occludin phosphorylation site, Ser490. Stable transfection of the phosphomimetic Ser490 (S490D) and the non-phosphorylatable (S490A) mutants along with the wild type (WT) and empty vector (EV) were used to determine the role of occludin Ser490 in growth control. Here, we show that occludin is present at centrosomes in interphase and occludin Ser490 is phosphorylated throughout mitosis and co-localized with mitotic centrosomes in MDCK cells. Occludin S490D expressing cells showed localization of occludin at centrosomes and increased proliferation. Conversely, occludin S490A impeded centrosome separation, lowered the mitotic index following release from S-phase synchronization, and reduced proliferation. This chapter of the dissertation provides evidence that occludin is present at centrosomes and potentially regulates cell cycle progression through Ser490 phosphorylation mediated centrosome separation and subsequent mitotic entry. In primary bovine retinal endothelial cells (BREC), phosphorylation of occludin Ser490 controls vascular endothelial growth factor (VEGF)-induced permeability. While this process is now well-defined in the retinal vasculature, an understanding of the occludin Ser490 phosphorylation contribution to small molecule permeability of epithelial cells and TJ assembly is lacking. Platelet-derived growth factor (PDGF) treatment of MDCK cells results in occludin phosphorylation and redistribution from the TJ concurrent with increases in permeability similar to the changes observed in endothelial cells treated with VEGF. Here, we verified conservation of occludin Ser490 phosphorylation in MDCK cells in response to PDGF treatment. Occludin expression was sufficient to reduce transcellular permeability to 467 Da tetramethylrhodamine (TAMRA) and arginine (Arg); however, occludin S490A further reduced TAMRA permeability while occludin S490D failed to reduce Arg permeability to the extent of wild-type (WT) and S490A. In addition, occludin S490D cells assembled the TJ in a manner similar to those expressing the empty vector, while expression of occludin WT accelerated TJ assembly. Interestingly, occludin S490A further accelerated TJ assembly. No construct affected protein content; however, in cells that stably express occludin S490A ZO-1 appeared at the TJ earlier during the recovery period. Collectively, this chapter of the dissertation provides evidence that inhibition of occludin Ser490 phosphorylation tightens the barrier and accelerates TJ assembly. Angiogenesis is a major feature of pathology of retinal diseases and complications including retinopathy of prematurity (ROP) and diabetic retinopathy. A common feature of these pathologies is an increase in VEGF. Here, we show that similarly to epithelial cells, occludin Ser490 phosphorylation is increased during mitosis. In addition, occludin Ser490 phosphorylation is increased during early angiogenesis in the oxygen-induced retinopathy model, which mimics ROP. Further, transient expression of occludin S490A in BREC reduced proliferation basally and in response to VEGF while occludin Ser490 phosphorylation state did not affect endothelial cell migration in a wound healing assay. Finally, in agreement with previous studies, reduction of occludin in human retinal endothelial cells increased proliferation. This chapter of the dissertation provides data that identifies occludin as a growth regulator in endothelial cells and suggests occludin Ser490 phosphorylation is important to retinal angiogenesis, likely as an early event, and while phosphorylation status is critical to VEGF-induced proliferation and permeability, it likely does not affect migration. The work presented here extends previous findings in regards to occludin Ser490 phosphorylation to state that this residue is critical to growth control as well as barrier function. The data described in this dissertation, taken together with published reports, strongly implicate occludin Ser490 phosphorylation as a potential point of therapeutic intervention in diseases of angiogenesis. Thus, this dissertation demonstrates that the tight junction protein occludin contributes to centrosome separation, cell cycle progression, and barrier function.