Abstract
Purpose :
IQGAP1, a scaffold protein with a Rac1 binding domain, regulates signaling leading to cell migration and adhesion. We previously found that VEGF-mediated Rac1 activation is required for choroidal endothelial cell (CEC) activation and migration, a necessary step in the development of choroidal neovascularization in neovascular age-related macular degeneration (AMD). We, therefore, tested the hypothesis that IQGAP1 mediates VEGF-induced Rac1 activation and CEC migration in cultured human primary CECs and also investigated the mechanisms involved.
Methods :
Primary human CECs treated with control PBS or VEGF (20 ng/ml) for 30 mins were assayed for: 1) Rac1 activation by immunoprecipitation (IP) with anti-GTP-Rac1 antibody and western blot of total Rac1, 2) co-IP of IQGAP1 and active Rac1 (GTP-Rac1) to determine their interaction, and 3) phosphorylation of IQGAP1 measured by IP with anti-IQGAP1 and western blot with anti-phospho-serine antibodies. To determine if serine phosphorylation of IQGAP1 is necessary for VEGF-mediated Rac1 activation and CEC migration, VEGF-induced VEGFR2 phosphorylation (p-VEGFR2) and Rac1 activation (GTP-Rac1), and CEC migration were measured in CECs co-transfected with IQGAP1 siRNA and plasmid DNA expressing either wild type IQGAP1 (p-EGFP-IQGAP1-WT) or IQGAP1 with serine 1441/1443 mutations into alanine (p-EGFP-IQGAP1-S1441A/S1443A). Statistics were performed using ANOVA and a minimum P value of <0.05 was considered statistically significant. Results were mean ±SE. Experiments included 6 samples/condition.
Results :
In CECs and compared to PBS, VEGF caused a 2 fold increase in GTP-Rac1, increased Rac1 interaction with IQGAP1, and promoted serine phosphorylation of IQGAP1. Expression of exogenous p-EGFP-IQGAP1-S1441A/S1443A in CECs with endogenous IQGAP1 knockdown inhibited VEGF-induced p-VEGFR2 and GTP-Rac1, and CEC migration (p< 0.01) compared to CECs transfected with p-EGFP-IQGAP1-WT.
Conclusions :
IQGAP1 mediates VEGF-induced CEC Rac1 activation and migration in a serine phosphorylation-dependent manner. Future studies are required to determine the role of CEC IQGAP1 in choroidal neovascularization in vivo. These data may provide insight into mechanisms underlying neovascular AMD.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.