Wang W et al. (2000), Ski represses bone morphogenic protein signalin...

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Proc Natl Acad Sci U S A 2000 Dec 19;9726:14394-9. doi: 10.1073/pnas.97.26.14394.

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Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells.

Wang W , Mariani FV , Harland RM , Luo K .

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The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells by directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-beta family members.

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Species referenced: Xenopus laevis
Genes referenced: ag1 bmp2 bmp4 bmpr1a evx1 ncam1 nog pdlim3 ski smad1 smad10 smad4 smad9 tbxt tgfbr1 ventx2.2 ventx3.2

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	Figure 1 (A–E) Human ski neuralizes Xenopus ectoderm. Explants were cultured until stage 20 for analysis of NCAM expression. (A) Embryos express NCAM only in the central nervous system. Anterior is to the left and dorsal is to the top. (B) Uninjected explants. (C) Explants expressing 500 pg of BF-2 mRNA. (D) Explants expressing 500 pg of human ski mRNA. (E) RT-PCR analysis. Lanes 1 and 2, whole embryo control (stage 20) followed by the RT minus negative control; lane 3, uninjected explants; lanes 4–6, explants injected with increasing amounts of ski mRNA. The elongation factor 1 alpha (EF1α) primers were used to determine RNA recovery. Primers for muscle actin were used to assess mesoderm development. W, whole embryo; RT−, no RT. (F–I) Ski inhibits Xvent2 expression in ectoderm. Ectodermal explants were cultured until the midgastrula stage (stage 10.5). (F) In the embryo, Xvent2 is expressed in the animal cap and the marginal zone (dorsal lip is indicated by the arrow). (G) Uninjected explants. (H) Explants expressing 500 pg of BF-2 mRNA. (I) Explants expressing 500 pg of ski mRNA. (J–M) Ski inhibits Xvent2 expression in the embryo. (J) Control embryos (ventral view) showing Xvent2 expression in the marginal zone (stage 10.5). (K) Embryos injected with 500 pg of ski mRNA; arrowheads point to where Xvent2 expression is inhibited. (L) Control embryos probed for noggin expression. (M) Embryos injected with 500 pg of ski mRNA probed for noggin expression. (N–Q) Ski does not inhibit BMP4 expression in the ectoderm. Explants were cultured until the midgastrula stage (stage 10.5). (N) In the embryo, BMP4 is expressed strongly in the ectoderm and the marginal zone (dorsal lip is indicated by the arrow). (O) Uninjected ectodermal explants. (P) Explants expressing 500 pg of BF-2. (Q) Explants expressing 500 pg of ski.
	Figure 2 Ski antagonizes BMP signaling in Xenopus explants. (A) Ski blocks BMP2 activity. Explants were cultured until stage 10.5 and analyzed for Xbra expression by RT-PCR. Lanes 1 and 2, whole embryo control and no RT (RT−); lane 3, uninjected explants; lanes 4 and 5, explants expressing AB2; lanes 6–8, explants expressing AB2 with increasing amounts of ski mRNA; lanes 9–11, explants expressing ski mRNA only. (B) Ski fails to neuralize in the presence of high levels of the constitutive active BMPRI (Alk3). Explants were analyzed at stage 20 for NCAM expression. Lanes 3–5, explants expressing AB2 mRNA only; lane 6–8, explants expressing ski and increasing amounts of Alk3 as indicated. Alk3*, (Q233D)Alk3. (C) Ski blocks the activity of Smad1. Lane 3, uninjected sample; lane 4, Smad1 stimulates the expression of the ventral markers, Xvex-1 and Xhox-3; lanes 5–7, explants expressing Smad1 mRNA in combination with increasing amounts of ski mRNA; Lane 8, ski alone.
	Figure 3 Ski represses BMP signaling in mammalian cells. (A) Ski inhibits BMP-induced ALP in W-20-17 cells. W-20-17 cells plated in a 96-well cluster were transfected with increasing amounts of c-ski as indicated. Twenty-four hours after transfection, the cells were treated with 250 ng/ml BMP2 for 40 h. ALP was measured by an Elisa reader and expressed as p-nitrophenol produced in nmol/min per mg protein. Ski represses BMP-induced transcriptional activation in W-20-17 (B) and Hep3B (C and D) cells. W-20-17 and Hep3B cells were cotransfected with 0.75 μg of 15xGCCG-Luc (B and C) or Xvent2-Luc (D), 0.5 μg of constitutive active type I receptors (Alk3* or Alk5), 0.75 μg of ski, and 0.15 μg of Smads. Luciferase activity was measured 48 h later. Alk3, (Q233D)Alk3; Alk5*, (T204D)Alk5; S1, Smad1; S4, Smad4.
	Figure 4 Ski interacts with Smad1 and Smad5. (A) Flag-tagged full-length or truncated Smad proteins were cotransfected into 293T cells together with HA-tagged Ski. The Smad-bound Ski was isolated by immunoprecipitation with anti-Flag M2 mAb and detected by Western blotting with an anti-HA mAb. Cell lysates were blotted directly as a control for HA-ski expression. (B) Ski associated with endogenous Smad1 and Smad4 in W-20-17 cells. W-20-17 cells were transfected with Flag-Ski together with or without the constitutively active type I BMP receptor. Ski-associated Smad complex was isolated by immunoprecipitation with anti-Flag agarose and detected by immunoblotting with an anti-Smad1 (T-20) or anti-Smad4 (C-20) antibody. (C) Ski binds to Smad1 directly in vitro. GST-Ski immobilized on glutathione-Sepharose was incubated with purified recombinant Smad1 or Smad4 as described in Materials and Methods. The Ski-bound Smads were eluted by glutathione and detected by immunoblotting with anti-Smad1 or anti-Smad4. GST alone was used as negative control. The amounts of wild-type or mutant GST-Ski used in the binding assay were shown by Coomassie staining. * indicates nonspecific background bands.
	Figure 5 Interactions between Ski and the Smads are required for repression of BMP signaling by Ski. (A) Interaction between mutant Ski proteins and the Smads. (Upper) Schematic drawings of the Ski mutants are shown. (Lower) HA-tagged, wild-type, or mutant Ski proteins were cotransfected into 293T cells together with Flag-tagged Smad1 or Smad4 as indicated. The Smad-bound Ski proteins were isolated by immunoprecipitation with anti-Flag M2 mAb and detected by Western blotting with an anti-HA mAb. Cell lysates were blotted directly as a control for HA-Ski expression. (B) Mutant analysis in Xenopus ectoderm. Explants were harvested at stage 22 and assayed for NCAM, XAG, and muscle actin expression. Lanes 1 and 2, whole embryo and no RT (RT−) control; lane 3, uninjected sample; lanes 4 and 5, explants expressing 500 pg of wild-type ski mRNA; lanes 6–8, explants expressing 500 pg of mRNA of ski mutants m1, m2, or m3. (C) To test the ability of the Ski mutants to repress BMP-induced ALP activity, various ski mutants were transfected into W-20-17 cells. ALP activity was assayed as described in Materials and Methods. (D). Hep3B cells were cotransfected with 0.75 μg of 15xGCCG-Luc, 0.5 μg of constitutive active BMPRI (Alk3), 0.75 μg of HA-tagged, wild-type, or mutant ski, and 0.15 μg of Smads. Luciferase activity was measured 48 h later. (Lower) The levels of Ski proteins present in the transfected cells were detected by Western blotting with the anti-HA antibody. indicates a nonspecific background band.

References [+] :

Akiyoshi, c-Ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads. 1999, Pubmed

	Figure 1 (A–E) Human ski neuralizes Xenopus ectoderm. Explants were cultured until stage 20 for analysis of NCAM expression. (A) Embryos express NCAM only in the central nervous system. Anterior is to the left and dorsal is to the top. (B) Uninjected explants. (C) Explants expressing 500 pg of BF-2 mRNA. (D) Explants expressing 500 pg of human ski mRNA. (E) RT-PCR analysis. Lanes 1 and 2, whole embryo control (stage 20) followed by the RT minus negative control; lane 3, uninjected explants; lanes 4–6, explants injected with increasing amounts of ski mRNA. The elongation factor 1 alpha (EF1α) primers were used to determine RNA recovery. Primers for muscle actin were used to assess mesoderm development. W, whole embryo; RT−, no RT. (F–I) Ski inhibits Xvent2 expression in ectoderm. Ectodermal explants were cultured until the midgastrula stage (stage 10.5). (F) In the embryo, Xvent2 is expressed in the animal cap and the marginal zone (dorsal lip is indicated by the arrow). (G) Uninjected explants. (H) Explants expressing 500 pg of BF-2 mRNA. (I) Explants expressing 500 pg of ski mRNA. (J–M) Ski inhibits Xvent2 expression in the embryo. (J) Control embryos (ventral view) showing Xvent2 expression in the marginal zone (stage 10.5). (K) Embryos injected with 500 pg of ski mRNA; arrowheads point to where Xvent2 expression is inhibited. (L) Control embryos probed for noggin expression. (M) Embryos injected with 500 pg of ski mRNA probed for noggin expression. (N–Q) Ski does not inhibit BMP4 expression in the ectoderm. Explants were cultured until the midgastrula stage (stage 10.5). (N) In the embryo, BMP4 is expressed strongly in the ectoderm and the marginal zone (dorsal lip is indicated by the arrow). (O) Uninjected ectodermal explants. (P) Explants expressing 500 pg of BF-2. (Q) Explants expressing 500 pg of ski.
	Figure 2 Ski antagonizes BMP signaling in Xenopus explants. (A) Ski blocks BMP2 activity. Explants were cultured until stage 10.5 and analyzed for Xbra expression by RT-PCR. Lanes 1 and 2, whole embryo control and no RT (RT−); lane 3, uninjected explants; lanes 4 and 5, explants expressing AB2; lanes 6–8, explants expressing AB2 with increasing amounts of ski mRNA; lanes 9–11, explants expressing ski mRNA only. (B) Ski fails to neuralize in the presence of high levels of the constitutive active BMPRI (Alk3). Explants were analyzed at stage 20 for NCAM expression. Lanes 3–5, explants expressing AB2 mRNA only; lane 6–8, explants expressing ski and increasing amounts of Alk3 as indicated. Alk3*, (Q233D)Alk3. (C) Ski blocks the activity of Smad1. Lane 3, uninjected sample; lane 4, Smad1 stimulates the expression of the ventral markers, Xvex-1 and Xhox-3; lanes 5–7, explants expressing Smad1 mRNA in combination with increasing amounts of ski mRNA; Lane 8, ski alone.
	Figure 3 Ski represses BMP signaling in mammalian cells. (A) Ski inhibits BMP-induced ALP in W-20-17 cells. W-20-17 cells plated in a 96-well cluster were transfected with increasing amounts of c-ski as indicated. Twenty-four hours after transfection, the cells were treated with 250 ng/ml BMP2 for 40 h. ALP was measured by an Elisa reader and expressed as p-nitrophenol produced in nmol/min per mg protein. Ski represses BMP-induced transcriptional activation in W-20-17 (B) and Hep3B (C and D) cells. W-20-17 and Hep3B cells were cotransfected with 0.75 μg of 15xGCCG-Luc (B and C) or Xvent2-Luc (D), 0.5 μg of constitutive active type I receptors (Alk3* or Alk5), 0.75 μg of ski, and 0.15 μg of Smads. Luciferase activity was measured 48 h later. Alk3, (Q233D)Alk3; Alk5*, (T204D)Alk5; S1, Smad1; S4, Smad4.
	Figure 4 Ski interacts with Smad1 and Smad5. (A) Flag-tagged full-length or truncated Smad proteins were cotransfected into 293T cells together with HA-tagged Ski. The Smad-bound Ski was isolated by immunoprecipitation with anti-Flag M2 mAb and detected by Western blotting with an anti-HA mAb. Cell lysates were blotted directly as a control for HA-ski expression. (B) Ski associated with endogenous Smad1 and Smad4 in W-20-17 cells. W-20-17 cells were transfected with Flag-Ski together with or without the constitutively active type I BMP receptor. Ski-associated Smad complex was isolated by immunoprecipitation with anti-Flag agarose and detected by immunoblotting with an anti-Smad1 (T-20) or anti-Smad4 (C-20) antibody. (C) Ski binds to Smad1 directly in vitro. GST-Ski immobilized on glutathione-Sepharose was incubated with purified recombinant Smad1 or Smad4 as described in Materials and Methods. The Ski-bound Smads were eluted by glutathione and detected by immunoblotting with anti-Smad1 or anti-Smad4. GST alone was used as negative control. The amounts of wild-type or mutant GST-Ski used in the binding assay were shown by Coomassie staining. * indicates nonspecific background bands.
	Figure 5 Interactions between Ski and the Smads are required for repression of BMP signaling by Ski. (A) Interaction between mutant Ski proteins and the Smads. (Upper) Schematic drawings of the Ski mutants are shown. (Lower) HA-tagged, wild-type, or mutant Ski proteins were cotransfected into 293T cells together with Flag-tagged Smad1 or Smad4 as indicated. The Smad-bound Ski proteins were isolated by immunoprecipitation with anti-Flag M2 mAb and detected by Western blotting with an anti-HA mAb. Cell lysates were blotted directly as a control for HA-Ski expression. (B) Mutant analysis in Xenopus ectoderm. Explants were harvested at stage 22 and assayed for NCAM, XAG, and muscle actin expression. Lanes 1 and 2, whole embryo and no RT (RT−) control; lane 3, uninjected sample; lanes 4 and 5, explants expressing 500 pg of wild-type ski mRNA; lanes 6–8, explants expressing 500 pg of mRNA of ski mutants m1, m2, or m3. (C) To test the ability of the Ski mutants to repress BMP-induced ALP activity, various ski mutants were transfected into W-20-17 cells. ALP activity was assayed as described in Materials and Methods. (D). Hep3B cells were cotransfected with 0.75 μg of 15xGCCG-Luc, 0.5 μg of constitutive active BMPRI (Alk3), 0.75 μg of HA-tagged, wild-type, or mutant ski, and 0.15 μg of Smads. Luciferase activity was measured 48 h later. (Lower) The levels of Ski proteins present in the transfected cells were detected by Western blotting with the anti-HA antibody. indicates a nonspecific background band.