Published online before print February 3, 2005, doi: 10.1161/01.RES.0000158286.51045.16
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© 2005 American Heart Association, Inc.
Molecular Medicine |
Novel Role of ARF6 in Vascular Endothelial Growth Factor–Induced Signaling and Angiogenesis
From the Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga.
Correspondence to Masuko Ushio-Fukai, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, Rm 319, Atlanta, GA 30322. E-mail mfukai{at}emory.edu
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.
Key Words: ARF6 • caveolin • vascular endothelial growth factor • endothelial cell • angiogenesis
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