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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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EGF treatment leads to lamellipodia formation, membrane ruffling, and cortical actin polymerization in NA but not  TA cells. Confocal micrographs of rhodamine-phalloidin stained  NA13 and TA37 cells before (a and b), 5 min (c and d), and 15  min (e and f) after the addition of EGF. The main micrograph in  c shows a z-section towards the top of the cells to reveal membrane ruffles (arrowheads), whereas the insert shows an enhanced z-section at the base of the cells to reveal lamellipodia  (arrowhead). No z-section through TA cells revealed ruffles or  lamellipodia (d) even when enhanced to the same degree as the  inset in c (d, inset). Bottom panels of a, b, e, and f show optical  sections made perpendicular to the substratum through the cells  of the colonies above, at the position of the colony indicated by  the arrow. (Cells were treated with 100 ng/ml EGF. Similar results were also obtained with 10 ng/ml.) Photomicrographs shown  are representative of four independent experiments. Bar, 10 μm.
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Figure 5: EGF treatment leads to lamellipodia formation, membrane ruffling, and cortical actin polymerization in NA but not TA cells. Confocal micrographs of rhodamine-phalloidin stained NA13 and TA37 cells before (a and b), 5 min (c and d), and 15 min (e and f) after the addition of EGF. The main micrograph in c shows a z-section towards the top of the cells to reveal membrane ruffles (arrowheads), whereas the insert shows an enhanced z-section at the base of the cells to reveal lamellipodia (arrowhead). No z-section through TA cells revealed ruffles or lamellipodia (d) even when enhanced to the same degree as the inset in c (d, inset). Bottom panels of a, b, e, and f show optical sections made perpendicular to the substratum through the cells of the colonies above, at the position of the colony indicated by the arrow. (Cells were treated with 100 ng/ml EGF. Similar results were also obtained with 10 ng/ml.) Photomicrographs shown are representative of four independent experiments. Bar, 10 μm.

Mentions: We investigated whether EGFR activation results in actin cytoskeletal rearrangements in TA cells, culminating in cell rounding. Serum-starved NA and TA cells displayed low levels of F-actin as judged by phalloidin staining (Fig. 5, a and b). EGF treatment of NA cells resulted in membrane ruffles (Fig. 5 c) and lamellipodial extensions (Fig. 5 c, insert) within 5 min, which was followed by a dramatic increase in polymerized cortical actin by 15 min (Fig. 5 e). No increase in actin stress fibers was detected at 5 or 15 min after EGF treatment (Fig. 5, c and e). When TA cells were treated with EGF, membrane ruffling, lamellipodia formation, and cortical actin polymerization were all greatly reduced (Fig. 5, d and f). The cortical actin polymerization occurring in NA cells 15 min after EGF treatment was associated with an increase in cell height. The bottom panels of Fig. 5, a, b, e, and f show confocal microscopy optical sections made perpendicular to the substratum through the cells at the position of the colony indicated by the arrow. As judged by comparing these bottom panels, 15 min of EGF treatment resulted in a significant increase in cell height in NA cells, which was much reduced in TA cells. The presence of membrane ruffles, lamellipodia, and polymerized cortical actin was tested in A431 cells and clones NA13, NA15, TA5, TA36, and TA37 with similar results as shown in Fig. 5 for clones NA13 and TA37. 40 min after treatment with EGF, the great majority of A431 and NA cells became rounded/contracted, whereas TA cells did not (Fig. 6). EGF-induced rounding was tested in eight NA and eight TA clones with results similar to those shown in Fig. 6 for A431 cells and clones NA13, TA37, and TA36. We conclude that sustained expression of TAM67 inhibits EGF-induced cytoskeletal rearrangements and cell rounding.


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)

EGF treatment leads to lamellipodia formation, membrane ruffling, and cortical actin polymerization in NA but not  TA cells. Confocal micrographs of rhodamine-phalloidin stained  NA13 and TA37 cells before (a and b), 5 min (c and d), and 15  min (e and f) after the addition of EGF. The main micrograph in  c shows a z-section towards the top of the cells to reveal membrane ruffles (arrowheads), whereas the insert shows an enhanced z-section at the base of the cells to reveal lamellipodia  (arrowhead). No z-section through TA cells revealed ruffles or  lamellipodia (d) even when enhanced to the same degree as the  inset in c (d, inset). Bottom panels of a, b, e, and f show optical  sections made perpendicular to the substratum through the cells  of the colonies above, at the position of the colony indicated by  the arrow. (Cells were treated with 100 ng/ml EGF. Similar results were also obtained with 10 ng/ml.) Photomicrographs shown  are representative of four independent experiments. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 5: EGF treatment leads to lamellipodia formation, membrane ruffling, and cortical actin polymerization in NA but not TA cells. Confocal micrographs of rhodamine-phalloidin stained NA13 and TA37 cells before (a and b), 5 min (c and d), and 15 min (e and f) after the addition of EGF. The main micrograph in c shows a z-section towards the top of the cells to reveal membrane ruffles (arrowheads), whereas the insert shows an enhanced z-section at the base of the cells to reveal lamellipodia (arrowhead). No z-section through TA cells revealed ruffles or lamellipodia (d) even when enhanced to the same degree as the inset in c (d, inset). Bottom panels of a, b, e, and f show optical sections made perpendicular to the substratum through the cells of the colonies above, at the position of the colony indicated by the arrow. (Cells were treated with 100 ng/ml EGF. Similar results were also obtained with 10 ng/ml.) Photomicrographs shown are representative of four independent experiments. Bar, 10 μm.
Mentions: We investigated whether EGFR activation results in actin cytoskeletal rearrangements in TA cells, culminating in cell rounding. Serum-starved NA and TA cells displayed low levels of F-actin as judged by phalloidin staining (Fig. 5, a and b). EGF treatment of NA cells resulted in membrane ruffles (Fig. 5 c) and lamellipodial extensions (Fig. 5 c, insert) within 5 min, which was followed by a dramatic increase in polymerized cortical actin by 15 min (Fig. 5 e). No increase in actin stress fibers was detected at 5 or 15 min after EGF treatment (Fig. 5, c and e). When TA cells were treated with EGF, membrane ruffling, lamellipodia formation, and cortical actin polymerization were all greatly reduced (Fig. 5, d and f). The cortical actin polymerization occurring in NA cells 15 min after EGF treatment was associated with an increase in cell height. The bottom panels of Fig. 5, a, b, e, and f show confocal microscopy optical sections made perpendicular to the substratum through the cells at the position of the colony indicated by the arrow. As judged by comparing these bottom panels, 15 min of EGF treatment resulted in a significant increase in cell height in NA cells, which was much reduced in TA cells. The presence of membrane ruffles, lamellipodia, and polymerized cortical actin was tested in A431 cells and clones NA13, NA15, TA5, TA36, and TA37 with similar results as shown in Fig. 5 for clones NA13 and TA37. 40 min after treatment with EGF, the great majority of A431 and NA cells became rounded/contracted, whereas TA cells did not (Fig. 6). EGF-induced rounding was tested in eight NA and eight TA clones with results similar to those shown in Fig. 6 for A431 cells and clones NA13, TA37, and TA36. We conclude that sustained expression of TAM67 inhibits EGF-induced cytoskeletal rearrangements and cell rounding.

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