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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 AP-1 transactivation and invasion of A431 cells. (a) Western blot  analysis of A431 cells, two NA  clones, and three TA clones using a  polyclonal antibody against c-Jun to  reveal expression of TAM67. (b)  Measurement of AP-1 directed CAT  expression in NA and TA cells as  described in Materials and Methods.  Columns show the average for three  independent experiments. (c) Quantitative analysis of chemotaxis and  invasion of A431, NA, and TA cells  in response to EGF as described in  Materials and Methods and elsewhere (Hennigan et al., 1994; Lamb  et al., 1997a). Columns show the average for four independent experiments. (d) Confocal images of propidium iodide stained cell nuclei of  NA13 and TA37 cells at the bottom  (0 μm) and at a distance 30 μm from  the bottom of the filter (i.e., 20 μm  into the Matrigel) after EGF stimulation.
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Figure 2: Expression of TAM67 inhibits AP-1 transactivation and invasion of A431 cells. (a) Western blot analysis of A431 cells, two NA clones, and three TA clones using a polyclonal antibody against c-Jun to reveal expression of TAM67. (b) Measurement of AP-1 directed CAT expression in NA and TA cells as described in Materials and Methods. Columns show the average for three independent experiments. (c) Quantitative analysis of chemotaxis and invasion of A431, NA, and TA cells in response to EGF as described in Materials and Methods and elsewhere (Hennigan et al., 1994; Lamb et al., 1997a). Columns show the average for four independent experiments. (d) Confocal images of propidium iodide stained cell nuclei of NA13 and TA37 cells at the bottom (0 μm) and at a distance 30 μm from the bottom of the filter (i.e., 20 μm into the Matrigel) after EGF stimulation.

Mentions: Previously, we have demonstrated that invasion of v-fos-transformed or EGF-treated fibroblasts requires functional AP-1 (Lamb et al., 1997a). To determine if AP-1 activity is required for EGF-dependent in vitro invasion of human SCC-derived cell lines, we generated A431 transfectants constitutively expressing the c-Jun deletion mutant, TAM67. A431 cells were selected because their response to EGF treatment is well characterized and because they demonstrate a marked invasion response to EGF. TAM67 has been clearly demonstrated to ify AP-1 transcriptional activity (Brown et al., 1993; Domann et al., 1994b; Dong et al., 1997; Li et al., 1998). G418-resistant A431 subclones were obtained and tested by Western blot analysis for expression of TAM67. Three A431 TAM67-expressing clones, TA5, TA36, and TA37, and two nonexpressing G418-resistant clones, NA13 and NA15, were used most extensively in subsequent experiments (Fig. 2 a). Sustained TAM67 expression was found to inhibit basal and EGF-induced AP-1 transcriptional transactivation using a collagenase promoter-CAT reporter construct (Fig. 2 b). As was previously demonstrated for malignant mouse epidermal cell lines (Domann et al., 1994b; Dong et al., 1997), introduction of TAM67 did not alter the growth rates of expressing clones (data not shown).


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 AP-1 transactivation and invasion of A431 cells. (a) Western blot  analysis of A431 cells, two NA  clones, and three TA clones using a  polyclonal antibody against c-Jun to  reveal expression of TAM67. (b)  Measurement of AP-1 directed CAT  expression in NA and TA cells as  described in Materials and Methods.  Columns show the average for three  independent experiments. (c) Quantitative analysis of chemotaxis and  invasion of A431, NA, and TA cells  in response to EGF as described in  Materials and Methods and elsewhere (Hennigan et al., 1994; Lamb  et al., 1997a). Columns show the average for four independent experiments. (d) Confocal images of propidium iodide stained cell nuclei of  NA13 and TA37 cells at the bottom  (0 μm) and at a distance 30 μm from  the bottom of the filter (i.e., 20 μm  into the Matrigel) after EGF stimulation.
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Related In: Results  -  Collection

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

Figure 2: Expression of TAM67 inhibits AP-1 transactivation and invasion of A431 cells. (a) Western blot analysis of A431 cells, two NA clones, and three TA clones using a polyclonal antibody against c-Jun to reveal expression of TAM67. (b) Measurement of AP-1 directed CAT expression in NA and TA cells as described in Materials and Methods. Columns show the average for three independent experiments. (c) Quantitative analysis of chemotaxis and invasion of A431, NA, and TA cells in response to EGF as described in Materials and Methods and elsewhere (Hennigan et al., 1994; Lamb et al., 1997a). Columns show the average for four independent experiments. (d) Confocal images of propidium iodide stained cell nuclei of NA13 and TA37 cells at the bottom (0 μm) and at a distance 30 μm from the bottom of the filter (i.e., 20 μm into the Matrigel) after EGF stimulation.
Mentions: Previously, we have demonstrated that invasion of v-fos-transformed or EGF-treated fibroblasts requires functional AP-1 (Lamb et al., 1997a). To determine if AP-1 activity is required for EGF-dependent in vitro invasion of human SCC-derived cell lines, we generated A431 transfectants constitutively expressing the c-Jun deletion mutant, TAM67. A431 cells were selected because their response to EGF treatment is well characterized and because they demonstrate a marked invasion response to EGF. TAM67 has been clearly demonstrated to ify AP-1 transcriptional activity (Brown et al., 1993; Domann et al., 1994b; Dong et al., 1997; Li et al., 1998). G418-resistant A431 subclones were obtained and tested by Western blot analysis for expression of TAM67. Three A431 TAM67-expressing clones, TA5, TA36, and TA37, and two nonexpressing G418-resistant clones, NA13 and NA15, were used most extensively in subsequent experiments (Fig. 2 a). Sustained TAM67 expression was found to inhibit basal and EGF-induced AP-1 transcriptional transactivation using a collagenase promoter-CAT reporter construct (Fig. 2 b). As was previously demonstrated for malignant mouse epidermal cell lines (Domann et al., 1994b; Dong et al., 1997), introduction of TAM67 did not alter the growth rates of expressing clones (data not shown).

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