Rab5 mediates caspase-8-promoted cell motility and metastasis - PubMed (original) (raw)
Rab5 mediates caspase-8-promoted cell motility and metastasis
Vicente A Torres et al. Mol Biol Cell. 2010.
Abstract
Caspase-8 is a key apical sensory protein that governs cell responses to environmental cues, alternatively promoting apoptosis, proliferation, and cell migration. The proteins responsible for integration of these pathways, however, have remained elusive. Here, we reveal that Rab5 regulates caspase-8-dependent signaling from integrins. Integrin ligation leads to Rab5 activation, association with integrins, and activation of Rac, in a caspase-8-dependent manner. Rab5 activation promotes colocalization and coprecipitation of integrins with caspase-8, concomitant with Rab5 recruitment to integrin-rich regions such as focal adhesions and membrane ruffles. Moreover, caspase-8 expression promotes Rab5-mediated internalization and the recycling of beta1 integrins, increasing cell migration independently of caspase catalytic activity. Conversely, Rab5 knockdown prevented caspase-8-mediated integrin signaling for Rac activation, cell migration, and apoptotic signaling, respectively. Similarly, Rab5 was critical for caspase-8-driven cell migration in vivo, because knockdown of Rab5 compromised the ability of caspase-8 to promote metastasis under nonapoptotic conditions. These studies identify Rab5 as a key integrator of caspase-8-mediated signal transduction downstream of integrins, regulating cell survival and migration in vivo and in vitro.
Figures
Figure 1.
Caspase-8 regulates Rab5 activation and association with β1 integrin. (A) Rab5-GTP loading assays were performed on NB7 neuroblastoma cells deficient for (−), or reconstituted with (+) caspase-8, that were either held in suspension (susp) or allowed to attach to fibronectin substrates (2 μg/ml) for 45 min (adh). Representative immunoblots as well as a quantification of three independent experiments is shown (mean ± SE; *p < 0.05). (B) Neuroblastoma cells, as described above, were allowed to attach onto plates coated with 10 μg/ml anti-β1 integrin or anti CD44 mAb (control, 10 mg/ml) for 45 min. Rab5 GTP levels were measured as indicated in A. One of two similar experiments is shown. (C) β1 integrin and associated complex was immunoprecipitated from the neuroblastoma cell lines. Rab5, caspase-8, and integrin were detected by immunoblotting in both immunoprecipitates (IP) and whole cell lysates. (D) Isolated focal adhesion (FA) and cytosolic (Cyt) fractions were analyzed by immunoblotting with caspase-8, actin, Rab5, and Rab7 antibodies, as shown. Data are representative of three independent experiments. (E) Quantification of Rac activation after cellular attachment to fibronectin coated plates (2 μg/ml); extracts were obtained and Rac-GTP levels were measured as indicated in A, by using a GST-PAK1 pull-down assay. Data shown are averaged from three independent experiments (mean ± SE; #p < 0.01). (F) Neuroblastoma cells stably expressing GFP (control), GFP-caspase-8 (C8), or GFP-caspase 8/C360A (C8i) were grown on glass coverslips, fixed, and analyzed by confocal microscopy. Rab5 (red channel) and actin (phalloidin, blue channel) staining are shown. Bar, 10 μm.
Figure 2.
Caspase-8–dependent regulation of β1 integrin trafficking. (A) NB7 neuroblastoma cells lacking (−casp8) or expressing caspase-8 (+casp8) were permitted to attach to fibronectin coated coverslips (2 μg/ml) for 60 min, fixed, and confocal analysis of Rab5 (red channel) and β1 integrin (green channel) was performed. A representative image from three independent experiments is shown. The right panels denote magnified regions from pictures on left: the perinuclear region (“central”) and the cell edge (“peripheral”). Bar, 10 μm. (B) Colocalization analysis of Rab5 and integrin was performed with ImageJ software (as described in Materials and Methods; the mean of three experiments ± SE is shown; *p < 0.01). (C) Neuroblastoma cells lacking (−) or expressing (+) caspase-8 were fractionated on discontinuous sucrose gradients, and integrin distribution in early or late endosome fractions quantified by immunoblotting and scanning densitometry. Integrin detected in the total pool of endosomes is shown in the left graph as percentage of total integrin in whole cell lysates; integrin associated with late endosomes fractions is shown in the right graph as percentage of total pool of endosomes (mean of three experiments ± SE). (D) Neuroblastoma cells lacking (−C8) or expressing caspase 8 (+C8) were analyzed by flow cytometry for expression of total integrin (left) or active integrin conformers (right) alone or in the presence of 200 mM MnCl2 (open histograms) to “activate” integrin. (E) Internalization and recycling of β1 integrin were measured in NB7 cells lacking (−C8) or reconstituted for caspase 8 (+C8) expression. For internalization studies (left), cells were labeled with 0.2 mg/ml NHS-SS-biotin for 30 min at 4°C, washed, and then brought to 37°C in serum-free medium; cell extracts were obtained and biotinylated integrin measured by immunoprecipitation, immunolotting, and scanning densitometry. Recycling experiments (right) permitted internalization for 30 min, and then surface biotin was removed and samples incubated at times as shown in serum-free medium 37°C. Data are shown as the mean ± SE of three experiments (*p < 0.05; **p < 0.1).
Figure 3.
Requirement of caspase-8 and Rab5 for A549 cell migration. (A) A549 cells were allowed to attach and spread onto fibronectin-coated coverslips (2 μg/ml) for 60 min, fixed, and analyzed by confocal microscopy for the presence of Rab5 (red channel), β1 integrin (green channel), and actin (phalloidin; blue channel). (B) A549 cells treated with control (left) or caspase-8–targeted (right) shRNA were attached to fibronectin and stained for Rab5, β1 integrin, and actin, as indicated above. Bar, 10 μm. (C) Rab5, caspase-8, and β1 integrin levels were assessed by immunoblot analysis of A549 cell lysates, by using lines expressing the control, caspase-8–, or Rab5-targeted shRNAs, as indicated. (D) Cells treated with control (Con), caspase-8– (C8), or Rab5 (R5)-targeted shRNA were attached to fibronectin-coated plates (2 μg/ml) by 45 min, and extracts were obtained. Rac-GTP levels were measured as indicated in Figure 1A, by using the GST-PAK1 pull-down assay. Numbers indicate averages from two independent experiments. (E) Ruffle quantification in A549 cells stably expressing control (Con), caspase-8– (C8), or Rab5-targeted (R5) shRNAs. Cells were allowed to attach to fibronectin substrates for 60 min, fixed, and analyzed by phalloidin staining. The percentage of cells with peripheral ruffles is shown (data from two independent experiments, n = 25 images; *p < 0.001 relative to control. (F) Cells attached to fibronectin-coated coverslips and analyzed by confocal microscopy, as in (A). Cell area was measured via NIH-Image J software, and cell area shown normalized to cells treated with control shRNA. Data were averaged from 25 pictures (mean ± SE). *p < 0.005 relative to control. (G) Cell migration was evaluated in Transwell plates coated with fibronectin (2 μg/ml) for 2 h, as indicated in the Materials and Methods, and data are normalized to controls. Data shown are from two independent experiments done in triplicate (mean ± SE). *p < 0.01 relative to control.
Figure 4.
Caspase-8–driven neuroblastoma cell adhesion and migration require Rab5. (A) NB7 cells stably expressing GFP (left) or GFP-caspase-8(C360A) (C8i-GFP, right) were plated at confluence on 2 μg/ml fibronectin-coated coverslips, the monolayer wounded, and cells allowed to migrate into the wound for 4 h. Cells were then fixed and stained for Rab5 (polyclonal antibody; red channel) and integrin β1 (blue channel). Bar, 10 μm. (B) NB7 cells deficient (−C8) or reconstituted for caspase 8 (+C8) or a mutant caspase 8 (C360A, ‘C8i’) and coexpressing either control (+) or Rab5 shRNA (−), as indicated, were lysed and assessed by immunoblotting for their relative expression of Rab5, Rab21, β1 integrin, caspase-8, and actin. (C) Cells, as described above, were permitted to attach to fibronectin- (2 μg/ml) or collagen (10 μg/ml)-coated substrates for 30 min, and the observed adhesion was normalized to controls. The mean ± SE of three experiments is shown (*p < 0.01, **p < 0.05, compared with casp8-; #p < 0.01, compared with shRNA-control). (D) Cells treated with control (+) or Rab5 shRNA (−), either lacking (−) or expressing caspase 8 (+) were permitted to attach to fibronectin-coated plates (2 μg/ml), and Rac-GTP levels were measured (as per Figure 1A) after 45 min. (E) Cell migration was assessed in Transwell chambers plates coated with fibronectin (2 μg/ml) after 2 h. Data are normalized to control cells, and representative of two independent experiments done in triplicate (mean ± SE). *p < 0.05, compared with casp8-; #p < 0.01, compared with shRNA-control. (F and G) shRNA control or shRNA Rab5-treated cells, either lacking (−) or expressing caspase 8 (+) or caspase 8/C360A (C8i) were allowed to attach onto fibronectin (2 μg/ml) at confluence. The monolayer was wounded, and cells were allowed to migrate for 16 h. (F) Representative phase contrast images are shown. (G) Quantification of three independent experiments each performed in triplicate. Data represent the mean ± SE; *p < 0.05, compared with casp8-; #p < 0.01, relative to shRNA control.
Figure 5.
Caspase-8 enhances neuroblastoma metastasis under nonapoptotic conditions in a Rab5-dependent manner. Control shRNA (black bars) or Rab5-shRNA–treated cells (open bars) either lacking (−) or expressing caspase 8/C360A (C8i) were seeded into the upper chorioallantoic membrane of 10-d-old chick embryos, and tumors were permitted to develop for 8 d. (A) Total cumulative incidence of detected human Alu sequences, (expresses as percentage) from genomic DNA isolated from lungs of the tumor-seeded chick cohorts was measured by polymerase chain reaction. Statistical comparisons between cohorts representing three experiments were done via unpaired t test. (#p = 0.30; **p = 0.09; *p = 0.07). (B) Tumors were resected from the chorioallantoic membrane, and the wet weight was determined (mean ± SE).
References
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