Limits...
Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases.

Wojciak-Stothard B, Ridley AJ - J. Cell Biol. (2003)

Bottom Line: Instead, Rho and Rac1 regulated directionality of cell movement.Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement.Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House St., London W1W 7BS, UK. beata@ludwig.ucl.ac.uk

ABSTRACT
Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

Show MeSH

Related in: MedlinePlus

Effects of RhoA, Rac1, Cdc42 and inhibitors on shear stress–induced changes in cell spreading, elongation, and alignment. HUVECs were stimulated with shear stress for 30 min (A) or for 4 h (B and C). After stimulation, the cells were fixed and stained for F-actin, and cell spreading (A), elongation (B), or alignment (C) parameters were calculated with ImageProPlus software. The cells were untreated (controls) or were infected with adenoviruses encoding dominant negative mutants of Rho GTPases, and then subjected to shear stress 16 h after infection. The expression levels of myc-tagged N19RhoA, N17Rac1, and N17Cdc42 are shown in D. Alternatively, the inhibitors Y-27632, ML-7, and LY294002 (LY) were applied 30 min before stimulation with shear stress. Cell alignment is shown as the percentage of cells that aligned within 10° of the shear stress direction. In A–C, black bars represent cells under shear stress, and striped bars represent cells in static conditions. *, P ≤ 0.05; **, P ≤ 0.01, t test, comparison with static controls.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172912&req=5

fig3: Effects of RhoA, Rac1, Cdc42 and inhibitors on shear stress–induced changes in cell spreading, elongation, and alignment. HUVECs were stimulated with shear stress for 30 min (A) or for 4 h (B and C). After stimulation, the cells were fixed and stained for F-actin, and cell spreading (A), elongation (B), or alignment (C) parameters were calculated with ImageProPlus software. The cells were untreated (controls) or were infected with adenoviruses encoding dominant negative mutants of Rho GTPases, and then subjected to shear stress 16 h after infection. The expression levels of myc-tagged N19RhoA, N17Rac1, and N17Cdc42 are shown in D. Alternatively, the inhibitors Y-27632, ML-7, and LY294002 (LY) were applied 30 min before stimulation with shear stress. Cell alignment is shown as the percentage of cells that aligned within 10° of the shear stress direction. In A–C, black bars represent cells under shear stress, and striped bars represent cells in static conditions. *, P ≤ 0.05; **, P ≤ 0.01, t test, comparison with static controls.

Mentions: In control cells in the absence of shear stress (static cultures), inhibition of RhoA with N19RhoA and inhibition of Rho-kinase with Y-27632 promoted cell spreading (Fig. 3 A). In contrast, N17Rac1 significantly decreased the cell spread area (P < 0.05), whereas N17Cdc42 and LY294002 did not have a significant effect on the cell spread area (Fig. 3 A). Cell elongation in static cultures was slightly reduced by Y-27632, but was not significantly changed by any of the other inhibitors (Fig. 3 B). Although N17Cdc42 expressed by adenoviral infection did not have significant effects on the shape of subconfluent endothelial cells, the protein was active as it inhibited thrombin-induced contractility in confluent cultures of endothelial cells as described previously (Wojciak-Stothard et al., 2001).


Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases.

Wojciak-Stothard B, Ridley AJ - J. Cell Biol. (2003)

Effects of RhoA, Rac1, Cdc42 and inhibitors on shear stress–induced changes in cell spreading, elongation, and alignment. HUVECs were stimulated with shear stress for 30 min (A) or for 4 h (B and C). After stimulation, the cells were fixed and stained for F-actin, and cell spreading (A), elongation (B), or alignment (C) parameters were calculated with ImageProPlus software. The cells were untreated (controls) or were infected with adenoviruses encoding dominant negative mutants of Rho GTPases, and then subjected to shear stress 16 h after infection. The expression levels of myc-tagged N19RhoA, N17Rac1, and N17Cdc42 are shown in D. Alternatively, the inhibitors Y-27632, ML-7, and LY294002 (LY) were applied 30 min before stimulation with shear stress. Cell alignment is shown as the percentage of cells that aligned within 10° of the shear stress direction. In A–C, black bars represent cells under shear stress, and striped bars represent cells in static conditions. *, P ≤ 0.05; **, P ≤ 0.01, t test, comparison with static controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172912&req=5

fig3: Effects of RhoA, Rac1, Cdc42 and inhibitors on shear stress–induced changes in cell spreading, elongation, and alignment. HUVECs were stimulated with shear stress for 30 min (A) or for 4 h (B and C). After stimulation, the cells were fixed and stained for F-actin, and cell spreading (A), elongation (B), or alignment (C) parameters were calculated with ImageProPlus software. The cells were untreated (controls) or were infected with adenoviruses encoding dominant negative mutants of Rho GTPases, and then subjected to shear stress 16 h after infection. The expression levels of myc-tagged N19RhoA, N17Rac1, and N17Cdc42 are shown in D. Alternatively, the inhibitors Y-27632, ML-7, and LY294002 (LY) were applied 30 min before stimulation with shear stress. Cell alignment is shown as the percentage of cells that aligned within 10° of the shear stress direction. In A–C, black bars represent cells under shear stress, and striped bars represent cells in static conditions. *, P ≤ 0.05; **, P ≤ 0.01, t test, comparison with static controls.
Mentions: In control cells in the absence of shear stress (static cultures), inhibition of RhoA with N19RhoA and inhibition of Rho-kinase with Y-27632 promoted cell spreading (Fig. 3 A). In contrast, N17Rac1 significantly decreased the cell spread area (P < 0.05), whereas N17Cdc42 and LY294002 did not have a significant effect on the cell spread area (Fig. 3 A). Cell elongation in static cultures was slightly reduced by Y-27632, but was not significantly changed by any of the other inhibitors (Fig. 3 B). Although N17Cdc42 expressed by adenoviral infection did not have significant effects on the shape of subconfluent endothelial cells, the protein was active as it inhibited thrombin-induced contractility in confluent cultures of endothelial cells as described previously (Wojciak-Stothard et al., 2001).

Bottom Line: Instead, Rho and Rac1 regulated directionality of cell movement.Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement.Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House St., London W1W 7BS, UK. beata@ludwig.ucl.ac.uk

ABSTRACT
Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

Show MeSH
Related in: MedlinePlus