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First published online 23 November 2004
doi: 10.1242/jcs.01566

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TNF induces sequential activation of Cdc42- and p38/p53-dependent pathways that antagonistically regulate filopodia formation

Centre de Recherche en Biochimie Macromoléculaire, CNRS FRE 2593, IFR 24, 1919 route de Mende, 34293 Montpellier CEDEX 5, France

View larger version (50K): [in a new window]   Fig. 1. Cdc42 is required for the rapid induction of TNF-mediated filopodia formation, but not for their subsequent decrease. (A) Representative actin staining of MEFs following TNF-treatment. Serum-starved MEFs were treated with 100 ng ml–1 TNF for the indicated time, then fixed and stained for F-actin. Arrows depict filopodia. Bar, 10 µm. (B) Quantification of MEFs having filopodia shown in (A). The percentages of filopodia-positive cells following TNF-treatment, relative to control cells, are shown. Values are means±s.d. of three independent experiments. Significance in induction of filopodia formation caused by TNF compared with untreated: P=0.016 (Student's t-test). (C) Effect of inhibition of Cdc42 activity on TNF-induced filopodia formation. MEFs were transfected with either GFP-tagged Cdc42-N17 (dominant negative form of Cdc42) (a and b) or with the control plasmid pEGFP (c and d). Serum-arrested transfected MEFs were treated with 100 ng ml–1 TNF for 10 minutes, then fixed and analysed for F-actin organisation (b and d) and GFP staining (a and c). Arrows depict filopodia. Bar, 10 µm. (D) Quantification of Cdc42-N17 transfected MEFs having filopodia. MEFs transfected with GFP-tagged Cdc42-N17 were treated as in (A) with TNF for the indicated times. The percentages of filopodia-positive cells following TNF-treatment are shown. Values are means±s.d. of three independent experiments. (E) Cdc42 activity in TNF-treated MEFs. Serum-arrested MEFs were treated with 100 ng ml–1 TNF for various times as indicated, then lysed and the GTP-bound form of Cdc42 was assayed as described in Materials and Methods.

View larger version (21K): [in a new window]   Fig. 2. p38 kinase is required for the inhibition of TNF-induced filopodia formation. (A) p38 kinase assay in TNF-treated MEFs. Serum-arrested cells were transfected with the HA-tagged p38 kinase, then treated with 100 ng ml–1 TNF for the indicated times. Immunoprecipitated p38 kinase activity was determined as described in Materials and Methods. Values are the means±s.d. of three independent experiments. Statistical significance of p38 kinase activation caused by TNF compared with untreated: P=0.061 (Student's t-test). (B) p38 kinase assay in transfected MEFs treated with TNF. MEFs were transfected with the HA-tagged p38 kinase in the presence or not of vectors expressing either Cdc42-N17, N-Wasp, Cdc42-V12, MKK3, KD-MKK3 or pCDNA3. Serum-arrested cells were treated or not with 100 ng ml–1 TNF for the indicated times. Immunoprecipitated p38 kinase activity was determined as described in Materials and Methods. The values are the means±s.d. of three independent experiments. Statistical significance of p38 kinase activation caused by TNF compared with untreated: P=0.061 (Student's t-test).

View larger version (46K): [in a new window]   Fig. 3. Inhibition of TNF-induced filopodia formation depends on p38 kinase activity. (A) Quantification of TNF-treated MEFs having filopodia in the presence of SB 203580. The percentages of filopodia-positive cells treated with TNF in the presence of the p38 kinase inhibitor SB 203580 relative to control cells are shown. Cells were fixed, stained for F-actin, then quantified for the presence of filopodia, as described in Materials and Methods. For each experiment, 100 cells were scored and results are expressed as the means±s.d. of three independent experiments. (B) Representative actin staining of transfected MEFs. MEFs were transiently transfected with GFP-labelled Cdc42-V12 (visualised in a and d) or with HA-tagged MKK3, or both. 20 hours later cells were observed for F-actin staining (c, e and g) and for MKK3 expression (b and f). Bar, 10 µm. (C) Representative actin staining of transfected MEFs following TNF treatment. MEFs were transiently transfected with HA-tagged MKK3, then co-treated with TNF and SB 203580. After fixation, cells were visualised and stained for MKK3 expression (a) and for F-actin (b). (D) Quantification of MEFs having filopodia shown in MEFs from B and C, transfected or not with MKK3, were pretreated or not with SB 203580 and/or with 100 ng ml–1 TNF for the indicated time. Cells were fixed, stained for F-actin then quantified for the presence of filopodia. MEFs were scored positively when presenting at least five filopodia. For each experiment, 100 cells were scored and values are the means±s.d. of three independent experiments. (E) Quantification of MEFs having filopodia shown in MEFs from B, transfected or not with Cdc42-V12, were pretreated or not with SB 203580 and/or with TNF for the indicated time. Scoring and analysis were performed as in D.

View larger version (13K): [in a new window]   Fig. 4. p53-dependent transcriptional activity in TNF-treated MEFs. MEFs were transfected with the firefly luciferase gene under the control of the p53 responsive element derived from the mdm2 gene in combination with empty vector alone (Control), or MKK3, or Cdc42-N17, or N-Wasp, or KD-MKK3 or p53 wt as positive control. As indicated, cells were treated with either TNF, or SB 203580, or both. The luciferase activity was assayed 24 hours later. The results are expressed as means±s.d. of four independent experiments.

View larger version (40K): [in a new window]   Fig. 5. TNF activates p53 and induces its phosphorylation at serine 18 through p38 kinase. (A) Abundance and status of phosphorylated p53 at serine 18, p53 and p21 in TNF-treated MEFs. Serum-arrested MEFs were treated with TNF for the indicated time. Total protein lysates were prepared and analysed by gel electrophoresis followed by immuno-blotting analysis using antibodies to p53 phosphorylated at serine 18 (pp53/Ser18), p53 and p21. Each lane contains equal amounts of loaded proteins (40 µg). (B) Abundance and status of phosphorylated p53 at serine 18, p53 and p21 in TNF-treated MEFs prealably treated with SB 203580. Serum-arrested cells were pre-treated with SB 203580, followed by a treatment with TNF for the indicated time. Lysates were analysed as in (A). (C) Abundance and status of phosphorylated p53 at serine 18, p53 and p21 in TNF-treated MEFs expressing Cdc42-N7. Cells were transfected with GFP-tagged Cdc42-N17 before serum-starvation, followed by a treatment with TNF for the indicated time. Lysates were analysed as in (A).

View larger version (46K): [in a new window]   Fig. 6. Wild-type p53 activity is required to mediate TNF-induced filopodia inhibition. (A) Effects of TNF on filopodia formation in actinomycin D-treated MEFs. Serum-arrested cells were pretreated or not with the transcription inhibitor actinomycin D, followed by a treatment with 100 ng ml–1 TNF for various times as indicated, fixed and stained for F-actin, then quantified for the presence of filopodia. For each experiment, 100 cells were scored and results are the mean±s.d. of three independent experiments. (B) Quantification of MEFs having filopodia. MEFs were transfected or not with plasmids encoding p53 wt, p53-H175, p53-H273 or pEGFP, then serum arrested before treatment or not with TNF for 1 hour as indicated. After fixation and staining for F-actin, cells were analysed for the presence of filopodia as indicated in Fig. 3. (C) Representative actin staining of MEFs transfected MEFs with p53-H273. Serum arrested cells were treated (a and b) or not (c and d) with TNF for 1 hour. Cells were fixed, visualised for p53 expression as revealed by GFP staining (a) and stained for F-actin (b). Arrows indicate filopodia. Bar, 10 µm. (D) Actin staining of p53–/–MEFs. Cells were either untreated (a) or treated with TNF for 1 hour (b), then fixed and stained for F-actin. In (c) and (d), p53–/–MEFs were transfected with HA-tagged MKK3 before staining for MKK3 (c) or F-actin (d). Arrows indicate filopodia. Bar, 10 µm. (E) Quantification of p53–/–MEFs having filopodia. The percentages of filopodia-positive cells relative to control cells are shown. p53–/–MEFs were transfected or not with plasmids encoding pEGFP (Control), p53 wt, MKK3, or treated with TNF for 1 hour, as indicated. Cells were fixed, stained for F-actin, then quantified for the presence of filopodia, as described in Materials and Methods. For each experiment, 100 cells were scored and results are expressed as the means±s.d. of three independent experiments. (F) Abundance of Cdc42 and p38 in MEFs expressing p53 mutants and in p53–/–MEFs. MEFs were transfected with pEGFP, p53H273, p53 H175, p53 wt and p53–/–MEFs before preparation of total protein lysates, and analysis by gel electrophores was followed by immunoblotting analysis using antibodies to Cdc42 and p38. Each lane contains equal amounts of loaded proteins (40 µg).