Smad3 protects β-catenin degradation in chondrocytes. A, Smad3 expression plasmid was co-transfected with β-TrCP into RCJ3.1C5.18 cells. 24 h after transfection, the cell lysates were collected, and Western blotting (WB) was performed using the anti-β-catenin antibody. Transfection of Smad3 prevented β-catenin degradation. B, RCJ3.1C5.18 cells were transfected with control or Smad3 siRNA or Smad3 expression plasmid. The cell lysates were collected 0, 30, 60, 120, or 300 min after cycloheximide treatment (10 μg/ml), and the β-catenin protein levels were detected by Western blotting. Silencing of Smad3 accelerated β-catenin degradation and transfection of Smad3 prevented β-catenin degradation. C, the silencing efficiency of Smad3 siRNA was determined by Western blotting. The efficiency is over 70%. D, RCJ3.1C5.18 cells were transiently transfected with Smad3 expression plasmid. 24 h after transfection, the cell lysates were collected, and Western blotting was performed using the anti-p-β-catenin antibody. Smad3 significantly inhibited β-catenin phosphorylation. E, Smad3 and HA-ubiquitin expression plasmids were co-transfected with β-TrCP expression plasmid into RCJ3.1C5.18 cells in the presence of proteasome inhibitor MG132 (10 μm, 4 h of incubation). 24 h after transfection, the cell lysates were collected, and ubiquitinated proteins were pulled down using an UbiQapture^TM-Q kit, and ubiquitinated β-catenin was detected using the anti-β-catenin antibody. Transfection of β-TrCP induced β-catenin ubiquitination (second lane) and co-transfection of Smad3 prevented β-catenin ubiquitination (fourth lane). Ub, ubiquitin; IP, immunoprecipitation.

Smad3 promotes β-catenin nuclear translocation. A, RCJ3.1C5.18 cells were treated with Wnt3a (100 ng/ml) for 24 h. The cells were then fixed and incubated with the anti-β-catenin antibody (Ab) followed by the incubation with a fluorescein isothiocyanate-conjugated secondary antibody. Treatment with Wnt3a induced β-catenin nuclear translocation and silencing of Smad3 inhibited β-catenin nuclear translocation. B, p3TP-Lux reporter construct was co-transfected with WT or mutant forms of Smad3 into RCJ3.1C5.18 cells. 24 h after transfection, the cells were treated with TGF-β (2 ng/ml) for 4 h. The cell lysates were collected, and luciferase assay was performed. Transfection of mutant Smad3 constructs significantly inhibited TGF-β reporter activity. C, WT or mutant forms of Smad3 were transfected into RCJ3.1C5.18 cells. 24 h after transfection, the cells were treated with TGF-β (2 ng/ml) for 4 h. The cells were then fixed and incubated with the anti-β-catenin or anti-Smad3 antibody followed by the incubation with fluorescein isothiocyanate-conjugated secondary antibody. Transfection of mutant Smad3 constructs inhibited TGF-β-induced Smad3 and β-catenin nuclear translocation. D, RCJ3.1C5.18 cells were transfected with WT or mutant Smad3 (K43N/K44Q and ΔK40K41) expression plasmids. 24 h after transfection, the cells were treated with TGF-β (2 ng/ml) for 4 h. Nuclear and cytoplasmic proteins were collected using NE-PER nuclear and cytoplasmic extraction reagents (Pierce). Western blotting was performed using the anti-β-catenin antibody and anti-FLAG antibody to detect changes in β-catenin and Smad3 protein levels. Transfection of Smad3 increased nuclear Smad3 as well as β-catenin protein levels. In contrast, transfection of mutant Smad3 constructs inhibited Smad3 as well as β-catenin protein levels even in the presence of TGF-β. E, RCJ3.1C5.18 cells were treated with TGF-β (2 ng/ml) for 4 h. The cells were then fixed and incubated with the anti-β-catenin antibody followed by the incubation with a fluorescein isothiocyanate-conjugated secondary antibody. Treatment with TGF-β induced β-catenin nuclear translocation and silencing of Smad3 inhibited β-catenin nuclear translocation.

Interaction domains of Smad3 and β-catenin. A, β-catenin expression plasmid was co-transfected with Myc-Smad1 or Myc-Smad3 into COS cells. 24 h after transfection, the cell lysates were collected. IP was performed using the anti-β-catenin antibody followed by Western blotting (WB) using an anti-Myc antibody (top panel). Although both Smad1 and Smad3 interact with β-catenin, Smad3 had stronger interaction with β-catenin. B, β-catenin protein structure and truncated β-catenin constructs. C, COS cells were transfected with truncated β-catenin constructs: FLAG-BD1 (amino acids 1–260), FLAG-BD2 (amino acids 261–500), and FLAG-BD3 (amino acids 501–781) with Myc-Smad3. 24 h after transfection, the cell lysates were collected. IP was performed using the anti-Myc antibody followed by Western blotting using the anti-FLAG antibody (top panel). The N-terminal domain and mid-region of β-catenin interacted with Smad3. D, RCJ3.1C5.18 cells were transfected with three truncated β-catenin constructs FLAG-BD1, -BD2, and -BD3. 24 h after transfection, the cells were treated with TGF-β (2 ng/ml) for 4 h. IP was performed using the anti-Smad3 antibody followed by Western blotting using the anti-FLAG antibody (top panel). The N-terminal domain and mid-region of β-catenin interacted with Smad3 in a TGF-β-dependent manner in RCJ3.1C5.18 cells. E, a diagram shows Smad3 structure and truncated Smad3 constructs. F, WT and truncated Smad3 constructs: FLAG-N-Smad3 (amino acids 1–145), FLAG-NL-Smad3 (amino acids 1–211), and FLAG-C-Smad3 (amino acids 200–425) were transfected into 293T cells. 24 h after transfection, the cell lysates were collected. IP was performed using the anti-FLAG or anti-Myc antibody followed by Western blotting using the anti-β-catenin antibody (top panel). The C-terminal domain of Smad3 interacted with β-catenin. IP, immunoprecipitation.

Smad4 is involved in the protection of β-catenin degradation. A, Smad3 expression plasmid or Smad3 siRNA were co-transfected with HA-Smad4 expression plasmid into RCJ3.1C5.18 cells. Immunoprecipitation was performed using the anti-β-catenin antibody followed by Western blotting (WB) using the anti-HA antibody (top panel). As a control, IP was performed using the anti-Smad3 antibody followed by Western blotting using the anti-HA antibody (middle panel). Smad4 interacted with β-catenin in a Smad3-dependent manner. B, cell lysates were collected from RCJ3.1C5.18 cells treated with or without TGF-β (2 ng/ml) for 4 h. IP was performed using the anti-β-catenin antibody followed by Western blotting using the anti-HA antibody (top panel). As a control, IP was also performed using the anti-Smad3 antibody followed by Western blotting using the anti-HA antibody (middle panel). Smad4 interacted with β-catenin in a TGF-β-dependent manner in RCJ3.1C5.18 cells. C, Smad4 siRNA significantly inhibited Smad4 mRNA expression (∼70% inhibition). D, RCJ3.1C5.18 cells were transfected with Smad3 and β-TrCP expression plasmids in the presence or absence of Smad4 siRNA. 24 h after transfection, the cell lysates were collected, and Western blotting was performed using the anti-β-catenin antibody. Smad4 siRNA inhibited the protective effect of Smad3 on β-catenin degradation. E, RCJ3.1C5.18 cells were co-transfected with Smad2 and β-TrCP expression plasmids with or without Smad3 siRNA. 24 h after transfection, the cell lysates were collected, and Western blotting was performed using the anti-β-catenin antibody. Smad2 showed a limited effect on protection of β-catenin degradation when Smad3 gene was silenced. IP, immunoprecipitation.

Smad3 enhances β-catenin activity in chondrocytes. A and B, RCJ3.1C5.18 cells were transfected with control (Cont) or Smad3 siRNA and treated with Wnt3a. Expression of cyclin D1 and Axin2 was examined by real time PCR. Treatment with Wnt3a significantly increased cyclin D1 and Axin2 expression. Silencing of Smad3 inhibited Wnt3a-induced expression of cyclin D1 and Axin2. C, RCJ3.1C5.18 cells were transfected with control or Smad3 siRNA and cyclin D1 promoter construct (−962CD1) and treated with Wnt3a. Wnt3a stimulated cyclin D1 promoter activity, and Smad3 siRNA significantly inhibited Wnt3a-induced cyclin D1 promoter activity. D, WT or NLS mutant forms of Smad3 (K43NK44Q or ΔK40K41) were co-transfected with the TOP-flash reporter construct into RCJ3.1C5.18 cells. 24 h after transfection, the cells were treated with Wnt3a (100 ng/ml). The cell lysates were collected 24 h later for luciferase assay. Wnt3a-stimulated TOP-flash reporter activity was enhanced by transfection of Smad3 and inhibited by transfection of mutant Smad3. E, WT or NLS mutant forms of Smad3 were co-transfected with β-catenin expression plasmid and the −962CD1 reporter construct into RCJ3.1C5.18 cells. Cell lysates were collected 24 h later for luciferase assay. β-Catenin-induced cyclin D1 promoter activity was enhanced by Smad3 and inhibited by mutant Smad3. F, WT (−962wtCD1) and mutant cyclin D1 (−962mCD1) (TCF binding site deletion) promoter constructs were transfected into RCJ3.1C5.18 cells. 24 h after transfection, the cells were treated with different concentrations of TGF-β (0, 0.2, 1, and 5 ng/ml). The cell lysates were collected 24 h later for luciferase assay. TGF-β stimulated cyclin D1 promoter activity in a dose-dependent manner. The effect of TGF-β was significantly reduced in cells transfected with mutant cyclin D1 promoter in which four TCF binding sites were deleted.