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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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Effects of constitutively activated RhoA, Rac1, and Cdc42 on cell alignment under shear stress. HUVECs were transfected with expression vectors encoding GFP-L63RhoA (A), GFP-L61Rac1 (B), or GFP-L61Cdc42 (C). After 18 h, cells were exposed to shear stress for 4 h. The cells expressing GFP-tagged proteins are green and indicated with arrowheads; F-actin staining is in red. Shear direction is indicated with an arrow. Bar, 20 μm.
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fig5: Effects of constitutively activated RhoA, Rac1, and Cdc42 on cell alignment under shear stress. HUVECs were transfected with expression vectors encoding GFP-L63RhoA (A), GFP-L61Rac1 (B), or GFP-L61Cdc42 (C). After 18 h, cells were exposed to shear stress for 4 h. The cells expressing GFP-tagged proteins are green and indicated with arrowheads; F-actin staining is in red. Shear direction is indicated with an arrow. Bar, 20 μm.

Mentions: The effects of constitutively activated RhoA, Rac1, and Cdc42 on actin organization and shear stress–induced alignment were consistent with Rho and Rac, but not with Cdc42, contributing to shear stress–induced cell orientation. Constitutively activated L63RhoA induced formation of stress fibers and prevented cell alignment (Fig. 5 A), in agreement with previous observations on bovine aortic endothelial cells (Tzima et al., 2001). L61Rac1 also inhibited HUVEC alignment and induced formation of lamellipodia (Fig. 5 B) similar to results on bovine aortic endothelial cells (Tzima et al., 2002). In contrast, L61Cdc42 did not have a significant effect on cell alignment (Fig. 3 C and Fig. 5 C) or elongation (not depicted), which is consistent with the lack of effect of N17Cdc42 on these responses.


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 constitutively activated RhoA, Rac1, and Cdc42 on cell alignment under shear stress. HUVECs were transfected with expression vectors encoding GFP-L63RhoA (A), GFP-L61Rac1 (B), or GFP-L61Cdc42 (C). After 18 h, cells were exposed to shear stress for 4 h. The cells expressing GFP-tagged proteins are green and indicated with arrowheads; F-actin staining is in red. Shear direction is indicated with an arrow. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Effects of constitutively activated RhoA, Rac1, and Cdc42 on cell alignment under shear stress. HUVECs were transfected with expression vectors encoding GFP-L63RhoA (A), GFP-L61Rac1 (B), or GFP-L61Cdc42 (C). After 18 h, cells were exposed to shear stress for 4 h. The cells expressing GFP-tagged proteins are green and indicated with arrowheads; F-actin staining is in red. Shear direction is indicated with an arrow. Bar, 20 μm.
Mentions: The effects of constitutively activated RhoA, Rac1, and Cdc42 on actin organization and shear stress–induced alignment were consistent with Rho and Rac, but not with Cdc42, contributing to shear stress–induced cell orientation. Constitutively activated L63RhoA induced formation of stress fibers and prevented cell alignment (Fig. 5 A), in agreement with previous observations on bovine aortic endothelial cells (Tzima et al., 2001). L61Rac1 also inhibited HUVEC alignment and induced formation of lamellipodia (Fig. 5 B) similar to results on bovine aortic endothelial cells (Tzima et al., 2002). In contrast, L61Cdc42 did not have a significant effect on cell alignment (Fig. 3 C and Fig. 5 C) or elongation (not depicted), which is consistent with the lack of effect of N17Cdc42 on these responses.

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