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Cancer Research 68, 8695, November 1, 2008. doi: 10.1158/0008-5472.CAN-08-0788
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Different Roles of Estrogen Receptors {alpha} and β in the Regulation of E-Cadherin Protein Levels in a Mouse Mammary Epithelial Cell Line

Luisa A. Helguero, Karolina Lindberg, Cissi Gardmo, Thomas Schwend, Jan-Åke Gustafsson and Lars-Arne Haldosén

Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden

Requests for reprints: Luisa A. Helguero, Unit for Nuclear Receptor Biology, Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Hälsovägen 7, 141 57 Stockholm, Sweden. Phone: 46-8-608-9277; Fax: 46-8-774-5538; E-mail: luisa.helguero{at}ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Two estrogen receptors (ER{alpha} and ERβ) are found throughout the mammary gland. Evidence indicates that, while ER{alpha} transduces proliferation signals, ERβ opposes this effect and is necessary for epithelial differentiation. Using mouse mammary epithelial cells, we have previously shown that activation of ERβ opposes ER{alpha}-induced proliferation and increases apoptosis. Furthermore, stable knockdown of ERβ resulted in loss of growth contact inhibition. In this work, we report that loss of ERβ is associated with a decrease of E-cadherin protein levels through different posttranscriptional regulatory mechanisms. Ligand activation of ER{alpha} induced E-cadherin extracellular shedding and internalization only in the absence of ERβ, followed by lysosomal degradation. Loss of ERβ also led to an increase of E-cadherin uptake in a ligand-independent manner through mechanisms that required caveolae formation. Proteasome activity was necessary for both mechanisms to operate. Increased E-cadherin internalization correlated with the up-regulation of β-catenin transcriptional activity and impaired morphogenesis on Engelbreth-Holm-Swarm matrix. Taken together, these results emphasize the role of epithelial ERβ in maintaining cell adhesion and a differentiated phenotype and highlight the potential importance of ERβ for the design of specific agonists for use in breast cancer therapy. [Cancer Res 2008;68(21):8695–704]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Estrogen receptors (ER) exist as two isoforms, ER{alpha} and ERβ, with similar affinity for the endogenous estrogen 17β-estradiol (E2) but considerable selectivity for other natural and synthetic compounds (1–3). ER{alpha} and ERβ regulate gene transcription by direct binding to estrogen-regulated elements (ERE) or indirectly by protein-protein interaction with transcription factor activator protein-1 (AP-1) and Sp1, wherein, depending on cell type, promoter context, and ligand, ERβ may antagonize ER{alpha}-induced transactivation (4, 5).

Estrogens are important for mammary gland morphogenesis and differentiation. Initially, they were thought to act through paracrine mechanisms, requiring stromal ER{alpha} and growth factor signaling (6–8). Yet, characterization of ER{alpha}–/– mice showed that, for proliferation and morphogenesis of the mouse mammary gland, the epithelial compartment is necessary and sufficient to transduce estrogen signaling through a paracrine mechanism (9). ERβ is also widespread throughout the mammary tissue, and studies in ERβ–/– mice have revealed its role in regulating cell adhesion proteins (10–12), such as E-cadherin, occludin, and connexin 32, in the lactating mammary gland (10). However, the mechanisms behind this regulation remain to be elucidated.

Recently, we showed that in HC11 cells, which are nonmalignant mouse mammary epithelial precursors with stem/progenitor cell characteristics (13–16) and express ER{alpha} and ERβ (17), the response to estrogens is dependent on the relative levels of both receptors (17). By stable expression of short inhibitory hairpin RNAs (shRNA), we generated cell lines expressing either higher or lower ER{alpha}-to-ERβ ratio (siERβ and siER{alpha}, respectively). When ER{alpha} was knocked down, cells underwent apoptosis in response to E2, whereas knockdown of ERβ resulted in increased proliferation and loss of contact growth inhibition measured as colony formation in soft agar (17).

Morphology and polarization of the epithelium is highly dependent on adequate expression and localization of the cell-cell adhesion protein E-cadherin. Through its cytoplasmic domain, E-cadherin interacts with the catenins and forms a complex with the actin cytoskeleton known as adherens junction (AJ; reviewed in ref. 18). Loss of membrane E-cadherin results in free β-catenin, which may enhance transcription of TCF/Lef-1–regulated genes (reviewed in ref. 19). Whereas E-cadherin levels do not vary in many well-differentiated tumors, E-cadherin is frequently lost during disease progression (reviewed in ref. 20). Regulation of E-cadherin may occur at both gene expression and posttranscriptional levels. Promoter methylation (21, 22) has been found in different types of cancers, whereas transcriptional repression occurs through corepressors, such as Snail (23). E-cadherin can be cleaved by metalloproteases (24–26) and internalized through clathrin-dependent and independent pathways, as well as by caveolin-1–mediated endocytosis (reviewed by ref. 27).

In human malignant and nonmalignant breast cancer cell lines, as well as in prostate cancer cell lines, E-cadherin may be regulated by estrogens (28–30). However, whereas some authors (28) showed that, in human breast cancer cells, E-cadherin expression is positively associated with ER{alpha}-dependent up-regulation of metastasis-associated protein 3 (MTA3; a transcriptional repressor for Snail expression), others used the same cell lines to show that there is a direct regulation through binding of ER{alpha} and associated transcriptional corepressors to the E-cadherin promoter, which results in repression of E-cadherin expression (30). It is noteworthy that all of these cells do not express ERβ, whereas studies on prostate cancer cells DU145, which express ERβ but lack ER{alpha} and androgen receptor, showed that activation of ERβ by 5{alpha}-androstane-3β,17β-diol, but not E2, results in up-regulation of E-cadherin at the transcriptional and protein levels (29).

In this report, we show that loss of ERβ from HC11 cells results in down-regulation of E-cadherin protein. Our data indicate that this is not a process regulated at the gene expression level but posttranscriptionally. It requires protein shedding, internalization and degradation and correlates with an increase in β-catenin transcriptional activity and impaired morphogenesis on the Engelbreth-Holm-Swarm (EHS) matrix.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
A detailed description of the procedures can be found as supplementary data (Supplementary Fig. S1).

Hormones and reagents. E2, MG132, biotin N-hydroxysuccinimide ester (NHS-biotin), and nystatin were from Sigma. Proteasome inhibitor 1 was from Calbiochem; 4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis (4-hydroxy-phenyl)-propionitrile (DPN) were from Tocris. Primary antibodies were obtained from BD Transduction Laboratories (mouse anti–E-cadherin, mouse anti–β-catenin, rabbit anti–caveolin 1, mouse anti–clathrin heavy chain, mouse anti–N-cadherin), Santa Cruz (rabbit anti–E-cadherin), AbCam (rabbit anti–LAMP-1, rabbit anti–Rab4, Rab5, Rab11, mouse anti-ERβ, and rabbit anti–β-actin), Sigma [mouse anti-vimentin, mouse anti–E-cadherin (DECMA-1), and rabbit anti-Rab7], and Calbiochem (rabbit anti-MTA3). Secondary antibodies were anti-mouse or anti-rabbit FitC, Cy3, or TritC (Sigma).

Cell culture. Generation of HC11 cells stably expressing shRNA to ER{alpha} (siER{alpha}), ERβ (siERβ), or control cells (ctrol-siRNA), as well as growth culture conditions for these and HC11 wild-type (HC11-wt) cells, have been described previously (17). Before the experiments, cells were starved for 24 h in serum-free medium [SFM; phenol red–free RPMI 1640, 50 µg/mL gentamicin, 5 µg/mL insulin, 2 mg/mL fetuin (Sigma), and 35 µg/mL human transferrin (Roche)].

Whole-cell and nuclear extracts. Cell pellets were resuspended in lysis buffer [1% NP40, 50 mmol/L Tris-HCl (pH 7.5), 140 mmol/L NaCl, 2 mmol/L EDTA, protease inhibitor cocktail (Roche), 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L Na3VO4], incubated for 20 min on ice, and centrifuged at 20,000 x g for 20 min at 4°C. Protein was quantified with Bradford reagent (Bio-Rad). Nuclear extracts were prepared, as described previously (31).

Western blot. Cell extracts or concentrated conditioned media were resolved on 7.5%, 10%, or 4% to 20% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The luminescent signal was detected with Enhanced Chemiluminescence Plus (Amersham) or West Pico (Pierce) kits. Experiments were repeated at least thrice in duplicate cultures.

Quantitative real-time PCR. cDNA was synthesized from 1 µg mRNA. Real-time PCR was carried out with SYBR-Green PCR Master Mix in an ABI PRISM 7500 apparatus (Applied Biosystems) with the following primers: 18S rRNA forward 5'-CCTGCGGCTTAATTTGACTCA-3', reverse 5'-AGCTATCAATCTGTCAATCCTGTCC-3; E-cadherin forward 5'-ATGGGGCACCACCATCAC-3', reverse 5'-CTGGGTACACGCTGGGAAAC-3'.

Generation of SP1-pGL3B, ERE-pGL3P, and SP1-EREpGL3B constructs. A GC-rich region in positions –178 to +92 of the mouse E-cadherin promoter containing Sp1 and AP-2 sites (32) was amplified from a pCAT construct, kindly supplied by Dr. J Behrens and subsequently ligated into the pGL3-Basic luciferase reporter plasmid (Promega; SP1-pGL3B). Next, the mouse E-cadherin gene, including 5' (–5127) and 3' (+7000) untranslated region (UTR) regions, was analyzed for putative AP-1, Sp1, and ERE elements. Two EREs were found in the 3' UTR region (Supplementary Fig. S2), and an 838-bp region was amplified from HC11 genomic DNA and subsequently ligated into a pGL3 promoter (ERE-pGL3P). SP1-EREpGL3B was obtained by inserting the 5' Sp-1 and the 3' 2 x ERE containing fragments into pGL3 basic plasmid.

Indirect immunofluorescence and image acquisition. Immunostaining was carried out as described previously (31). Confocal analysis was performed using a laser scanning confocal microscope (Leica CLSM), with the following lenses: 40x (HCX PL APO 1.25–0.75, oil immersion) and 60x (HCX PL APO 1.32–0.6, oil immersion). Images were acquired with LSM image browser under the same settings, and when edited with Adobe Photoshop 6.0, the same adjustments were applied to all images. Manders' overlap coefficient (0 corresponds to nonoverlapping images and 1 to 100% colocalization) and M1 (the ratio of the summed intensity of pixels from channel 1 for which the intensity of E-cadherin is above 0/total intensity in channel 1) were obtained using Image J and JACoP application (33). Phase-contrast images were captured using an inverted microscope (Leica) and video camera (VersaDoc Imaging system).

Transient transfections and luciferase assay. HC11 cells at 70% confluence were transfected in SFM with luciferase reporter constructs (3 x ERE; ref. 34) SP1-pGL3B, ERE-pGL3P, SP1-EREpGL3B, Top or FopFlash (Upstate) and Renilla luciferase pRL-TK (Promega) using Fugene 6 (Roche). After 24 h, cells were treated with 10 nmol/L E2, PPT, or DPN. Luciferase activity was measured with dual luciferase kit (Promega). Firefly luciferase units were normalized to those of Renilla. Experiments were repeated at least twice and carried out in triplicates.

Zymography. Twenty micrograms of concentrated conditioned media were separated in 10% SDS-PAGE gels copolymerized with 1 mg/mL porcine gelatin type A (Sigma) or 1.5 mg/mL {alpha}-casein (Sigma). The gels were washed in 0.5% Triton-H2O and incubated overnight at 37°C in digestion buffer [50 mmol/L Tris (pH 7.5), 200 mmol/L NaCl, 5 mmol/L CaCl2, 0.05% Na3N]. After incubation, the gels were stained with Coomassie blue and destained in 30% methanol and 10% acetic acid solution.

Morphogenesis assay. Growth on EHS matrix was studied according to refs. (35, 36). EHS matrix was prepared from the EHS sarcoma propagated in C57Bl/6 female mice according to Schuetz and colleagues (37) and was a kind gift from Prof. A. Mode (Karolinska Institutet).

Statistical analysis. Differences between more than two groups in the same experiments were compared using one-way ANOVA and Dunnett's posttest, whereas differences between two groups were calculated using one-tailed Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
ERβ knockdown affects E-cadherin protein expression in HC11 mouse mammary epithelial cells. Confluent cells were incubated for 24 h with 10 nmol/L E2 or the ER{alpha} selective agonist PPT. Immunoblotting was used to quantify E-cadherin in whole cell extracts. In HC11-wt, ctrol-siRNA, and siER{alpha} cells, E-cadherin was up-regulated by E2, whereas no effect was observed with PPT. On the contrary, in siERβ cells, E-cadherin was down-regulated by both E2 and PPT and E2-induced down-regulation was reversed by the antiestrogen ICI 182 780 (Fig. 1A ). The efficiency of the siRNA in knockdown of ERβ protein and inhibition of its transcriptional activity but not that of ER{alpha} is shown (Supplementary Figs. S3A and B, respectively).


Figure 1
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  		Figure 1. ER regulation of E-cadherin levels. A and B, immunoblotting analysis of whole cell extract from confluent cells treated for 24 h with 10 nmol/L E2, PPT (ER{alpha} agonist), or DPN (ERβ agonist) alone or in combination with 10 nmol/L ICI 164 384 or 100 nmol/L ICI 182 780. The bar graphs show densitometry values for each treatment, wherein values corresponding to untreated cells were arbitrarily set to 1. A, for each cell type, changes between E2-treated and untreated cells were analyzed with Student's t test. *, P < 0.05. B, differences between groups were analyzed with one-way ANOVA and Dunnett's posttest. *, P < 0.05. C, quantitative real-time PCR of E-cadherin mRNA expression was carried out on mRNA from confluent HC11-wt cells incubated with 10 nmol/L E2, PPT, or DPN for different time points. The experiment is representative of three and carried out in triplicates. Statistical analysis was carried out for each time point using one-way ANOVA and Dunnett's posttest. *, P < 0.05. D, conditioned medium from confluent cells treated for 24 h with 10 nmol/L E2 was concentrated six times. Twenty micrograms of protein from each concentrate were resolved by SDS-PAGE, and membranes were blotted with DECMA-1 E-cadherin ectodomain antibody. The experiment is representative of three, and the bar graph shows the relative intensity change compared with the untreated cells of which the intensity was arbitrarily set to 1. For each clone, changes between treated and untreated cells were analyzed with Student's t test. *, P < 0.05.

 
Using the selective agonist DPN (Fig. 1B), only ERβ was activated. Similar to E2, DPN up-regulated E-cadherin protein expression whereas coincubation with the antiestrogen ICI 164 384 reversed this effect. These effects were specific for E-cadherin as neither β-catenin, {alpha}-catenin, nor p120 catenin showed a differential regulation by E2 treatment (not shown).

These data suggest that ligand activation of ERβ up-regulates E-cadherin and that ligand activation of ER{alpha} down-regulates E-cadherin protein levels but only when ERβ expression is reduced.

ER regulation of E-cadherin gene expression does not correlate with changes at the protein level. ER{alpha}, through up-regulation of MTA3 expression, has been positively correlated with increased E-cadherin expression in MCF-7 breast cancer cells. Thus, we investigated a possible correlation between MTA3 and E-cadherin expression in nonmalignant HC11-wt cells after incubation with E2 and ER-selective agonists. Surprisingly, in HC11-wt cells, MTA3 expression did not change after 1, 3, or 6 hours of treatment with E2, PPT, or DPN (not shown) and a tendency toward reduction was observed after 24 hours of incubation with all agonists (Supplementary Fig. S4A). Because our results were generated using another system than the study referred to, we also analyzed MTA3 expression in MCF-7 breast cancer cells, which were used in that study (28) and grown under the same conditions as used in our experiments (SFM). MTA3 was indeed up-regulated by E2 and PPT, but this did not correlate to E-cadherin expression (Supplementary Fig. S4B). Next, we incubated both MCF-7 and HC11 cells in 10% DCC–fetal bovine serum (FBS) for 5 days before ligand treatment, similar to the protocol used in the above referenced study (28). Using these conditions, E2 and PPT up-regulated both MTA3 and E-cadherin expression in MCF-7 cells (Supplementary Fig. S4C). As expected, because these cells do not express ERβ (not shown), DPN had no effect. On the contrary, in HC11 cells, E2 and DPN up-regulated E-cadherin expression but only DPN up-regulated MTA3, indicating that in these cells E2 regulation of E-cadherin does not involve MTA3 pathway.

It has been shown that ER{alpha} may down-regulate E-cadherin gene transcription (30). Therefore, quantitative real-time PCR was used to measure E-cadherin mRNA in confluent HC11-wt cells treated for 2, 4, 8, 12, 18, and 24 hours with 10 nmol/L E2, PPT, or DPN (Fig. 1C). Treatment with PPT or DPN only induced a slight increase in E-cadherin mRNA after 2 hours (P < 0.05), whereas E2 had a similar effect after 2 and 12 hours of incubation. A cause for the E2-induced increase could be cyclical accumulation of mRNA. However, this effect was not reproduced by either PPT or DPN, although, at the protein level, DPN up-regulated E-cadherin protein in a similar way as E2. To study if the slight mRNA induction after 2 hours of treatment was transduced into increased protein levels, immunoblots were carried out after 3 and 6 hours of incubation with E2, PPT, or DPN. No significant differences were observed except for a tendency to reduce E-cadherin levels in the PPT-treated cells (Supplementary Fig. S5). These data suggest that in HC11 cells, ER regulation of E-cadherin gene expression does not explain the above observations on E-cadherin protein levels.

To further test if ER could regulate E-cadherin transcription, we used reporter assays. In the mouse E-cadherin gene, a GC-rich region containing an Sp1 site in position –50 (32) seems as one candidate regulatory element for mediating ER action. Using Dragon ERE Finder Version 2.0,1 we also identified two putative EREs at positions +68679 and +68763 in the 3' UTR region (Supplementary Fig. S2). We tested the ability of 10 nmol/L E2, PPT, or DPN to activate transcription from these sites using SP1-pGL3B, ERE-pGL3E, and SP1-EREpGL3B luciferase reporter plasmids. After 4 and 24 hours of incubation with E2, PPT, or DPN, no induction of the reporter gene was observed (data not shown).

Ligand activation of ER{alpha} in cells with ERβ knockdown induces E-cadherin shedding. To study if E2-induced down-regulation of E-cadherin protein levels in siERβ cells was due to extracellular shedding, E-cadherin fragments in the conditioned media from cells treated for 24 hours with 10 nmol/L E2 were analyzed by immunoblotting (Fig. 1D). For this purpose, we used two antibodies against the extracellular domain of E-cadherin, one monoclonal and one polyclonal (DECMA-1 and H-108, respectively) with similar results (H-108 data not shown). An 88-kDa fragment was found in all cell lines. Under basal conditions, the amount of this fragment from siERβ cells was lower than in the controls. However, after E2 stimulation, the level of this fragment increased only in samples from siERβ cells (Fig. 1D).

These results were supported by immunofluorescence experiments, wherein E-cadherin was detected with an antibody to the extracellular domain (H-108; E-cad/ecto); membrane staining was reduced after E2 incubation (Supplementary Fig. S6). Instead, cytoplasmic staining was observed. Taking into account that the epitope for H-108 antibody spans from aa 600 to 707, which is very close to the beginning of the mouse E-cadherin transmembrane domain (aa 710–733), it is probable that the cytoplasmic staining corresponds to internalized truncated E-cadherin fragment product of E2/ER{alpha}-induced shedding.

These results indicate that one mechanism responsible for E-cadherin down-regulation observed in the absence of ERβ is E2/ER{alpha}-induced shedding.

Loss of ERβ in HC11 cells induces differential E-cadherin cellular localization. In HC11-wt and ctrol-siRNA cells, E-cadherin staining was carried out using an antibody to the cytoplasmic domain (E-cad/cyto) and was detected in the cell-cell contacts (Fig. 2A ). On the other hand, siERβ cells showed less attachment and increased cytoplasmic staining with a granular pattern (Fig. 2A, arrows). siERβ cells were also larger, and scanning of the cells through the Z axis indicated that they were flatter than the controls. Incubation of siERβ cells with 10 nmol/L E2 for 24 hours yielded a similar picture as seen in untreated cells, and incubation with 100 nmol/L ICI 182 780 had no effect (not shown), indicating that the cytoplasmic staining and granular pattern observed in siERβ cells are not ER{alpha}-mediated effects but results of reduced ERβ levels.


Figure 2
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  		Figure 2. E-cadherin subcellular localization. A, staining of HC11 cells grown for 48 h in SFM was carried out with E-cadherin antibody against the cytoplasmic domain. Images show one stack of 3.3 µm. Note the granular cytoplasmic staining in siERβ cells (arrows). Bars, 30 µm (top) and 10 µm (bottom). B, loss of ERβ induces E-cadherin internalization. Cells were incubated with NHS-biotin for 1 h in D-PBS, washed, and allowed to internalize the biotinylated proteins for 4 h in SFM. Cells were fixed and stained with an E-cadherin intracellular epitope antibody (red) and fluorescein-coupled streptavidin (green). Arrows, granular colocalization pattern; arrowheads, membrane biotin. Images show one stack of 3.3 µm. Bars, 15 µm.

 
Loss of ERβ results in increased E-cadherin internalization. Posttranscriptional processes involving E-cadherin endocytosis, endosomal sorting, and lysosomal-mediated degradation play a major role in dynamically modulating cell surface E-cadherin levels. To test if E-cadherin internalization was related to the cytoplasmic granular staining of E-cadherin observed in siERβ cells (Fig. 2A), the extracellular domain of membrane proteins was biotinylated 4 hours before fixation. Biotin was detected using fluorescein-conjugated streptavidin (Fig. 2B; split of merged image in two channels is shown in Supplementary Fig. S7A). Internalized biotinylated proteins were clearly observed in siERβ cells, whereas in HC11-wt or ctrol-siRNA cells higher biotin staining was associated with the cellular membrane (Fig. 2B, arrowhead; ctrol-siRNA cells not shown). Overlap (ov) and M1 coefficients were as follows: for untreated siERβ cells, ov 0.63 ± 0.07 and M1 0.25 ± 0.02; for E2-treated cells, ov 0.58 ± 0.01, M1 0.23 ± 0.04 (indicating that although reduced ERβ levels were associated with higher internalization, this was not enhanced by E2). However, given that internalized proteins can recycle or transit toward degradation, we used different vesicle markers to characterize the granules observed and to study whether E2 could influence the fate of internalized E-cadherin.

Clathrin-dependent and independent mechanisms have been postulated for E-cadherin internalization. Therefore, we carried out double immunofluorescence stainings with clathrin and E-cad/ecto antibodies on E2-treated and untreated siERβ cells (Fig. 3A ). Overall colocalization was significantly higher after E2 treatment (ov 0.71 ± 0.05, M1 0.38 ± 0.04 versus ov 0.81 ± 0.02, M1 0.44 ± 0.07, respectively; P < 0.05), and partial colocalization in cytoplasmic granules was observed more clearly in E2-treated cells (Fig. 3A and Supplementary Fig. S8A, arrow). To investigate the fate of the intracellular granules observed in siERβ cells, cells were stained with the following markers: Rab5 for early endosomes, Rab7 for late endosomes, Rab4 for recycling vesicles, Rab11 for exocytic vesicles, and LAMP-1 for lysosomal vesicles. E-cad/cyto antibody partially colocalized only with Rab5, and E2 enhanced this effect (ov 0.63 ± 0.05, M1 0.16 ± 0.03 versus ov 0.74 ± 0.04, M1 0.22 ± 0.06, respectively; P < 0.05; Fig. 3B and Supplementary Fig. S8B, arrow). E2 effect on increased colocalization with LAMP-1 was also significant (ov 0.45 ± 0.01, M1 0.19 ± 0.02 versus ov 0.52 ± 0.04, M1 0.24 ± 0.04, respectively; P < 0.05; Fig. 3C and Supplementary Fig. S9A, arrow). Taken together, the results presented thus far indicate that when ERβ levels are reduced and cells are treated with E2, ER{alpha} induces shedding and the resulting cytoplasmic domain is likely to be internalized and degraded in the lysosome. However, the results do not explain why siERβ cells still showed E-cadherin cytplasmic staining even in the absence of ligand.


Figure 3
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  		Figure 3. E2 induces E-cadherin internalization through clathrin-dependent pathway. siERβ cells were incubated with or without 10 nmol/L E2 for 24 h, fixed, and processed for immunocytochemistry. E-cadherin colocalization was evaluated by double staining with clathrin (red; A), Rab5 (green; B), or Lamp-1 (green; C). Bar, 30 µm. Images show one stack of 3.3 µm, and colocalization is indicated by the arrows.

 
Given that caveolin-1–mediated endocytosis is another mechanism for E-cadherin uptake (reviewed in ref. 27), we carried out colocalization studies on siERβ cells using E-cad/cyto and caveolin-1 antibodies. E-cadherin–positive granules partially colocalized with caveolin-1 under basal conditions, as well as after E2 treatment (Fig. 4A ; E2 data not shown). Colocalization, measured as overlap and M1 coefficients, was significantly higher in siERβ cells compared with HC11-wt and ctrol-siRNA cells and was not enhanced by E2 treatment (Supplementary Table S1).


Figure 4
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  		Figure 4. Caveolin-1 is associated with E-cadherin membrane localization. A, untreated HC11-wt, siERβ, and ctrol-siRNA cells were fixed and double-stained with anti–caveolin-1 (green) and E-cadherin (red) cytoplasmic epitope antibodies. Bar, 30 µm. The arrows indicate colocalization. Images show one stack of 3.3 µm. B, inhibition of caveolae formation restores membrane E-cadherin in siERβ cells. After 20-h treatment with 10 nmol/L E2, 25 µg/mL nystatin (nys) were added to the medium and cells were incubated for an additional 4 h. siERβ cells were fixed and processed for immunofluorescence staining with an antibody against E-cadherin cytoplasmic domain. Confocal images show one stack of 3.3-µm thickness. Bar, 30 µm. C, proteasome inhibition increases E-cadherin membrane localization in siERβ cells. After 20-h treatment with 10 nmol/L E2, 10 µmol/L MG132 or 0.15 µg/mL proteasome inhibitor 1 (PI 1) was added to the medium, and cells were incubated for an additional 4 h. Cells were fixed and processed for E-cadherin immunofluorescence staining with antibodies against the intracellular or the extracellular domain. Confocal images show one stack of 3.3-µm thickness. Bar, 30 µm. D, proteasome inhibition does not affect E-cadherin protein levels in siERβ cells. Cells were treated as described in C, and total E-cadherin protein levels were quantified by immunoblotting with intracellular E-cadherin antibody.

 
These results suggest that, upon loss of ERβ, caveolae might influence internalization of E-cadherin in a ligand-independent manner. To test this hypothesis, we incubated cells with nystatin, which selectively disrupts caveolae but does not affect clathrin-coated pits or actin fibers (38). Nystatin partially restored membrane E-cadherin under basal conditions but not in E2-treated siERβ cells (Fig. 4B), indicating that E2/ER{alpha}-induced down-regulation of E-cadherin protein levels occurs independent of the formation of caveolae and, as shown previously, is related to extracellular shedding and lysosomal degradation.

The c-Cbl–like E3 ubiquitin ligase Hakai mediates ubiquitination of E-cadherin, leading to destabilization of AJs, internalization of the E-cadherin complex, and further degradation (39). Although ubiquitin-mediated internalization of membrane proteins is generally associated with lysosomal degradation (40), some evidence suggests that the proteasome might also be involved (41, 42). Thus, siERβ cells were treated with two proteasome inhibitors (MG132 or the proteasome inhibitor 1) during the last 4 hours of experiment, and E-cadherin localization was analyzed by immunostaining with E-cad/cyto or E-cad/ecto antibodies, and total cellular E-cadherin levels were analyzed by immunoblotting (Fig. 4C and D). Under basal conditions, inhibition of the proteasome resulted in the retention of membrane E-cadherin detected with both antibodies. After E2 incubation, a similar picture to that seen in untreated cells was observed with E-cad/cyto. However, membrane signal was not detected with E-cad/ecto antibody. Instead, and similar to the results shown in Supplementary Fig. S6, some cytoplasmic stainings were observed. Coincubation with E2 and MG132 resulted in E-cadherin membrane retention was observed with E-cad/cyto antibody, but only a partial retention was observed with E-cad/ecto (Fig. 4C; only MG132 is shown). Contrary to the expected result, the proteasome inhibitors did not prevent E2-induced decrease of E-cadherin protein levels, as shown previously (compare Fig. 1A with Fig. 4D).

Taken together, these results indicate that, although inhibition of the proteasome favors membrane localization of full-length and/or truncated E-cadherin, it does not influence E2-induced shedding.

Loss of ERβ is associated with E2-induced activation of β-catenin. Influence of estrogen signaling on transcriptional activation of β-catenin was evaluated using a TCF/Lef-1 regulated luciferase reporter gene. Transiently transfected HC11-wt, ctrl-siRNA, and siERβ cells were incubated with 10 nmol/L E2 for 24 hours. Luciferase activity did not significantly change in either HC11-wt or ctrl-siRNA cells. In siERβ cells, reporter gene transcription was increased by E2 (Fig. 5A, top ). This effect was reversed by cotransfection with hERβ expression vector (Fig. 5A, top, insert). The E2-induced reporter gene activation correlated with increased nuclear β-catenin, whereas no effect was seen on total cellular β-catenin levels (Fig. 5A, bottom). Immunofluorescence showed that in siERβ cells, β-catenin (in a similar way as E-cadherin; Fig. 2A), is also found in cytoplasmic granules (Supplementary Fig. S10 and Fig. 5B, arrow). Furthermore, E2 treatment resulted in more diffuse staining throughout the cytoplasm (Fig. 5B).


Figure 5
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  		Figure 5. Loss of ERβ induces β-catenin transcriptional activation. A, HC11 cells were transiently transfected with β-catenin reporter plasmid. Activity of TopFlash and the mutated control FopFlash-driven luciferase reporter gene was measured and normalized to Renilla activity. Then, FopFlash background levels were subtracted from TopFlash activity. The experiment is representative of three. *, P < 0.05. The insert shows siERβ cells cotransfected with β-catenin reporter plasmid and human ERβ expression plasmid. Bottom, β-catenin nuclear and total levels in cells treated with 10 nmol/L E2 for 24 h. B, siERβ cells were fixed and stained with an anti–β-catenin antibody. Confocal images show one stack of 3.3-µm thickness. Bar, 20 µm. C, loss of ERβ results in impaired morphogenesis. Cells were cultured on EHS matrix for 10 d. Phase contrast images are shown. Experiment is representative of three.

 
Expression of mesenchymal markers. HC11-wt cells do not express N-cadherin, neither do ctrol-siRNA nor siERβ cells, as analyzed by immunoblotting and immunofluorescence (data not shown). However, we did observe some vimentin-positive cells in all cell lines (data not shown), indicating that this is not associated with loss of ERβ. Furthermore, in wound assays, we observed that incubation of HC11-wt cells with the ER{alpha} agonist PPT, but not the ERβ agonist DPN, increased closure of the wound (Supplementary Fig. S11), indicating that activation of ER{alpha} may result in increased migration.

Morphogenesis is hindered by knockdown of ERβ. The formation of mature AJs, of which E-cadherin is a key player, is directly linked to cell polarity. HC11 cells can grow on reconstituted basement membrane, such as EHS, and form polarized acini (36). Formation of acini occurs through a period of 10 days, with active proliferation, followed by arrest and apoptosis to form the lumen (36). Acini were observed in HC11-wt and ctrl-siRNA cells, although the latter was not as efficient in forming a lumen. Nevertheless, in siERβ cells, the growth was disorganized (Fig. 5C), cells did not arrest but kept growing, giving origin to larger structures. Incubation with 100 nmol/L ICI 182 780 throughout the 10-day period did not prevent this effect (not shown), indicating that this is not an ER{alpha}-mediated effect but is due to the loss of ERβ, which may be related to ER regulation of E-cadherin cellular localization, as well as increased β-catenin transcriptional activity.


   Discussion
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 Discussion
Disclosure of Potential...
 References
 
There is a connection between estrogen signaling and E-cadherin expression both in vivo and in vitro. Administration of E2 causes rapid E-cadherin mRNA up-regulation in mouse ovary (43), whereas studies in uterus and lactating mammary gland from ERβ–/– mice have shown lower E-cadherin staining (10, 12). Furthermore, estrogens regulate E-cadherin expression in cancer cell lines (28–30). Yet, the molecular mechanism and the final outcome of the response to E2 remain ambiguous. Fujita and colleagues have postulated that E-cadherin expression is dependent on ER{alpha} through its induction of MTA3 expression (28). However, because the authors did not use E2 but 10% FBS, it is not clear if this effect is mediated through ligand-dependent or independent activation of ER{alpha}. In contrast, Oesterreich and colleagues (30) showed that ER{alpha} overexpression resulted in E2-induced repression of E-cadherin gene expression through binding of ER{alpha} and transcriptional corepressors N-CoR and SAFB1 to a regulatory sequence in the mouse E-cadherin promoter region. We cloned this sequence (32) into pGL3B luciferase plasmid and observed no ER regulation. However, in our case, we used SFM, which might not allow E2 repressive activity in breast cancer cells (30).

Interestingly, the cell lines used in the above-mentioned studies express very low or undetectable levels of ERβ. But in DU145 prostate cancer cells which do express ERβ, it was shown that, while E2 induced E-cadherin mRNA, it inhibited E-cadherin protein expression and that 5{alpha}-androstane-3β,17β-diol, which is a ligand with higher affinity for ERβ than for ER{alpha}, had the opposite effect, induced both E-cadherin mRNA and protein, and inhibited migration (29). Our results with a mouse mammary epithelial cell model are in agreement with this study. We have found that E2 influences E-cadherin protein levels in different directions, dependent on ERβ expression, and that this occurs through posttranscriptional mechanisms. Thus, in the case where both ERs are expressed in nonmalignant mammary epithelial cells, E2 will have a positive effect on E-cadherin level through ERβ; but if this receptor is lost, E2 will have a negative effect on E-cadherin through ER{alpha}. Because E-cadherin mRNA did not correlate with E-cadherin protein levels, it seems that the activities of these two receptors oppose each other at the posttranscriptional level.

PPT or E2 induced E-cadherin down-regulation only when ERβ was knocked down, but PPT had no effect on HC11-wt cells. This indicates that ERβ activation induces E-cadherin expression and also protects it from ER{alpha}-induced down-regulation.

Posttranscriptional regulation of E-cadherin may occur through different pathways, such as degradation by extracellular proteases as metalloproteases (24, 25, 36) and serine proteases (44). We found a correlation between ligand activation of ER{alpha} and an increase in soluble 88-kDa E-cadherin fragment in conditioned media from siERβ cells. We have measured gelatinase and caseinase activity using substrate gel zymography and found no bands corresponding to matrix metalloproteinase-3 (MMP-3) or MMP-7 (not shown), whereas MMP-9 and MMP-2 activity were higher in siERβ compared with HC11-wt cells. However, the intensity of the active bands decreased after E2 treatment (Supplementary Fig. S12). As a result, we were not successful in identifying the molecular species associated with E2/ER{alpha}-induced shedding.

The fact that, after E2 incubation in siERβ cells, E-cadherin staining was disorganized with cytoplasmic granular staining, which partially colocalized with clathrin, Rab5, and LAMP-1, suggests that internalization and subsequent lysosomal degradation is another mechanism whereby E-cadherin is regulated by ER{alpha} in a context where ERβ is absent.

Ubiquitin-dependent endocytosis is strongly linked to proteasomal activity. In siERβ cells, inhibition of proteasome resulted in membrane retention of E-cadherin but did not prevent E2-induced down-regulation. One possibility is that proteasome inhibition depletes the cells of free pools of ubiquitin (45), enabling a shorter fragment of E-cadherin to remain in the membrane.

In siERβ cells, independent of any ligand, caveolin-1 colocalized with E-cadherin. Caveolin-1 is the structural component of caveolae, which are thought to function as tumor suppressors due to their capacity to concentrate Src kinase and other signal transduction proteins. In addition, it is speculated that a subpopulation of membrane and caveolae-associated ERs are the effectors of nongenomic estrogen effects (reviewed in ref. 46). ERs colocalize with caveolin-1 and are necessary for ligand-independent extracellular signal-regulated kinase activation in osteocytic and osteoblastic cells (47). However, it remains to be established if physical association of ERs to caveolae occurs in HC11 cells.

Loss of ERβ also correlated with E2-induced activation of β-catenin. Due to the loss of E-cadherin in the membrane, β-catenin would become available to enhance transcription from TCF/Lef-1–regulated genes. This could be the result of a direct or indirect interaction between ER{alpha} and β-catenin, enhancing transactivation of cognate reporter genes (48, 49). β-Catenin activation is commonly associated with epithelial mesenchymal transition; however, siERβ cells did not express N-cadherin, and vimentin staining was not increased. Thus, the phenotypic change induced by the loss of ERβ did not correlate with these EMT characteristics, although it affected morphogenesis on EHS.

Finally, the regulation of MTA3 protein was different in MCF-7 compared with HC11 cells. HC11 cells are undifferentiated mouse mammary epithelial precursors with stem cell properties and can be induced to differentiate (through epidermal growth factor withdrawal) into different lineages according to hormone conditions (13–16). HC11 cells also endogenously express ER{alpha} and ERβ. On the other hand, MCF-7 cells are human breast cancer cells that only express ER{alpha}. Thus, many differences exist between these two systems, and it is premature to say that the differences observed with regard to the regulation of E-cadherin are due to cell transformation, ER expression, or species differences. However, the fact that adhesion, proliferation, and morphogenesis are impaired in a cell line with stem/progenitor cell characteristics highlights the importance of ERβ in maintaining epithelial homeostasis.

In this work, we have presented evidence indicating that at least two mechanisms are responsible for E-cadherin regulation (Fig. 6 ). One mechanism would take place in the absence of ERβ (1). Ligand activation of ER{alpha} results in the shedding and increase of free E-cadherin soluble fragments, which may inhibit cell adhesion in a paracrine way (44, 50). The resulting truncated E-cadherin protein in the membrane is internalized and degraded in the lysosome. Due to loss of membrane E-cadherin, β-catenin would become available to enhance transcription from TCF/Lef-1–regulated genes. This, in turn, results in increased cellular proliferation (17) and migration (Supplementary Fig. S4), respectively. Expression of ERβ would counteract ER{alpha}-mediated effects and, upon ligand activation, up-regulate E-cadherin protein levels. Through a different mechanism (2), which does not require a ligand, ERβ inhibits caveolin-1–mediated internalization of E-cadherin. In both mechanisms 1 and 2, a role for the proteasome remains to be elucidated.


Figure 6
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  		Figure 6. Schematic interpretation of the results presented in this study. Pathway 1: in the absence of ERβ, ligand activation of ER{alpha} induces E-cadherin shedding. This results in formation of free, soluble E-cadherin fragments, which may inhibit cell adhesion in a paracrine way. The resulting shorter membrane protein is internalized through the clathrin-dependent pathway and degraded in the lysosome. Due to the loss of E-cadherin membrane localization, β-catenin is available to activate transcription from TCF/Lef-1–regulated genes. This would, in turn, result in increased cellular proliferation and migration. ERβ—although not yet known if dependent or not on ligand binding—inhibits these effects of ER{alpha}. Ligand activation of ERβ also results in up-regulation of E-cadherin protein levels. Pathway 2: independent of a ligand, ERβ inhibits caveolae-mediated internalization of E-cadherin. This mechanism needs to be worked out in detail, but data presented in this report suggest that the proteasome is involved in this process.

 
Together, these results suggest that ERβ may act as a tumor suppressor in mammary progenitor cells and highlight cellular pathways, which may be targeted in treatment of breast cancer by ERβ agonists.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
J-Å. Gustafsson: Commercial research grant, consultancy honorarium, and ownership interest, KaroBio AB. The other authors disclosed no potential conflicts of interest.


   Acknowledgments
 
Grant support: David and Astrid Hageléns Postdoctoral Fellowship (L.A. Helguero), Lars-Hiertas Minne, KaroBio AB, and Swedish Cancer Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the past and present members of the Unit for Nuclear Receptor Biology for providing reagents and supportive discussions, Dr. A. Ström for Flag-hERβ plasmid, Dr. J. Behrens for Sp1-pCAT plasmid, Dr. A. Mode for kindly supplying us with EHS matrix, and Drs. S. Strömblad and J. Lock for instructive discussions during the completion of this work.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

1 http://sdmc.lit.org.sg/ERE-V2/index Back

Received 2/29/08. Revised 8/ 5/08. Accepted 8/22/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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