Semester

Spring

Date of Graduation

2008

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Fred Minnear

Abstract

Sphingosine 1-phosphate (S1P), a biologically active lipid, induces a myriad of cellular events including enhancement of the endothelial barrier and assembly of adherens junction proteins, VE-cadherin and catenins. The over all goal in this set of studies was to investigate what cellular mechanisms contribute to S1P-induced enhancement of the vascular endothelial barrier.;Since VE-cadherin contributes to the stabilization of the endothelial barrier, I determined in the first study if the rapid, barrier-enhancing activity of S1P requires VE-cadherin. Ca2+-dependent, homophilic VE-cadherin binding of endothelial cells, derived from human umbilical veins and grown as monolayers, was disrupted with EGTA, an antibody to the extracellular domain of VE-cadherin, or gene silencing of VE-cadherin with small interfering RNA (siRNA). All three protocols caused a reduction in the immunofluorescent localization of VE-cadherin at intercellular junctions, the separation of adjacent cells, and a decrease in basal, endothelial electrical resistance. In all three conditions, S1P rapidly increased endothelial electrical resistance. These findings demonstrate that S1P enhances the endothelial barrier independently of homophilic VE-cadherin binding. Junctional localization of VE-cadherin, however, was associated with the sustained activity of S1P. Imaging with phase-contrast and differential interferencecontrast (DIC) optics revealed that S1P induced cell spreading and closure of intercellular gaps. Pretreatment with Latrunculin B, an inhibitor of actin polymerization, or Y-27632, a Rho kinase inhibitor, attenuated cell spreading and the rapid increase in electrical resistance induced by S1P. I conclude that S1P rapidly closes intercellular gaps, resulting in an increased electrical resistance across endothelial cell monolayers, via cell spreading and Rho kinase and independently of VE-cadherin.;Based on the previous observation that S1P increases the localization of junctional VE-cadherin as early as 10 min, I determined in the second study what cellular mechanisms contributed to this increase. Because VE-cadherin dynamically traffics between cell surface and cytoplasmic vesicles, I hypothesized that S1P can regulate VE-cadherin trafficking. Immunofluorescence microscopy and a biotinylated cell impermeable reagent were used to demonstrate internalized and cell surface VE-cadherin. Endocytosis of VE-cadherin was induced by EGTA or VEGF. S1P decreased EGTA-induced endocytosis and co-localization of internalized VE-cadherin with EEA 1, a marker of early endosomes, and prevented and reversed the effect of VEGF. S1P also increased the recovery of cell surface VE-cadherin after endocytosis by EGTA or VEGF and increased the co-localization of internalized VE-cadherin with Rab11, a marker of the recycling endosome. Bafilomycin A1, an inhibitor of recycling, and microtubule inhibitors, taxol and colchicine, blocked the increase in junctional VE-cadherin and the sustained increase in endothelial electrical resistance induced by S1P, the latter using the Ca2+-switch protocol. Bafilomycin A1 also prevented the increase in recovery of cell surface VE-cadherin induced by S1P. When co-treated or post-treated with VEGF, S1P blocked or reversed the increased interaction of VE-cadherin with beta-arrestin2, an endocytic adapter protein. I conclude that S1P stabilizes homophilic VE-cadherin binding by decreasing endocytosis and increasing recycling of VE-cadherin, the latter requiring microtubules.;In the third study, I investigated the cellular mechanisms contributing to the regulatory effects of S1P on VE-cadherin trafficking. S1P increased co-localization and interaction of p120 with VE-cadherin and increased biotinylated, cell surface VE-cadherin and p120 with or without EGTA pretreatment. In addition, S1P enhanced the co-localization of p120 with EGFP-tagged Rab11, a maker of recycling endosomes. VEGF decreased the interaction of VE-cadherin with p120, and co-treatment or posttreatment with S1P blocked or reversed, respectively, this effect of VEGF. Interestingly, S1P increased the interaction of p120 with kinesin, a motor protein that moves along microtubules to transport vesicles.;In summary, I conclude that S1P enhances the endothelial barrier through multiple ways: the rapid increase in endothelial barrier function is independent of VEcadherin, but requires Rho kinase and actin cytoskeleton-based cell spreading; sustained enhancement of the barrier is related to an increase in junctional VE-cadherin on which S1P has a novel effect---regulation of VE-cadherin trafficking, i.e. decreasing endocytosis and increasing recycling of VE-cadherin. p120 possibly is involved in this novel regulatory effect of S1P. Augmented interaction of p120 with kinesin by S1P may facilitate transport of recycling VE-cadherin via microtubules.

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