Semester

Summer

Date of Graduation

2006

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Fred L Minnear

Abstract

IQGAP1 overexpression inhibits E-cadherin-mediated epithelial cell-cell adhesion by distabilizing the adherens junctions, and activated Rac1 and Cdc42 may prevent these effects by removing IQGAP1 from the adherens junction complex. In the present study, we determined if IQGAP1 associates with the adherens junction of endothelial cells and affects endothelial barrier function. In human umbilical vein endothelial cells (HUVECs), IQGAP1 associated with VE-cadherin, the catenins, beta, gamma, alpha, and p120, but not with N-cadherin or the tight junction proteins, occludin, claudin-5, and ZO-1. Detergent extracted most of the IQGAP1 associated with VE-cadherin. Treatment of endothelial cells with sphingosine-1-phosphate (S1P), which increases the activity of Rac1, increased the association of IQGAP1 with Rac1, and the amount of insoluble VE-cadherin and beta-catenin at intercellular junctions. To determine if the increased localization of junctional VE-cadherin induced by S1P occurred via the removal of IQGAP1, the protein level of IQGAP1 was reduced with small interfering RNA or siRNA. Reduction of IQGAP1 by transfection of siRNA resulted in a higher endothelial electrical resistance in HUVECs as compared to transfection of a scrambled siRNA. Reduction of IQGAP1 also induced an increase and a decrease, respectively, in the protein levels of VE-cadherin and N-cadherin. Also, more VE-cadherin and less N-cadherin were associated with p120 and beta-catenin. Furthermore, more insoluble (cytoskeletal-associated) VE-cadherin was localized at intercellular junctions and less insoluble N-cadherin was present in HUVECs. Overexpression of a VE-cadherin-alpha-catenin fusion protein, which lacked the binding sites for beta-catenin on VE-cadherin and alpha-catenin, diminished the localization of junctional IQGAP1. These findings suggest that IQGAP1 knockdown positively influences the endothelial barrier by increasing the protein level of VE-cadherin and the interaction of VE-cadherin with the cytoskeleton, possibly by enhancing the p120-VE-cadherin association.

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