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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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(A) Rac translocates to membrane ruffles in NA13 but  not TA37 cells after 5 min treatment with EGF. Confocal micrographs are shown for NA13 and TA37 cells stained using a mouse  anti-Rac mAb. (a and b) NA13 and TA37 cells untreated; (c and e)  NA13 cells treated with 100 ng/ml EGF for 5 min; (d and f) TA37  cells treated with 100 ng/ml EGF for 5 min. A representative field  of cells for at least three independent experiments is shown. (B)  Western blot analysis demonstrating equivalent levels of expression of Rac and Rho in A431, NA, and TA cells. Bar, 10 μm.
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Figure 9: (A) Rac translocates to membrane ruffles in NA13 but not TA37 cells after 5 min treatment with EGF. Confocal micrographs are shown for NA13 and TA37 cells stained using a mouse anti-Rac mAb. (a and b) NA13 and TA37 cells untreated; (c and e) NA13 cells treated with 100 ng/ml EGF for 5 min; (d and f) TA37 cells treated with 100 ng/ml EGF for 5 min. A representative field of cells for at least three independent experiments is shown. (B) Western blot analysis demonstrating equivalent levels of expression of Rac and Rho in A431, NA, and TA cells. Bar, 10 μm.

Mentions: No assay is presently available to directly measure the activation of Rho. However, in mouse dermal fibroblasts Rac has been shown to be recruited from the cytoplasm to the plasma membrane upon growth factor stimulation concomitant with its activation (Azuma et al., 1998). In serum-deprived NA13 and TA37 cells Rac as detected by immunofluorescence was not found associated with the plasma membrane (Fig. 9 A, a and b). Upon EGF stimulation though Rac was detected in the plasma membrane of NA13 but not TA37 cells (compare Fig. 9 A, c and e with d and f), indicating that EGF stimulation does not activate Rac in TA cells.


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)

(A) Rac translocates to membrane ruffles in NA13 but  not TA37 cells after 5 min treatment with EGF. Confocal micrographs are shown for NA13 and TA37 cells stained using a mouse  anti-Rac mAb. (a and b) NA13 and TA37 cells untreated; (c and e)  NA13 cells treated with 100 ng/ml EGF for 5 min; (d and f) TA37  cells treated with 100 ng/ml EGF for 5 min. A representative field  of cells for at least three independent experiments is shown. (B)  Western blot analysis demonstrating equivalent levels of expression of Rac and Rho in A431, NA, and TA cells. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132955&req=5

Figure 9: (A) Rac translocates to membrane ruffles in NA13 but not TA37 cells after 5 min treatment with EGF. Confocal micrographs are shown for NA13 and TA37 cells stained using a mouse anti-Rac mAb. (a and b) NA13 and TA37 cells untreated; (c and e) NA13 cells treated with 100 ng/ml EGF for 5 min; (d and f) TA37 cells treated with 100 ng/ml EGF for 5 min. A representative field of cells for at least three independent experiments is shown. (B) Western blot analysis demonstrating equivalent levels of expression of Rac and Rho in A431, NA, and TA cells. Bar, 10 μm.
Mentions: No assay is presently available to directly measure the activation of Rho. However, in mouse dermal fibroblasts Rac has been shown to be recruited from the cytoplasm to the plasma membrane upon growth factor stimulation concomitant with its activation (Azuma et al., 1998). In serum-deprived NA13 and TA37 cells Rac as detected by immunofluorescence was not found associated with the plasma membrane (Fig. 9 A, a and b). Upon EGF stimulation though Rac was detected in the plasma membrane of NA13 but not TA37 cells (compare Fig. 9 A, c and e with d and f), indicating that EGF stimulation does not activate Rac in TA cells.

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