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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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Activated forms of Rac and Rho function equally in  NA13 and TA37 cells. Confocal micrographs of cells microinjected with expression constructs encoding myc-tagged versions  of either V12Rac (a–d) or V14Rho (e–h) and stained for the myc-tag using a myc specific mAb (9E10; to detect the injected cells)  and phalloidin (for the visualization of polymerized actin). Arrowheads in a and c indicate colocalization of Rac and F-actin at  sites of cell–cell contacts. A representative field of cells for at  least three independent experiments is shown for each treatment.  Bar, 10 μm.
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Figure 8: Activated forms of Rac and Rho function equally in NA13 and TA37 cells. Confocal micrographs of cells microinjected with expression constructs encoding myc-tagged versions of either V12Rac (a–d) or V14Rho (e–h) and stained for the myc-tag using a myc specific mAb (9E10; to detect the injected cells) and phalloidin (for the visualization of polymerized actin). Arrowheads in a and c indicate colocalization of Rac and F-actin at sites of cell–cell contacts. A representative field of cells for at least three independent experiments is shown for each treatment. Bar, 10 μm.

Mentions: The inability of EGF to induce membrane ruffles, lamellipodia, and cortical F-actin in TA cells suggests that EGFR signaling to the actin cytoskeleton is compromised in these cells. This could be due to a failure to activate Rac and Rho, the absence or functional inactivity of certain downstream effectors of Rac and Rho, or a combination of both. To determine whether the downstream effectors of Rac and Rho responsible for the EGF-induced cytoskeletal rearrangements are present and functional in TA cells, plasmids directing the expression of myc-epitope-tagged activated forms of Rac1 and RhoA (RacV12 and RhoV14) were introduced by microinjection into NA13 and TA37 cells. Expression of RacV12 resulted in the appearance of F-actin at the site of cell to cell contacts, where RacV12 also accumulated (Fig. 8, a–d), as has also been reported for MDCK cells (Ridley et al., 1995; Hordijk et al., 1997; Takaishi et al., 1997). Expression of RhoV14 resulted in an increase in F-actin around the cell cortex and rounded morphology resembling that which occurred after EGF treatment (Fig. 8, e–h). It also induced a low but detectable increase in actin stress fibers, which did not occur after EGF treatment (data not shown). These results indicate that the main cytoskeletal rearrangement in A431 cells mediated by Rho is the polymerization of cortical actin rather than the formation of stress fibers. Most significantly, however, actin structures formed in both NA13 cells and TA37 cells after microinjection of either RacV12 or RhoV14 were equivalent (compare Fig. 8 a with c and e with g). These results indicate that the downstream effectors of Rac and Rho required for the specific EGF-induced actin cytoskeletal and morphological changes are functional in TA cells, which suggests that EGF activation of Rac and Rho is compromised 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)

Activated forms of Rac and Rho function equally in  NA13 and TA37 cells. Confocal micrographs of cells microinjected with expression constructs encoding myc-tagged versions  of either V12Rac (a–d) or V14Rho (e–h) and stained for the myc-tag using a myc specific mAb (9E10; to detect the injected cells)  and phalloidin (for the visualization of polymerized actin). Arrowheads in a and c indicate colocalization of Rac and F-actin at  sites of cell–cell contacts. 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 8: Activated forms of Rac and Rho function equally in NA13 and TA37 cells. Confocal micrographs of cells microinjected with expression constructs encoding myc-tagged versions of either V12Rac (a–d) or V14Rho (e–h) and stained for the myc-tag using a myc specific mAb (9E10; to detect the injected cells) and phalloidin (for the visualization of polymerized actin). Arrowheads in a and c indicate colocalization of Rac and F-actin at sites of cell–cell contacts. A representative field of cells for at least three independent experiments is shown for each treatment. Bar, 10 μm.
Mentions: The inability of EGF to induce membrane ruffles, lamellipodia, and cortical F-actin in TA cells suggests that EGFR signaling to the actin cytoskeleton is compromised in these cells. This could be due to a failure to activate Rac and Rho, the absence or functional inactivity of certain downstream effectors of Rac and Rho, or a combination of both. To determine whether the downstream effectors of Rac and Rho responsible for the EGF-induced cytoskeletal rearrangements are present and functional in TA cells, plasmids directing the expression of myc-epitope-tagged activated forms of Rac1 and RhoA (RacV12 and RhoV14) were introduced by microinjection into NA13 and TA37 cells. Expression of RacV12 resulted in the appearance of F-actin at the site of cell to cell contacts, where RacV12 also accumulated (Fig. 8, a–d), as has also been reported for MDCK cells (Ridley et al., 1995; Hordijk et al., 1997; Takaishi et al., 1997). Expression of RhoV14 resulted in an increase in F-actin around the cell cortex and rounded morphology resembling that which occurred after EGF treatment (Fig. 8, e–h). It also induced a low but detectable increase in actin stress fibers, which did not occur after EGF treatment (data not shown). These results indicate that the main cytoskeletal rearrangement in A431 cells mediated by Rho is the polymerization of cortical actin rather than the formation of stress fibers. Most significantly, however, actin structures formed in both NA13 cells and TA37 cells after microinjection of either RacV12 or RhoV14 were equivalent (compare Fig. 8 a with c and e with g). These results indicate that the downstream effectors of Rac and Rho required for the specific EGF-induced actin cytoskeletal and morphological changes are functional in TA cells, which suggests that EGF activation of Rac and Rho is compromised 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