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Papers In Press, published online ahead of print November 28, 2001
Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215
Corresponding Author: dmukhopa{at}caregroup.harvard.edu
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) promotes its function primarily by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2). Recently, it has been shown that KDR is responsible for VPF/VEGF-stimulated endothelial cell (EC) proliferation and migration, while Flt-1 activation down-modulates KDR-mediated EC proliferation. Whereas KDR-mediated EC proliferation and migration have been extensively studied, much less is known about Flt-1-mediated antiproliferation. Here, we demonstrate that Flt-1-mediated antiproliferative activity can be blocked completely by the dominant negative mutant of CDC42 (CDC42-17N), and partially by a Rac1 dominant negative mutant (Rac1-17N), but is not affected by a RhoA dominant negative mutant (RhoA-19N). Both CDC42-17N and Rac1-17N increase the intracellular Ca2+ mobilization in response to VPF/VEGF but have no effect on KDR and MAPK phosphorylation. Utilizing the chimeric-receptor (EGLT) in which the extra cellular domain of epidermal growth factor receptor (EGFR) was fused to the transmembrane and intracellular domains of Flt-1, we also demonstrate that CDC42 and Rac1 are activated by EGLT. Previously, we showed that phosphatidylinositol 3-kinase (PI3K) is required for Flt-1-mediated antiproliferative activity but phospholipase C (PLC) is not. As expected, CDC42 and Rac1 activation mediated by EGLT can be completely inhibited by PI3K inhibitors (wortmannin and LY294002) and the p85 dominant negative mutant (p85DN), but not by either PLC inhibitor (U73122) or an intracellular Ca2+ chilator BAPTA/AM. Surprisingly, pertussis toxin and overexpression of the free Gbg specific sequestering minigene hbARK1(495) also completely inhibit EGLT-mediated CDC42 and Rac1 activation. Moreover, pertussis toxin treatment also increases the intracellular Ca2+ mobilization and inhibits the antiproliferation activity in HUVEC stimulated with VPF/VEGF, thus suggesting that pertussis toxin-sensitive G proteins and the Gbg subunits are involved in the signaling pathway of Flt-1 that down-regulates HUVEC proliferation. Taken together, these results further expand our understanding of Flt-1-mediated antiproliferative activity in VPF/VEGF-stimulated endothelium.
J. Biol. Chem, 10.1074/jbc.M110842200
Submitted on November 12, 2001
Revised on November 28, 2001
Accepted on November 27, 2001
Flt-1-mediated down-regulation of endothelial cell proliferation through pertussis toxin-sensitive G proteins, bg subunits, small GTPase CDC42 and partly by Rac-1
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