Smad7 is required for TGF-β-induced association between β-catenin and LEF1. (A) Lysates from PC-3U (upper panel) and HaCaT cells (lower panel), transiently transfected with control (C-siRNA) or specific Smad7 siRNAs (S7-siRNA), were treated or not with TGF-β for the indicated time periods and the total cell lysates were subjected to coimmunoprecipitation with a polyclonal antibody for β-catenin or a nonimmune, control (C) antibody and then analyzed for coprecipitated LEF1. Cell lysates from 293T cells transiently transfected with HA-LEF1 were used as a positive control (Pos.Ctrl.) to verify the identity of endogenous LEF1. The cell lysates were investigated for the amount of endogenous LEF1, β-catenin, and Smad7. The filters were then stripped, blocked, and reprobed with actin antibodies to confirm equal loading of proteins. (B) The same cell lysates from PC-3U and HaCaT cells were also investigated for the amount of endogenous Smad7 and c-Myc by immunoblotting. Total cell lysates from cells ectopically expressing Flag-Smad7 (PC-3U/pMEP4-Flag-S7) were used as positive control for Smad7 (Pos. Ctrl). The filters then were stripped, blocked, and reprobed with actin antibodies to confirm equal loading of proteins.

Smad7 is required for TGF-β-induced phosphorylation of p38 and the TGF-β-induced nuclear accumulation of β-catenin is associated with p38. (A) Lysates from PC-3U cells transiently transfected with control siRNA (C-siRNA) or specific Smad7 siRNA (S7-siRNA) were treated or not with TGF-β for the indicated time periods and the total cell lysates were investigated for the amount of endogenous p-p38 and Smad7. The filters were then stripped and reprobed with antibodies against total p38 to show the specificity of the band for p38 and again stripped and reprobed with actin antibodies to confirm equal loading of proteins. Total cell lysates from cells ectopically expressing Flag-Smad7 (PC-3U/pMEP4S-7) were used as the positive control for Smad7 (Ctrl). (B) The subcellular localization of β-catenin in PC-3U cells treated with TGF-β in the absence or presence of 10 μM SB203580, the p38 inhibitor, for the indicated time periods were analyzed by immunofluorescence staining. Note the absence of TGF-β-induced redistribution in cells simultaneously treated with SB203580. SB203580 was added to cells 1 h before the initiation of the experiment. Nuclei were counterstained with DAPI. (C) Mouse embryonic fibroblasts derived from wild-type (wt) or p38α^−/− knockout mice were treated with TGF-β for the indicated time periods and subjected to immunofluorescence staining for endogenous β-catenin and Smad7. Nuclei were counterstained with DAPI. Note the lack of TGF-β-induced effects on β-catenin and Smad7 colocalization in p38α^−/− mouse embryonic fibroblasts compared to wild-type mouse embryonic fibroblasts.

Transfection of siRNA for β-catenin reduces TGF-β induced apoptosis. (A) PC-3U and HaCaT cells were transfected or not with specific or unspecific (ctrl) siRNA for β-catenin and treated with TGF-β for 24 h. The localization and amount of β-catenin and number of apoptotic cells, shown by M30 staining, were examined by immunofluorescence. Nuclei were counterstained with DAPI. PC-3U cells transfected with specific siRNA for β-catenin and treated with 1 μM staurosporine (STS) for 8 h served as the control. The number of apoptotic cells was determined in triplicate samples in three different experiments, and at least 1,000 nuclei/sample were counted. The mean values are given in Table 1. Note the correlation between M30 staining and high levels of β-catenin. (B) Lysates from PC-3U and HaCaT cells transiently transfected with control (Ctrl) siRNA or specific β-catenin siRNA were treated or not with TGF-β for the indicated time periods, and the total cell lysates were investigated for the amount of endogenous β-catenin. The filters were then stripped and reprobed with actin antibodies to confirm equal loading of proteins.

TGF-β-induced association between β-catenin, LEF1, and Smad7. (A) Coprecipitation of endogenous β-catenin with wild-type (wt) or different Flag-Smad7 deletion mutants in transiently transfected COS1 cells. Lysates of transfected cells were subjected to immunoprecipitation with Flag M2 (F) or nonimmune, control (C) antibodies and then analyzed for coprecipitated Flag-Smad7 by immunoblotting with a β-catenin antibody. (B) Coprecipitation of HA-LEF1 or HA-TCF-4 with wild-type Flag-Smad7 or a Flag-Smad7 deletion mutant (delta-N terminal; C) in transiently transfected 293T cells. Lysates of transfected cells were subjected to immunoprecipitation with polyclonal Flag antibodies and then analyzed for coprecipitated HA-LEF1/HA-TCF-4 by immunoblotting with a LEF1/TCF-4 mouse monoclonal antibody. (C and D) Coprecipitation of endogenous β-catenin and LEF1/TCF with endogenous Smad7 in PC-3U or PC-3U/AS-S7 cells treated with TGF-β for the indicated time periods. Lysates from cells were subjected to immunoprecipitation with antisera against Smad7 or control antibodies (C) and then analyzed by immunoblotting for coprecipitated β-catenin (C) or coprecipitated LEF1 (D). Immunoblots from the corresponding total cell lysate were incubated with antibodies specific for β-catenin, Smad7, LEF1/TCF-4, c-Myc, or actin. Lysates from PC-3U cells treated with TGF-β in the absence or presence of cycloheximide for the indicated time periods were investigated for the amount of endogenous c-Myc (E) or LEF1 (F). The same filters were stripped and reprobed with antibodies against actin to confirm equal loading of proteins.

Smad7 is required for the TGF-β-induced redistribution of β-catenin to the perinuclear-nuclear compartment. (A) Subcellular localization of endogenous Smad7 and β-catenin in PC-3U or PC-3U/AS-S7 cells after treatment with TGF-β for 2 h, 6 h, or 12 h as investigated by immunofluorescence with antibodies against Smad7 and β-catenin, together with additional staining of the nuclei by DAPI. Note that β-catenin is localized in the cytoplasm and in cell-cell contacts in untreated cells. After 2 h of TGF-β treatment, a portion of Smad7 is perinuclear or nuclear as β-catenin. At longer time points (12 h) after TGF-β treatment, both proteins predominantly localize in the perinuclear and nuclear compartment (left panel). In contrast, in PC-3U/AS-S7 cells, where the levels of endogenous Smad7 is reduced, no significant change of the subcellular localization of β-catenin was observed (right panel). (B) Quantification of the effect of TGF-β treatment on the subcellular localization of endogenous β-catenin in PC-3U and PC-3U/AS-S7 cells is shown in Fig. 2A. (C) PC-3U cells were transiently transfected with control (C) or specific Smad7 (S7) siRNAs and treated or not with TGF-β for the indicated times. The subcellular localizations of β-catenin and Smad7 were analyzed as described above. The values for the quantification of the subcellular localization of β-catenin in the transfected cells are shown in panel D. (F) HaCaT cells were transiently transfected with control (C) or specific Smad7 (S7) siRNAs and treated or not for the indicated time periods. The distribution of endogenous β-catenin and Smad7 was investigated by immunofluorescence and shown in panel E. The quantification of the differences in distribution of β-catenin is shown in panel F. Approximately 300 cells from each condition were counted under the microscope at 40× magnification. (G) Immunoblots from subcellular fractions of PC-3U and HaCat; nucleus (N) and cytoplasm (CP), derived from cells treated or not with TGF-β for the indicated time periods. Lamin A and β-tubulin were used as markers for the nucleus and cytoplasmic fractions, respectively. Total cell lysate from cells ectopically expressing Flag-Smad7 (PC-3U/pMEP4-Flag-S7) were used as the positive control for Smad7.

TGF-β regulates the levels of β-catenin in PC-3U cells via phosphorylation of GSK-3β on Ser9 and Akt on Ser473 in a Smad7-, p38 MAP kinase-, and phosphatidylinositol 3-kinase-dependent manner. Total cell lysates were prepared from PC-3U and PC-3U/AS-S7 cells, treated or not with TGF-β, and used for immunoblotting. (A) Levels of β-catenin and Smad7 and of GSK-3β and Akt phosphorylated at Ser9 and Ser473, respectively, in PC-3U cells treated with TGF-β for different time periods. In the upper and lower panels, the phosphorylated and nonphosphorylated forms, respectively, of GSK-3β and Akt, are shown. A similar analysis with PC-3U/AS-S7 cells treated with TGF-β is also shown. Equal amounts of proteins were loaded for each cell line. The filters were subjected to immunoblotting simultaneously under equal conditions. (B) Lysates from PC-3U cells transiently transfected with control (C-siRNA) or specific Smad7 siRNAs (S7-siRNA) were treated or not with TGF-β for the indicated time periods, and the total cell lysates were investigated for the amount of endogenous pGSK-3β Ser9, phospho-Akt Ser473, and Smad7. The filters were then stripped and reprobed with actin antibodies to confirm equal loading of proteins. Total cell lysate from cells ectopically expressing Flag-Smad7 (PC-3U/pMEP4S-7) were used as the positive control for Smad7 (Pos. Ctrl). (C) Effects of the p38 inhibitor SB203580 and the phosphatidylinositol 3-kinase inhibitor LY294002 on TGF-β-induced phosphorylation of GSK-3β on Ser9. Total cell lysates were prepared from PC-3U cells treated or not with TGF-β, in the presence or absence of SB203580 and LY294002, andsubjected to immunoblotting. The phosphorylation of GSK-3β on Ser9 and amount of β-catenin are shown in the upper two panels, and the phosphorylation of Akt on Ser473 is shown in the lower panel. Both filters were stripped, blocked, and reprobed with actin antibodies, which served as the control for equal loading of proteins.

Schematic illustration of the molecular mechanisms involved in the TGF-β-Smad7 signaling pathway resulting in activation of Akt and p38, followed by phosphorylation and inactivation of GSK-3β. The inactivated GSK-3β thus cannot phosphorylate and cause degradation of β-catenin, which leads to its accumulation. The figure summarizes the result of the current study and of a previously published study (18). For further explanation and details, see the text.