Vascular endothelial growth factor receptor-2 activates ADP-ribosylation factor 1 to promote endothelial nitric-oxide synthase activation and nitric oxide release from endothelial cells.

Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates endothelial function via production and release of nitric oxide (NO), an important signaling molecule. The molecular basis leading to NO production involves phosphatidylinositiol-3 kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) activation. In this study, we have examined whether small GTP-binding proteins of the ADP-ribosylation factor (ARF) family act as molecular switches to regulate signaling cascades activated by VEGF in endothelial cells. Our results show that this growth factor can promote the rapid and transient activation of ARF1. In endothelial cells, this GTPase is present on dynamic plasma membrane ruffles. Inhibition of ARF1 expression, using RNA interference, markedly impaired VEGF-dependent eNOS phosphorylation and NO production by preventing the activation of the PI3K/Akt signaling axis. Furthermore, our data indicate that phosphorylation of Tyr(801), on VEGF receptor 2, is essential for activating Src- and ARF1-dependent signaling events leading to NO release from endothelial cells. Lastly, this mediator is known to regulate a broad variety of endothelial cell functions. Depletion of ARF1 markedly inhibits VEGF-dependent increase of vascular permeability as well as capillary tubule formation, a process important for angiogenesis. Taken together, our data indicate that ARF1 is a novel modulator of VEGF-stimulated NO release and signaling in endothelial cells.

Endothelin-1 promotes migration of endothelial cells through the activation of ARF6 and the regulation of FAK activity.

Several proteins act in concert to promote remodeling of the actin cytoskeleton during migration. This process is highly regulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family of proteins. Here, we show that endothelin-1 (ET-1) can promote the activation of ARF6 and migration of endothelial cells through the activation of ET(B) receptors. Inhibition of ARF6 expression using RNA interference markedly impairs basal and ET-1 stimulated cell migration. In contrast, depletion of ARF1 has no significant effect. In order to delineate the underlying mechanism, we examined the signaling events activated in endothelial cells following ET-1 stimulation. Here, we show that this hormone promotes the phosphorylation of focal adhesion kinase (FAK), Erk1/2, and the association of FAK to Src, as well as of FAK to GIT1. These have been shown to be important for the formation and turnover of focal adhesions. In non-stimulated cells, depletion of ARF6 leads to increased FAK and Erk1/2 phosphorylation, similar to what is observed in ET-1 treated cells. In these conditions, FAK is found constitutively associated with the soluble tyrosine kinase, Src. In contrast, depletion of ARF6 impairs the ability of GIT1 to form an agonist promoted complex with FAK, thereby preventing disassembly of focal adhesions. As a consequence, ARF6 depleted endothelial cells are impaired in their ability to form capillary tubes. Taken together, our data suggest that ARF6 is central in regulating focal adhesion turnover in endothelial cells. Our study provides a molecular mechanism by which, this small GTPase regulates cell motility, and ultimately angiogenesis.

Autocrine activation of the hepatocyte growth factor receptor/met tyrosine kinase induces tumor cell motility by regulating pseudopodial protrusion.

The multiple beta-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFalpha antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFalpha antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line.