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Expression and localization of Ski determine cell type-specific TGFbeta signaling effects on the cell cycle.

Jacob C, Grabner H, Atanasoski S, Suter U - J. Cell Biol. (2008)

Bottom Line: We show that the protooncogene Ski (Sloan-Kettering viral oncogene homologue) is an important regulator of these effects.Additionally, both Ski and Rb move to the cytoplasm, where they partially colocalize.In vivo, Ski and phospho-Rb (pRb) appear to interact in the Schwann cell cytoplasm of developing sciatic nerves.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Cell Biology, ETH Zurich, CH-8093 Zurich, Switzerland. claire.jacob@cell.biol.ethz.ch

ABSTRACT
Transforming growth factor beta (TGFbeta) promotes epithelial cell differentiation but induces Schwann cell proliferation. We show that the protooncogene Ski (Sloan-Kettering viral oncogene homologue) is an important regulator of these effects. TGFbeta down-regulates Ski in epithelial cells but not in Schwann cells. In Schwann cells but not in epithelial cells, retinoblastoma protein (Rb) is up-regulated by TGFbeta. Additionally, both Ski and Rb move to the cytoplasm, where they partially colocalize. In vivo, Ski and phospho-Rb (pRb) appear to interact in the Schwann cell cytoplasm of developing sciatic nerves. Ski overexpression induces Rb hyperphosphorylation, proliferation, and colocalization of both proteins in Schwann cell and epithelial cell cytoplasms independently of TGFbeta treatment. Conversely, Ski knockdown in Schwann cells blocks TGFbeta-induced proliferation and pRb cytoplasmic relocalization. Our findings reveal a critical function of fine-tuned Ski levels in the control of TGFbeta effects on the cell cycle and suggest that at least a part of Ski regulatory effects on TGFbeta-induced proliferation of Schwann cells is caused by its concerted action with Rb.

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Ski down-regulation in Schwann cells prevents TGFβ-induced proliferation and ser780pRb localization in the cytoplasm. (A and C) Western blot analysis of Ski expression (A) and immunostaining of Ski (red) and DAPI (blue) labeling in Schwann cells infected with a control shRNA (control) or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and kept in growing medium (for Western blot analysis) or treated with TGFβ (for immunostaining; C). (B) Graph representing the percentage of BrdU-labeled rat Schwann cells infected with a control shRNA or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM or treated with TGFβ for 24 h. (D) Immunostaining of ser780pRb (pRb; green) and DAPI (blue) labeling (pRb appears turquoise when nuclear) in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and treated with TGFβ for 24 h. (E) Western blot analysis of P0 in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM. Lysates of cells infected with the control shRNA and the Ski-specific shRNA lentiviruses have been run on the same gel but not on consecutive lanes. (F) Western blot analysis of P0 in rat Schwann cells infected with a Ski-specific shRNA (Ski-0%) and cultured in DM or treated with TGFβ or dbCAMP for 24 h. For A, E, and F, β-actin was used as loading control, and the graphs represent the densitometry of the bands of the protein of interest normalized to the loading control. Statistical analyses were performed using two-tailed t tests on at least three independent experiments. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig7: Ski down-regulation in Schwann cells prevents TGFβ-induced proliferation and ser780pRb localization in the cytoplasm. (A and C) Western blot analysis of Ski expression (A) and immunostaining of Ski (red) and DAPI (blue) labeling in Schwann cells infected with a control shRNA (control) or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and kept in growing medium (for Western blot analysis) or treated with TGFβ (for immunostaining; C). (B) Graph representing the percentage of BrdU-labeled rat Schwann cells infected with a control shRNA or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM or treated with TGFβ for 24 h. (D) Immunostaining of ser780pRb (pRb; green) and DAPI (blue) labeling (pRb appears turquoise when nuclear) in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and treated with TGFβ for 24 h. (E) Western blot analysis of P0 in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM. Lysates of cells infected with the control shRNA and the Ski-specific shRNA lentiviruses have been run on the same gel but not on consecutive lanes. (F) Western blot analysis of P0 in rat Schwann cells infected with a Ski-specific shRNA (Ski-0%) and cultured in DM or treated with TGFβ or dbCAMP for 24 h. For A, E, and F, β-actin was used as loading control, and the graphs represent the densitometry of the bands of the protein of interest normalized to the loading control. Statistical analyses were performed using two-tailed t tests on at least three independent experiments. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: To perform loss-of-function experiments, we transduced Schwann cells with a lentivirus carrying a puromycin selection marker and a Ski-specific short hairpin RNA (shRNA) that was able to down-regulate endogenous Ski to either nondetectable levels (Ski-0%) or to 40% of its endogenous expression (Ski-40%) in puromycin-selected Schwann cells (Fig. 7, A and C). Concerning Ski-0%, all experiments were performed with two different Ski shRNAs with comparable efficiencies for down-regulation, yielding similar results in our assays. Ski levels of expression were quantified in Schwann cells cultured in growing medium and selected with puromycin.


Expression and localization of Ski determine cell type-specific TGFbeta signaling effects on the cell cycle.

Jacob C, Grabner H, Atanasoski S, Suter U - J. Cell Biol. (2008)

Ski down-regulation in Schwann cells prevents TGFβ-induced proliferation and ser780pRb localization in the cytoplasm. (A and C) Western blot analysis of Ski expression (A) and immunostaining of Ski (red) and DAPI (blue) labeling in Schwann cells infected with a control shRNA (control) or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and kept in growing medium (for Western blot analysis) or treated with TGFβ (for immunostaining; C). (B) Graph representing the percentage of BrdU-labeled rat Schwann cells infected with a control shRNA or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM or treated with TGFβ for 24 h. (D) Immunostaining of ser780pRb (pRb; green) and DAPI (blue) labeling (pRb appears turquoise when nuclear) in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and treated with TGFβ for 24 h. (E) Western blot analysis of P0 in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM. Lysates of cells infected with the control shRNA and the Ski-specific shRNA lentiviruses have been run on the same gel but not on consecutive lanes. (F) Western blot analysis of P0 in rat Schwann cells infected with a Ski-specific shRNA (Ski-0%) and cultured in DM or treated with TGFβ or dbCAMP for 24 h. For A, E, and F, β-actin was used as loading control, and the graphs represent the densitometry of the bands of the protein of interest normalized to the loading control. Statistical analyses were performed using two-tailed t tests on at least three independent experiments. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Related In: Results  -  Collection

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fig7: Ski down-regulation in Schwann cells prevents TGFβ-induced proliferation and ser780pRb localization in the cytoplasm. (A and C) Western blot analysis of Ski expression (A) and immunostaining of Ski (red) and DAPI (blue) labeling in Schwann cells infected with a control shRNA (control) or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and kept in growing medium (for Western blot analysis) or treated with TGFβ (for immunostaining; C). (B) Graph representing the percentage of BrdU-labeled rat Schwann cells infected with a control shRNA or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM or treated with TGFβ for 24 h. (D) Immunostaining of ser780pRb (pRb; green) and DAPI (blue) labeling (pRb appears turquoise when nuclear) in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and treated with TGFβ for 24 h. (E) Western blot analysis of P0 in rat Schwann cells infected with a control or a Ski-specific shRNA (Ski-0% or Ski-40%) lentivirus and cultured in DM. Lysates of cells infected with the control shRNA and the Ski-specific shRNA lentiviruses have been run on the same gel but not on consecutive lanes. (F) Western blot analysis of P0 in rat Schwann cells infected with a Ski-specific shRNA (Ski-0%) and cultured in DM or treated with TGFβ or dbCAMP for 24 h. For A, E, and F, β-actin was used as loading control, and the graphs represent the densitometry of the bands of the protein of interest normalized to the loading control. Statistical analyses were performed using two-tailed t tests on at least three independent experiments. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: To perform loss-of-function experiments, we transduced Schwann cells with a lentivirus carrying a puromycin selection marker and a Ski-specific short hairpin RNA (shRNA) that was able to down-regulate endogenous Ski to either nondetectable levels (Ski-0%) or to 40% of its endogenous expression (Ski-40%) in puromycin-selected Schwann cells (Fig. 7, A and C). Concerning Ski-0%, all experiments were performed with two different Ski shRNAs with comparable efficiencies for down-regulation, yielding similar results in our assays. Ski levels of expression were quantified in Schwann cells cultured in growing medium and selected with puromycin.

Bottom Line: We show that the protooncogene Ski (Sloan-Kettering viral oncogene homologue) is an important regulator of these effects.Additionally, both Ski and Rb move to the cytoplasm, where they partially colocalize.In vivo, Ski and phospho-Rb (pRb) appear to interact in the Schwann cell cytoplasm of developing sciatic nerves.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Cell Biology, ETH Zurich, CH-8093 Zurich, Switzerland. claire.jacob@cell.biol.ethz.ch

ABSTRACT
Transforming growth factor beta (TGFbeta) promotes epithelial cell differentiation but induces Schwann cell proliferation. We show that the protooncogene Ski (Sloan-Kettering viral oncogene homologue) is an important regulator of these effects. TGFbeta down-regulates Ski in epithelial cells but not in Schwann cells. In Schwann cells but not in epithelial cells, retinoblastoma protein (Rb) is up-regulated by TGFbeta. Additionally, both Ski and Rb move to the cytoplasm, where they partially colocalize. In vivo, Ski and phospho-Rb (pRb) appear to interact in the Schwann cell cytoplasm of developing sciatic nerves. Ski overexpression induces Rb hyperphosphorylation, proliferation, and colocalization of both proteins in Schwann cell and epithelial cell cytoplasms independently of TGFbeta treatment. Conversely, Ski knockdown in Schwann cells blocks TGFbeta-induced proliferation and pRb cytoplasmic relocalization. Our findings reveal a critical function of fine-tuned Ski levels in the control of TGFbeta effects on the cell cycle and suggest that at least a part of Ski regulatory effects on TGFbeta-induced proliferation of Schwann cells is caused by its concerted action with Rb.

Show MeSH
Related in: MedlinePlus