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Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin.

Kjøller L, Hall A - J. Cell Biol. (2001)

Bottom Line: In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression.We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses.This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research Campaign Oncogene and Signal Transduction Group, University College London, United Kingdom.

ABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

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Effect of p130CasΔSD and Mac-1 on uPAR-induced protrusions. (A) Swiss 3T3 cells in growth medium were coinjected with pRC/CMV-uPAR (100 μg/ml) and either one of the expression plasmids pSSRα-p130CasΔSD/pEBG-GST-p130CasΔSD or both of the expression plasmids pRK5-CD11b and pRK5-CD18 (α- and β chain of Mac-1). After 4 h of expression, cells were fixed, stained, and the number of uPAR-expressing cells with clearly identifiable protrusions was determined as described above. Expression of inhibitor constructs was verified by immunofluorescence staining using anti-GST (rabbit polyclonal) or anti-CD11b (clone ICRF 44) as primary antibodies. Expression of untagged and GST-tagged p130CasΔSD gave identical results and the data were averaged for presentation. Data are average ± SD for at least three experiments, each examining 100 injected cells. (B) NIH 3T3 cells transfected with pEGFP-C1 and empty vector or pRC/CMV-uPAR and/or pRK5-CD11b and pRK5-CD18 as indicated were subjected to detachment assays (see Materials and Methods for details) in the presence of 5 mM EDTA. Results are expressed as the fraction of adherent cells relative to the number uPAR-transfected adherent cells under these conditions. Results are mean ± SD of three experiments each performed in triplicate.
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Figure 8: Effect of p130CasΔSD and Mac-1 on uPAR-induced protrusions. (A) Swiss 3T3 cells in growth medium were coinjected with pRC/CMV-uPAR (100 μg/ml) and either one of the expression plasmids pSSRα-p130CasΔSD/pEBG-GST-p130CasΔSD or both of the expression plasmids pRK5-CD11b and pRK5-CD18 (α- and β chain of Mac-1). After 4 h of expression, cells were fixed, stained, and the number of uPAR-expressing cells with clearly identifiable protrusions was determined as described above. Expression of inhibitor constructs was verified by immunofluorescence staining using anti-GST (rabbit polyclonal) or anti-CD11b (clone ICRF 44) as primary antibodies. Expression of untagged and GST-tagged p130CasΔSD gave identical results and the data were averaged for presentation. Data are average ± SD for at least three experiments, each examining 100 injected cells. (B) NIH 3T3 cells transfected with pEGFP-C1 and empty vector or pRC/CMV-uPAR and/or pRK5-CD11b and pRK5-CD18 as indicated were subjected to detachment assays (see Materials and Methods for details) in the presence of 5 mM EDTA. Results are expressed as the fraction of adherent cells relative to the number uPAR-transfected adherent cells under these conditions. Results are mean ± SD of three experiments each performed in triplicate.

Mentions: To initiate an investigation into the mechanism by which uPAR activates Rac, we have focused on the possibility that an integrin might function as a transmembrane adaptor for uPAR signaling. The docking protein p130Cas is phosphorylated upon integrin activation and has been proposed to participate in Rac activation (for review see O'Neill et al. 2000). We therefore tested the effect of a dominant negative version of p130Cas (p130CasΔSD) on uPAR-induced protrusions. Coexpression of p130CasΔSD, which lacks the substrate domain for phosphorylation, strongly inhibited the uPAR-induced effect (Fig. 8 A), suggesting that it plays a role in the pathway leading to cytoskeletal changes.


Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin.

Kjøller L, Hall A - J. Cell Biol. (2001)

Effect of p130CasΔSD and Mac-1 on uPAR-induced protrusions. (A) Swiss 3T3 cells in growth medium were coinjected with pRC/CMV-uPAR (100 μg/ml) and either one of the expression plasmids pSSRα-p130CasΔSD/pEBG-GST-p130CasΔSD or both of the expression plasmids pRK5-CD11b and pRK5-CD18 (α- and β chain of Mac-1). After 4 h of expression, cells were fixed, stained, and the number of uPAR-expressing cells with clearly identifiable protrusions was determined as described above. Expression of inhibitor constructs was verified by immunofluorescence staining using anti-GST (rabbit polyclonal) or anti-CD11b (clone ICRF 44) as primary antibodies. Expression of untagged and GST-tagged p130CasΔSD gave identical results and the data were averaged for presentation. Data are average ± SD for at least three experiments, each examining 100 injected cells. (B) NIH 3T3 cells transfected with pEGFP-C1 and empty vector or pRC/CMV-uPAR and/or pRK5-CD11b and pRK5-CD18 as indicated were subjected to detachment assays (see Materials and Methods for details) in the presence of 5 mM EDTA. Results are expressed as the fraction of adherent cells relative to the number uPAR-transfected adherent cells under these conditions. Results are mean ± SD of three experiments each performed in triplicate.
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Related In: Results  -  Collection

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Figure 8: Effect of p130CasΔSD and Mac-1 on uPAR-induced protrusions. (A) Swiss 3T3 cells in growth medium were coinjected with pRC/CMV-uPAR (100 μg/ml) and either one of the expression plasmids pSSRα-p130CasΔSD/pEBG-GST-p130CasΔSD or both of the expression plasmids pRK5-CD11b and pRK5-CD18 (α- and β chain of Mac-1). After 4 h of expression, cells were fixed, stained, and the number of uPAR-expressing cells with clearly identifiable protrusions was determined as described above. Expression of inhibitor constructs was verified by immunofluorescence staining using anti-GST (rabbit polyclonal) or anti-CD11b (clone ICRF 44) as primary antibodies. Expression of untagged and GST-tagged p130CasΔSD gave identical results and the data were averaged for presentation. Data are average ± SD for at least three experiments, each examining 100 injected cells. (B) NIH 3T3 cells transfected with pEGFP-C1 and empty vector or pRC/CMV-uPAR and/or pRK5-CD11b and pRK5-CD18 as indicated were subjected to detachment assays (see Materials and Methods for details) in the presence of 5 mM EDTA. Results are expressed as the fraction of adherent cells relative to the number uPAR-transfected adherent cells under these conditions. Results are mean ± SD of three experiments each performed in triplicate.
Mentions: To initiate an investigation into the mechanism by which uPAR activates Rac, we have focused on the possibility that an integrin might function as a transmembrane adaptor for uPAR signaling. The docking protein p130Cas is phosphorylated upon integrin activation and has been proposed to participate in Rac activation (for review see O'Neill et al. 2000). We therefore tested the effect of a dominant negative version of p130Cas (p130CasΔSD) on uPAR-induced protrusions. Coexpression of p130CasΔSD, which lacks the substrate domain for phosphorylation, strongly inhibited the uPAR-induced effect (Fig. 8 A), suggesting that it plays a role in the pathway leading to cytoskeletal changes.

Bottom Line: In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression.We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses.This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research Campaign Oncogene and Signal Transduction Group, University College London, United Kingdom.

ABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

Show MeSH
Related in: MedlinePlus