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RhoA is required for monocyte tail retraction during transendothelial migration.

Worthylake RA, Lemoine S, Watson JM, Burridge K - J. Cell Biol. (2001)

Bottom Line: We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration.We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction.Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. becky_worthylake@med.unc.edu

ABSTRACT
Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

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(A) Transendothelial migration of monocytes is enhanced by MCP-1. The number of monocytes that transmigrated through an IL-1 activated, and endothelial monolayer increased more than fivefold for THP-1 cells and twofold for primary monocytes when MCP-1 was present in the lower chamber of a transwell chamber. The average number of transmigrated cells per field is plotted as the average from three separate experiments. (B) RhoA is required for transendothelial migration of monocytes. Transendothelial migration of primary monocytes electroporated with C3 is plotted as a percent of transmigration activity achieved by monocytes loaded with GST (control).
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fig1: (A) Transendothelial migration of monocytes is enhanced by MCP-1. The number of monocytes that transmigrated through an IL-1 activated, and endothelial monolayer increased more than fivefold for THP-1 cells and twofold for primary monocytes when MCP-1 was present in the lower chamber of a transwell chamber. The average number of transmigrated cells per field is plotted as the average from three separate experiments. (B) RhoA is required for transendothelial migration of monocytes. Transendothelial migration of primary monocytes electroporated with C3 is plotted as a percent of transmigration activity achieved by monocytes loaded with GST (control).

Mentions: To study the interaction between monocytes and endothelial cells, we first optimized a transmigration assay (see Materials and methods for details). Primary human umbilical vein endothelial cells (HUVECs) were seeded on transwells at a confluent density 3 d before the assay. The HUVECs were stimulated with the inflammatory cytokine, IL-1, overnight to activate the endothelial cells and upregulate the surface expression of cell adhesion molecules crucial for monocyte adhesion. Either primary monocytes or THP-1 cells, a monocytic cells line, were added to the upper chamber and allowed to transmigrate for 1 or 3 h, respectively. The number of cells that crossed the endothelial monolayer were counted, and the results were plotted in Fig. 1 A. The presence of the chemokine MCP-1 in the lower chamber increased the number of primary monocytes and THP-1 cells that transmigrated. These results show that our cell culture system supported transendothelial migration and was suitable for studying the function of the monocyte cytoskeleton during this process. To assess the requirement of RhoA in transmigration, we electroporated C3 exoenzyme into primary monocytes immediately after their isolation. C3 treatment resulted in decreased RhoA activity, but had no affect on the activity of Rac1 or Cdc42 as determined by affinity precipitation assays (data not shown) (Bagrodia et al., 1998). Fig. 1 B shows that transmigration of cells electroporated with C3 was only 34% as efficient as control cells electroporated with glutathione S-transferase (GST).


RhoA is required for monocyte tail retraction during transendothelial migration.

Worthylake RA, Lemoine S, Watson JM, Burridge K - J. Cell Biol. (2001)

(A) Transendothelial migration of monocytes is enhanced by MCP-1. The number of monocytes that transmigrated through an IL-1 activated, and endothelial monolayer increased more than fivefold for THP-1 cells and twofold for primary monocytes when MCP-1 was present in the lower chamber of a transwell chamber. The average number of transmigrated cells per field is plotted as the average from three separate experiments. (B) RhoA is required for transendothelial migration of monocytes. Transendothelial migration of primary monocytes electroporated with C3 is plotted as a percent of transmigration activity achieved by monocytes loaded with GST (control).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196864&req=5

fig1: (A) Transendothelial migration of monocytes is enhanced by MCP-1. The number of monocytes that transmigrated through an IL-1 activated, and endothelial monolayer increased more than fivefold for THP-1 cells and twofold for primary monocytes when MCP-1 was present in the lower chamber of a transwell chamber. The average number of transmigrated cells per field is plotted as the average from three separate experiments. (B) RhoA is required for transendothelial migration of monocytes. Transendothelial migration of primary monocytes electroporated with C3 is plotted as a percent of transmigration activity achieved by monocytes loaded with GST (control).
Mentions: To study the interaction between monocytes and endothelial cells, we first optimized a transmigration assay (see Materials and methods for details). Primary human umbilical vein endothelial cells (HUVECs) were seeded on transwells at a confluent density 3 d before the assay. The HUVECs were stimulated with the inflammatory cytokine, IL-1, overnight to activate the endothelial cells and upregulate the surface expression of cell adhesion molecules crucial for monocyte adhesion. Either primary monocytes or THP-1 cells, a monocytic cells line, were added to the upper chamber and allowed to transmigrate for 1 or 3 h, respectively. The number of cells that crossed the endothelial monolayer were counted, and the results were plotted in Fig. 1 A. The presence of the chemokine MCP-1 in the lower chamber increased the number of primary monocytes and THP-1 cells that transmigrated. These results show that our cell culture system supported transendothelial migration and was suitable for studying the function of the monocyte cytoskeleton during this process. To assess the requirement of RhoA in transmigration, we electroporated C3 exoenzyme into primary monocytes immediately after their isolation. C3 treatment resulted in decreased RhoA activity, but had no affect on the activity of Rac1 or Cdc42 as determined by affinity precipitation assays (data not shown) (Bagrodia et al., 1998). Fig. 1 B shows that transmigration of cells electroporated with C3 was only 34% as efficient as control cells electroporated with glutathione S-transferase (GST).

Bottom Line: We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration.We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction.Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. becky_worthylake@med.unc.edu

ABSTRACT
Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

Show MeSH
Related in: MedlinePlus