Novel role of ARF6 in vascular endothelial growth factor-induced signaling and angiogenesis.
Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts- and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.
Ikeda
Satoshi
S
Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga 30322, USA.
Ushio-Fukai
Masuko
M
Zuo
Lian
L
Tojo
Taiki
T
Dikalov
Sergey
S
Patrushev
Nikolay A
NA
Alexander
R Wayne
RW
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D019943:Amino Acid Substitution; D000818:Animals; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002458:Cell Fractionation; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D004789:Enzyme Activation; D005260:Female; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D006614:Hindlimb / Q000098:blood supply; D006801:Humans; D007511:Ischemia / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D017354:Point Mutation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000302:isolation & purification / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000302:isolation & purification / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D011505:Protein-Tyrosine Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse; D020830:rac1 GTP-Binding Protein