Abstract 532: Protein Phosphatase 1B as an Endogenous Negative Regulator for VEGF Signaling Linked to Angiogenesis in Endothelial Cells.
Vascular endothelial growth factor (VEGF) stimulates angiogenesis through increasing endo-thelial cell (EC) proliferation and migration primarily through the VEGFR2 receptor. Tyrosine phosphorylation events are regulated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs). PTP inhibitors have been shown to promote neovascularization in ischemia hindlimb models. The role of endogenous PTPs in VEGF signaling is incompletely understood. Here we show that PTP1B is dramatically upregulated (10.3-fold) in a mouse hindlimb ischemia model of angiogenesis, which is associated with an increase in the expression of VEGF and phospho-VEGFR2, and capillary density. Functional consequence of upregulated PTP1B in VEGF signaling is demonstrated by the observation that overexpression of WT-PTP1B, but not catalytically inactive mutant PTP1B-C/S, significantly inhibits VEGFR2 autophopsphorylation, phosphorylation of ERK1/2 and paxillin as well as EC proliferation (95.8%, 85.8%, 97.6%, 58.2%, respectively) without affecting either EC migration or phosphorylation of p38MAP kinase in ECs. Co-immunoprecipitation of PTP1B with VEGFR2 in EC as well as co-transfection of myc-tagged VEGFR2 cytosolic domain and flag-tagged PTP1B in HEK293 cells demonstrate that PTP1B directly binds to VEGFR2 in vivo. In vivo dephosphor-ylation assay using recombinant active PTP1B protein and VEGF-stimulated EC lysates immunoprecitated with VEGFR2 shows that PTP1B dephosphorylates activated VEGFR2. VEGF stimulation transiently increases phosphatase activity of PTP1B (1.4-fold), which is cotempo-raneous with the tyrosine phosphorylation of VEGFR2. Knockdown of PTP1B by siRNA enhances VEGF-induced VEGFR2 autophosphorylation and EC proliferation (2.1-fold and 1.5-fold, respectively) without affecting EC migration. In summary, these results suggest that PTP1B negatively regulates VEGFR2 receptor activation via direct binding to the VEGFR2, which in turn selectively inhibits VEGF-stimulated EC proliferation. This mechanism should provide insights into a novel role of PTP1B, as an endogenous negative regulator, in participating in an acute cellular adaptation to an increase in VEGF signaling and postnatal angiogenesis in vivo.