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.
Daher
Zeinab
Z
Department of Biochemistry, Membrane Protein Study Group (GEPROM), Faculty of Medicine, University of Montréal, PO Box 6128, Downtown station, Montréal, Canada H3C 3J7.
Noël
Josette
J
Claing
Audrey
A
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002465:Cell Movement; D042783:Endothelial Cells / Q000201:enzymology / Q000502:physiology*; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1 / Q000494:pharmacology*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D018715:Microscopy, Video; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D034741:RNA, Small Interfering; D044023:Receptor, Endothelin B / Q000378:metabolism; D013997:Time Factors; D019061:src-Family Kinases / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1; C485647:GIT1 protein, human; D009842:Oligopeptides; D010880:Piperidines; D034741:RNA, Small Interfering; D044023:Receptor, Endothelin B; C086539:BQ 788; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human