ERα induces the ubiquitination and degradation of Smad. A–C, estrogen reduced steady-state Smad protein levels via ERα. A, ERα-positive breast cancer cell line MCF-7 was treated with TGF-β (1 ng/ml), estrogen (10⁻⁸ m), ICI182,780 (10⁻⁶ m), or MG132 (10⁻⁶ m) or left untreated. We then determined the protein levels of Smad2/3, phosphorylated Smad2/3, ERα, and β-actin by Western blotting using appropriate antibodies. B, MCF-7 cells were transfected with control or ERα siRNA and cultured in the presence or absence of TGF-β or estrogen. Phosphorylated Smad2, Smad3 levels were examined by Western blotting. C, expression plasmids encoding FLAG-tagged Smad2 and Smad3 were co-transfected into 293 cells with ERα and a constitutively active TGF-β type I receptor ALK5 TD expression plasmids. These cells were then incubated in the presence or absence of estrogen or MG132. The protein levels of Smad2, Smad3, pSmad2, pSmad3, and ERα were examined by Western blotting using the indicated antibodies. D, DNA binding ability of ERα was not required for induction of Smad degradation. FLAG-tagged Smad2, Smad3, and ALK5 TD expression plasmids were transfected into 293 cells with or without expression plasmids encoding ERα or ERα(mC). These cells were then incubated in the presence or absence of estrogen and MG132. We then determined the protein levels of Smad2, Smad3, ERα, and ERα(mC) by Western blotting using protein-specific or anti-FLAG-M2 antibodies. E, estrogen facilitated Smads degradation. MCF-7 cells were cultured in medium containing cycloheximide (100 μg/ml). Cells were harvested at the indicated times. Smad protein levels in untreated and estrogen-treated cells were examined by immunoblotting (left). The bands were quantified and represented by graphs (right). F, estrogen induced Smad2 ubiquitination. Plasmids encoding Myc-tagged Smad2, HA-tagged ubiquitin, and ALK5 TD were transfected into 293 cells in the presence or absence of an ERα expression plasmid. After culturing transfected cells in the presence of MG132 with or without estrogen, ubiquitinated proteins were immunoprecipitated with an anti-HA antibody. Ubiquitinated Smad was detected by immunoblotting these immunoprecipitates with an anti-Myc antibody (upper panel). To confirm Smad ubiquitination, Myc-tagged Smad2 was immunoprecipitated using an anti-Myc antibody; the ubiquitination of the immunoprecipitated protein was confirmed by immunoblotting with an anti-HA antibody (second panel). Smad2 and ERα were detected in whole cell lysates by immunoblotting using anti-Myc (third panel) and anti-ERα (fourth panel) antibodies, respectively.

ERα inhibits TGF-β signaling via Smad degradation. A and B, Smurf1 enhanced estrogen-dependent Smad degradation. Plasmids encoding FLAG-tagged Smad2, Smad3, and ALK5 TD were transfected with or without those encoding ERα, HA-tagged Smurf1, or HA-tagged Smurf1 CA. After culturing transfected cells with or without estrogen (10⁻⁸ m) or MG132 (10⁻⁶ m), we evaluated the indicated protein levels by Western blotting using appropriate antibodies. C, Smurf1 enhanced Smad2 ubiquitination. Vectors encoding Myc-tagged Smad2, HA-tagged ubiquitin, and ALK5 TD were transfected into 293 cells with or without plasmids encoding ERα, Smurf1, or Smurf1 CA. After incubation in the presence of MG132 with or without estrogen, Myc-tagged Smad2 or HA-tagged ubiquitin was immunoprecipitated with anti-Myc or anti-HA antibodies, respectively. The ubiquitination status of Smad2 was assessed by Western blotting probed with anti-HA antibody or anti-Myc antibody, respectively. D, ubiquitin-proteasome pathway was involved in ERα-mediated inhibition of TGF-β-dependent transcription. Expression plasmids encoding ALK5 KR or ALK5 TD, the 9xCAGA luciferase reporter plasmid, and pRSVβGAL were transfected into 293 cells with or without plasmids encoding ERα or Smurf1 CA. Transfected cells were cultured in the presence or absence of MG132 prior to examination of cell extracts by luciferase assay. E, stably ERα-expressing MDA-MB-231 cells were transfected with control or the mixture of Smurf1 and Smurf2 siRNAs and cultured in the presence or absence of estrogen. Smad2, Smad3, and Smurf1 protein levels were examined by Western blotting.

The degradation of Smad protein and inhibition of TGF-β-dependent transcription are mediated by identical regions within ERα. A, series of ERα truncations. B, binding of Smad2 or Smurf1 to the ERα truncations. The in vitro-translated ERα mutants were combined with purified, glutathione-Sepharose-bound GST-Smad2 or GST-Smurf1. After incubation, we analyzed the Sepharose:protein complexes by SDS-PAGE and autoradiography. C, Smad2 degradation was induced by truncated forms of ERα. Expression plasmids encoding FLAG-tagged Smad2 and the truncated ERα forms were transfected into 293 cells. After culturing the transfected cells in the presence or absence of estrogen (10⁻⁸ m), FLAG-tagged Smad2 was detected in cell extracts by immunoblotting with an anti-FLAG-M2 antibody. D, TGF-β-mediated transcription was inhibited by the truncated forms of ERα. Expression plasmids encoding ERα truncations and ALK5 TD were co-transfected into 293 cells with the 9×CAGA luciferase reporter plasmid and ph-RL-TK. Transfected cells were cultured in the presence or absence of estrogen prior to examination of cell extracts by luciferase assay.

Estrogen inhibits TGF-β signaling. A, MCF-7 cells transfected with a reporter plasmid encoding nine tandem CAGA repeats (9×CAGA) were cultured in the absence or presence of TGF-β (1 ng/ml) or estrogen (+, 10⁻⁹ m; ++, 10⁻⁸ m). Cell extracts were analyzed in luciferase assays. B, MCF-7 cells were cultured in the absence or presence of TGF-β or estrogen (10⁻⁸ m), and PAI-1 mRNA levels were examined by real-time RT-PCR. C, MCF-7 cells were transfected with control or ERα siRNA and cultured in the presence or absence of TGF-β or estrogen. PAI-1 mRNA levels were examined. D, gene expression profiles from TGF-β-treated and/or estrogen-treated MCF-7 cells were analyzed using a DNA microarray. E, combinations of indicated expression plasmids were co-transfected with the 9×CAGA reporter plasmid and ph-RL-TK into 293 cells. Cell extracts derived from cultures incubated in the presence or absence of estrogen were examined in luciferase assays.

ERα induces the ubiquitination and degradation of Smurf. A and B, estrogen reduced the steady-state levels of Smurf1 and Smurf2. A, ERα-positive MCF-7 breast cancer cells were incubated in the presence or absence of estrogen (10⁻⁸ m) or MG132 (10⁻⁶ m). We evaluated Smurf1 and Smurf2 protein levels by Western blotting. B, expression plasmids encoding FLAG-tagged Smurf1 or Smurf1 CA, and ALK5 TD were transfected into 293 cells with or without an ERα expression plasmid. Transfected cells were incubated in the presence or absence of estrogen or MG132. We then determined Smurf1 and Smurf1 CA protein levels by Western blotting using an anti-FLAG-M2 antibody. C, estrogen induced Smurf1 ubiquitination. Expression plasmids encoding Myc-tagged Smurf1, HA-tagged ubiquitin, and ALK5 TD were transfected into 293 cells with or without an ERα expression plasmid. After incubation in the presence of MG132 with or without estrogen, Myc-tagged Smurf1, and HA-tagged ubiquitin were immunoprecipitated using anti-Myc and anti-HA antibodies, respectively. We examined the ubiquitination status of Smurf1 by Western blotting using anti-Myc (upper panel) or anti-HA (second panel) antibodies, respectively.

ERα, Smad, and Smurf form a ternary complex. A, ERα associated with Smad. Expression plasmids encoding FLAG-tagged Smad2 or Smad3 were co-transfected with or without an ERα expression plasmid into 293 cells. Smad proteins were immunoprecipitated from the cell extracts using an anti-FLAG-M2 antibody. ERα was detected in precipitates by Western blotting using an antibody against ERα. *, antibody. B, ERα associated with Smurf. We co-transfected 293 cells with a Myc-tagged Smurf1 CA expression plasmid in the presence or absence of an ERα expression plasmid. Smurf1 CA protein was immunoprecipitated from cell lysates using an anti-Myc antibody. ERα was detected in precipitates by Western blotting using an antibody against ERα. C, endogenous ERα, Smad, and Smurf proteins formed a complex. After lysis of MCF-7 cells, proteins were immunoprecipitated from cell lysates using an anti-ERα antibody in the absence or presence of TGF-β with or without estrogen (10⁻⁸ m). Precipitates were examined by Western blotting using antibodies against Smad2 and Smurf1. D, ERα formed a ternary complex with Smad and Smurf. FLAG-tagged Smad2 and/or Myc-tagged Smurf1 expression plasmids were co-transfected into 293 cells with or without an ERα expression plasmid. Smad2 was immunoprecipitated from cell lysates using an anti-FLAG-M2 antibody. Proteins were eluted from the beads using FLAG peptide, then re-immunoprecipitated with an anti-Myc antibody. ERα was detected in the precipitate by Western blotting using an antibody against ERα.

Effects of ERα truncations on the migratory and invasive potential of cancer cells. A and B, expression plasmid encoding one of the ERα mutants or an empty vector (control) was transfected into ERα-negative MDA-MB-231 breast cancer cells. After selection in G-418, transfected cells were seeded in the transwell chambers (A) or Matrigel-coated upper chambers (B). Cells were incubated for 20 h in the absence or presence of TGF-β or estrogen (10⁻⁸ m). Cells penetrating the filters were stained with crystal violet and counted under a microscope. * and N.S. indicate p < 0.05 and p > 0.05 for estrogen-treated cells versus control cells, respectively.