Interaction with Smad4 is indispensable for suppression of BMP signaling by c-Ski

Abstract

c-Ski is a transcriptional corepressor that interacts strongly with Smad2, Smad3, and Smad4 but only weakly with Smad1 and Smad5. Through binding to Smad proteins, c-Ski suppresses signaling of transforming growth factor-beta (TGF-beta) as well as bone morphogenetic proteins (BMPs). In the present study, we found that a mutant of c-Ski, termed c-Ski (ARPG) inhibited TGF-beta/activin signaling but not BMP signaling. Selectivity was confirmed in luciferase reporter assays and by determination of cellular responses in mammalian cells (BMP-induced osteoblastic differentiation of C2C12 cells and TGF-beta-induced epithelial-to-mesenchymal transdifferentiation of NMuMG cells) and Xenopus embryos. The ARPG mutant recruited histone deacetylases 1 (HDAC1) to the Smad3-Smad4 complex but not to the Smad1/5-Smad4 complex. c-Ski (ARPG) was unable to interact with Smad4, and the selective loss of suppression of BMP signaling by c-Ski (ARPG) was attributed to the lack of Smad4 binding. We also found that c-Ski interacted with Smad3 or Smad4 without disrupting Smad3-Smad4 heteromer formation. c-Ski (ARPG) would be useful for selectively suppressing TGF-beta/activin signaling.

Figure 1.

c-Ski (ARPG) selectively suppressed TGF-β signaling. Luciferase reporter assay was conducted using (CAGA)₉-MLP-Luc (A, top) and 3GC2-Lux (B, bottom). Cells were stimulated with TGF-β (2.5 ng/ml) (A) or BMP-7 (500 ng/ml) (B). □, unstimulated samples; ▪, ligand-stimulated samples. Expression of recombinant proteins was determined by immunoblotting analysis of the cell lysates using anti-Flag M2 antibody (bottom).

Figure 2.

c-Ski (ARPG) failed to inhibit BMP-induced osteoblastic differentiation of C2C12 cells. C2C12 cells were infected with adenoviruses carrying various cDNAs, followed by BMP-6 treatment (200 ng/ml) for 96 h. Osteoblastic differentiation was monitored by induction of alkaline phosphatase activity (top). □, unstimulated samples; ▪, BMP-6–stimulated samples. Expression of recombinant proteins was determined by immunoblotting analysis of the cell lysates using anti-Flag M2 antibody (bottom).

Figure 3.

Inhibition of TGF-β–induced EMT in NMuMG cells by c-Ski and c-Ski (ARPG). NMuMG cells were infected with adenoviruses carrying various cDNAs, followed by TGF-β treatment (5 ng/ml). Twenty-four hours after ligand stimulation, cell morphology was observed under microscopy. Control cells (left panels) and TGF-β–treated cells (right panels) are shown for noninfected cells (A) and cells infected with adenoviruses carrying LacZ (B), Smad7 (C), c-Ski (D), and c-Ski (ARPG) (E).

Figure 4.

Effects of c-Ski (ARPG) on signaling of TGF-β superfamily in Xenopus embryos. (A) Effect on endogenous BMP signaling using whole embryos. Equivalent amounts (500 pg) of RNAs encoding c-Ski, c-Ski (ARPG), or β-globin were injected near the ventral midline of four-cell Xenopus embryos. Resultant phenotypes in a representative experiment are shown (left, c-Ski; middle, c-Ski (ARPG); and right, β-globin). (B) Effect on endogenous BMP signaling in animal caps. RNA encoding c-Ski or c-Ski (ARPG) was injected into the animal pole of four-cell embryos. Animal caps were excised at brastulla stage 8 and cultured until stage 23. RNAs were then extracted from the animal caps and expression of marker genes (muscle actin and NCAM) was analyzed by RT-PCR. Histone H4 was used as a loading control. (C) Effect on activin signaling in animal caps. RNA encoding c-Ski or c-Ski (ARPG) was injected together with RNA encoding activin into the animal pole of four-cell embryos, and expression of marker genes was analyzed as described above.

Figure 5.

Interaction of c-Ski (ARPG) with Smad proteins. (A) Recruitment of HDAC1 to activated Smad complexes by c-Ski and c-Ski (ARPG) and (B) binding of c-Ski and c-Ski (ARPG) to each of the Smad proteins. COS-7 cells were transfected with indicated plasmids. Smad proteins (A) or c-Ski (B) was immunoprecipitated from cell lysates and coprecipitated proteins were visualized by immunoblotting. ALK-5TD and ALK-6QD are constitutively active forms of ALK-5 and ALK-6, respectively. (C) c-Ski (ARPG) suppression of TGF-β signaling was dependent on R-Smad binding. c-Ski (Δ40) lacks the N-terminal 40 amino acid residues and therefore cannot bind Smad2/3. c-Ski (ARPGΔ40) is a double mutant of (ARPG) and (Δ40). Luciferase reporter assay was conducted in HepG2 cells stimulated with TGF-β (2.5 ng/ml) using (CAGA)₉-MLP-Luc. □, unstimulated samples; ▪, TGF-β–stimulated samples.

Figure 6.

Selective loss of suppression of BMP signaling by c-Ski W274E mutant. (A) Luciferase reporter assay in HepG2 cells stimulated with TGF-β or BMP-7 using (CAGA)₉-MLP-Luc (left) and 3GC2-Lux (right), respectively. □, unstimulated samples; ▪, ligand-stimulated samples. (B) Recruitment of HDAC1 to activated Smad complexes. COS-7 cells were transfected with indicated plasmids. Smad proteins were immunoprecipitated from cell lysates and coprecipitated proteins (c-Ski and HDAC1) were visualized by immunoblotting. ALK-5TD and ALK-6QD are constitutively active forms of ALK-5 and ALK-6, respectively.

Figure 7.

Effects of c-Ski proteins on complex formation of Smad proteins. COS-7 cells were transfected with various combination of cDNAs (Smad3, Smad4, c-Ski, and ALK-5TD). c-Ski (wild-type or mutant) or Smad4 was immunoprecipitated and coprecipitated proteins were visualized by immunoblotting. Effect of c-Ski W274E on complex formation was examined by immunoprecipitating c-Ski W274E (A) or Smad4 (B). Effect of c-Ski (ARPG) (C), c-Ski ΔS2/3 (D), or wild-type c-Ski (E) was examined by immunoprecipitating c-Ski proteins. Effects of increasing amounts of c-Ski proteins on heteromer formation were also examined by immunoprecipitating Smad4 (F).

Figure 8.

Schematic model of inhibition of Smad signaling by c-Ski (A), c-Ski (ARPG) (B), v-Ski (C), and v-Ski (ARPG) (D). c-Ski possesses a Smad2/3 binding region as well as Smad4 binding region, whereas c-Ski (ARPG) lacks a Smad4 binding region, causing failure of c-Ski (ARPG) to inhibit BMP signaling. v-Ski, which has truncated structure in the N- and C- termini of c-Ski, lacks a Smad2/3 binding region and one of the mSin3A binding regions. v-Ski inhibits TGF-β/activin signaling as well as BMP signaling through interaction with Smad4. v-Ski (ARPG) does not inhibit Smad signaling because it no longer interacts with Smad proteins.