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Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERα and ERβ, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-β acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERα and TGF-β/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERα inhibits TGF-β signaling by decreasing Smad protein levels. ERα-mediated reductions in Smad levels did not require the DNA binding ability of ERα, implying that ERα opposes the effects of TGF-β via a novel non-genomic mechanism. Our analysis revealed that ERα formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERα.
Exposure to estrogen, which regulates cellular growth, proliferation, and differentiation, is an important risk factor for the development of breast cancers. The effects of estrogens are mediated by their ligation of estrogen receptors (ER),4 ERα and ERβ, members of the nuclear receptor (NR) superfamily that function as ligand-induced transcription factors (1,–3). Upon binding of estrogen, the receptor ligand-binding domain (LBD) undergoes a characteristic conformational change that induces receptor dimerization. The dimer then binds DNA to stimulate gene expression. ERα activity is stimulated by two distinct regions, activation function-1 (AF-1) and AF-2 domains; the latter, located in the LBD, exerts ligand-dependent transcriptional activity (4). Crystal structure analysis of ERs and other NRs revealed 12 conserved helices within their LBDs (5). Helix 12, the C-terminal helix, forms the critical core (AD core) of AF-2 function for the receptor. This region plays an important role in the binding of coactivators to the ligand-bound receptor (6). Estrogen-dependent ERα transactivation is also regulated by the ubiquitin-proteasome pathway (7,–10). ERα and its coactivators cycle onto and off of estrogen-responsive promoters in a ligand-dependent manner (11,–13). ERα is ubiquitinated after each round of transcription, facilitating release from the promoter; this event may be essential for subsequent ERα-dependent transcription.
The transforming growth factor β (TGF-β) superfamily is a large, evolutionarily conserved family of secreted multifunctional peptides involved in almost every aspect of cellular behavior (14). TGF-β regulates cellular processes through three high-affinity cell surface TGF-β receptors, termed type I (TβRI), type II (TβRII), and type III (TβRIII) (15, 16). TβRI and TβRII contain serine/threonine protein kinases within their intracellular domains (17, 18). Ligation of TβRI triggers the phosphorylation of members of the Smad family of transcription factors (19, 20). Smad2 and Smad3 are receptor-activated Smads (R-Smad) downstream of TβRI activation. Smad4 serves as a common partner (Co-Smad) for all receptor-activated Smads (21,–23). Smad6 and Smad7 are inhibitory Smads (I-Smad) that block Smad2 and Smad3 phosphorylation, thus inhibiting TGF-β signaling (24, 25). The protein levels of Smad are regulated by Smurf1 and Smurf2, members of a family of HECT E3 ubiquitin ligases (26,–28). Smurfs are able to induce R-Smad degradation. Smurfs also bind I-Smads, facilitating their recruitment to TGF-β receptors, which mediates the rapid turnover of Smads (29, 30). In this way, Smurf activity leads to the termination of TGF-β signaling.
TGF-β superfamily (TGF-β, activin, and bone morphogenic protein (BMP))/Smad signaling is reportedly regulated by estrogen/ER signaling. In MCF-7 cells, TGF-β-induced transcription and migratory potential are inhibited by estrogen (31), and activin and estrogen signaling are reciprocally suppressed their signaling (32). Smad3-dependent transcription is inhibited by ERα through binding to Smad3, and the inhibition is abrogated by the expression of AP-1 transcription factors (33, 34). A complex of Smad3/4 mediates the inhibition of ERα-mediated estrogenic activity of gene transcription in breast cancer cells (35). In addition, ERα interacts with Smad1, which is a downstream signal transducer of BMP signaling, to inhibit BMP-induced transcription (36, 37). These observations suggest the regulatory interaction between Smad and ERα; however, it remains poorly understood at the molecular level.
Here, we show that ERα inhibits TGF-β signaling by decreasing Smad protein levels. ERα-mediated reductions in Smad levels did not require the DNA-binding ability of ERα, implying that ERα opposes the effects of TGF-β via a novel non-genomic mechanism. Our analysis revealed that ERα formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERα.
cDNAs encoding ERα, Smad2/3, Smurf1/2, ALK5 TD/KR, and the ERα deletion mutants were cloned into pcDNA3 or pCMV5 expression vector. The 9×CAGA luciferase reporter plasmid have been described previously (38). The pGL3-Basic, pGL3-Control, and ph-RL-TK vectors were purchased from Promega. Mouse anti-FLAG-M2 (Sigma), mouse anti-HA (Roche), mouse anti-Myc (Nacalai Tesque), anti-human-ERα (Santa Cruz Biotechnology), anti-human-ERβ (Chemicon), anti-human-Smad2/3 (BD), anti-human-phospho-Smad2 (Cell Signaling), anti-human-Smad3 (Cell Signaling), anti-human-phospho-Smad3 (BIOSOURCE), and mouse anti-β-actin (Sigma) antibodies were used according to the manufacturers' instructions.
MCF-7 and MDA-MB-231 breast cancer cells, and 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Twenty-four hours before transfection, cells were shifted to phenol red-free DMEM containing 4% charcoal-stripped FBS. Transfection was performed with either PerFectin Transfection Reagent (Gene Therapy Systems) or TransFast Transfection Reagent (Promega) according to the manufacturers' protocols. Cells were treated with or without TGF-β (1 ng/ml) and estrogen (10−8 m). Twenty-four hours after the addition of estrogen, cells were harvested and analyzed by Western blotting using appropriate antibodies.
After 293 cells were transfected with the designated plasmids, cells were lysed in TNE buffer (10 mm Tris-HCl (pH 7.8), 0.5% Nonidet P-40 (Nonidet P-40), 0.15 m NaCl, 1 mm EDTA, and 1 μm phenylmethylsulfonyl fluoride). Extracted proteins were immunoprecipitated with antibody-coated protein A/G-Sepharose (Amersham Biosciences) or anti-FLAG M2 agarose (Sigma) beads. Bound proteins were separated using SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore), and detected with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized using an enhanced chemiluminescence (ECL) Western blot detection system (Amersham Biosciences).
The 9×CAGA-Luc plasmid was co-transfected into 293 cells with expression vector encoding either wild-type or mutant ERα. We co-transfected the ph-RL-TK vectors into all cell lines as a reference plasmid, which was used to normalize transfection efficiency. Twenty-four hours after transfection, we added either estrogen (10−8 m) or vehicle alone (ethanol) to the cells with fresh medium containing 1% Charcoal-stripped FBS for an additional 24 h of incubation. Luciferase assays were performed using cell extracts according to the manufacturer's protocol (Promega). Individual transfections were assessed in triplicate and repeated at least three times.
MCF-7 cells were cultured in medium containing 4% charcoal-stripped FBS for 48 h, which was then replaced with fresh medium containing 4% FBS. After 24 h, fresh culture medium containing 0.2% charcoal-stripped FBS, TGF-β (1 ng/ml), and cycloheximide (100 μg/ml) with or without estrogen (10−8 m) was added. Cells were harvested at several time points after the addition of cycloheximide.
293 cells were transiently co-transfected with vectors encoding HA-tagged ubiquitin (Ub) and Myc-tagged Smad2, Myc-tagged Smurf1, or ERα as indicated. Cells were cultured in the presence of MG132 (1 μm) with or without estrogen (10−8 m), and lysed in radioimmune precipitation assay buffer (RIPA). After clarification by centrifugation, soluble proteins were immunoprecipitated from the cell extracts using anti-Myc (Nacalai Tesque) or anti-HA (Sigma) agarose for 4 h at 4 °C. The resin was washed in RIPA buffer, and immunoprecipitates were immunoblotted using anti-HA or anti-Myc antibodies.
We prepared GST fusion protein constructs of full-length Smad2 or Smurf1 by subcloning the protein-coding regions into pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ). Constructs were individually transformed into Escherichia coli BL21 cells; recombinant protein expression was induced for 6 h with 0.1 mm isopropyl-thio-β-d-galactopyranoside. We used glutathione-Sepharose (GE) to purify the GST fusion proteins according to the manufacturer's instructions. In vitro translated, [35S]methionine-labeled ERα constructs were prepared using the TNT coupled transcription/translation system (Promega, Madison, WI). Five microliters of labeled protein was mixed with ~1 μg of purified GST-Smad2 or GST-Smurf1 bound to glutathione-Sepharose (GE). This mixture was incubated for 1 h in GST pull-down buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, and 5 mm EDTA). Sepharose-protein complexes were washed five times in GST pull-down buffer and analyzed using SDS-PAGE and autoradiography.
Total RNA was prepared from samples using an RNeasy RNA isolation kit (Qiagen). The preparation of in vitro transcription products and the hybridization and scanning of the oligonucleotide array were performed according to Affymetrix protocols (2001 Affymetrix Genechip Technical Manual; Santa Clara, CA).
Suspensions (0.5 ml) containing 1.25 × 105 cells (invasion assay) or 0.5 × 105 cells (migration assay) were layered in the upper compartments of rehydrated Matrigel Invasion chambers or transwell chambers, respectively. These cells were then incubated for 20 h (invasion assay) or 12 h (migration assay) at 37 °C with 0.75 ml of DMEM in the lower chambers. After incubation, cells on the upper surface of the filter were removed, and invading or migrating cells were fixed in methanol. Fixed cells were stained with crystal violet and counted under a microscope.
Significance of differences was determined by Student's t test analyses, using Microsoft Excel.
To examine the effects of estrogen on TGF-β signal transduction, we performed a transcription reporter assay. A reporter plasmid encoding a TGF-β response element (9×CAGA) was transfected into MCF-7 cells. TGF-β treatment enhances transcription from the reporter plasmid, which was profoundly inhibited by estrogen treatment (Fig. 1A, lanes 3 and 4). We also examined plasminogen activator inhibitor-1 (PAI-1) mRNA level, which is one of the target genes of TGF-β/Smad signaling, using real-time RT-PCR. TGF-β-induced PAI-1 mRNA expression level was significantly reduced by the treatment with estrogen (Fig. 1B). To examine whether estrogen inhibits TGF-β signaling via ERα, we reduced the endogenous level of ERα in MCF-7 cells using siRNA. In ERα-deficient cells, we did not observe an estrogen-dependent inhibitory effect on PAI-1 mRNA levels (Fig. 1C). Next, we used microarray analysis to determine the effects of estrogen on several endogenous TGF-β-induced gene expression. This approach allowed us to compare the gene expression profiles of untreated (Fig. 1D, lane 1), TGF-β-treated (Fig. 1D, lane 2), and TGF-β and estrogen-treated MCF-7 cells (Fig. 1D, lane 3). Of the 54,675 genes represented on the microarray, the expression levels of 956 genes increased greater than 2-fold following TGF-β treatment (Fig. 1D, lane 2). Estrogen reduced the expression of 683 of these 956 genes up-regulated by TGF-β treatment (Fig. 1D, lane 3). These data suggest that estrogen antagonizes TGF-β-dependent transcriptional regulation via ERα.
We further investigated the molecular mechanism of ERα-mediated transcriptional suppression by transfecting the reporter plasmid (9×CAGA) into the human kidney cell line 293 in the presence of either a constitutively active TGF-β type I receptor (mutation of Thr-204 to Asp) or a catalytically inactive receptor (ALK5 KR; mutation of Lys-378 to Arg). ALK5 TD, but not ALK5 KR, induced transcription from the reporter plasmid promoter (Fig. 1E, lanes 2 and 3). Co-expression of ERα in ALK5 TD-expressing cells reduced transcriptional activity; further reduction was observed following estrogen treatment (Fig. 1E, lanes 5 and 6). We obtained similar results following co-transfection of ALK5 TD with ERα(mC), a form of ERα bearing three amino acid substitutions in the DNA-binding domain (10) that eliminates the interaction of the receptor with DNA (Fig. 1E, lanes 7 and 8). These results suggest that inhibition of TGF-β signaling does not require ERα binding to estrogen-responsive elements (ERE) within the nucleus.
As Smad proteins are key transducers in TGF-β signaling, it is possible that estrogen inhibits TGF-β signaling by preventing the recruitment of Smad to promoter region of TGF-β responsive genes. Therefore, we performed the chromatin immunoprecipitation experiment to examine whether estrogen interferes with DNA binding of Smad. As shown in supplemental Fig. S1, the binding of Smad3 on PAI-1 promoter was reduced by treatment with estrogen. These data suggest that ERα inhibits TGF-β signaling by reducing the recruitment of Smad protein on the promoter region. Thus, we examined the endogenous protein levels of Smad2 and -3 by Western blotting. In MCF-7 cells, the protein levels of both Smad2 and 3 were reduced by co-treatment with TGF-β and estrogen (Fig. 2A). Following TGF-β receptor ligation, phosphorylated Smads translocate into the nucleus. In addition to reductions in total protein, phosphorylated Smad2 and 3 levels were significantly decreased by estrogen treatment (Fig. 2A). In contrast to estrogen, treatment with the pure ER antagonist ICI182,780, which attenuates receptor dimerization, effecting rapid degradation of the ER protein and inhibition of transcription, did not markedly affect Smad protein levels (Fig. 2A). The protein levels of ERα were reduced in the presence of estrogen or ICI182,780 (Fig. 2A). The estrogen-dependent reduction in Smad2 and 3 could be abrogated by treatment with the proteasome inhibitor MG132 (Fig. 2A, lane 7). In addition, Smad mRNA levels remained unchanged by estrogen treatment (data not shown). These results indicated that estrogen induces Smad proteolysis. To examine whether estrogen induces Smad degradation via ERα, we reduced the endogenous levels of ERα in MCF-7 cells using siRNA. In ERα-deficient cells, we did not observe an estrogen-dependent reduction of Smad protein levels (Fig. 2B), indicating that estrogen induces Smad proteolysis via ERα.
Next, we transfected expression vectors encoding Smad and either ERα or ERα(mC) into 293 cells. We observed estrogen-dependent degradation of both Smad and phosphorylated Smad proteins following co-expression of ERα (Fig. 2C). Consistent with the results of the transcription reporter assay (Fig. 1E), ERα(mC) also induced Smad protein degradation (Fig. 2D). These data raise the possibility that Smad protein degradation does not require ERα binding to ERE within the nucleus and ERα-dependent transcription. To obtain further evidence for this hypothesis, we inhibited ERα-dependent transcription using transcriptional inhibitor, α-amanitin, and tested its effects on estrogen-dependent Smad degradation. As expected, the degradation of Smad by estrogen and ERα was not affected by treatment with α-amanitin (supplemental Fig. S2). These findings indicate that ERα-dependent transcription was not necessary for the estrogen-dependent Smad degradation. We also examined the effects of ERβ on TGF-β signaling and Smad degradation. As shown in supplemental Fig. S3, ERβ also inhibited TGF-β signaling and enhanced Smad degradation in an estrogen-dependent manner, similar to ERα.
We confirmed that estrogen enhances Smad degradation using pulse-chase experiments. In the absence of estrogen, the half-lives of the Smad proteins exceeded 7.5 h (Fig. 2E). Estrogen treatment reduced the Smad protein half-lives to less than 4.5 h (Fig. 2E). We also examined the effect of estrogen on Smad2 ubiquitination. Exogenous expression of ERα resulted in broad bands on Western blots, which were consistent with ubiquitin-conjugated Smad2 (Fig. 2F). These broad bands intensified in samples treated with estrogen (Fig. 2F), suggesting that estrogen enhances Smad2 ubiquitination.
Smurf1 and -2 are known as an E3-type ubiquitin ligase for Smad proteins. Therefore, we next determined the protein levels of Smurf. Interestingly, treatment with both TGF-β and estrogen reduced Smurf protein levels (Fig. 3A). This reduction was inhibited by MG132 (Fig. 3A, lane 5), suggesting Smurf levels were also decreased via proteasomal degradation in an estrogen-dependent manner. We next transfected into 293 cells either Smurf1 or Smurf1 CA, a mutant form of the protein bearing an amino acid substitution that abolishes ubiquitin ligase activity, in the presence or absence of ERα. Steady state levels of Smurf1 were reduced by ERα expression (Fig. 3B, lane 3 of upper panel); this reduction was enhanced by estrogen treatment (Fig. 3B, lane 4 of upper panel). In contrast, Smurf1 CA levels were unaffected by ERα co-expression (Fig. 3B, lanes 3 and 4 of lower panel), suggesting that estrogen-dependent Smurf1 degradation requires its ubiquitin ligase activity. We next examined the effect of estrogen on Smurf1 ubiquitination. The broad band representing multiple ubiquitin-conjugated Smurf1 products intensified following ERα expression (Fig. 3C, lane 4); estrogen treatment enhanced the intensity of this signal (Fig. 3C, lane 5), indicating that estrogen-bound ERα enhances Smurf1 ubiquitination.
Next, we assessed the effect of Smurf1 expression on the estrogen-dependent degradation of Smad2 and 3. Under condition in which Smurf1 expression exerted little effect on Smad2 and -3 protein levels in the absence of ERα (Fig. 4A, lanes 3 and 4); co-expression with ERα enhanced the reduction of Smad2 and -3 in the presence of estrogen (Fig. 4A, lanes 6 and 8). Smurf1 also enhanced estrogen-dependent ERα down-regulation. In contrast, Smurf1 CA expression inhibited the estrogen-dependent reduction in ERα and Smad levels (Fig. 4A, lanes 9 and 10). The reduction in these proteins was abrogated by treatment with MG132 (Fig. 4B). These results confirmed the participation of Smurf1 in ERα-mediated Smad degradation. Whereas co-expression of Smurf1 with ERα and Smad2 enhanced estrogen-dependent Smad2 ubiquitination (Fig. 4C, lanes 7 and 8), Smurf1 CA expression reduced ubiquitinated Smad2 levels (Fig. 4C, lane 9), indicating that Smurf1 functions in estrogen-mediated Smad ubiquitination. Smurf2 exerted similar effects to those seen for Smurf1 on Smad ubiquitination and degradation (data not shown). Therefore, we next attempted to determine the effect of Smurf1 expression on the estrogen-dependent inhibition of TGF-β-induced transcription using a reporter assay. Estrogen-dependent suppression of reporter transcription was abrogated by either Smurf1 CA overexpression or MG132 treatment (Fig. 4D). As both of these treatments inhibit Smad degradation, it is likely that Smad protein degradation mediates ERα-induced inhibition of TGF-β signaling. To confirm that ERα inhibits TGF-β signaling by inducing Smad degradation, we reduced endogenous levels of Smurf1 and Smurf2 using siRNA. In Smurf-deficient cells, we did not observe estrogen-dependent Smad degradation (Fig. 4E).
Estrogen treatment induced the simultaneous degradation of Smad2, Smurf1, and ERα in cells co-expressing these proteins (Fig. 4A, lane 8), suggesting that ERα, Smad2, and Smurf1 form a complex within the nucleus. We examined the interactions between these proteins by co-immunoprecipitation from 293 cells transfected with ERα and either FLAG-tagged Smad2 or 3 (Fig. 5A) or Myc-tagged Smurf1 CA (Fig. 5B). ERα was detected in anti-FLAG and anti-Myc immunoprecipitates by protein immunoblotting with antibodies against ERα (Fig. 5, A and B), suggesting that Smad2, Smad3, and Smurf1 interact with ERα. These interactions were detected in both the absence and presence of estrogen. To confirm the interaction of endogenous proteins, we immunoprecipitated proteins from MCF-7 cell extracts using an anti-ERα antibody. By immunoblotting using antibodies against either Smad2 or Smurf1, we determined that the precipitated proteins included Smad2 and Smurf1 in the presence of TGF-β (Fig. 5C), confirming an interaction between ERα and these proteins in vivo. To investigate if ERα forms a ternary complex containing both Smad and Smurf, we transfected ERα, FLAG-tagged Smad2, and Myc-tagged Smurf1 into 293 cells. Proteins were initially immunoprecipitated with an anti-FLAG antibody. After elution with excess FLAG peptide, we performed a secondary immunoprecipitation with anti-Myc antibody. Immunoblotting of the precipitate with an anti-ERα antibody demonstrated the presence of ERα in the final immunoprecipitate (Fig. 5D), suggesting that ERα formed a ternary complex with both Smad and Smurf. To show further evidence that ERα forms a ternary complex with Smad and Smurf, and enhances simultaneous ubiquitination and degradation of these proteins, we examined if the ERα ubiquitination level is enhanced by the co-expression of Smad and Smurf in an estrogen-dependent manner. As shown in supplemental Fig. S4, ubiquitinated ERα were significantly increased by co-expression of Smad2 and Smurf1. These data indicate that ERα formed a ternary complex with both Smad and Smurf, and induces simultaneous degradation of these proteins to inhibit TGF-β pathways.
Note that estrogen-dependent Smad degradation of exogenously expressed Smad proteins occurred even in the absence of ALK5 TD in 293 cells (supplemental Fig. S5; left), indicating that TGF-β-dependent Smad phosphorylation is not necessary for its degradation. Immunohistochemical analysis of 293 cells revealed that Smad2 localized in the nucleus in the absence of ALK5 TD (supplemental Fig. S5; right), further indicating that the co-localization of Smad, Smurf, and ERα is necessary for ternary complex formation and subsequent Smad degradation.
As phosphorylated Smad2 and 3 bind to Smad4 and the complex translocates into the nucleus to stimulate the transcription of target genes, we examined whether ERα interacts with Smad4 (supplemental Fig. S6). In contrast to Smad2 and 3, Smad4 was not co-immunoprecipitated with ERα. In addition, we found that the protein levels of Smad4 did not change by treatment with estrogen (supplemental Fig. S7). These data suggest that Smad4 is absent from the complex containing ERα, R-Smad, and Smurf in the nucleus.
To identify the regions of ERα responsible for Smad degradation, we generated several truncations of ERα (Fig. 6A). GST pull-down experiments using this series of truncated ERα proteins demonstrated that the C region alone was sufficient to bind both Smad2 and Smurf1 (Fig. 6B). ERαABC and ERαCDEF were both able to bind Smad2 and Smurf1 and subsequently stimulate Smad2 degradation (Fig. 6, B and C). In contrast, ERαAB and ERαDEF, neither of which could bind Smad or Smurf, had no effect on Smad protein levels, indicating that the interaction of ERα, Smad, and Smurf is necessary to induce degradation. Whereas both ERαC and ERαCDE(ΔAD) were able to bind Smad and Smurf, expression of either construct could not induce degradation of Smad (Fig. 6, B and C). These results imply that the binding of ERα to Smad and Smurf is necessary, but not sufficient, for degradation. All ERα deletion mutants capable of inducing Smad2 degradation contained the transactivation domain (Fig. 6, B and C). As ERαC and ERαCDE(ΔAD) did not possess transcriptional activity, coactivator recruitment to the receptor may be necessary to induce Smad degradation. Next, we tested the effect of individual ERα deletion mutants on TGF-β-dependent transcription. We co-transfected truncated forms of ERα into 293 cells with a plasmid encoding ALK5 TD and the 9×CAGA reporter plasmid. The ERα mutants capable of inducing Smad protein degradation also abrogated TGF-β-dependent transcription (Fig. 6D), indicating that ERα-induced inhibition of TGF-β signaling arises from Smad degradation.
It is well known that TGF-β promotes cellular migration and invasion of breast cancer cells by increasing several target gene expressions. Therefore, we tested the effects of estrogen on TGF-β–induced migration and invasion. As shown in Fig. 7A, the migration of ERα-expressing MDA-MB-231 cells was increased by treatment with TGF-β, and significantly reduced by co-treatment with estrogen. Invasion of these cells was also reduced by co-treatment with estrogen (Fig. 7B). The expression of ERα mutants capable of reducing TGF-β signaling decreased the migratory and invasive potential of MDA-MB-231 cells (Fig. 7, A and B). In contrast, the ERα mutants incapable of reducing TGF-β signaling had no effect on migration or invasiveness (Figs. 6, B–D and and7,7, A and B). These findings indicate that estrogen/ERα inhibits cellular migration and invasion via inhibition of TGF-β signaling. Taken together, our results indicated that ERα forms a ternary complex with Smad and Smurf, enhances ubiquitination and degradation of Smad proteins, and inhibits TGF-β signaling pathway (Fig. 8).
In summary, ERα forms a protein complex with Smad and Smurf, and induces simultaneous degradation of these proteins to inhibit TGF-β pathways in an estrogen-dependent manner. As estrogen-dependent suppression was abrogated by inhibiting Smad degradation, it is likely that Smad protein degradation mediates ERα-induced inhibition of TGF-β signaling. This ERα-mediated degradation and inhibition of TGF-β-dependent transcription did not require the DNA binding ability of ERα, and are mediated by identical regions within ERα, implying that ERα opposes the effects of TGF-β via a novel non-genomic mechanism.
To the best of our knowledge, this is the first study showing that ERα enhances the degradation of Smad proteins via a ubiquitin-proteasome pathway. ERα-dependent Smad degradation and inhibition of TGF-β-induced transcription did not require the binding of ERα to an ERE. We have already identified several proteins that are degraded via an ER-dependent pathway.4 In addition, it was reported that aryl hydrocarbon receptor (AhR) induces the degradation of ERα (39). Considering these results together, ERα-dependent protein degradation appears to be a novel non-genomic function of ERα. Our data indicate that ERα non-genomically induces Smad degradation and that Smad degradation makes a major contribution to the ERα-mediated inhibition of TGF-β signaling.
The dependence of ERα-mediated Smad degradation on the presence of the ERα transactivation domain raises the possibility that coactivator binding to the receptor may be necessary for degradation. Several recent reports suggest that Smad is acetylated by CBP/p300 and that the acetylation status of Smad affects its ubiquitination and degradation. Overexpression of mutated CBP, which does not possess an acetyltransferase activity, inhibits estrogen-dependent Smad and ERα degradation (data not shown), supporting a role for coactivators in degradation. Therefore, we determined the acetylation status of Smad and Smurf using antibodies specific for acetylated proteins. The acetylation status of these proteins, however, was not altered by estrogen treatment (data not shown). A ubiquitin-conjugating enzyme (E2) also may be part of the coactivator complex (40). One report showed that an E2, UbcH7, interacts with SRC-1 to regulate the transcriptional activity of NRs (41). Such an interaction between ERα and an E2 may be necessary for Smad degradation. Here, we first demonstrated the novel non-genomic ERα function, by which TGF-β signaling is inhibited via Smad degradation. Our observations provide new insights into the non-genomic functions of ERα.
*This work was supported by a nuclear system to decipher operation code (DECODE) and Targeted Proteins Research Program (TPRP) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
4J. Yanagisawa, unpublished data.
4The abbreviations used are: