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First published online 11 November 2008
doi: 10.1242/jcs.031286

Journal of Cell Science 121, 3960-3970 (2008)
Published by The Company of Biologists 2008
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BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity

Cristina Gamell1,*, Nelson Osses1,2,*, Ramon Bartrons1, Thomas Rückle3, Montserrat Camps3, José Luis Rosa1 and Francesc Ventura1,{ddagger}

1 Departament de Ciències Fisiològiques II, Universitat de Barcelona, IDIBELL, L'Hospitalet de Llobregat, Spain
2 Instituto de Química, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Chile
3 Departments of Chemistry and Signal Transduction, Merck Serono SA, Research Center Geneva, 9, chemin des Mines, 1211 Geneva, Switzerland


Figure 1
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  		Fig. 1. BMP2 induces chemotactic C2C12 cell migration. (A) Representative images of propidium-iodide-stained C2C12 migrated cells are shown on the left. Quantitative analysis of eight random fields from three independent experiments is shown on the right (mean ± s.e.m.; *P<0.001, paired t-test). (B) Wounded C2C12 cell monolayers were allowed to migrate in the presence or absence of 3 nM BMP2 for different times. A quantitative analysis of the invaded area was obtained from three photographed fields obtained in at least four independent experiments and represented as mean ± s.e.m.

 

Figure 2
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  		Fig. 2. BMP2 induces actin cytoskeleton reorganization. (A) C2C12 cells were stimulated with 3 nM BMP2 for the indicated times. Actin was visualized with TRITC-conjugated phalloidin using a confocal microscope. Arrows indicate actin cortical protrusions. Scale bar: 20 µm. (B) Serum-starved C2C12 cells were pretreated with 2 µM cytochalasin D (Cyto D) for 20 minutes and allowed to recover in the absence or presence of 3 nM BMP2 for 1 hour. Cell lysates were analyzed by immunoblotting with anti-phospho-Smad1 and reprobed with anti-{alpha}-tubulin antibodies. (C) Cells treated as above were allowed to recover in the absence or presence of 3 nM BMP2. Upper row shows cells before and after 2 µM Cyto D treatment. Middle and lower rows show recovery in the absence or presence of BMP2, respectively. Scale bar: 20 µm. A quantitative analysis of the formation of stress fibers in the absence of BMP2 or cells showing cortical actin protrusions as a percent of total in the presence of BMP2 are shown. Data are mean ± s.e.m. of at least 200 cells obtained in different fields from five independent experiments. (D) Serum-starved C2C12 cells (Control) were pre-treated with 2 µM cytochalasin D (Cyto D) for 20 minutes and after washing, were allowed to recover in the presence of 3 nM BMP2 for different times. Actin was visualized with TRITC-conjugated phalloidin using a confocal microscope. Arrows indicate actin cortical protrusions that were evident within the first 60 minutes and cells then recovered their initial aspect showing abundant stress fibers. Scale bar: 20 µm.

 

Figure 3
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  		Fig. 3. BMP2-induced actin reorganization and cell migration is mediated by Cdc42. (A) Swiss 3T3 cells were serum-starved for 7 days and stimulated with 3 nM BMP2. Arrows indicate spike-like filopodia (see detail at 30 minutes). As a control, cells were stimulated with EGF 20 ng/ml for 10 minutes (upper row). Scale bar, 20 µm. (B) Serum-starved C2C12 cells were stimulated with 3 nM BMP2. Levels of active GTP-bound Cdc42 and Rac were determined using the PBD domain of PAK1 followed by immunoblotting with anti-Cdc42 and anti-Rac antibodies. (C) C2C12 cells were transfected with the indicated GFP-tagged expression vectors. Cells were serum-starved for 16 hours (control), pre-treated with cytochalasin D and allowed to recover for 1 hour in the absence or presence of 3 nM BMP2. Merged images of phalloidin (red) and GFP signal (green) are shown. Cells showing cortical actin protrusions (arrows) are indicated. Scale bar: 20 µm. Transfected cells were counted and those showing cortical actin protrusions represented as % of total (graph on right). Mean ± s.e.m. of at least 80 transfected cells obtained from three independent experiments (*P<0.001, paired t-test). (D) C2C12 cells were transfected with the GFP-tagged expression vectors indicated and analyzed by chemotaxis assay after 2 hours (left panel) and by wound-healing migration assay performed for 12 hours (right panel) in the presence or absence of BMP2. Mean ± s.e.m. of three independent experiments (*P<0.001 compared with the GFP-transfected control cells and in the absence of BMP2, One-way ANOVA followed by Bonferroni's multiple comparison test).

 

Figure 4
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  		Fig. 4. PI3K activity is required for BMP2-induced actin reorganization and cell migration in C2C12 cells. (A) Cells were serum-starved for 16 hours prior to preincubation with 15 µM LY294002 for 60 minutes and then stimulated with 3 nM BMP2 for the indicated times. Cell lysates were analyzed by immunoblotting with anti-Akt-P(Ser473) and reprobed with anti-Akt total antibody. (B) Cells treated as above were fixed and stained with TRITC-conjugated phalloidin. Scale bar: 20 µm. Cells with actin protrusions were quantified as shown in graph below. Mean ± s.e.m. from four independent experiments (*P<0.001, paired t-test). (C) Cell monolayers were wounded and allowed to migrate for 12 hours in the presence or absence of 3 nM BMP2 and/or LY294002. The graph in lower panel indicates the invaded area. Mean ± s.e.m. from three independent experiments (*P<0.001, paired t-test).

 

Figure 5
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  		Fig. 5. PI3K{alpha} is required for BMP2-induced cell migration. (A) Cells were serum-starved for 16 hours. The isoform-selective inhibitors of class I PI3K were added to the medium 1 hour before stimulation of cells with BMP2 and used at a final concentration of 1 µM. Cell lysates were analyzed by immunoblotting with anti-Akt-P(Ser473) and membranes reprobed with anti-Akt total antibody. (B) Quantitative analysis of phase-contrast images of a wound-healing assay performed for 12 hours in the presence of 3 nM BMP2 and the PI3K-isoform-selective inhibitors. Mean ± s.e.m. from three independent experiments (*P<0.001, paired t-test).

 

Figure 6
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  		Fig. 6. BMP2 activates Cdc42 and PI3K pathways independently. (A) C2C12 cells treated with 3 nM BMP2 and/or LY294002. Levels of GTP-bound Cdc42 were determined using GST pull-down assay. (B) C2C12 cells were transfected with GFP-tagged plasmids for wild-type Cdc42 (GFP-Cdc42wt) or a dominant-negative form (GFP-Cdc42N17). Cells were stimulated with 3 nM BMP2 and cell lysates were analyzed by immunoblotting with anti-Akt-P(Ser473), and reprobed with anti-Akt antibody and anti-Cdc42 antibody.

 

Figure 7
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  		Fig. 7. BMP2 stimulates LIMK1 activity. (A) Serum-starved C2C12 cells were stimulated with 3 nM BMP2 and cell lysates analyzed by immunoblotting with anti-LIMK1-P and anti-LIMK1 antibody. The bottom panel indicates the relative LIMK1-P levels. Mean ± s.e.m. from three independent experiments (*P<0.05, paired t-test). (B) C2C12 cells were treated as above for 40 minutes but in the absence or presence of LY294002. Cell lysates were analyzed by immunoblotting with the indicated antibodies. The bottom panel indicates the relative LIMK1-P levels. Mean ± s.e.m. from three independent experiments (*P<0.05, paired t-test). (C) C2C12 cells were treated with BMP2 and LY294002 as indicated. Endogenous LIMK1 was subjected to an in vitro kinase assay with GST-cofilin as a substrate. Values of cofilin phosphorylation with respect to the LIMK1 present in cell extracts are shown.

 

Figure 8
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  		Fig. 8. BMP2 stimulates PAK1 and PAK4 activity. (A) C2C12 cells were stimulated with 3 nM BMP2 for different times and cell lysates were analyzed with the indicated phospho-specific antibodies, and membranes reprobed with anti-PAK1 and anti-PAK4 antibodies. Graphs indicate the relative PAK1-P and PAK4-P levels. Mean ± s.e.m. from three independent experiments (*P<0.05, paired t-test). (B) C2C12 cells were serum-starved, incubated with LY294002 and stimulated with 3 nM BMP2 for 40 minutes. Cell lysates were analyzed as above. Graph indicates the relative PAK1-P and PAK4-P levels. Mean ± s.e.m. from three independent experiments (*P<0.05, paired t-test). (C) C2C12 cells stimulated with 3 nM BMP2 and LY294002 were analyzed for PAK1 or PAK4 in vitro kinase activity using MBP as a substrate. The graph indicates the relative PAK1 and PAK4 activity. Mean ± s.e.m. from three independent experiments (*P<0.05, paired t-test).

 

Figure 9
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  		Fig. 9. Cdc42 is required for BMP2-dependent PAK1 and PAK4 phosphorylation. (A) C2C12 cells transfected with the indicated expression vectors were serum-starved for 16 hours and stimulated with 3 nM BMP2 for 40 minutes. Cell lysates were analyzed with the indicated phospho-specific antibodies, and membranes reprobed with anti-PAK1, anti-PAK4 and anti-Cdc42 antibodies. Graph indicates PAK1-P and PAK4-P levels of BMP2-treated cells relative to their respective untreated controls. Mean ± s.e.m. of three independent experiments (*P<0.05, unpaired t-test). (B) PAK activity is required for BMP2 to phosphorylate the LIMK1 associated to the BMPRII tail. Immunoblot analysis of C2C12 cells overexpressing His-tagged BMPRII under a tetracyclin (Tet-off)-regulated promoter (left panel). Cells overexpressing His-tagged BMPRII were transfected with the expression constructs indicated, serum-starved for 16 hours and stimulated with 3 nM BMP2 for 40 minutes. Cell extracts were subjected to immunoblotting to detect the total and phosphorylated LIMK1 after Ni2+-NTA-agarose purification (right panel).

 

Figure 10
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  		Fig. 10. Role of BMPRII in BMP2-dependent actin cytoskeleton reorganization and cell migration. (A) Chemotaxis assay. Serum-starved C2C12 cells were allowed to migrate for 2 hours in the presence or absence of 30 pM BMP7 as described in Materials and Methods. Quantitative analysis of eight random fields from three independent experiments (Mean ± s.e.m.; *P<0.0001, paired t-test). (B) Wound-healing migration assay. Serum-starved wounded C2C12 cell monolayers were allowed to migrate in the presence or absence of 3 nM BMP2 or 3 nM BMP7 for 24 hours. The graph indicates the invaded area. Mean ± s.e.m. of three independent experiments (*P<0.001, paired t-test). (C) Immunoblot analysis of BMPRII expression in C2C12 cells transfected with the indicated siRNA for 48 hours. (D) C2C12 cells were transfected with the indicated siRNA for 48 hours and serum-starved for 16 hours. Then, cells were pretreated with 2 µM cytochalasin D for 20 minutes and allowed to recover in the absence or presence of 3 nM BMP2 for 1 hour. Merged images of phalloidin and GFP signals are shown. Cells showing cortical actin protrusions (arrows) are indicated. Scale bar: 20 µm. Transfected cells were counted and those showing cortical actin protrusions represented as % of total (graph on right). Mean ± s.e.m. of at least 80 transfected cells obtained from three independent experiments (*P<0.05, paired t-test compared with the condition in the absence of BMP2). (E) C2C12 cells were transfected with the indicated siRNA for 48 hours and serum-starved for 16 hours. Cell monolayers were wounded and allowed to migrate for 24 hours in the presence or absence of 3 nM BMP2. The graph indicates the invaded area. Mean ± s.e.m. from three independent experiments (*P<0.001 compared with the condition in the absence of BMP2, one-way ANOVA followed by Bonferroni's multiple comparison test).

 

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© The Company of Biologists Ltd 2008