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First published online March 29, 2004
doi: 10.1242/10.1242/jcs.01036

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Smad7 is required for TGF-ß-induced activation of the small GTPase Cdc42

Ludwig Institute for Cancer Research, Biomedical Center, Box 595, 751 24 Uppsala, Sweden

View larger version (89K): [in a new window]   Fig. 1. Smad7-dependent activation of the actin filament system. PC-3U/pMEP4-S7 cells were serum-starved in 1% FBS for 12 hours and treated with 1 µM CdCl2 for 12 hours or 24 hours, or with 1 µM CdCl2 for 12 hours together with TGF-ß for 30 minutes and 12 hours or 24 hours. The expression of Flag-tagged Smad7 was visualized by a Flag-specific mouse antibody (M5) followed by a FITC-labeled anti-mouse antibody. Filamentous actin was visualized by TRITC-labeled phalloidin. Arrowheads indicate cells with lamellipodia. Bars, 20 µm.

View larger version (49K): [in a new window]   Fig. 2. Quantification of the Smad7-dependent activation of the actin filament system. (A) Confocal image of a representative PC-3U/pMEP4-S7 cell treated with 1 µM CdCl2 for 12 hours. Flag-Smad7 and filamentous actin was detected as in Fig. 1. The fluorescence intensity changes across the yellow line in the direction of the arrowhead were plotted as the line intensities in the histogram in the lower panel. The red channel (Ch1-T1) represents filamentous actin and the green channel (Ch2-T2) represents Flag-Smad7. (B) Quantification of the effect of CdCl2 and TGF-ß treatment of the PC-3U/pMEP4-S7 cells in Fig. 1 on membrane ruffling and stress-fiber formation. At least 300 cells were counted under the microscope for each condition. (C) PC-3U/pMEP4-S7 cells were serum-starved in 1% FBS for 12 hours and stimulated with 5 ng/ml TGF-ß for 30 minutes, 12 hours and 24 hours to show the effect on the actin filament system in the absence of CdCl2-induced Smad7 expression. Parental PC-3U cells were mock-treated with 1 µM CdCl2 for 12 hours or stimulated with 5 nM TGF-ß for 12 hours to show that CdCl2 alone did not induce any effect on the actin filament system. Bars, 20 µm.

View larger version (36K): [in a new window]   Fig. 3. Smad7-dependent activation of the Rho GTPases. (A) PC-3U/pMEP4-S7 cells were serum-starved in 1% FBS for 12 hours and treated with 1 µM CdCl2 for 12 hours or with 1 µM CdCl2 together with 5 ng/ml TGF-ß for 15 minutes up to 48 hours. The control cells, PC-3U, were stimulated with 5 ng/ml of TGF-ß for 12 hours as positive control, and with CdCl2 for 12 hours as negative control. The amount of active, GTP-loaded Rac1, Cdc42 and RhoA was determined by GST pull-down assays with GST-PAK-CRIB, GST-WASP-CRIB or GST-Rhotekin, respectively. Rac1, Cdc42 and RhoA were detected by immunoblotting using antibodies specific for the respective GTPase. (B) Immunoblots were analyzed by densitometry and the data were combined into diagrams showing the activation of Rac1, Cdc42 and RhoA. Each column represents the mean + s.e.m. of three independent experiments.

View larger version (93K): [in a new window]   Fig. 4. Effects of Smad7 depletion on actin organization and activation of Rho GTPases. (A) PC-3U/AS-S7 cells (upper panel) or parental PC-3U cells (lower panel) were serum-starved in 1% FBS for 12 hours and treated with 5 ng/ml TGF-ß for 15 minutes, 30 minutes, 12 hours and 24 hours. Actin filaments were visualized by TRITC-labeled phalloidin. Arrowheads in the lower panel indicate parental cells with membrane ruffles, a response absent in PC-3U/AS-S7 cells. Bar, 20 µm. (B) Quantification of the effect of TGF-ß treatment of the PC-3U/AS-S7 and PC-3U cells in panel A on membrane ruffling and stress-fiber formation. At least 300 cells were counted under the microscope for each condition. (C) PC-3U/AS-S7 cells were serum-starved in 1% FBS for 12 hours and treated with 5 ng/ml TGF-ß for 5 minutes to 48 hours. As a positive control, PC-3U cells were stimulated with 5 ng/ml of TGF-ß for 15 minutes and 12 hours. The amount of active, GTP-loaded Rac1, Cdc42 and RhoA was determined by GST pull-down assays with GST-PAK-CRIB, GST-WASP-CRIB, or GST-Rhotekin, respectively. Rac1, Cdc42 and RhoA were detected by immunoblotting using antibodies specific for the respective GTPase. (D) Immunoblots were analyzed by densitometry and the data are combined into diagrams showing the activation of Rac1, Cdc42, and RhoA. Each column represents the mean + s.e.m. from three independent experiments.

View larger version (40K): [in a new window]   Fig. 5. Involvement of phosphatidylinositol-3 kinase in the TGF-ß-induced activation of actin filament system. (A) PC-3U cells were serum-starved in 1% FBS for 12 hours, treated or not for 1 hour with 10 µM of the phosphatidylinositol-3 kinase inhibitor LY204009, before stimulation with 10 ng/ml of TGF-ß for 30 minutes or 24 hours. Filamentous actin was visualized by TRITC-labeled phalloidin. Arrowheads indicate cells with lamellipodia (30 minutes) and stress fibers (24 hours). Bar, 20 µm. (B) PC-3U cells were starved in 1% FBS for 12 hours and stimulated with 10 ng/ml of TGF-ß for different time periods as depicted in the panel. The total cell lysates were subjected to SDS-PAGE followed by immunoblotting using specific Akt antibodies. In the upper and lower panels, the phosphorylated and the nonphosphorylated forms of Akt are shown, respectively. (C) PC-3U cells were starved in 1% FBS for 12 hours, pretreated for 1 hour with or without 10 µM of the phosphatidylinositol-3 kinase inhibitor and stimulated with 10 ng/ml of TGF-ß for 30 minutes and 24 hours. The total cell lysates were subjected to SDS-PAGE followed by immunoblotting with specific Akt antibodies. (D) PC-3U cells were starved in 1% FBS for 12 hours, pretreated for 1 hour with or without 10 µM of the phosphatidylinositol-3 kinase inhibitor and stimulated with 10 ng/ml TGF-ß for up to 48 hours. The total cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies specific for Smad2 phosphorylated on serine residues 465 and 467 or Smad2.

View larger version (25K): [in a new window]   Fig. 6. The effect on activation of Rho GTPases of inhibitors of p38 MAPK and phosphatidylinositol-3 kinase. (A) PC-3U/pMEP4-S7 were starved in 1% FBS for 12 hours, pretreated with or without either 10 µM SB203580 or 10 µM LY294002 for 1 hour, and stimulated with 1 µM of CdCl2 for 12 hours. The parental cells, PC-3U, were stimulated with 5 ng/ml of TGF-ß for 12 hours as positive control and CdCl2 for 12 hours as negative control. The amount of active, GTP-loaded Rac1, Cdc42 and RhoA was determined by GST pull-down assays with GST-PAK-CRIB, GST-WASP-CRIB or GST-Rhotekin, respectively. Rac1, Cdc42 and RhoA were detected by immunoblotting using antibodies specific for the respective GTPase. (B) Immunoblots were analyzed by densitometry and the data are combined into diagrams showing the activation of Rac1, Cdc42, and RhoA. Each column represents the mean + s.e.m. of three independent experiments.

View larger version (106K): [in a new window]   Fig. 7. The effect of p38 inhibitors MAPK and PI3K on Smad7-dependent activation of the actin filament system. (A) PC-3U/pMEP4-S7 cells were starved in 1% FBS for 12 hours and pretreated for 1 hour with 10 µM SB203580, before the addition of 1 µM of CdCl2 for 12 hours or 24 hours, or 1 µM CdCl2 for 12 hours together with 30 minutes, 12 hours or 24 hours of TGF-ß. Flag-tagged Smad7 was visualized by a Flag-specific antibody followed by an FITC-labeled anti-mouse antibody. Filamentous actin was visualized by TRITC-labeled phalloidin. Arrowheads indicate cells with stress fibers (24 hours, upper panel) and cells lacking stress fibers (24 hours, lower panel). Bars, 20 µm. (B) PC-3U/pMEP4-S7 cells were transiently transfected with dominant negative mutant p38 MAPK and starved in 1% FBS for 12 hours. The cells were treated with 1 µM CdCl2 for 12 hours. The expression of dominant negative p38 MAPK was visualized with a mouse monoclonal anti-p38 MAPK antibody followed by a TRITC-labeled anti-mouse antibody. Smad7 was detected by a goat anti-Smad7 antibody followed by an Alexa Fluor 488-labeled anti-goat antibody. Arrowheads denote transfected cells. Bar, 20 µm.

View larger version (31K): [in a new window]   Fig. 8. The signaling components involved in TGF-ß-induced activation of Cdc42 in PC-3U cells.

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