Flt-1-mediated down-regulation of endothelial cell proliferation through pertussis toxin-sensitive G proteins, βγ subunits, small GTPase CDC42, and partly by Rac-1

Huiyan Zeng, Dezheng Zhao, Debabrata Mukhopadhyay

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Abstract

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 Ca2+ 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, U73122, or an intracellular Ca2+ chilator BAPTA/AM. Surprisingly, pertussis toxin and overexpression of the free Gβγ-specific sequestering minigene hβARK1(495) also inhibit EGLT-mediated CDC42 and Rac1 activation completely. Moreover, pertussis toxin treatment also increases the intracellular Ca2+ mobilization and inhibits the antiproliferation activity, thus suggesting that pertussis toxin-sensitive G proteins and the Gβγ 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.

Original languageEnglish (US)
Pages (from-to)4003-4009
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number6
DOIs
StatePublished - Feb 8 2002
Externally publishedYes

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Monomeric GTP-Binding Proteins
Pertussis Toxin
Endothelial cells
Protein Subunits
Cell proliferation
GTP-Binding Proteins
Vascular Endothelial Growth Factor A
Down-Regulation
Endothelial Cells
Cell Proliferation
Chemical activation
Type C Phospholipases
Cell Movement
Phosphatidylinositol 3-Kinase
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factor Receptor-2
Phosphorylation
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Receptor Protein-Tyrosine Kinases
Phosphatidylinositol 3-Kinases

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{074588e130fa4c1a8de0b02ca561663c,
title = "Flt-1-mediated down-regulation of endothelial cell proliferation through pertussis toxin-sensitive G proteins, βγ subunits, small GTPase CDC42, and partly by Rac-1",
abstract = "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 Ca2+ 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, U73122, or an intracellular Ca2+ chilator BAPTA/AM. Surprisingly, pertussis toxin and overexpression of the free Gβγ-specific sequestering minigene hβARK1(495) also inhibit EGLT-mediated CDC42 and Rac1 activation completely. Moreover, pertussis toxin treatment also increases the intracellular Ca2+ mobilization and inhibits the antiproliferation activity, thus suggesting that pertussis toxin-sensitive G proteins and the Gβγ 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.",
author = "Huiyan Zeng and Dezheng Zhao and Debabrata Mukhopadhyay",
year = "2002",
month = "2",
day = "8",
doi = "10.1074/jbc.M110842200",
language = "English (US)",
volume = "277",
pages = "4003--4009",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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TY - JOUR

T1 - Flt-1-mediated down-regulation of endothelial cell proliferation through pertussis toxin-sensitive G proteins, βγ subunits, small GTPase CDC42, and partly by Rac-1

AU - Zeng, Huiyan

AU - Zhao, Dezheng

AU - Mukhopadhyay, Debabrata

PY - 2002/2/8

Y1 - 2002/2/8

N2 - 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 Ca2+ 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, U73122, or an intracellular Ca2+ chilator BAPTA/AM. Surprisingly, pertussis toxin and overexpression of the free Gβγ-specific sequestering minigene hβARK1(495) also inhibit EGLT-mediated CDC42 and Rac1 activation completely. Moreover, pertussis toxin treatment also increases the intracellular Ca2+ mobilization and inhibits the antiproliferation activity, thus suggesting that pertussis toxin-sensitive G proteins and the Gβγ 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.

AB - 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 Ca2+ 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, U73122, or an intracellular Ca2+ chilator BAPTA/AM. Surprisingly, pertussis toxin and overexpression of the free Gβγ-specific sequestering minigene hβARK1(495) also inhibit EGLT-mediated CDC42 and Rac1 activation completely. Moreover, pertussis toxin treatment also increases the intracellular Ca2+ mobilization and inhibits the antiproliferation activity, thus suggesting that pertussis toxin-sensitive G proteins and the Gβγ 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.

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