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Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells

Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR6543, Centre Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice, France

View larger version (23K): [in a new window]   Fig. 1. Temporal activation of Rho A by thrombin and S1P. Confluent monolayers of HUVECs were cultivated as indicated in Materials and Methods and, after overnight starvation, were treated with thrombin (10 nM) or S1P (0.5 µM) for the times indicated. The amount of RBD-bound and total RhoA in cell extracts was determined by western analysis. A minor band, not regulated by agonists, was often detected above total RhoA, which likely corresponds to a geranyl-geranylated form of the protein. The results shown are representative of three independent experiments in which thrombin and S1P effects were directly compared. Fold stimulation of GTP-bound Rho was determined following normalization against total Rho.

View larger version (41K): [in a new window]   Fig. 2. Temporal activation of Cdc42 and Rac1 by thrombin and S1P. Confluent monolayers of serum-starved HUVECs were treated for the times indicated with 10 nM thrombin or 0.5 µM SIP. Western blot analyses of active CRIB-bound Rac1 and Cdc42 are shown in the upper panels, and total proteins are shown in the lower panels. The results shown are representative of two independent experiments in which the effects of thrombin and S1P were directly compared. Fold stimulation of GTP-bound Rho proteins was determined following normalization against total GTPases.

View larger version (103K): [in a new window]   Fig. 3. S1P induces co-localization of cortactin and actin in lamellipodia. Serum-starved HUVECs were treated, or not (control), with S1P (0.5 µM) or thrombin (10 nM) for 2 minutes. After fixation and permeabilization, cells were stained with anti-cortactin antibody (left panels) and phalloidin-FITC (middle panels) and analyzed by confocal microscopy. Merged image pairs (right) highlight regions of co-localization (orange).

View larger version (112K): [in a new window]   Fig. 7. Src inhibitor inhibits S1P-induced cortical localization of cortactin. Serum-starved HUVECs were pretreated with the Src kinase inhibitor, PP2 (5 µM), or Y27632 (7 µM), a Rho kinase inhibitor, for 30 minutes at 37°C; S1P was added for an additional 2 minutes before fixing and staining cells for cortactin (red) and actin (green). Confocal images of stained cells are shown.

View larger version (52K): [in a new window]   Fig. 4. Rac is involved in cortactin relocalization. HUVECs were electroporated with the pMT90-myc Rac1 N17 expression vector. Eighteen hours after electroporation, cells were detached, plated onto fibronectin-coated coverslips and allowed to spread for 8 hours before overnight serum starvation. Cells were then stimulated for 2 minutes with 0.5 µM S1P, fixed and stained for detection of myc expression (left panel) and cortactin localization (right panel). Arrows show myc-tagged RacN17-expressing cells.

View larger version (31K): [in a new window]   Fig. 5. S1P induces activation of Src kinase. Serum-starved confluent monolayers were treated with S1P (0.5 µM) or thrombin (10 nM) for the indicated times and an immunoprecipitation kinase assay was performed using anti-Src family kinase antibody (cst-1) as described in Materials and Methods. Src autophosphorylation is shown in the upper panel. Equal loading was verified by blotting aliquots of supernatants from each sample with cst-1.

View larger version (77K): [in a new window]   Fig. 6. S1P induces cortical targeting of pSrc. (A) Serum-starved HUVECs were treated for 1 minute with S1P (0.5 µM) or thrombin (10 nM), fixed and co-stained for active, phosphorylated Src (pSrc, right panels) and actin (left panels). The arrow in photos from S1P-treated cells depicts sites at the edge of membrane ruffles where pSrc appears (magnification, 1000x). (B) Cells treated with S1P (0.5 µM) or thrombin (10 nM) were fixed and stained with anti-phosphotyrosine antibody (pTyr). The arrow indicates enriched staining of tyrosine-phosphorylated residues in membrane ruffles.

View larger version (36K): [in a new window]   Fig. 8. Src kinase inhibition does not block Rac activation by S1P. Serum-starved HUVECs were pretreated with 5 µM PP2 for 20 minutes and 0.5 µM S1P was added for the indicated times. Active, CRIB-bound Rac1 is shown in the upper panel and the total amount of proteins in the middle panel. Fold stimulation of GTP-bound Rac was determined following normalization against total Rac. Blotting of the same samples with an anti-active-Erk1/2 antibody (pErk1/2) is shown below. The results shown are representative of three independent experiments.

View larger version (112K): [in a new window]   Fig. 9. S1P-induced HUVEC migration is inhibited by PP2. (A) Confluent monolayers of HUVECs were scraped with a 26G needle and the effect of 0.5 µM S1P or 10 nM thrombin on cell migration into the wounded area was observed in the presence or absence of 5 µM PP2 or 10 µM U0126. Two hours after agonist addition, cultures were fixed and the actin cytoskeleton was stained with phalloidin-FITC (bar, 40 µm). (B) Serum-starved HUVECs were treated with 0.5 µM S1P for the indicated times prior to analysis of Erk activation by western blotting using an anti-phospho Erk antibody (pErk1/2).