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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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Inactivation of Rac and Rho in NA13 cells inhibits  EGF-induced actin rearrangements. Confocal micrographs of  cells microinjected with an expression construct encoding a myc-tagged version of RacN17 (a–d) or with C3 transferase and FITC-dextran (e and f), treated with EGF for either 5 min (a and b) or  15 min (c–f), and costained either for myc and phalloidin (cells in  a–d) or for phalloidin alone (e and f). Microinjected cells were  detected either by myc-tag specific mAb (9E10)-staining (b and d)  or the presence of FITC-dextran (f). Arrowheads in a point to  lamellipodia and membrane ruffles in noninjected cells, and in  c and e indicate noninjected cells with cortical F-actin. A representative field of cells for at least three independent experiments  is shown for each treatment. Bar, 10 μm.
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Figure 7: Inactivation of Rac and Rho in NA13 cells inhibits EGF-induced actin rearrangements. Confocal micrographs of cells microinjected with an expression construct encoding a myc-tagged version of RacN17 (a–d) or with C3 transferase and FITC-dextran (e and f), treated with EGF for either 5 min (a and b) or 15 min (c–f), and costained either for myc and phalloidin (cells in a–d) or for phalloidin alone (e and f). Microinjected cells were detected either by myc-tag specific mAb (9E10)-staining (b and d) or the presence of FITC-dextran (f). Arrowheads in a point to lamellipodia and membrane ruffles in noninjected cells, and in c and e indicate noninjected cells with cortical F-actin. A representative field of cells for at least three independent experiments is shown for each treatment. Bar, 10 μm.

Mentions: To study the involvement of Rho-like GTPases in EGF-induced cytoskeletal changes, Rac1 was selectively inhibited by microinjection of NA13 cells with a plasmid encoding a myc-epitope-tagged dominant negative mutant of Rac1 (DN-Rac) and Rho by microinjection of Clostridium botulinum exoenzyme C3 transferase. After an overnight period, following microinjection of DN-Rac, or 15 min after the microinjection of C3 transferase, cells were treated with EGF for 5 or 15 min and stained for F-actin with phalloidin. Expression of DN-Rac inhibited membrane ruffles and lamellipodia formation (Fig. 7, a and b), indicating that Rac activation is required for the formation of these membrane protrusions in EGF-treated NA13 cells. No accumulation of polymerized cortical actin nor cell rounding was detected in the C3 transferase-microinjected NA13 cells after 15 min of EGF treatment (Fig. 7, e and f), although membrane ruffles were observable after 5 min EGF treatment (data not shown), indicating that C3 transferase was not inhibiting Rac activity. This observation demonstrates that Rho activity is required for EGF-induced cortical actin polymerization and cell rounding. Furthermore, treatment of NA13 cells microinjected with DN-Rac with EGF for 15 min blocked cortical actin polymerization (Fig. 7, c and d). This suggests that Rac activation is upstream of Rho activation in the EGF-dependent pathway regulating cortical actin polymerization in A431 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)

Inactivation of Rac and Rho in NA13 cells inhibits  EGF-induced actin rearrangements. Confocal micrographs of  cells microinjected with an expression construct encoding a myc-tagged version of RacN17 (a–d) or with C3 transferase and FITC-dextran (e and f), treated with EGF for either 5 min (a and b) or  15 min (c–f), and costained either for myc and phalloidin (cells in  a–d) or for phalloidin alone (e and f). Microinjected cells were  detected either by myc-tag specific mAb (9E10)-staining (b and d)  or the presence of FITC-dextran (f). Arrowheads in a point to  lamellipodia and membrane ruffles in noninjected cells, and in  c and e indicate noninjected cells with cortical F-actin. A representative field of cells for at least three independent experiments  is shown for each treatment. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Inactivation of Rac and Rho in NA13 cells inhibits EGF-induced actin rearrangements. Confocal micrographs of cells microinjected with an expression construct encoding a myc-tagged version of RacN17 (a–d) or with C3 transferase and FITC-dextran (e and f), treated with EGF for either 5 min (a and b) or 15 min (c–f), and costained either for myc and phalloidin (cells in a–d) or for phalloidin alone (e and f). Microinjected cells were detected either by myc-tag specific mAb (9E10)-staining (b and d) or the presence of FITC-dextran (f). Arrowheads in a point to lamellipodia and membrane ruffles in noninjected cells, and in c and e indicate noninjected cells with cortical F-actin. A representative field of cells for at least three independent experiments is shown for each treatment. Bar, 10 μm.
Mentions: To study the involvement of Rho-like GTPases in EGF-induced cytoskeletal changes, Rac1 was selectively inhibited by microinjection of NA13 cells with a plasmid encoding a myc-epitope-tagged dominant negative mutant of Rac1 (DN-Rac) and Rho by microinjection of Clostridium botulinum exoenzyme C3 transferase. After an overnight period, following microinjection of DN-Rac, or 15 min after the microinjection of C3 transferase, cells were treated with EGF for 5 or 15 min and stained for F-actin with phalloidin. Expression of DN-Rac inhibited membrane ruffles and lamellipodia formation (Fig. 7, a and b), indicating that Rac activation is required for the formation of these membrane protrusions in EGF-treated NA13 cells. No accumulation of polymerized cortical actin nor cell rounding was detected in the C3 transferase-microinjected NA13 cells after 15 min of EGF treatment (Fig. 7, e and f), although membrane ruffles were observable after 5 min EGF treatment (data not shown), indicating that C3 transferase was not inhibiting Rac activity. This observation demonstrates that Rho activity is required for EGF-induced cortical actin polymerization and cell rounding. Furthermore, treatment of NA13 cells microinjected with DN-Rac with EGF for 15 min blocked cortical actin polymerization (Fig. 7, c and d). This suggests that Rac activation is upstream of Rho activation in the EGF-dependent pathway regulating cortical actin polymerization in A431 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