Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) receptor-1 down-modulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF ) achieves its multiple functions by activating two receptor tyrosine kinas es, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2), both of which are se lectively expressed on primary vascular endothelium. To dissect the respect ive signaling pathways and biological functions mediated by these receptors in primary endothelial cells with these two receptors intact, we developed a chimeric receptor system in which the N terminus of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular do main of KDR (EGDR) and Flt-1 (EGLT). We observed that KDR, but not Flt-1, w as responsible for VPF/VEGF-induced human umbilical vein endothelial cell ( HUVEC) proliferation and migration. Moreover, Flt-1 showed an inhibitory ef fect on KDR-mediated proliferation, but not migration. We also demonstrated that the inhibitory function of Flt-1 was mediated through the phosphatidy linositol 3-kinase (PI-3K)-dependent pathway because inhibitors of PI-3K as well as a dominant negative mutant of p85 (PI-3K subunit) reversed the inh ibition, whereas a constitutively activated mutant of p110 introduced the i nhibition to HUVEC-EGDR. We also observed that, in VPF/VEGF-stimulated HUVE Cs, the Flt-1/EGLT-mediated down-modulation of KDR/EGDR signaling was at or before intracellular Ca2+ mobilization, but after KDR/EGDR phosphorylation . By mutational analysis, we further identified that the tyrosine 794 resid ue of Flt-1 was essential for its antiproliferative effect. Taken together, these studies contribute significantly to our understanding of the signali ng pathways and biological functions triggered by KDR and Flt-1 and describ e a unique mechanism in which PI-3K acts as a mediator of antiproliferation in primary vascular endothelium.