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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 uPAR interaction with VN in the induction of protrusions and uPAR localization. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) and returned to growth medium without additions (none) or containing PIPLC (2 U/ml), mouse monoclonal antibody (Mab) against human uPAR clones R2, R3, and R9 (30 μg/ml), or mouse monoclonal antibody clone 13H1 (30 μg/ml) against VN as indicated. Cells were fixed and stained as described above and 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) uPAR-expressing cells treated with anti-uPAR R9 or anti-VN 13H1 were fixed, and uPAR localization was visualized as described in the legend to Fig. 1 A. Typical examples of uPAR distribution are shown. Bar, 10 μm.
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Figure 3: Role of uPAR interaction with VN in the induction of protrusions and uPAR localization. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) and returned to growth medium without additions (none) or containing PIPLC (2 U/ml), mouse monoclonal antibody (Mab) against human uPAR clones R2, R3, and R9 (30 μg/ml), or mouse monoclonal antibody clone 13H1 (30 μg/ml) against VN as indicated. Cells were fixed and stained as described above and 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) uPAR-expressing cells treated with anti-uPAR R9 or anti-VN 13H1 were fixed, and uPAR localization was visualized as described in the legend to Fig. 1 A. Typical examples of uPAR distribution are shown. Bar, 10 μm.

Mentions: To confirm that the uPAR-induced cytoskeletal changes and protrusions are due to surface expression of the receptor, PIPLC which removes GPI-linked proteins from the cell surface was included in the assay. The PIPLC treatment removed all surface-expressed human uPAR as assessed by immunofluorescence studies on nonpermeabilized cells (data not shown). PIPLC treatment blocked the appearance of protrusions induced by uPAR expression (Fig. 3 A). The effect of PIPLC was not due to a general effect on protrusive activity, since similar protrusions, induced by activated Rac, were not inhibited by PIPLC treatment (see below). Further evidence for cell surface activity of uPAR in the induction of protrusions was obtained by examining the effect of domain-specific monoclonal antibodies against uPAR. The monoclonal antibody R2, which recognizes an epitope within domains D2 and D3 (Rønne et al. 1991; Gårdsvoll et al. 1999), had no effect, whereas the antibodies R3 and R9, which both recognize the D1 domain, inhibited protrusion induction by 80–100% (Fig. 3 A). We conclude that uPAR-induced protrusions require cell surface expression of uPAR and availability of uPAR D1.


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 uPAR interaction with VN in the induction of protrusions and uPAR localization. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) and returned to growth medium without additions (none) or containing PIPLC (2 U/ml), mouse monoclonal antibody (Mab) against human uPAR clones R2, R3, and R9 (30 μg/ml), or mouse monoclonal antibody clone 13H1 (30 μg/ml) against VN as indicated. Cells were fixed and stained as described above and 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) uPAR-expressing cells treated with anti-uPAR R9 or anti-VN 13H1 were fixed, and uPAR localization was visualized as described in the legend to Fig. 1 A. Typical examples of uPAR distribution are shown. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Role of uPAR interaction with VN in the induction of protrusions and uPAR localization. (A) Swiss 3T3 cells were injected with pRc/CMV-uPAR (100 μg/ml) and returned to growth medium without additions (none) or containing PIPLC (2 U/ml), mouse monoclonal antibody (Mab) against human uPAR clones R2, R3, and R9 (30 μg/ml), or mouse monoclonal antibody clone 13H1 (30 μg/ml) against VN as indicated. Cells were fixed and stained as described above and 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) uPAR-expressing cells treated with anti-uPAR R9 or anti-VN 13H1 were fixed, and uPAR localization was visualized as described in the legend to Fig. 1 A. Typical examples of uPAR distribution are shown. Bar, 10 μm.
Mentions: To confirm that the uPAR-induced cytoskeletal changes and protrusions are due to surface expression of the receptor, PIPLC which removes GPI-linked proteins from the cell surface was included in the assay. The PIPLC treatment removed all surface-expressed human uPAR as assessed by immunofluorescence studies on nonpermeabilized cells (data not shown). PIPLC treatment blocked the appearance of protrusions induced by uPAR expression (Fig. 3 A). The effect of PIPLC was not due to a general effect on protrusive activity, since similar protrusions, induced by activated Rac, were not inhibited by PIPLC treatment (see below). Further evidence for cell surface activity of uPAR in the induction of protrusions was obtained by examining the effect of domain-specific monoclonal antibodies against uPAR. The monoclonal antibody R2, which recognizes an epitope within domains D2 and D3 (Rønne et al. 1991; Gårdsvoll et al. 1999), had no effect, whereas the antibodies R3 and R9, which both recognize the D1 domain, inhibited protrusion induction by 80–100% (Fig. 3 A). We conclude that uPAR-induced protrusions require cell surface expression of uPAR and availability of uPAR D1.

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