FIG. 6.

Smad7 inhibits the transcriptional activities of Smad3/4, Smad2/4, and Smad1/4. (A) Smad7 inhibits the expression of CAGA-luciferase induced by overexpression of Smad3 and Smad4 in R1B/L17 cells. TβRI-deficient R1B/L17 cells were transfected with CAGA-luciferase (0.5 μg), TβRI (0.1 μg), Smad3 and Smad4 (0.1 μg), and Smad7 (0.1 μg and 0.2 μg) as indicated. At 24 h posttransfection, cells were treated with TGF-β1 (50 pM) for 20 h or left untreated and then harvested for luciferase assay and immunobloting. Protein expression is shown below. (B) Smad7 inhibits the expression of ARE-luciferase induced by overexpression of Smad2 and Smad4 in R1B/L17 cells. TβRI-deficient R1B/L17 cells were transfected with ARE-luciferase (0.5 μg), TβRI (0.1 μg), Smad2 and Smad4 (0.2 μg and 0.1 μg, respectively), and Smad7 (0.1 μg and 0.3 μg) as indicated. At 24 h posttransfection, cells were treated with TGF-β1 (50 pM) for 20 h or left untreated and then harvested for luciferase assay. Protein expression is shown below. (C) Smad7 represses the expression of the BMP-responsive reporter GCCG₁₂-luciferase induced by overexpression of Smad1 and Smad4 in Hep3B cells. Smad7 (0.3 μg) as indicated and GCCG₁₂-luciferase (0.4 μg) were coexpressed with and without Smad1 (0.2 μg) and Smad4 (0.1 μg) in Hep3B cells. At 24 h posttransfection, cells were treated with BMP4 (1.6 nM) or noggin (2 nM) for another 20 h or left untreated and then harvested for luciferase assay. Protein expression is shown below. All reporter assays were performed in triplicate, and the data represent the means and standard deviations of three independent experiments after normalization to Renilla luciferase activity.