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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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Role of RGD-binding integrins in uPAR-induced protrusions and adhesion. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) and incubated in growth medium containing GRGDdSP (RGDd) (1.2 mM), GPenGRGDSPCA (cRGD) (0.05 mM), or hamster anti–mouse β3 integrin (clone 2C9.G2) (20 μg/ml) as indicated for 4 h before fixing and staining. The number of uPAR-expressing cells exhibiting clearly identifiable protrusions was determined. Data are average ± SD for at least three experiments, each examining 100 injected cells. (B) Swiss 3T3 cells were injected with FITC-dextran or pRC/CMV-uPAR and treated with cRGD (0.05 mM). The organization of the actin cytoskeleton was visualized as described above. Typical morphologies of uninjected (no inj.) or uPAR-expressing cells are shown. (C) NIH 3T3 cells transfected with pEGFP-C1 and empty vector or pRC/CMV-uPAR as indicated were subjected to detachment or attachment assays (see Materials and Methods for details) in the presence of 5 mM EDTA, 0.05 mM cRGD, and 20 μg/ml anti-uPAR R3 as indicated. Results are expressed as fraction of adherent cells relative to identically transfected cells in the absence of additions. In the absence of additions, the adhesion efficiencies of uPAR-transfected and control cells were indistinguishable (data not shown). Data are average ± SD for at least two experiments performed in triplicate. Bars, 10 μm.
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Figure 4: Role of RGD-binding integrins in uPAR-induced protrusions and adhesion. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) and incubated in growth medium containing GRGDdSP (RGDd) (1.2 mM), GPenGRGDSPCA (cRGD) (0.05 mM), or hamster anti–mouse β3 integrin (clone 2C9.G2) (20 μg/ml) as indicated for 4 h before fixing and staining. The number of uPAR-expressing cells exhibiting clearly identifiable protrusions was determined. Data are average ± SD for at least three experiments, each examining 100 injected cells. (B) Swiss 3T3 cells were injected with FITC-dextran or pRC/CMV-uPAR and treated with cRGD (0.05 mM). The organization of the actin cytoskeleton was visualized as described above. Typical morphologies of uninjected (no inj.) or uPAR-expressing cells are shown. (C) NIH 3T3 cells transfected with pEGFP-C1 and empty vector or pRC/CMV-uPAR as indicated were subjected to detachment or attachment assays (see Materials and Methods for details) in the presence of 5 mM EDTA, 0.05 mM cRGD, and 20 μg/ml anti-uPAR R3 as indicated. Results are expressed as fraction of adherent cells relative to identically transfected cells in the absence of additions. In the absence of additions, the adhesion efficiencies of uPAR-transfected and control cells were indistinguishable (data not shown). Data are average ± SD for at least two experiments performed in triplicate. Bars, 10 μm.

Mentions: Since uPAR has been described to modulate integrin affinity (Chapman et al. 1999; Ossowski and Aguirre-Ghiso 2000; Preissner et al. 2000), we tested whether de novo binding of integrins to RGD-containing extracellular matrix proteins was necessary for the uPAR-induced response. The peptides GRGDdSP (RGDd) and GPenGRGDSPCA (cRGD), known to preferably interfere with integrin-mediated cell adhesion to FN and VN, respectively (Pierschbacher and Ruoslahti 1987), were added to cells before injection of the uPAR expression plasmid. Uninjected cells were less spread and contained fewer stress fibers than untreated cells (compare Fig. 4 B and Fig. 1 A), but the peptides did not inhibit the uPAR-mediated induction of protrusions, indicating the involvement of an adhesion receptor different from RGD-binding integrins (Fig. 4A and Fig. B). In addition, uPAR-induced protrusions were not affected by a function-blocking antibody to the integrin β3 subunit. In contrast, the cRGD and the anti-β3 antibody inhibited Rac-induced protrusions (see below).


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)

Role of RGD-binding integrins in uPAR-induced protrusions and adhesion. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) and incubated in growth medium containing GRGDdSP (RGDd) (1.2 mM), GPenGRGDSPCA (cRGD) (0.05 mM), or hamster anti–mouse β3 integrin (clone 2C9.G2) (20 μg/ml) as indicated for 4 h before fixing and staining. The number of uPAR-expressing cells exhibiting clearly identifiable protrusions was determined. Data are average ± SD for at least three experiments, each examining 100 injected cells. (B) Swiss 3T3 cells were injected with FITC-dextran or pRC/CMV-uPAR and treated with cRGD (0.05 mM). The organization of the actin cytoskeleton was visualized as described above. Typical morphologies of uninjected (no inj.) or uPAR-expressing cells are shown. (C) NIH 3T3 cells transfected with pEGFP-C1 and empty vector or pRC/CMV-uPAR as indicated were subjected to detachment or attachment assays (see Materials and Methods for details) in the presence of 5 mM EDTA, 0.05 mM cRGD, and 20 μg/ml anti-uPAR R3 as indicated. Results are expressed as fraction of adherent cells relative to identically transfected cells in the absence of additions. In the absence of additions, the adhesion efficiencies of uPAR-transfected and control cells were indistinguishable (data not shown). Data are average ± SD for at least two experiments performed in triplicate. Bars, 10 μm.
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Figure 4: Role of RGD-binding integrins in uPAR-induced protrusions and adhesion. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) and incubated in growth medium containing GRGDdSP (RGDd) (1.2 mM), GPenGRGDSPCA (cRGD) (0.05 mM), or hamster anti–mouse β3 integrin (clone 2C9.G2) (20 μg/ml) as indicated for 4 h before fixing and staining. The number of uPAR-expressing cells exhibiting clearly identifiable protrusions was determined. Data are average ± SD for at least three experiments, each examining 100 injected cells. (B) Swiss 3T3 cells were injected with FITC-dextran or pRC/CMV-uPAR and treated with cRGD (0.05 mM). The organization of the actin cytoskeleton was visualized as described above. Typical morphologies of uninjected (no inj.) or uPAR-expressing cells are shown. (C) NIH 3T3 cells transfected with pEGFP-C1 and empty vector or pRC/CMV-uPAR as indicated were subjected to detachment or attachment assays (see Materials and Methods for details) in the presence of 5 mM EDTA, 0.05 mM cRGD, and 20 μg/ml anti-uPAR R3 as indicated. Results are expressed as fraction of adherent cells relative to identically transfected cells in the absence of additions. In the absence of additions, the adhesion efficiencies of uPAR-transfected and control cells were indistinguishable (data not shown). Data are average ± SD for at least two experiments performed in triplicate. Bars, 10 μm.
Mentions: Since uPAR has been described to modulate integrin affinity (Chapman et al. 1999; Ossowski and Aguirre-Ghiso 2000; Preissner et al. 2000), we tested whether de novo binding of integrins to RGD-containing extracellular matrix proteins was necessary for the uPAR-induced response. The peptides GRGDdSP (RGDd) and GPenGRGDSPCA (cRGD), known to preferably interfere with integrin-mediated cell adhesion to FN and VN, respectively (Pierschbacher and Ruoslahti 1987), were added to cells before injection of the uPAR expression plasmid. Uninjected cells were less spread and contained fewer stress fibers than untreated cells (compare Fig. 4 B and Fig. 1 A), but the peptides did not inhibit the uPAR-mediated induction of protrusions, indicating the involvement of an adhesion receptor different from RGD-binding integrins (Fig. 4A and Fig. B). In addition, uPAR-induced protrusions were not affected by a function-blocking antibody to the integrin β3 subunit. In contrast, the cRGD and the anti-β3 antibody inhibited Rac-induced protrusions (see below).

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