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The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, Ozanne BW - J. Cell Biol. (1998)

Bottom Line: Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression.However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells.Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

View Article: PubMed Central - PubMed

Affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD, United Kingdom.

ABSTRACT
Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

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Expression of TAM67 inhibits motility of A431 cells.  (a) Colonies of cells were treated with EGF as described in Materials and Methods. Representative A431, NA, and TA colonies,  photographed using a phase contrast microscope, are shown before and 48 h after the addition of 10 ng/ml EGF. (EGF was also  used at concentrations of 2 and 5 ng/ml with similar results obtained.) Insets show higher magnification images of two different  motile cells. (b) Wounding assays for NA13 and TA37 cells.  Wounds were created in monolayers of NA13 or TA37 cells as  described in Materials and Methods and photographed immediately and 48 hours later. (c) Wounds created in monolayers of  NA13 and TA37 cells grown on glass coverslips were fixed and  stained for F-actin with phalloidin 24 after wounding. Photographs show cells at the edge of the wound. Results for one of  four independent experiments are shown. Bars: (a, c) 20 μm; (b)  100 μm.
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Figure 4: Expression of TAM67 inhibits motility of A431 cells. (a) Colonies of cells were treated with EGF as described in Materials and Methods. Representative A431, NA, and TA colonies, photographed using a phase contrast microscope, are shown before and 48 h after the addition of 10 ng/ml EGF. (EGF was also used at concentrations of 2 and 5 ng/ml with similar results obtained.) Insets show higher magnification images of two different motile cells. (b) Wounding assays for NA13 and TA37 cells. Wounds were created in monolayers of NA13 or TA37 cells as described in Materials and Methods and photographed immediately and 48 hours later. (c) Wounds created in monolayers of NA13 and TA37 cells grown on glass coverslips were fixed and stained for F-actin with phalloidin 24 after wounding. Photographs show cells at the edge of the wound. Results for one of four independent experiments are shown. Bars: (a, c) 20 μm; (b) 100 μm.

Mentions: The greatly reduced chemotactic response of TA cells to EGF stimulation suggested that they might display impaired motility in response to EGF. A431, NA, and TA cells normally grow as packed colonies. Colonies of A431 cells respond to HGF/SF ligation of its receptor, c-Met, by scattering, which requires the disruption of cell–cell junctions and an increase in cell motility (Tajima et al., 1992). Like the EGFR, c-Met is a ligand stimulated protein tyrosine kinase. We found that EGF, in addition to HGF/SF, induced scattering in A431 cells, as had previously been demonstrated for SCC-derived cells HSC-1, which also overexpress EGFR (Fujii et al., 1996). To assess whether inhibition of AP-1 activity directly inhibits motility of A431 cells, we tested both EGF and HGF/SF for colony scattering activity on A431 cells, two NA clones, and three TA clones. EGF efficiently induced scattering of A431 and NA colonies within 48 h, but did not induce scattering of TA colonies (Fig. 4 a). Treatment of A431 or NA cells with the EGFR inhibitor CGP52411 inhibited EGF-stimulated colony scattering (data not shown). Similar results were obtained with HGF/SF treatment even though A431, NA, and TA cells all expressed equivalent levels of c-Met (data not shown). These results suggest that TAM67 inhibits growth factor-stimulated cell–cell disruption and/or cell motility.


The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, Ozanne BW - J. Cell Biol. (1998)

Expression of TAM67 inhibits motility of A431 cells.  (a) Colonies of cells were treated with EGF as described in Materials and Methods. Representative A431, NA, and TA colonies,  photographed using a phase contrast microscope, are shown before and 48 h after the addition of 10 ng/ml EGF. (EGF was also  used at concentrations of 2 and 5 ng/ml with similar results obtained.) Insets show higher magnification images of two different  motile cells. (b) Wounding assays for NA13 and TA37 cells.  Wounds were created in monolayers of NA13 or TA37 cells as  described in Materials and Methods and photographed immediately and 48 hours later. (c) Wounds created in monolayers of  NA13 and TA37 cells grown on glass coverslips were fixed and  stained for F-actin with phalloidin 24 after wounding. Photographs show cells at the edge of the wound. Results for one of  four independent experiments are shown. Bars: (a, c) 20 μm; (b)  100 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132955&req=5

Figure 4: Expression of TAM67 inhibits motility of A431 cells. (a) Colonies of cells were treated with EGF as described in Materials and Methods. Representative A431, NA, and TA colonies, photographed using a phase contrast microscope, are shown before and 48 h after the addition of 10 ng/ml EGF. (EGF was also used at concentrations of 2 and 5 ng/ml with similar results obtained.) Insets show higher magnification images of two different motile cells. (b) Wounding assays for NA13 and TA37 cells. Wounds were created in monolayers of NA13 or TA37 cells as described in Materials and Methods and photographed immediately and 48 hours later. (c) Wounds created in monolayers of NA13 and TA37 cells grown on glass coverslips were fixed and stained for F-actin with phalloidin 24 after wounding. Photographs show cells at the edge of the wound. Results for one of four independent experiments are shown. Bars: (a, c) 20 μm; (b) 100 μm.
Mentions: The greatly reduced chemotactic response of TA cells to EGF stimulation suggested that they might display impaired motility in response to EGF. A431, NA, and TA cells normally grow as packed colonies. Colonies of A431 cells respond to HGF/SF ligation of its receptor, c-Met, by scattering, which requires the disruption of cell–cell junctions and an increase in cell motility (Tajima et al., 1992). Like the EGFR, c-Met is a ligand stimulated protein tyrosine kinase. We found that EGF, in addition to HGF/SF, induced scattering in A431 cells, as had previously been demonstrated for SCC-derived cells HSC-1, which also overexpress EGFR (Fujii et al., 1996). To assess whether inhibition of AP-1 activity directly inhibits motility of A431 cells, we tested both EGF and HGF/SF for colony scattering activity on A431 cells, two NA clones, and three TA clones. EGF efficiently induced scattering of A431 and NA colonies within 48 h, but did not induce scattering of TA colonies (Fig. 4 a). Treatment of A431 or NA cells with the EGFR inhibitor CGP52411 inhibited EGF-stimulated colony scattering (data not shown). Similar results were obtained with HGF/SF treatment even though A431, NA, and TA cells all expressed equivalent levels of c-Met (data not shown). These results suggest that TAM67 inhibits growth factor-stimulated cell–cell disruption and/or cell motility.

Bottom Line: Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression.However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells.Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

View Article: PubMed Central - PubMed

Affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD, United Kingdom.

ABSTRACT
Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

Show MeSH
Related in: MedlinePlus