Abstract 230: Regulation of Cell-Cell Adhesion and ROS-dependent VEGF Signaling by Protein Tyrosine Phosphatase 1B in Endothelial Cells
VE-cadherin-mediated cell-cell adhesion is important for preserving endothelial integrity. Tyrosine phosphorylation of VE-cadherin is required for loss of cell-cell contact which is initial event to promote angiogenesis. We demonstrated that VEGF stimulates endothelial cell (EC) proliferation through reactive oxygen species (ROS) which mediate tyrosine phosphorylation of VEGFR2 and VE-cadherin. Little is known underlying molecular mechanisms. Tyrosine phosphorylation events are regulated by tyrosine kinases and protein tyrosine phosphatases (PTPs) which are inactivated by ROS. In this study, we found that PTP1B is markedly increased (10.8-fold) in a mouse hindlimb ischemia model of angiogenesis. In ECs VEGF stimulation rapidly inhibits PTP1B activity (28%), in a ROS-dependent manner, within 5 min, which is associated with VEGFR2 autophosphorylation. Knockdown of PTP1B by siRNA enhances VEGF-induced VEGFR2 autophosphorylation (2.1-fold) and EC proliferation (1.5-fold), suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. Consistent with this, overexpression of wild-type (WT)-PTP1B, but not catalytically inactive, substrate-trapping mutant PTP1B-C/S, inhibits phosphorylation of VEGFR2, ERK1/2 and paxillin as well as EC proliferation (95.8%, 85.8%, 97.6%, 58.2%, respectively). Furthermore, PTP1B-C/S, but not PTP1B-WT, is co-precipitated with VEGFR2 in ECs, suggesting that their association is tyrosine phosphorylation-dependent. In vivo dephosphorylation assay reveals that PTP1B directly dephosphorylates activated VEGFR2. Of note, overexpression of PTP-1B-C/S increases, while PTP1B-WT decreases VE-cadherin tyrosine phosphorylation in basal confluent ECs, suggesting that PTP1B functions to stabilize cell-cell adhesions. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2 and stabilizes cell-cell adhesion through reducing tyrosine phosphorylation of VE-cadherin. VEGF-induced ROS inactivate PTP1B to promote ROS-dependent VEGFR2 signaling linked to EC proliferation, which may contribute to angiogenesis.