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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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The effects of inhibitors on shear-induced remodeling of actin cytoskeleton. Cells were untreated (A) or infected with adenoviruses to express β-gal (B), N17Cdc42 (C), N17Rac1 (D), or N19RhoA (E) 16 h before stimulation with shear stress for 2 h. Alternatively, cells were treated with 5 μM Y-27632 (F), 10 μM ML-7 (G), or 10 μM LY294002 (H) 30 min before and during exposure to shear stress for 4 h. Shear direction is indicated with an arrow (B). The arrowhead (A) points to a lamellipodium formed at the downstream part. Cells were stained for F-actin (red) and anti– c-myc 9E10 antibody (green) to visualize myc epitope–tagged N19RhoA, N17Rac1, and N17Cdc42 (B–D). Bar, 20 μm.
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fig4: The effects of inhibitors on shear-induced remodeling of actin cytoskeleton. Cells were untreated (A) or infected with adenoviruses to express β-gal (B), N17Cdc42 (C), N17Rac1 (D), or N19RhoA (E) 16 h before stimulation with shear stress for 2 h. Alternatively, cells were treated with 5 μM Y-27632 (F), 10 μM ML-7 (G), or 10 μM LY294002 (H) 30 min before and during exposure to shear stress for 4 h. Shear direction is indicated with an arrow (B). The arrowhead (A) points to a lamellipodium formed at the downstream part. Cells were stained for F-actin (red) and anti– c-myc 9E10 antibody (green) to visualize myc epitope–tagged N19RhoA, N17Rac1, and N17Cdc42 (B–D). Bar, 20 μm.

Mentions: Rac1 normally controls formation of membrane ruffles and lamellipodia, whereas Cdc42 controls formation of filopodia (Ridley, 2001a). We did not observe an increased formation of filopodia or lamellipodia at 5–30 min of exposure to shear stress as compared with static controls (Fig. 1). Static HUVECs had lamellipodia before shear stress but as described above, after cell retraction, lamellipodia were predominantly oriented at the downstream side of cells (see Fig. 1 F and Fig. 4 A). Filopodia were not observed under any conditions; in fact, introduction of constitutively active Cdc42 (V12Cdc42) into HUVECs results in the formation of very few filopodia followed by stress fibers and cell contraction (Wojciak-Stothard et al., 1998).


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)

The effects of inhibitors on shear-induced remodeling of actin cytoskeleton. Cells were untreated (A) or infected with adenoviruses to express β-gal (B), N17Cdc42 (C), N17Rac1 (D), or N19RhoA (E) 16 h before stimulation with shear stress for 2 h. Alternatively, cells were treated with 5 μM Y-27632 (F), 10 μM ML-7 (G), or 10 μM LY294002 (H) 30 min before and during exposure to shear stress for 4 h. Shear direction is indicated with an arrow (B). The arrowhead (A) points to a lamellipodium formed at the downstream part. Cells were stained for F-actin (red) and anti– c-myc 9E10 antibody (green) to visualize myc epitope–tagged N19RhoA, N17Rac1, and N17Cdc42 (B–D). Bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172912&req=5

fig4: The effects of inhibitors on shear-induced remodeling of actin cytoskeleton. Cells were untreated (A) or infected with adenoviruses to express β-gal (B), N17Cdc42 (C), N17Rac1 (D), or N19RhoA (E) 16 h before stimulation with shear stress for 2 h. Alternatively, cells were treated with 5 μM Y-27632 (F), 10 μM ML-7 (G), or 10 μM LY294002 (H) 30 min before and during exposure to shear stress for 4 h. Shear direction is indicated with an arrow (B). The arrowhead (A) points to a lamellipodium formed at the downstream part. Cells were stained for F-actin (red) and anti– c-myc 9E10 antibody (green) to visualize myc epitope–tagged N19RhoA, N17Rac1, and N17Cdc42 (B–D). Bar, 20 μm.
Mentions: Rac1 normally controls formation of membrane ruffles and lamellipodia, whereas Cdc42 controls formation of filopodia (Ridley, 2001a). We did not observe an increased formation of filopodia or lamellipodia at 5–30 min of exposure to shear stress as compared with static controls (Fig. 1). Static HUVECs had lamellipodia before shear stress but as described above, after cell retraction, lamellipodia were predominantly oriented at the downstream side of cells (see Fig. 1 F and Fig. 4 A). Filopodia were not observed under any conditions; in fact, introduction of constitutively active Cdc42 (V12Cdc42) into HUVECs results in the formation of very few filopodia followed by stress fibers and cell contraction (Wojciak-Stothard et al., 1998).

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