Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells.
Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.
Vouret-Craviari
Valérie
V
Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR6543, Centre Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice, France. vouret@unice.fr
Bourcier
Christine
C
Boulter
Etienne
E
van Obberghen-Schilling
Ellen
E
D000199:Actins / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D051356:Cortactin; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000254:growth & development*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007231:Infant, Newborn; D008246:Lysophospholipids; D008840:Microfilament Proteins / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D014471:Umbilical Veins; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
C412373:AG 1879; D000199:Actins; C495868:CTTN protein, human; D051356:Cortactin; D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D008840:Microfilament Proteins; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; C060506:sphingosine 1-phosphate; D019061:src-Family Kinases; D013917:Thrombin; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D013110:Sphingosine