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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.

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Regulation of cell migration speed by Rho signaling pathways. HUVECs cultured under static conditions (A) or under shear stress (B) were monitored by timelapse videomicroscopy for 5 h. ML-7, LY294002 (LY), and Y-27632 were added 30 min before the experiment or cells were infected with adenoviruses expressing N19RhoA, N17Rac1, and N17Cdc42 16 h before the experiment. Cells were tracked with Kinetic Imaging software and the trajectories were statistically analyzed using Mathematica software (see Materials and methods). *, P ≤ 0.05; **, P ≤ 0.01, comparison with controls, t test.
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fig6: Regulation of cell migration speed by Rho signaling pathways. HUVECs cultured under static conditions (A) or under shear stress (B) were monitored by timelapse videomicroscopy for 5 h. ML-7, LY294002 (LY), and Y-27632 were added 30 min before the experiment or cells were infected with adenoviruses expressing N19RhoA, N17Rac1, and N17Cdc42 16 h before the experiment. Cells were tracked with Kinetic Imaging software and the trajectories were statistically analyzed using Mathematica software (see Materials and methods). *, P ≤ 0.05; **, P ≤ 0.01, comparison with controls, t test.

Mentions: In static cultures, HUVECs migrated randomly at an average speed of 48.36 ± 7 μm/h (n = 120). The cells expressing N17Rac1 and N17Cdc42 moved significantly slower than control cells (Fig. 6 A), whereas the cells expressing N19RhoA or treated with Y-27632 did not show a reduction in the cell speed. LY294002 and ML-7 also decreased cell speed (Fig. 6 A).


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)

Regulation of cell migration speed by Rho signaling pathways. HUVECs cultured under static conditions (A) or under shear stress (B) were monitored by timelapse videomicroscopy for 5 h. ML-7, LY294002 (LY), and Y-27632 were added 30 min before the experiment or cells were infected with adenoviruses expressing N19RhoA, N17Rac1, and N17Cdc42 16 h before the experiment. Cells were tracked with Kinetic Imaging software and the trajectories were statistically analyzed using Mathematica software (see Materials and methods). *, P ≤ 0.05; **, P ≤ 0.01, comparison with controls, t test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172912&req=5

fig6: Regulation of cell migration speed by Rho signaling pathways. HUVECs cultured under static conditions (A) or under shear stress (B) were monitored by timelapse videomicroscopy for 5 h. ML-7, LY294002 (LY), and Y-27632 were added 30 min before the experiment or cells were infected with adenoviruses expressing N19RhoA, N17Rac1, and N17Cdc42 16 h before the experiment. Cells were tracked with Kinetic Imaging software and the trajectories were statistically analyzed using Mathematica software (see Materials and methods). *, P ≤ 0.05; **, P ≤ 0.01, comparison with controls, t test.
Mentions: In static cultures, HUVECs migrated randomly at an average speed of 48.36 ± 7 μm/h (n = 120). The cells expressing N17Rac1 and N17Cdc42 moved significantly slower than control cells (Fig. 6 A), whereas the cells expressing N19RhoA or treated with Y-27632 did not show a reduction in the cell speed. LY294002 and ML-7 also decreased cell speed (Fig. 6 A).

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