Flt-1-mediated down-regulation of endothelial cell proliferation through pertussis toxin-sensitive G proteins, beta gamma subunits, small GTPase CDC42, and partly by Rac-1.
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, whereas Flt-1 activation down-modulates KDR-mediated EC proliferation. Although 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 Ca(2+) mobilization in response to VPF/VEGF but have no effect on KDR and MAPK phosphorylation. Using the chimeric-receptor EGLT in which the extracellular domain of epidermal growth factor receptor 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 is required for Flt-1-mediated antiproliferative activity, but phospholipase C is not required. 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 but not by either the phospholipase C inhibitor, or an intracellular Ca(2+) chilator BAPTA/AM. Surprisingly, pertussis toxin and overexpression of the free Gbetagamma-specific sequestering minigene hbetaARK1(495) also inhibit EGLT-mediated CDC42 and Rac1 activation completely. Moreover, pertussis toxin treatment also increases the intracellular Ca(2+) mobilization and inhibits the antiproliferation activity, thus suggesting that pertussis toxin-sensitive G proteins and the Gbetagamma subunits are involved in the signaling pathway of Flt-1 that down-regulates EC proliferation. Taken together, these results further expand our understanding of Flt-1-mediated antiproliferative activity in VPF/VEGF-stimulated endothelium.
Zeng
Huiyan
H
Department of Pathology, Beth Israel Deaconess Medical Center and Harvard University School of Medicine, Boston, Massachusetts 02215, USA.
Zhao
Dezheng
D
Mukhopadhyay
Debabrata
D
D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000502:physiology*; D002460:Cell Line; D015536:Down-Regulation / Q000502:physiology*; D016228:Endothelial Growth Factors / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology*; D016326:Extracellular Matrix Proteins / Q000502:physiology*; D006801:Humans; D008222:Lymphokines / Q000502:physiology; D009154:Mutation; D037342:Pertussis Toxin; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000502:physiology*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000502:physiology*
D016228:Endothelial Growth Factors; D016326:Extracellular Matrix Proteins; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D002118:Calcium