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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 Rho, Rac, and Cdc42 activity by shear stress. HUVECs were subjected to shear stress for the indicated times. GTP-loading assays for RhoA, Rac1, and Cdc42 were performed as described in Materials and methods. Panels on the left show fold increase of RhoA, Rac1, and Cdc42 activity during stimulation and were calculated as the amount of GTP-bound protein relative to whole cell lysates. Corresponding representative examples of Western blots of GTP-bound proteins and total cell lysates are shown on the right. Values are means ± SD from four to five independent experiments. *, P ≤ 0.05; **, P ≤ 0.01, comparisons with static control, t test.
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fig2: Regulation of Rho, Rac, and Cdc42 activity by shear stress. HUVECs were subjected to shear stress for the indicated times. GTP-loading assays for RhoA, Rac1, and Cdc42 were performed as described in Materials and methods. Panels on the left show fold increase of RhoA, Rac1, and Cdc42 activity during stimulation and were calculated as the amount of GTP-bound protein relative to whole cell lysates. Corresponding representative examples of Western blots of GTP-bound proteins and total cell lysates are shown on the right. Values are means ± SD from four to five independent experiments. *, P ≤ 0.05; **, P ≤ 0.01, comparisons with static control, t test.

Mentions: To investigate the involvement of Rho, Rac, and Cdc42 in shear stress responses, we first measured their activity during endothelial cell adaptation to shear stress. RhoA activity increased threefold at 5 min after exposure to shear stress, and then decreased sharply below control levels within the next 10 min (Fig. 2) . RhoA activity gradually increased to 1.6-fold above basal level at 2 h after stimulation with shear stress (Fig. 2) and returned to basal levels by 4 h (not depicted). The rapid increase in RhoA activity at 5 min correlated with the formation of numerous stress fibers (Fig. 1 B) and the subsequent decrease in activity 15–30 min after stimulation correlated with the loss of stress fibers and cell rounding (Fig. 1, C, G, and H). The small increase in RhoA activity at 2 h corresponded to the stage when most of the cells were realigned within the direction of shear stress.


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 Rho, Rac, and Cdc42 activity by shear stress. HUVECs were subjected to shear stress for the indicated times. GTP-loading assays for RhoA, Rac1, and Cdc42 were performed as described in Materials and methods. Panels on the left show fold increase of RhoA, Rac1, and Cdc42 activity during stimulation and were calculated as the amount of GTP-bound protein relative to whole cell lysates. Corresponding representative examples of Western blots of GTP-bound proteins and total cell lysates are shown on the right. Values are means ± SD from four to five independent experiments. *, P ≤ 0.05; **, P ≤ 0.01, comparisons with static control, t test.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Regulation of Rho, Rac, and Cdc42 activity by shear stress. HUVECs were subjected to shear stress for the indicated times. GTP-loading assays for RhoA, Rac1, and Cdc42 were performed as described in Materials and methods. Panels on the left show fold increase of RhoA, Rac1, and Cdc42 activity during stimulation and were calculated as the amount of GTP-bound protein relative to whole cell lysates. Corresponding representative examples of Western blots of GTP-bound proteins and total cell lysates are shown on the right. Values are means ± SD from four to five independent experiments. *, P ≤ 0.05; **, P ≤ 0.01, comparisons with static control, t test.
Mentions: To investigate the involvement of Rho, Rac, and Cdc42 in shear stress responses, we first measured their activity during endothelial cell adaptation to shear stress. RhoA activity increased threefold at 5 min after exposure to shear stress, and then decreased sharply below control levels within the next 10 min (Fig. 2) . RhoA activity gradually increased to 1.6-fold above basal level at 2 h after stimulation with shear stress (Fig. 2) and returned to basal levels by 4 h (not depicted). The rapid increase in RhoA activity at 5 min correlated with the formation of numerous stress fibers (Fig. 1 B) and the subsequent decrease in activity 15–30 min after stimulation correlated with the loss of stress fibers and cell rounding (Fig. 1, C, G, and H). The small increase in RhoA activity at 2 h corresponded to the stage when most of the cells were realigned within the direction of shear stress.

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