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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 V12Rac expression in growing Swiss 3T3 cells. Swiss 3T3 cells in growth medium were injected with pRK5–myc-V12Rac (100 μg/ml). Cells were then incubated in the absence or presence of 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. Rac-expressing cells were identified with mouse anti-myc (clone 9E10) followed by FITC goat anti–mouse antibodies, and the number of cells expressing clearly identifiable protrusions was determined. Data are average ± SD for at least three experiments, each examining 100 injected cells. The inset shows the typical morphology of the actin cytoskeleton in a V12Rac-expressing cell. Bar, 10 μm.
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Figure 9: Effect of V12Rac expression in growing Swiss 3T3 cells. Swiss 3T3 cells in growth medium were injected with pRK5–myc-V12Rac (100 μg/ml). Cells were then incubated in the absence or presence of 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. Rac-expressing cells were identified with mouse anti-myc (clone 9E10) followed by FITC goat anti–mouse antibodies, and the number of cells expressing clearly identifiable protrusions was determined. Data are average ± SD for at least three experiments, each examining 100 injected cells. The inset shows the typical morphology of the actin cytoskeleton in a V12Rac-expressing cell. Bar, 10 μm.

Mentions: We have found that expression of activated Rac in growing Swiss 3T3 cells also leads to the induction of protrusions (Fig. 9) similar to those induced by uPAR (although they have a less prominent actin meshwork at the leading edge and the cell body is almost totally devoid of stress fibers). Due to the morphological similarities between uPAR- and Rac-induced protrusions, we investigated whether they shared other characteristics. Rac-induced protrusions were not inhibited by p130CasΔSD or Mac-1 coexpression, indicating that these inhibitors function upstream of Rac (data not shown). In addition, Rac-induced protrusions were not sensitive to PIPLC treatment, but they were inhibited by treatment with the cyclic RGD peptide GPenSPRGDCA or by a function-blocking antibody to the integrin αVβ3 (Fig. 9). We conclude that in contrast to the uPAR-induced protrusions, where uPAR activates Rac and also acts as a direct adhesion receptor, the Rac-induced protrusions are dependent on αVβ3 binding to the extracellular matrix.


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 V12Rac expression in growing Swiss 3T3 cells. Swiss 3T3 cells in growth medium were injected with pRK5–myc-V12Rac (100 μg/ml). Cells were then incubated in the absence or presence of 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. Rac-expressing cells were identified with mouse anti-myc (clone 9E10) followed by FITC goat anti–mouse antibodies, and the number of cells expressing clearly identifiable protrusions was determined. Data are average ± SD for at least three experiments, each examining 100 injected cells. The inset shows the typical morphology of the actin cytoskeleton in a V12Rac-expressing cell. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 9: Effect of V12Rac expression in growing Swiss 3T3 cells. Swiss 3T3 cells in growth medium were injected with pRK5–myc-V12Rac (100 μg/ml). Cells were then incubated in the absence or presence of 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. Rac-expressing cells were identified with mouse anti-myc (clone 9E10) followed by FITC goat anti–mouse antibodies, and the number of cells expressing clearly identifiable protrusions was determined. Data are average ± SD for at least three experiments, each examining 100 injected cells. The inset shows the typical morphology of the actin cytoskeleton in a V12Rac-expressing cell. Bar, 10 μm.
Mentions: We have found that expression of activated Rac in growing Swiss 3T3 cells also leads to the induction of protrusions (Fig. 9) similar to those induced by uPAR (although they have a less prominent actin meshwork at the leading edge and the cell body is almost totally devoid of stress fibers). Due to the morphological similarities between uPAR- and Rac-induced protrusions, we investigated whether they shared other characteristics. Rac-induced protrusions were not inhibited by p130CasΔSD or Mac-1 coexpression, indicating that these inhibitors function upstream of Rac (data not shown). In addition, Rac-induced protrusions were not sensitive to PIPLC treatment, but they were inhibited by treatment with the cyclic RGD peptide GPenSPRGDCA or by a function-blocking antibody to the integrin αVβ3 (Fig. 9). We conclude that in contrast to the uPAR-induced protrusions, where uPAR activates Rac and also acts as a direct adhesion receptor, the Rac-induced protrusions are dependent on αVβ3 binding to the extracellular matrix.

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