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Molecular Endocrinology, doi:10.1210/me.2007-0129
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Molecular Endocrinology 21 (10): 2427-2439
Copyright © 2007 by The Endocrine Society

Glycogen Synthase Kinase-3 Protects Estrogen Receptor {alpha} from Proteasomal Degradation and Is Required for Full Transcriptional Activity of the Receptor

Jean Grisouard, Senad Medunjanin, Alexander Hermani, Ashish Shukla and Doris Mayer

Hormones and Signal Transduction Group, German Cancer Research Center, 69120 Heidelberg, Germany

Address all correspondence and requests for reprints to: Doris Mayer, Hormones and Signal Transduction Group, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: d.mayer{at}dkfz.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glycogen synthase kinase-3 (GSK-3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-{alpha} (ER{alpha}) as substrate for GSK-3, the impact of GSK-3 on ER{alpha} function and activity upon 17ß-estradiol (E2)-dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK-3{alpha} or GSK-3ß results in the reduction of ER{alpha} levels and transcriptional activity in ER{alpha}-positive breast cancer cells. Using MCF-7 cells we demonstrate that reduction of ER{alpha} levels upon GSK-3 silencing was due to increased proteasomal degradation of ER{alpha} rather than inhibition of ER{alpha} protein synthesis. Indeed, under this condition, ER{alpha} protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ER{alpha} was obtained after GSK-3 silencing in the presence of MG132. We conclude that GSK-3 protects ER{alpha} from proteasomal degradation and plays a crucial role in ER{alpha} protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK-3ß phosphorylates ER{alpha} at Ser-118. GSK-3 silencing resulted in decrease of E2-induced nuclear ER{alpha} phosphorylation at Ser-118 and E2-induced estrogen response element-dependent luciferase reporter gene expression. Neither Ser-118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogen-responsive genes (pS2 and progesterone receptor) was decreased upon GSK-3 silencing. These findings demonstrated that GSK-3 is required for E2-induced ER{alpha} phosphorylation at Ser-118 and full transcriptional activity of the receptor upon E2 stimulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN RECEPTORS (ERs) (1) are members of the class I nuclear hormone receptor superfamily of proteins acting as transcription factors. Two subtypes, estrogen receptor-{alpha} (ER{alpha}) and ERß, have been identified. Whereas ERß exhibits a limited expression pattern (1), ER{alpha} has been detected in almost all tissues. In the breast, ER{alpha} is the predominant subtype expressed (2) and has long been known to play an important role in the development of the mammary gland and in the formation of breast cancer (3, 4). The structure of ER{alpha} consists of six functionally and physically independent domains, a variable amino-terminal A/B domain that contains a ligand-independent activation function (AF)-1; a C domain that is required for DNA binding; a D domain or hinge region that contains the nuclear localization signal; an E domain that harbors the ligand-binding domain and the ligand-dependent AF-2, and a C-terminal F domain, the function of which is still unclear (5). AF-1 and -2 can activate transcription independently and synergistically act in a promoter- and cell type-specific manner (6, 7).

Recent work suggests that kinase-specific phosphorylation of ER{alpha} alters receptor functions such as interaction with ligands, DNA, and coregulators (8, 9). A number of serine residues relevant for ER{alpha} activation by protein kinases have been identified. Most of these serine residues are located in the AF-1 domain (Ser-102/104/106, Ser-118, Ser-167) and are phosphorylated in estrogen-dependent or estrogen-independent manner. Phosphorylation of ER{alpha} in response to 17ß-estradiol (E2) treatment has been observed at Ser-104, Ser-106, Ser-118 (10, 11, 12), and Ser-167 (9), with Ser-118 showing the most prominent phosphorylation. Although these phosphorylation sites are well established, the protein kinases involved are controversial (9, 11). Some serine residues appear to be targeted by various protein kinases in response to E2. Cyclin A-Cdk2 phosphorylated Ser-104 and Ser-106 (13), and Ser-118 was phosphorylated by TFIIH kinase Cdk7 (14) and glycogen synthase kinase-3 (GSK-3) (12). Activated MAPK also phosphorylated Ser-118, but controversy exists regarding the dependence on E2 treatment (15). Ser-167 is a target for AKT/PKB and RSK (9, 11). Overall, Ser-118 has been suggested to represent the major phosphorylation site in response to E2 and to be required for full ER{alpha} activation (11, 16, 17).

GSK-3 is a proline-directed serine/threonine kinase regulated by phosphorylation, the unphosphorylated form being the enzymatically active form (18). Two isoforms GSK-3{alpha} and GSK-3ß have been identified in mammals. Although highly homologous within their kinase domains, these isoforms were not functionally identical. In mice, ablation of the GSK-3ß isoform resulted in an embryonic lethal phenotype (19) indicating the inability of GSK-3{alpha} to rescue the GSK-3ß-null mice. GSK-3ß has been described as the major isoform that not only regulates the function of numerous metabolic, signaling and structural proteins, but is the key regulator of many transcription factors involved in cellular differentiation and proliferation, cell death and immune regulation (20, 21). Recently, evidence was provided that GSK-3ß may affect ER{alpha} phosphorylation and activity. In rat hippocampus, E2 regulates interaction of ER{alpha}, GSK-3ß, and ß-catenin (22). GSK-3 also influences ER{alpha} activity in neuroblastoma cells (23). Moreover, we recently reported that GSK-3 plays an important role in both ligand-independent (24) and E2-dependent activation of ER{alpha} (12). ER{alpha} was especially depicted as a substrate for GSK-3ß. We further suggested that complex formation between ER{alpha} and GSK-3ß stabilizes the receptor under resting conditions in the cytoplasm and that GSK-3ß modulates ER{alpha} transcriptional activity upon ligand binding via phosphorylation of the nuclear receptor at Ser-118 (12). In this report, we investigated the involvement of GSK-3 regarding ER{alpha} function using RNA interference (RNAi) technology. We demonstrate that GSK-3 protects ER{alpha} from proteasomal degradation and stabilizes the receptor. We also show that silencing of GSK-3 results in decrease of both E2-induced ER{alpha} phosphorylation at Ser-118 and E2-induced ER{alpha} transcriptional activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GSK-3{alpha}/ß Silencing Causes Decrease of ER{alpha} Protein Content in ER{alpha}-Positive Human Breast Carcinoma Cell Lines
To investigate the involvement of GSK-3 in ER{alpha} function, MCF-7 cells were transfected with small interfering RNA (siRNA) targeting GSK-3{alpha}/ß to silence GSK-3 expression. In the presence of siRNA, GSK-3{alpha} and GSK-3ß protein levels were reduced by 60–70% in comparison with the untransfected and control siRNA-transfected cells (Fig. 1Go, A and B). The addition of 100 nM E2 for 48 h did not affect the silencing of GSK-3{alpha}/ß.


Figure 1
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  		Fig. 1. Decrease of ER{alpha} Protein in MCF-7 Cells after GSK-3{alpha}/ß Silencing

Cells were left untransfected (CT) or were transfected either with 50 nM GL3 control siRNA (CT siRNA) or with 50 nM siRNA targeting GSK-3{alpha}/ß (GSK-3{alpha}/ß siRNA) and treated or not with 100 nM E2 for 48 h. A, Immunoblot (IB) showing silencing of GSK-3{alpha}/ß. ß-Actin was used as loading control. B, Quantitative analysis of GSK-3{alpha} (black), GSK-3ß (white) or both isoforms (gray) from three independent RNAi experiments. C, Immunoblot showing down-regulation of ER{alpha} protein after GSK-3{alpha}/ß silencing. D, Quantitative analysis of ER{alpha} protein level given as fold of control and corrected from potential loading variations using ß-actin. Three independent experiments were analyzed (*, P < 0.05 determined by t test).

 
E2 treatment of untransfected cells for 48 h resulted in an approximately 50% decrease of ER{alpha} protein level (Fig. 1Go, C and D), which agreed with previous reports that prolonged treatment with E2 down-regulates the steady-state level of ER{alpha} in MCF-7 cells (25, 26). In cells not treated with E2, silencing of GSK-3{alpha}/ß caused a significant decrease of ER{alpha} protein level (~35%). Combination of GSK-3{alpha}/ß silencing and treatment with 100 nM E2 for 48 h, led to an even more pronounced and significant ER{alpha} down-regulation (~90%) in MCF-7 cells.

Similar results as described for MCF-7 cells were also observed in T47D and BT-474 human breast carcinoma cells (the figure published as Supplemental Data 1 on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

Effect of GSK-3 Silencing on ER{alpha} mRNA Expression in MCF-7 Cells
GSK-3 silencing-related decrease of ER{alpha} protein level described above might be induced by a decrease of ER{alpha} gene transcription leading to down-regulation of ER{alpha} biosynthesis. To investigate the hypothetical regulation of ER{alpha} transcription by GSK-3, quantitative real-time PCR experiments were performed using ER{alpha} primers with cDNA synthesized from RNA extracted from MCF-7 cells that had been treated or not with 10 nM E2 for 6, 24, or 48 h (Fig. 2Go). Basal ER{alpha} mRNA expression was significantly decreased at 6 h in GSK-3{alpha}/ß siRNA-treated cells, whereas basal ER{alpha} mRNA expression was similar for the control siRNA and GSK-3{alpha}/ß siRNA-treated cells at 24 h and 48 h. It has been reported that E2 treatment induces decrease of ER{alpha} mRNA expression in MCF-7 cells (25, 27, 28, 29). In our experiments, E2-related decrease of ER{alpha} mRNA expression was confirmed and was similar in control siRNA and GSK-3{alpha}/ß siRNA-transfected cells. However, after 48 h of E2 treatment, the decrease of ER{alpha} mRNA expression was slightly stronger upon GSK-3 silencing, although this was not statistically significant. Taken together, the results show that GSK-3 silencing has a transient effect on basal ER{alpha} mRNA expression, whereas GSK-3 silencing does not significantly alter E2-related decrease of ER{alpha} mRNA levels.


Figure 2
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  		Fig. 2. Effect of GSK-3{alpha}/ß Silencing on ER{alpha} mRNA Expression in MCF-7 Cells

Cells were transfected either with GL3 control siRNA (CT siRNA) or with siRNA targeting GSK-3{alpha}/ß (GSK-3{alpha}/ß siRNA) and treated or not with 10 nM E2 for 6 h, 24 h and 48 h. Total RNA was extracted and used to synthesize cDNA by reverse transcription. Quantitative real-time PCR using ER{alpha} primers showed that E2-related decrease of ER{alpha} mRNA expression was not significantly altered upon GSK-3{alpha}/ß silencing. ß-Actin was used as an internal control. Three independent experiments were analyzed (*, P < 0.05 determined by t test).

 
Increase of ER{alpha} Proteasomal Degradation Rather Than Reduction of ER{alpha} Protein Synthesis Down-Regulates ER{alpha} Protein upon GSK-3{alpha}/ß Silencing
Experiments using the translation inhibitor cycloheximide (CHX) and the proteasome inhibitor MG132 were performed to investigate the role of GSK-3 regarding ER{alpha} protein synthesis at the translational level and ER{alpha} proteasomal degradation, respectively (Fig. 3Go). In cells transfected with control siRNA and not stimulated with E2 (Fig. 3Go, left panel), CHX slightly decreased ER{alpha} protein content whereas MG132 slightly increased ER{alpha} protein content. In cells transfected with GSK-3{alpha}/ß siRNA and not stimulated with E2, the proteasome inhibitor MG132 significantly rescued GSK-3{alpha}/ß silencing related-ER{alpha} protein decrease. Such significant rescue was also observed when MG132 was used in combination with CHX. In E2-treated cells (Fig. 3Go, right panel), MG132 treatment rescued E2-induced- as well as GSK-3{alpha}/ß silencing related-ER{alpha} protein decrease. In GSK-3{alpha}/ß silenced cells treated with both CHX and E2, a significant MG132 mediated rescue was still observed. Taken together, these results suggest that down-regulation of ER{alpha} upon GSK-3{alpha}/ß silencing either in unstimulated or in E2-treated cells is due to increase of ER{alpha} proteasomal degradation rather than alteration of ER{alpha} protein synthesis.


Figure 3
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  		Fig. 3. ER{alpha} Protein Decrease Caused by GSK-3{alpha}/ß Silencing Is Due to an Increase of Proteasomal Degradation Rather Than Decreased ER{alpha} Protein Synthesis

Cells were transfected with siRNA targeting GSK-3{alpha}/ß (GSK-3{alpha}/ß siRNA). After 1 h pretreatment with either 5 µM MG132 or 50 µg/ml CHX or a combination of both, cells were treated (right panel) or not (left panel) with 10 nM E2 for 6 h. A, Immunoblots (IB) showing rescue of GSK-3{alpha}/ß silencing-related ER{alpha} protein decrease by inhibition of proteasomal degradation even where translation was blocked with CHX. ß-actin was used as loading control. B, Quantitative analysis of ER{alpha} protein level given as fold of control and corrected for potential loading variations using ß-actin. Three independent experiments were analyzed (*, P < 0.05 determined by t test).

 
GSK-3 Prevents ER{alpha} Ubiquitination and Proteasomal Degradation
Several studies recently described the involvement of the ubiquitin-proteasome pathway in the E2-induced degradation of ER{alpha} (30, 31, 32). From the results shown in Fig. 1Go, we hypothesized that GSK-3{alpha}/ß may be involved in ER{alpha} stabilization, preventing its ubiquitination and proteasomal degradation. To analyze this, ER{alpha} was immunoprecipitated from cells transfected with control or GSK-3{alpha}/ß siRNA followed by immunoblotting with anti-ubiquitin antibody (Fig. 4AGo). After GSK-3{alpha}/ß silencing, we detected an increase in ubiquitination of ER{alpha} in cells treated or not with E2 compared with the control siRNA-transfected cells. When proteasomal degradation was inhibited using MG132, further accumulation of ubiquitinated forms of ER{alpha} was observed. Detection of ER{alpha} protein in these samples confirmed ER{alpha} down-regulation caused either by E2 treatment or by GSK-3{alpha}/ß silencing (Fig. 4Go, B and C). Addition of MG132 rescued the down-regulation of ER{alpha} caused by E2 in control siRNA-transfected cells and, more importantly, in cells after GSK-3{alpha}/ß silencing both in presence or absence of E2. These results suggest an important role of GSK-3 regarding ER{alpha} stabilization.


Figure 4
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  		Fig. 4. ER{alpha} Ubiquitination and Proteasomal Degradation Is Increased upon GSK-3 Silencing

Cells were transfected either with GL3 control siRNA (CT siRNA) or with siRNA targeting GSK-3{alpha}/ß (GSK-3{alpha}/ß siRNA). After 1 h pretreatment with 5 µM MG132 where indicated, cells were treated or not with 10 nM E2 for 6 h. A, ER{alpha} immunoprecipitates (IP) from MCF-7 cells were subjected to immunoblotting (IB) detecting ubiquitinated forms of ER{alpha} that were found strongly increased upon silencing of GSK-3{alpha}/ß. B, Detection of ER{alpha} on the same membrane shown in panel A. C, Quantification of ER{alpha} protein levels given as fold of control from three independent experiments including that shown in panel B (*, P < 0.05 determined by t test; ns, not significant).

 
ER{alpha} Is a Substrate for GSK-3 and Is Phosphorylated at Ser-118
Because Ser-118 phosphorylation is an important step in ER{alpha} activation, we investigated whether this site is a target for GSK-3. By in vitro kinase assay, using recombinant human ER{alpha} (rhER{alpha}), GSK-3ß and an antibody detecting specifically phosphorylation of ER{alpha} at Ser-118, we demonstrated that ER{alpha} can be phosphorylated by GSK-3ß at Ser-118 (Fig. 5Go).


Figure 5
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  		Fig. 5. GSK-3 Phosphorylates ER{alpha} at Ser-118 in Vitro Kinase Assay Using Dephosphorylated rhER{alpha} and Purified GSK-3ß Shows that GSK-3ß Phosphorylates ER{alpha} at Ser-118

IB, Immunoblot.

 
Next, we analyzed the effects of GSK-3{alpha}/ß silencing on the phosphorylation of ER{alpha} after 100 nM E2 stimulation for 20 min. In untransfected and control siRNA-transfected cells, short-term E2 treatment-induced ER{alpha} phosphorylation at Ser-118 (Fig. 6AGo). This phosphorylation was significantly inhibited after GSK-3{alpha}/ß silencing (Fig. 6BGo). Furthermore, in accordance with our results shown in Fig. 1Go, ER{alpha} protein content was reduced in these cells (Fig. 6Go, A and B). In addition, a clear although not significant decrease of the pS118/ER{alpha} ratio was observed (Fig. 6BGo), suggesting that ER{alpha} down-regulation was not the only reason for the reduction of ER{alpha} phosphorylation level at Ser-118.


Figure 6
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  		Fig. 6. GSK-3 Phosphorylates ER{alpha} at Ser-118 in MCF-7 Cells

A, Cells were left untransfected (CT) or were transfected either with GL3 control siRNA (CT siRNA) or with siRNA targeting GSK-3{alpha}/ß (GSK-3{alpha}/ß siRNA) and treated or not with 100 nM E2 for 20 min. Immunoblots (IB) show reduction of E2-induced ER{alpha} phosphorylation at Ser-118 upon GSK-3 silencing. B, Quantitative analysis of pS118 and ER{alpha} protein signals given as fold of control. Data from three independent experiments including those shown in panel A revealed reduction of Ser-118 phosphorylation, of ER{alpha} protein, as well as of pS118/ER{alpha} ratio (*, P < 0.05 determined by t test; ns, not significant). C, Cells were transfected either with GL3 control siRNA or with siRNA targeting GSK-3{alpha}/ß. Where indicated, cells were pretreated with 5 µM MG132 for 6 h and then treated or not with 100 nM E2 for 20 min. D, Quantification of data shown in C and of two additional experiments carried out under the same conditions (*, P < 0.05 determined by t test). E2-induced and GSK-3{alpha}/ß silencing-related down-regulation of ER{alpha} was rescued by MG132; but reduction of E2-induced Ser-118 phosphorylation in cells transfected with GSK-3{alpha}/ß siRNA was not rescued.

 
Furthermore, we investigated the phosphorylation of ER{alpha} at Ser-118 after inhibition of the proteasome. Under this condition, we confirmed that ER{alpha} degradation due to GSK-3{alpha}/ß silencing was partly prevented by MG132 addition (Fig. 6Go, C and D, right). Importantly, ER{alpha} protein rescue was not accompanied by a rescue of E2-induced Ser-118 phosphorylation in GSK-3{alpha}/ß silenced cells (Fig. 6CGo), which is also evident from the reduced pS118/ER{alpha} ratio (Fig. 6DGo). These results provide further evidence that Ser-118 of ER{alpha} is a target for E2-induced phosphorylation by GSK-3{alpha}/ß in MCF-7 cells. Interestingly, E2-induced phosphorylation of ER{alpha} at Ser-118 was also decreased in MG132-treated cells transfected with control siRNA, showing that ER{alpha} was less phosphorylated at Ser-118 after inhibition of the proteasome.

Previous work from our group (12, 24) showed that GSK-3 and ER{alpha} are detected in both the cytoplasm and the nucleus of MCF-7 cells. Figure 7AGo suggests cytoplasmic as well as nuclear localization of ER{alpha} in serum-starved cells and shows a much stronger nuclear signal for ER{alpha} after E2 treatment, indicating a nuclear translocation of ER{alpha}. This agrees with published work (33). In GSK-3-silenced cells, the overall ER{alpha} fluorescence signal was markedly weaker whether the cells were treated with E2 or not. Moreover, GSK-3 silencing did not seem to affect E2-induced nuclear translocation of ER{alpha}. Cell fractionation studies (Fig. 7BGo) showed similar GSK-3{alpha}/ß silencing in both compartments. In agreement with the results shown in Fig. 7AGo, E2-induced ER{alpha} nuclear translocation was still occurring in GSK-3 silenced cells. Furthermore, Fig. 7BGo shows that GSK-3 silencing resulted in reduced E2-induced ER{alpha} phosphorylation at Ser-118 in the nucleus. Taken together, these data suggest a direct role of nuclear GSK-3 regarding ER{alpha} phosphorylation at Ser-118.


Figure 7
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  		Fig. 7. GSK-3{alpha}/ß Silencing Did Not Alter E2-Induced Nuclear Translocation of ER{alpha}

Cells were transfected either with GL3 control siRNA or with siRNA targeting GSK-3{alpha}/ß and treated or not with 100 nM E2 for 20 min. A, Immunofluorescence staining of ER{alpha} protein (red) shows that cytoplasmic and nuclear ER{alpha} levels were reduced after GSK-3{alpha}/ß silencing. E2-induced ER{alpha} translocation from the cytoplasm to the nucleus of MCF-7 cells was still occurring after GSK-3{alpha}/ß silencing. The same settings were used for all fluorescence micrographs. B, Cellular fractionation was performed and immunoblots (IB) show silencing of GSK-3{alpha}/ß in both cytoplasmic and nuclear compartments upon RNAi. After GSK-3{alpha}/ß silencing, E2-induced ER{alpha} phosphorylation at Ser-118 in the nucleus was decreased. ß-Actin and replication protein A/p34 were used as loading controls for cytoplasmic and nuclear extracts, respectively.

 
Decrease of ER{alpha} Activity Due to GSK-3 Silencing Is Not Rescued by Inhibition of the Proteasome
To assess ER{alpha} activity, MELN cells were transfected with control siRNA or GSK-3{alpha}/ß siRNA and treated with 10 nM E2 for different time periods before luciferase activity was measured. In control siRNA-transfected cells, luciferase activity was increased up to 7-fold after E2 treatment for 3, 6, and 8 h and thereafter declined (Fig. 8AGo). As expected, ER{alpha} was down-regulated over time upon E2 treatment. In presence of GSK-3{alpha}/ß siRNA, the basal luciferase activity (without E2 treatment) was significantly lower compared with the control at any time point analyzed. Moreover, E2-induced luciferase activity was significantly reduced by about 40% in comparison with the respective controls when cells were treated with E2 from 3–12 h. At 24 h, the luciferase activity after GSK-3 silencing was similar to the one obtained with control siRNA. Detection of ER{alpha} protein in the same samples used for luciferase reporter assay revealed reduction of ER{alpha} protein levels in cells transfected with GSK-3{alpha}/ß siRNA compared with control siRNA-transfected cells. Further reduction of ER{alpha} protein content was observed by E2 treatment after GSK-3{alpha}/ß silencing.


Figure 8
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  		Fig. 8. ER{alpha} Transcriptional Activity Is Decreased upon GSK-3{alpha}/ß Silencing

A, MELN cells transfected either with GL3 control siRNA (CT siRNA) or with siRNA targeting GSK-3{alpha}/ß (GSK-3{alpha}/ß siRNA) were treated with 10 nM E2 for 3, 6, 8, 12, or 24 h and ERE-dependent luciferase expression was measured in cell lysates. Relative luciferase activity given as fold of control was evaluated from three independent experiments (histogram). Immunoblot (IB) from the same lysates shows E2-induced and GSK-3{alpha}/ß silencing-related down-regulation of ER{alpha} (Western blot). B, Cells were transfected as above, pretreated with 5 µM MG132 for 1 h and treated or not with 10 nM E2 for 6 h. Relative luciferase activity was evaluated from three independent experiments (*, P < 0.05 determined by t test). GSK-3{alpha}/ß silencing-related reduction of E2-induced luciferase activity was not rescued by the addition of MG132. C, Immunoblot showing that ER{alpha} protein in samples analyzed in panel B was restored by MG132 treatment.

 
Next, we examined the effects of ER{alpha} rescue by proteasome inhibition on luciferase activity (Fig. 8Go, B and C). In agreement with results shown in Fig. 8AGo, treatment of cells with 10 nM E2 for 6 h resulted in E2-induced luciferase activity, which was significantly inhibited in the presence of GSK-3{alpha}/ß siRNA (Fig. 8BGo). In control siRNA-transfected cells, addition of MG132 slightly increased basal luciferase activity but did not alter E2-induced luciferase activity. After GSK-3{alpha}/ß silencing, the reduced E2-triggered luciferase activity was not rescued by MG132 treatment. However, ER{alpha} protein content in these cells was restored by MG132 treatment (Fig. 8CGo). These findings show that GSK-3{alpha}/ß is required for full ER{alpha} activation.

GSK-3{alpha}/ß Silencing Decreases E2-Induced Expression of Endogenous ER{alpha} Target Genes
Quantitative real-time PCR was performed using primers specific for pS2 and progesterone receptor (PR), which are both well-known estrogen-responsive genes (34). After GSK-3{alpha}/ß silencing, E2-induced expression of these genes was significantly decreased (Fig. 9Go, A and B). These results, in agreement with the luciferase assay performed in MELN cells, confirm the involvement of GSK-3{alpha}/ß in the regulation of ER{alpha} target genes expression and demonstrate the necessity of GSK-3{alpha}/ß for full ligand-dependent activity of ER{alpha}.


Figure 9
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  		Fig. 9. E2-Induced Expression of ER{alpha} Target Genes (pS2 and PR) Was Down-Regulated upon GSK-3{alpha}/ß Silencing

MCF-7 cells were transfected either with GL3 control siRNA (CT siRNA) or with siRNA targeting GSK-3{alpha}/ß (GSK-3{alpha}/ß siRNA) and treated or not with 10 nM E2 for 1, 3, 6, 8, and 24 h. Total RNA was extracted and used to synthesize cDNA by reverse transcription. Quantitative real-time PCR of the cDNA using pS2 primers (panel A) and PR primers (panel B) showed that E2-induced mRNA expression of these ER{alpha} target genes was decreased upon GSK-3{alpha}/ß silencing. ß-Actin was used as an internal control. Three independent experiments were analyzed (*, P < 0.05 determined by t test).

 
Silencing of Either GSK-3{alpha} or GSK-3ß Isoform Results in ER{alpha} Down-Regulation and Reduced Transcriptional Activation
Because most reports in the literature claim that the GSK-3ß isoform is of greater importance for biological functions in comparison with GSK-3{alpha} (19, 20, 21), we were interested in the role of the two individual GSK-3 isoforms regarding ER{alpha} signaling. The impact of individual GSK-3 isoforms on ER{alpha} protein level and activity were assessed using siRNAs specifically targeting either GSK-3{alpha} or GSK-3ß (Fig. 10Go, A–C). Silencing of GSK-3{alpha} resulted in ER{alpha} down-regulation in the absence of E2 (Fig. 10Go, A and B), in decrease of ER{alpha} phosphorylation at Ser-118 induced after 20 min E2 treatment (Fig. 10AGo), in a more pronounced ER{alpha} down-regulation after 6 h E2 treatment (Fig. 10BGo) and in decrease of E2-induced luciferase activity (Fig. 10CGo). Thus, the effects caused by GSK-3{alpha} silencing were similar to those described for GSK-3{alpha}/ß silencing. Moreover, silencing of GSK-3ß had similar effects on ER{alpha} protein level, Ser-118 phosphorylation and ER{alpha} activity like GSK-3{alpha} or GSK-3{alpha}/ß silencing (Fig. 10Go, A–C). Surprisingly, specific targeting of the GSK-3ß isoform resulted also in partial silencing of GSK-3{alpha}. The lack of specificity of siRNA targeting GSK-3ß is so far unclear. The results shown in Fig. 10Go were obtained with the validated siRNA targeting GSK-3ß from Ambion (Austin, TX). Furthermore, similar results (data not shown) were obtained using the GSK-3ß-specific siRNA sequence described in (35). We performed an additional experiment in which GSK-3{alpha}/ß silencing was compared with the combined silencing of GSK-3{alpha} and GSK-3ß individual isoforms (the figure published as Supplemental Data 2). Both approaches yielded similar results regarding GSK-3{alpha}/ß down-regulation (about 60% at 50 nM final siRNA concentrations), GSK-3{alpha}/ß silencing-related ER{alpha} down-regulation as well as GSK-3{alpha}/ß silencing-related decrease of E2-induced ER{alpha} activity. To summarize, similar results were obtained using different siRNA sequences specific for GSK-3{alpha}/ß or the individual isoforms regarding ER{alpha} signaling.


Figure 10
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  		Fig. 10. ER{alpha} Level and Activity Are Decreased upon Silencing GSK-3{alpha} or GSK-3ß Individually

Cells were transfected with 50 nM GL3 control siRNA (CT siRNA) or with 50 nM siRNA targeting either GSK-3{alpha}/ß (GSK-3{alpha}/ß siRNA), or GSK-3{alpha} (GSK-3{alpha} siRNA), or GSK-3ß (GSK-3ß siRNA). A, Immunoblots (IB) showing ER{alpha} levels and Ser-118 phosphorylation in MCF-7 cells treated or not with 100 nM E2 for 20 min. B, Immunoblots showing GSK-3{alpha}, GSK-3ß and ER{alpha} levels in MCF-7 cells treated with 10 nM E2 for 6 h. C, Relative luciferase activity given as fold of control was evaluated from three independent experiments in MELN cells treated with 10 nM E2 for 6 h (*, P < 0.05 determined by t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Phosphorylation events targeting the AF-1 domain is one of the most important features that regulate ER{alpha} activity. We previously established functional links between GSK-3 activity and E2-dependent ER{alpha} activation and provided evidence that GSK-3 modulates ER{alpha} function by phosphorylation of relevant serine residues (12). In the present report, we used RNAi technology to silence GSK-3 and to demonstrate that GSK-3 is required for ER{alpha} stabilization in MCF-7 breast cancer cells, for phosphorylation at Ser-118 and for full transcriptional activity of ER{alpha}. The stabilizing effect of GSK-3 on ER{alpha} was further shown using ER{alpha}-positive T47D and BT-474 cells.

Under resting conditions, a tightly regulated protein turnover allows the cells to maintain a balance between ER{alpha} protein synthesis and degradation leading to constant cellular level of the receptor (36). However, efficient silencing of GSK-3 in MCF-7 cells provokes a decrease of ER{alpha} protein by deregulating protein turnover. After GSK-3 silencing, when a decrease of ER{alpha} protein level was observed, a decrease in ER{alpha} synthesis was first hypothesized. According to our results, an early transient decrease of basal ER{alpha} mRNA expression was observed (Fig. 2Go). Adaptation of the cells to GSK-3 silencing on the one hand and to changes of the cell culture medium due to the experimental protocol on the other hand seemed to compensate this transient ER{alpha} mRNA decrease. Furthermore, GSK-3 silencing did not significantly alter E2-related decrease of ER{alpha} mRNA levels at the time points investigated. An autologous down-regulation pathway of ER{alpha}, which involves binding of liganded ER{alpha} to ER{alpha} genes, has been described (25, 37, 38). By this mechanism, estrogens induce a decline in both ER{alpha} protein and mRNA (25). Our data on E2-related decrease of ER{alpha} mRNA in nonsilenced cells can be explained by this mechanism. Surprisingly, the GSK-3 silencing-related decrease of ER{alpha} protein did not affect the autologous down-regulation of ER{alpha} mRNA, although a tendency to a more pronounced decrease of ER{alpha} mRNA expression, which we cannot explain, was observed in cells treated with E2 for 48 h. Moreover, the use of the translation inhibitor CHX in combination with the proteasome inhibitor MG132 showed a significant rescue of ER{alpha} protein upon GSK-3 silencing both in unstimulated and E2-stimulated cells demonstrating a direct impact of GSK-3 on ER{alpha} proteasomal degradation. Although these results do not exclude any modulation of ER{alpha} translation by GSK-3, GSK-3 is suggested to have an effect on ER{alpha} proteasomal degradation rather than on ER{alpha} biosynthesis.

Previous findings on direct interaction of the docking kinase GSK-3 with ER{alpha} raised the hypothesis that formation of an ER{alpha}/GSK-3 complex stabilizes ER{alpha} in the cytoplasm (12). This hypothesis is corroborated by the observation presented in this report that GSK-3 silencing using specific siRNA results in reduction of cellular ER{alpha} protein levels. Use of the proteasome inhibitor MG132 resulted in accumulation of ubiquitinated ER{alpha} and in rescue of ER{alpha} protein levels, showing that ER{alpha} degradation observed after GSK-3 silencing was due to proteasomal proteolysis. These findings permit the conclusion that GSK-3 protects ER{alpha} from proteasomal degradation and that complex formation between GSK-3 and ER{alpha} plays a crucial role in ER{alpha} protein stabilization and turnover. Complex formation involving GSK-3 has been shown to be important for stabilization of other proteins as well. In the Wnt signaling pathway, active GSK-3 appears in a multiprotein complex that includes the transcription factor ß-catenin and the scaffold protein axin (39). Axin binding to GSK-3 and subsequent phosphorylation leads to stabilization of this protein (40). In analogy to axin, the interaction of ER{alpha} with GSK-3 appears to be required for stabilization of the receptor.

A second important role of GSK-3 regarding ER{alpha} function is phosphorylation of the receptor. In vitro kinase assay showed that ER{alpha} is a GSK-3 substrate being phosphorylated at Ser-118. Cellular fractionation studies showed that Ser-118 phosphorylation mainly occurred in the nucleus. Because silencing of GSK-3 occurred both in the cytoplasm and the nucleus, and resulted in decrease of E2-induced ER{alpha} phosphorylation at Ser-118, we conclude that GSK-3 may phosphorylate this serine residue in the nuclei of MCF-7 cells. Another important aspect is that the ratio of pS118/ER{alpha} in GSK-3 silenced cells treated with E2 was reduced and this could not be rescued by inhibition of the proteasome. This clearly indicates that the reduction of Ser-118 phosphorylation observed after GSK-3 silencing was not the consequence of down-regulation of ER{alpha} protein but particularly due to lack of GSK-3 kinase activity. However, residual pS118 signal was observed after GSK-3 silencing (Figs. 6Go, 7Go, and 10Go). Although this was probably due to GSK-3 protein remaining after siRNA silencing, activity of another protein kinase cannot be excluded.

Another interesting finding was that treatment of MCF-7 cells with the proteasome inhibitor alone resulted in inhibition of Ser-118 phosphorylation. It has been reported that phosphorylation at Ser-118 contributes to E2-induced proteasomal proteolysis of ER{alpha}, both S118A and S118E ER{alpha} mutants being resistant to E2-induced degradation (41). In this context, we expected an accumulation of Ser-118 phosphorylated ER{alpha} in presence of MG132. Our results, however, clearly showed a decrease of Ser-118 phosphorylation after proteasome inhibition. A rational but hypothetical explanation may consider ER{alpha} phosphorylation at Ser-118 as a transient event, tagging ER{alpha} for ubiquitination which is followed by ER{alpha} dephosphorylation.

Proteasome inhibition reinforced the GSK-3 silencing-related decrease of E2-induced ER{alpha} phosphorylation at Ser-118. Because accumulation of ubiquitinated ER{alpha} was also enhanced under these conditions, pS118 could not be the only signal triggering ubiquitination and proteasomal degradation of ER{alpha}. Recently, different conformational states adopted by ER{alpha} in presence of different ligands were described to influence transcriptional activity indirectly by modulating receptor stability (42). Conformational change may similarly be involved in GSK-3 modulation of ER{alpha} stability. After GSK-3 silencing, the absence of complex formation between GSK-3 and ER{alpha} may result in altered conformation leading to destabilization of the receptor. This suggests that GSK-3 modulates ER{alpha} stabilization without requirement of Ser-118 phosphorylation and supports our hypothesis of receptor stabilization by GSK-3 in the cytoplasm (12).

Data presented in this study further showed that GSK-3 silencing resulted in a decrease of E2-induced estrogen response element (ERE)-dependent luciferase activity in MELN cells, suggesting that GSK-3 silencing altered ERE-dependent transcriptional activity of ER{alpha}. After inhibition of the proteasome, the decrease of E2-induced luciferase expression was not restored, whereas ER{alpha} protein level was rescued. This again suggests that particularly lack of GSK-3 kinase activity regarding ER{alpha} phosphorylation at Ser-118 may provoke a decrease of ER{alpha} transcriptional activity upon E2 treatment. Indeed, a recent report (41), in agreement with the fact that Ser-118 phosphorylation was required for full ER{alpha} transcriptional activity (11), demonstrated that Ser-118 phosphorylation regulates transcriptional efficiency through the differential recruitment of coactivators and transcriptional machinery to estrogen-responsive promoters. Importantly, MG132 treatment did not significantly alter E2-induced luciferase activity in cells transfected either with control siRNA or GSK-3 siRNA. These results are at variance with a study published previously (36), which reported a reduction of E2-induced ERE-dependent luciferase expression by MG132 treatment. The different findings can be explained by the different experimental settings. To minimize the side effects of MG132 in our study, we used a low dose of proteasome inhibitor for a short treatment period when measuring E2-induced ERE-luciferase activity in MELN cells. Moreover, GSK-3 silencing resulted in a decrease of E2-induced expression of pS2 and PR, suggesting that GSK-3 is required for full transcriptional expression of estrogen-responsive genes.

In previous work, a model for the potential function of GSK-3 in ER{alpha} activation was proposed (12). This model is complemented by including the present findings (Fig. 11AGo) and represents our current knowledge regarding GSK-3/ER{alpha} interactions. In unstimulated cells, a complex between active GSK-3 and ER{alpha} stabilizes the receptor. This complex formation was shown to occur mainly in the cytoplasm (12). Upon E2 treatment, phosphorylation of GSK-3{alpha}/ß at Ser-21/9 residues results in inhibition of the kinase. Consequently, ER{alpha} is released and translocates into the nucleus where it is phosphorylated by active nuclear GSK-3 at Ser-118 leading to full transcriptional activity. Eventually, E2 signal is switched off by ubiquitination and proteasomal degradation of ER{alpha}. The effects of GSK-3 silencing are described in Fig. 11BGo. The lack of GSK-3 results in reduction of ER{alpha} stabilization triggering its ubiquitination and proteasomal degradation. As a consequence, ER{alpha} protein content is decreased. Moreover, E2-induced Ser-118 phosphorylation of the remaining ER{alpha} is reduced due to lack of GSK-3 kinase activity in the nucleus, which results in decrease of E2-induced ER{alpha} transcriptional activity.


Figure 11
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  		Fig. 11. Model for the Effects of GSK-3 Silencing Regarding ER{alpha}

A, In unstimulated cells ER{alpha} is stabilized by interaction with active GSK-3 mainly in the cytoplasm (1 ). Treatment of cells with E2 results in phosphorylation and inactivation of GSK-3, ER{alpha} release (2 ), translocation of ER{alpha} into the nucleus where it is phosphorylated by active nuclear GSK-3 at Ser-118 (3 ). Eventually, E2 signal is switched off by ubiquitination (Ub) and proteasomal degradation of ER{alpha} (4 ). B, Lack of GSK-3 results in reduction of ER{alpha} stabilization triggering its ubiquitination and proteasomal degradation and in inhibition of E2-induced ER{alpha} phosphorylation at Ser-118.

 
In conclusion, GSK-3 on the one hand stabilizes ER{alpha} protecting the receptor from proteasomal degradation and on the other hand regulates E2-induced ER{alpha} activity by phosphorylation of the receptor at Ser-118. Therefore, GSK-3 appears to be essential for full transcriptional activity of ER{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Treatment
MCF-7, T47D, and BT-474 human breast carcinoma cell lines were obtained from American Type Culture Collection (Manassas, VA). MELN cells were obtained from wild-type MCF-7 cells by stable transfection with an ERE-controlled luciferase reporter plasmid (43). MCF-7 and MELN cells were maintained routinely (5% CO2, 37 C) in phenol red-free DMEM (4.5 g/liter glucose) (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin (Biochrom). Before experimental use, the cells were grown for 72 h in medium supplemented with 10% dextran-coated charcoal-treated (DCC)-FCS prepared as described (44). Thereafter, 3 x 105 cells/well were plated in six-well plates and grown for 24 h in medium with 10% DCC-FCS. Before hormonal treatment with 10 nM or 100 nM E2 (Sigma, München, Germany), serum-free medium was added to the cells for 24 h and cells were kept in this medium until the end of the experiment. At 1 or 6 h before long-term (6 h) and short-term (20 min) E2 treatment, respectively, a 5 µM final concentration of proteasome inhibitor MG132 (EMD Biosciences, Darmstadt, Germany) was added. A 50 µg/ml final concentration of translation inhibitor CHX (Sigma) was added 1 h before hormonal treatment.

RNAi
MCF-7, MELN, T47D, and BT-474 cells were transfected with 50 nM siRNA using Oligofectamine (Invitrogen). Luciferase GL3 duplex siRNA from Dharmacon (Lafayette, CO) was used as a control. Experiments have shown that GL3 duplex siRNA did not interfere with luciferase expression in MELN cells. Signal Silencing GSK-3{alpha}/ß siRNA (New England Biolabs, Frankfurt, Germany) were used to target both GSK-3 isoforms. GSK-3{alpha} isoform was specifically targeted by Signal Silence GSK-3{alpha} siRNA (New England Biolabs) and GSK-3ß isoform was specifically targeted using either siRNA duplex targeting the following sequence: 5'-AUCUUUGGAGCCACUGAUU-3' (35) synthesized by Dharmacon or validated siRNA (No. 42839) from Ambion. At 18 h after transfection, the medium was replaced with 10% DCC-FCS supplemented medium. Then, the cells were starved in medium without FCS and they were treated 24 h later with E2 or inhibitors. After short-term (20 min) or long-term treatment (3–48 h), lysis of the cells, cytoplasmic and nuclear extractions, or total RNA isolation were performed.

Cell Lysates
Cell pellets obtained by trypsinization were resuspended in ice-cold lysis buffer (12) and incubated on ice for 30 min. Cell lysates were cleared by centrifugation (10,000 x g, 10 min).

Cellular Fractionation
Cells were incubated on ice for 15 min in hypo-osmotic Buffer I (12), homogenized using a glass potter and centrifuged (2,000 x g, 5 min). The resulting pellets were used later to generate nuclear extracts. The supernatants were centrifuged (10,000 x g, 30 min) and the resulting supernatants were used as the cytoplasmic extracts. The nuclear pellets obtained after the first centrifugation step were resuspended in hyperosmotic Buffer II (12). After 15 min incubation on ice, nuclear debris were pelleted by centrifugation (10,000 x g, 30 min) and the resulting supernatants represented the nuclear protein extracts.

Immunofluorescence
MCF-7 cells were grown on poly-D-lysine-coated glass coverslips for 24 h in DMEM/10% DCC-FCS. Then, cells were transfected with control siRNA or GSK-3{alpha}/ß siRNA, and the medium was replaced by serum-free DMEM, 24 h before E2 treatment (20 min, 100 nM). Cells were fixed for 15 min with 4% paraformaldehyde, washed with PBS and permeabilized for 5 min with 0.2% Triton X-100 in PBS. After washing with PBS and blocking (PBS + 1% BSA for 15 min), cells were incubated [1 h at room temperature (RT)] with anti-ER{alpha} (HC-20) polyclonal antibody (1:200 in PBS) from Santa Cruz Biotechnology (Santa Cruz, CA). After washing with PBS, coverslips were incubated for 1 h at RT with Cy3-conjugated antirabbit antibody (1:300 in PBS) from Invitrogen and mounted in Elvanol (Merck, Darmstadt, Germany). Fluorescence was viewed under an Axiovert S100 TV microscope (Zeiss, Oberkochen, Germany) at wavelengths of 550 nm (Cy3).

Western Blot Analysis
Protein concentrations of lysates, cytoplasmic and nuclear extracts were determined with the DC protein assay kit from Bio-Rad (München, Germany). Lysates, cytoplasmic and nuclear extracts containing equal amounts of proteins (20 µg/sample) were boiled in 1x sodium dodecyl sulfate (SDS) sample buffer (5 min, 95 C), subjected to 12% SDS-PAGE and blotted on an Immobilon-P membrane (Millipore, Eschwege, Germany). After blocking of the membrane (1 h, RT) with 5% nonfat dry milk in Tris-buffered saline supplemented with 0.1% Tween 20, the membrane was incubated with primary antibody (overnight, 4 C), washed and incubated (45 min, RT) with horseradish peroxidase-conjugated goat antimouse or antirabbit antibodies from Dianova (Hamburg, Germany). Immunoreactive protein bands were detected with ECL-plus system from Amersham Pharmacia Biotech (Freiburg, Germany). Phosphoproteins and respective proteins were detected on the same membrane after stripping [20 min, 60 C in 1 M Tris-HCl (pH 6.7), 0.01% ß-mercaptoethanol and 10% SDS]. The intensities of the bands were quantified by the Image J software (National Institutes of Health, Bethesda, MD). Results were expressed as relative phosphoprotein or protein levels standardized such that values obtained in cells treated with vehicle only were set to 1. Data represent the mean and SEM from a minimum of three independent experiments.

Monoclonal antibodies to pSer118-ER{alpha} and to ER{alpha} (NCL-L-6F11) were from New England Biolabs and Novocastra (Newcastle, UK), respectively. Anti-ER{alpha} (HC-20) polyclonal antibody was from Santa Cruz Biotechnology and anti-GSK-3{alpha}/ß monoclonal antibody from Biosource International (Solingen, Germany). Anti-ß-actin, anti-ß-tubulin and anti-replication protein A/p34 monoclonal antibodies were from Abcam (Cambridge, UK), Upstate (Lake Placid, NY) and Lab Vision (Fremont, CA), respectively.

Ubiquitination Assay
MCF-7 cells were treated with 5 µM MG132 and/or 10 nM E2 in presence or absence of siRNA targeting GSK-3{alpha}/ß. Cell lysates were produced, and 200 µg protein were used for ubiquitination assay. After preclearing the lysates for 2 h with protein A-Sepharose beads (Sigma), overnight immunoprecipitation using anti-ER{alpha} antibody (Novocastra) and protein A-Sepharose beads was performed. Beads were centrifuged, washed and boiled in 1x SDS sample buffer for 10 min. After centrifugation, the supernatants were processed by SDS-PAGE and Western blotting. Ubiquitinated ER{alpha} was detected using anti-ubiquitin polyclonal antibody from Dako (Glostrup, Denmark). After stripping of the membrane, immunoblotting using anti-ER{alpha} (HC-20) polyclonal antibody was performed as loading control.

Firefly Luciferase Reporter Gene Assay
MELN cells were seeded at a density of 3 x 105 cells/well in six-well plates. After treatment for 3–24 h, cells were washed with PBS (Mg2+- and Ca2+-free) and lysed (10 min, RT) with 150 µl/well of Luciferase Cell Culture Lysis Reagent (Promega, Mannheim, Germany). After centrifugation (10,000 x g, 10 min), the supernatant was collected and luciferase activity was analyzed using the firefly luciferase assay system from Promega.

RNA Isolation and Quantification
After treatment, cell pellets were collected and total RNA was isolated with the RNeasy kit (QIAGEN, Hilden, Germany). One microgram of RNA was reverse transcribed using oligo(deoxythymidine) primers (QIAGEN) and SuperScript II reverse transcriptase (Invitrogen). cDNA was purified with the QIAquick PCR purification kit (QIAGEN). Quantitative real-time PCRs using the iQ SYBR Green supermix (Bio-Rad) were performed on PTC-200 Peltier Thermal Cycler (MJ Research, Miami, FL) using the following primers: ER{alpha} forward, 5'-TTACTGACCAACCTGGCAGA-3' and ER{alpha} reverse, 5'-ATCATGGAGGGTCAAATCCA-3'; pS2 forward, 5'-ATACCATCGACGTCCCTCCA-3'; pS2 reverse: 5'-AAGCGTGTCTGAGGTGTCCG-3'; PR forward: 5'-GGCATGGTCCTTGGAGGT-3'; PR reverse: 5'-CAATGGCTGTGGGAGAGC-3'; ß-actin forward, 5'-CCAACCGCGAGAAGATGA-3' and ß-actin reverse, 5'-CCAGAGGCGTACAGGGATAG-3'.

In Vitro Kinase Assay
rhER{alpha} was from Invitrogen. This rhER{alpha} turned out to be highly phosphorylated. The first step was therefore to dephosphorylate the receptor. The rhER{alpha} was incubated for 30 min at 30 C in 50 mM Tris (pH 7.5), 5 mM dithiothreitol, 2 mM MnCl2 and 20 U/µg rhER{alpha} of {lambda}-protein phosphatase (EMD Biosciences). The reaction was stopped by the addition of 1 mM molybdate and 10 mM p-nitrophenyl phosphate and 10 min incubation at RT. The mix was stored at –20 C or directly used for in vitro kinase assay. For this reaction, a volume corresponding to 100 ng of dephosphorylated rhER{alpha} was added to 1x GSK-3ß kinase buffer and to 1 µl of recombinant rabbit GSK-3ß (New England BioLabs; 250,000 U/ml or 50 ng/µl). After 10 min incubation at 30 C, the reaction was stopped by the addition of 1x SDS sample buffer. The samples were subjected to SDS-PAGE, and the kinase effects were detected by an antibody specific for the ER{alpha} phosphorylation site.

Statistical Analysis
For quantitative analysis of Western blots, signal intensities were determined with the Image J software and were normalized with ß-actin used as loading control. Luciferase analysis was performed using Berthold LB 9505 C (version 4.08) software (Bad Wildbad, Germany). For quantitative PCRs, MJ opticon monitor analysis software (version 3.1) from Bio-Rad was used and loading variations were normalized by ß-actin.

For each set of data, mean ± SEM was calculated evaluating three independent experiments. Differences between groups were statistically evaluated using t test. A P value < 0.05 was considered significant.


   ACKNOWLEDGMENTS
 
We thank Damir Krunic (German Cancer Research Center, Heidelberg, Germany) for helping us with Western blot quantification software. Special thanks to Dr. Milen Kirilov (German Cancer Research Center) for his valuable advice concerning the quantitative PCR analysis and to Raphael Bleiler (German Cancer Research Center) for his valuable technical assistance regarding cell culture and RNAi experiments.


   FOOTNOTES
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online July 3, 2007

Abbreviations: AF-1 or -2, Activation function-1 or -2; CHX, cycloheximide; DCC, dextran-coated charcoal-treated; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; FCS, fetal calf serum; GSK-3, glycogen synthase kinase-3; PFA, paraformaldehyde; PR, progesterone receptor; pS118, phosphorylated ER{alpha} serine-118 residue; rhER{alpha}, recombinant human ER{alpha}; RNAi, RNA interference; RT, room temperature; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA.

Received for publication March 7, 2007. Accepted for publication June 29, 2007.


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Nuclear Receptors:   ERα
Ligands:   17β-Estradiol



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