TGF-β1 induces nuclear translocation of β-catenin without affecting the steady-state protein level of β-catenin and independent of canonical Wnt signaling pathway. (A) Cytosolic and nuclear fractions of protein lysates were isolated from human MSCs treated or untreated with TGF-β1 for 2 h or Wnt3A for 6 h. Western blot was probed with an anti-β-catenin monoclonal antibody. (B) β-catenin localization was detected by immunofluorescence with the anti-β-catenin antibody after cells were treated or untreated with TGF-β1 for 1 h. (C) Cytosolic and nuclear fraction of protein lysates were isolated from MDCK cells treated or untreated with TGF-β1 for 2 h. Western blots were probed with anti-β-catenin antibody. (D) Cytosolic and nuclear fraction of protein lysates were isolated from MDCK cells treated or untreated with Wnt3A. Western blots were probed with anti-β-catenin antibody. (E) Total cell lysates were prepared from MSCs after treatment with TGF-β1 or Wnt3A for 24 h. Western blots were probed with anti-β-catenin antibody. (F) MSCs were pretreated with protein translation inhibitor cyclohexmide for 1 h before TGF-β1 treatment for 2 h. Cytosolic and nuclear fractions of β-catenin were detected by anti-β-catenin antibody. (G) MSCs were pretreated with CHX for 1 h before TGF-β1 treatment for 6 h. Protein levels of β-catenin and PAI-1 in whole-cell lysates were determined by Western blot with anti-β-catenin and PAI-1 antibodies. (H) MSCs were pretreated with Fz8CRD containing conditioned medium for 6 h before TGF-β1 treatment for 2 h. Nuclear fractions of β-catenin were detected by anti-β-catenin antibody. (I) The levels of nuclear β-catenin in vector control and DVL-ΔPDZ adenovirus-infected MSCs treated with TGF-β1 for 2 h were determined by Western blot analysis.

TGF-β1 and nuclear β-catenin have similar biological effects on MSCs. (A) Proliferation of human MSCs was examined by ³H-thymidine incorporation after the cells were untreated or treated with TGF-β1 for 24 h and relative proliferation activities are shown. Error bars were calculated from three duplicate experiments. (B) ALP activity was probed for osteogenic differentiation after MSCs were cultured in OS medium in the absence or presence of TGF-β1 for 10 d. (C) MSCs were infected with MSCV-mutant β-catenin or vector control retroviruses. Localization and ectopic expression of the mutant β-catenin were shown. (D) Proliferation of MSCs infected with the mutant β-catenin or vector control retroviruses was examined by ³H-thymidine incorporation. Relative proliferation activities are shown and error bars were calculated for standard deviation from three duplicate experiments. (E) ALP activity was probed for osteogenic differentiation after MSCs were infected with the mutant β-catenin or vector control retroviruses and cultured in OS medium for 10 d.

β-catenin nuclear translocation is mediated by the TGF-β signaling pathway. (A) Whole-cell lysates of MSCs untreated or treated with TGF-β1 and the type I TGF-β receptor inhibitor SD208 for 30 min were blotted with a polyclonal antiphospho-Smad2 antibody. (B) Nuclear fractions of MSCs untreated or treated with TGF-β1 and SD208 were detected by anti-β-catenin antibody. The concentrations of SD208 used in this assay were 50 and 100 nM. (C) MSCs were infected with control (pRS) or Smad3 siRNA (pRS-Smad3) expressing retrovirus and selected by puromycin. Whole-cell lysates from stable cell populations were blotted with an anti-Smad3 rabbit polyclonal antibody. Equal loading was confirmed by the presence of β-catenin. (D) Nuclear fractions from pRS or pRS-Smad3 retroviruse-infected MSCs untreated or treated with TGF-β1 for 2 h or Wnt3A for 6 h were blotted with the anti-β-catenin antibody. (E) Whole-cell lysates from MSCs untreated or treated with TGF-β1 for 1 or 2 h were subjected to immunoprecipitation with an anti-Smad3 polyclonal antibody. An anti-Flag antibody was used as the Ab control for the immunopre-cipitation. The Western blots were carried out using antibodies against β-catenin. (IP) Immunoprecipitation; (IB) immunoblotting. (F) Immunoprecipitation was performed as in E with lysates from MSCs untreated or treated with TGF-β for 2 h. GSK-3β was detected by a mouse monoclonal antibody.

Nuclear β-catenin is required for the primary effects of TGF-β1 on MSCs through regulation of specific downstream target genes. (A) 293T cells were cotransfected with pcDNA3-Myc-β-catenin and pcDNA3-HA-LEF1 or pcDNA3-HA-LEF1ΔC, and coimmunoprecipitations were performed with anti-HA and immunoblotted with anti-Myc. (WT) Wild type; (ΔC) LEF1ΔC. (B) 293T cells were cotransfected with pRK5-Flag-Smad3 and pcDNA3-HA-LEF1 or pcDNA3-HA-LEF1ΔC, and coimmunoprecipitations were performed with anti-HA and immunoblotted with anti-Flag. (C) MSCs were infected with vector control or LEF1ΔC adenoviruses. Ectopic expression of LEF1ΔC was detected with an anti-HA antibody. (D) Cytosolic and nuclear fraction of protein lysates were isolated from LEF1ΔC or vector control adenovirus-infected MSCs untreated or treated with TGF-β for 2 h. Western blots were performed with anti-β-catenin antibody or anti-HA antibody. (E) Proliferation of human MSCs infected with the LEF1ΔC or the vector control adenoviruses was examined by ³H-thymidine incorporation after the cells were untreated or treated with TGF-β1 for 24 h. Relative proliferation activities are shown and error bars were calculated for standard deviation from three duplicate experiments. (F) ALP activity was probed for osteogenic differentiation after MSCs were infected with LEF1ΔC or the vector control adenoviruses and cultured in OS medium for 5 d. (G) BLK mRNA levels in vector control and LEF1ΔC adenovirus-infected MSCs treated or untreated with TGF-β1 for 2 h were quantified by real-time PCR. Fold induction is shown and error bars were calculated for standard deviation from three duplicate experiments. (H) BAX mRNA levels in vector control and LEF1ΔC adenovirus-infected MSCs treated or untreated with TGF-β1 for 2 h were quantified by real-time PCR. Fold induction is shown and error bars were calculated for standard deviation from three duplicate experiments. (I) Protein levels of PAI-1 in vector control and LEF1ΔC adenovirus-infected MSCs untreated or treated with TGF-β1 for indicated time were determined by Western blot with anti-PAI-1 antibody.