Interaction and Dissociation by Ligands of Estrogen Receptor and Hsp90: The Antiestrogen RU 58668 Induces a Protein Synthesis-Dependent Clustering of the Receptor in the Cytoplasm

Jocelyne Devin-Leclerc, Xia Meng, Francine Delahaye, Philippe Leclerc, Etienne-Emile Baulieu and Maria-Grazia Catelli

Institut National de la Santé et de la Recherche Médicale U33 Lab Hormones 94276 Le Kremlin Bicêtre Cedex, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The in vivo interaction of estrogen receptor (ER) and Hsp90, demonstrated in the absence of hormone by a nuclear cotranslocation assay of the cytoplasmic Hsp90 with the karyophilic receptor, was disrupted by agonist and antagonist ligands, which, after dissociating the Hsp90, allowed the chaperone protein to be relocalized in the cytoplasm. The pure antiestrogen RU 58668 (RU), which was unable to stimulate an estrogen-dependent reporter gene and completely inhibited its estradiol-induced activity, also profoundly modified the subcellular distribution of ER in a specific time- and dose-dependent manner; ER appeared as speckled fluorescent clusters mainly located in the perinuclear region of the cytoplasm. The kinetics of appearance and reversal of the RU-dependent ER mislocalization in the presence or absence of cycloheximide demonstrated 1) that this effect was reversed by RU withdrawal or estradiol (E2) treatment, and 2) that cycloheximide with RU inhibited and reversed the ER cytoplasmic mislocalization induced by RU alone. These results point to a protein synthesis-dependent step in the mechanism of action of this antiestrogen. After RU treatment, a large portion of ER was found in the particulate fraction of the cytoplasm. However, confocal and electron microscopic analysis showed that ER clusters were not associated with specific cytoplasmic organelles or compartments. Using ER mutants, it was found that the ligand binding domain was sufficient for RU to produce receptor mislocalization, while the constitutive nuclear localization signals were dispensable. We propose that the antiestrogenic properties of RU are primarily due to the induction of an aggregation-prone receptor conformation that cannot undertake the constitutive and the ligand-induced nuclear localization function of the receptor because it is sequestered in the cytoplasm by fast turning over protein(s). We predict that antiestrogens able to block ER nuclear localization will behave as pure antihormones and will inhibit all the nuclear action of ER elicited by agonistic ligands or by ligand-independent mechanisms such as growth factor stimulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) is a ligand-dependent transcriptional activator that belongs to the nuclear receptor superfamily. The specific natural ligand, estradiol (E2), induces formation of receptor homodimers that bind tightly to the estrogen-responsive elements (ERE), leading to activation of gene expression (1, 2, 3). The nuclear localization of ER in the absence of hormone results from the function of three constitutive nuclear localization proto-signals (p-NLS), that cooperate with the nuclear localization function of the ligand-binding domain (LBD) in the presence of hormone (4, 5). Although ER is located predominantly in the nucleus, it actually shuttles between the nucleus and the cytoplasm (6).

ER can be recovered from the hormone-free target cell cytosols in the inactive non-DNA binding 8–9S heterooligomeric form that includes, among others, the 90-kDa heat shock protein (Hsp90), the only protein of the complex that has the intrinsic ability for selective receptor binding (7, 8, 9, 10). The Hsp90 molecular chaperone (11), required for viability in eukaryotes (12), is found in all tissues or cells as an abundant dimer, essentially localized in the cytoplasm but also present at low levels in the nucleus (13, 14, 15). Biochemical, cell biological, genetic, and pharmacological approaches have been extensively used to study the interactions of Hsp90 and steroid receptors and their consequences for receptor function upon ligand binding. Results strongly support a dual role of Hsp90 in regulating steroid receptor activity: Hsp90 helps to maintain steroid receptors inactive in the absence of hormone and to ensure efficient hormonal responses (10, 16).

Concerning the in vivo interaction between Hsp90 and steroid receptors, a nuclear cotranslocation assay demonstrated that, in the absence of hormone, nuclear-targeted Hsp90 was able to shift cytoplasmic glucocorticoid receptor or progesterone receptor mutants into the nucleus (17). Likewise, the interaction between wild-type Hsp90 and two nuclear steroid receptors, progesterone receptor and ER, shifted Hsp90 into the nucleus (15, 17), implying that nuclear steroid receptors are, at least in part, complexed with Hsp90, which dissociates from the receptors upon ligand binding. Moreover, the transcriptional properties and subcellular localization of human or mouse ER and their mutants in the presence of E2 and mixed agonist-antagonists such as tamoxifen or pure antiestrogens like ICI 164 384 and ICI 182 780 have been determined. While tamoxifen only abolished the activating function of the LBD (AF-2), without interfering with the nuclear localization of ER, the ICI compounds were also able to inhibit AF-1 (located in the N-terminal domain of ER); ER bound to pure antiestrogens, although possessing DNA-binding capacity in vitro, was incapable of nucleo-cytoplasmic shuttling (6, 18, 19).

In the search for specific estrogen antagonists for ER-positive breast cancer therapy (20), RU 58668 (thereafter called RU) which, in vitro, displays affinities for human and murine ERs equivalent to those of E2, was selected for its complete antiestrogenic properties, in vivo, on the uterotrophy induced by E2 in mice as well as for its unique ability to induce a long-term regression of MCF-7 tumors implanted in nude mice (21, 22).

In view of these results, we investigated the effect of E2 and RU on the in vivo interaction between ER and Hsp90 using the nuclear translocation assay. In vivo dissociation of Hsp90 from ER was found after agonist or antagonist treatment. In addition, upon RU treatment, ER was segregated into cytoplasmic clusters that appeared and were maintained only in the presence of protein synthesis. Moreover, RU was unable to stimulate transcription of an estrogen-dependent reporter and completely repressed the estrogen-induced activity. These findings led us to propose that the antiestrogenic effects of this compound are primarily due to a protein synthesis-dependent mislocalization of ER concomitant to a disruption of its constitutive and ligand-induced nuclear localization functions.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The in Vivo Interaction between Hsp90 and ER Is Disrupted by E2 and RU
To investigate the in vivo interaction between chicken Hsp90 and human ER, double indirect immunofluorescence and confocal laser microscopic analyses were performed after transfection of plasmid pSVK390-HEGO in Cos-7 cells. ER and Hsp90 were detected with the antibodies JS 32/34 (23) and BF4 (7), respectively. The percentage of transfected cells expressing a high level of Hsp90 and ER was constantly above 50%. No staining was seen in nontransfected Cos-7 cells or in cells treated with the fluorescent second antibodies alone.

Hsp90 and ER, when expressed alone, were predominantly cytoplasmic and nuclear, respectively (4, 5, 17). With the plasmid pSVK390-HEGO, we confirmed our previous results (15), demonstrating that, in the absence of hormone, Hsp90 was in part or completely shifted to the nuclear compartment as a consequence of its interaction with ER (Fig. 1Go, a and b). The effects of the agonist, E2 (10 nM) (Fig. 1Go, c and d), and the antagonist, RU (100 nM) (Fig. 1Go, e–h), which were added to Cos-7 cells 24 h after transfection and incubated for a further 24 h, were demonstrated by the disruption of the in vivo interaction between ER and Hsp90 in 70% of cells expressing both proteins, Hsp90 showing a predominant diffuse cytoplasmic staining. Moreover, in the presence of RU, ER, which is nuclear localized in the absence and in the presence of E2, was found to form perinuclear cytoplasmic clusters of various size. Scatter diagram analysis revealed that ER and Hsp90 did not colocalize after treatment with E2 or RU. Upon treatment with tamoxifen, a mixed agonist-antagonist ligand, the subcellular localization of ER and Hsp90 was similar to that obtained by E2, while ICI 164384, another pure antiestrogen, produced the same effect as RU (not shown).



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Figure 1. E2 and RU Disrupt the in Vivo Hsp90-ER Interaction

Subcellular localization of Hsp90 (a, c, e, and g) and ER (b, d, f, and h) coexpressed in Cos-7 cells, as revealed by double indirect immunofluorescence staining and laser confocal microscopy. ER and Hsp90 were detected by JS 32/34 and BF4 antibodies, respectively. E2 or RU was added 24 h after transfection. a and b, Control; c and d, E2 24 h; e to h, RU 24 h.

 
To confirm the results observed after transient transfection experiments, the same in vivo interaction and its disruption by specific ligands were investigated in the ERC3 cell line, which stably expresses a high level of human ER (24). ER was revealed with the monoclonal antibody H222 (25) and the endogenous Hsp90 was revealed with a polyclonal antibody (26). Treatment with various ER ligands did not exceed 16 h because E2 inhibited growth and caused lysis of ERC3 cells (24).

The confocal laser microscopic analysis of untreated ERC3 cells revealed a partial nuclear cotranslocation of Hsp90 with ER, confirming an in vivo interaction between the endogenous Hsp90 and ER (Fig. 2Go, a and b). The subcellular localization of Hsp90 in the parental Chinese hamster ovary (CHO) cell line, with no detectable endogenous ER, in the absence or presence of hormone, was essentially cytoplasmic as shown in Fig. 2kGo. The treatment of ERC3 cells with 10 nM E2 (Fig. 2Go, c and d) or 100 nM RU (Fig. 2Go, e and f) disrupted the Hsp90-ER interaction, the nucleus being almost devoid of Hsp90. In the presence of cycloheximide (CHX, 50 µg/ml), the Hsp90-ER interaction was still detectable, Hsp90 being found in part in the nucleus (Fig. 2Go, g and h) and was disrupted by E2 (Fig. 2Go, i and j), indicating that the loss of nuclear localization of Hsp90 was actually a consequence of its dissociation from ER and not due to new Hsp90 synthesis. Moreover, after RU treatment, the ER was localized in cytoplasmic clusters as already observed in transfected Cos-7 cells (Fig. 1Go, f and h). Again, scatter diagram analysis did not reveal any preferential colocalization of Hsp90 and ER in the presence of E2 and RU.



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Figure 2. Interaction and Dissociation of Endogenous Hsp90 and ER in the ERC3 Cell Line

Subcellular localization of Hsp90 (a, c, e, g, and i) and ER (b, d, f, h, and j). ER was detected by H222 monoclonal antibody and endogenous Hsp90 by polyclonal antibody. a and b, Control; c and d, E2 16 h; e and f, RU 16 h; g and h, CHX 8 h; i and j, E2 + CHX 8 h; k, endogenous Hsp90 in CHO cells, with no detectable ER.

 
Even though Hsp90-steroid receptor dissociation occurs rapidly after in vivo hormonal treatment (10), the change in Hsp90 subcellular distribution, as a consequence of dissociation, takes place after 8–16 h of hormone treatment, according to cell line. Therefore, the in vivo Hsp90-ER interaction and their dissociation in the presence of hormone, as well as the cytoplasmic clustering of ER by RU, can be documented independently of transient transfection procedures.

RU-Dependent ER Mislocalization Is Specific and Requires Protein Synthesis
To investigate the mechanism by which RU acted on ER localization and to avoid differences in subcellular distribution due to new receptor synthesis, transfected Cos-7 cells were treated with RU in the presence of CHX. As shown in Fig. 3Go, the cytoplasmic ER clusters, observed in the presence of RU (Fig. 3cGo), were not detected in the presence of RU + CHX (Fig. 3dGo), the receptor being found in the nucleus as in control (Fig. 3aGo) or in E2-treated cells (Fig. 3bGo). This result demonstrated that RU was able to mislocalize the receptor only in the presence of continuous protein synthesis, indicating that its clustering effect is exerted on newly synthesized receptor and/or that it is dependent on the synthesis of other protein(s).



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Figure 3. The Effect of RU on Subcellular Localization of ER Depends on Protein Synthesis

ER was detected by H222 antibody. E2 or RU was added 24 h after transfection. a, Control; b, E2 24 h; c, RU 24 h; d, RU + CHX 24 h.

 
RU treatment did not interfere with the subcellular nuclear localization of transfected progesterone receptor that possesses a functional NLS or with the localization of nuclear-targeted Hsp90 (17) (data not shown), indicating that RU specifically acted on ER and did not interfere with the general NLS-dependent nuclear localization mechanism.

Kinetics of Appearance and Reversal of RU-Dependent ER Mislocalization
Even though the clustering effect was detectable at 10 nM RU, transfection of Cos-7 cells and RU treatment at 100 nM were used for easy detection and classification of the immunofluorescent cells, to study the kinetics of appearance (type I experiments) and reversal (type II experiments) of RU effect on ER.

In the type I experiments (Fig. 4Go), the antiestrogen was added 24 h after transfection for times varying from 1–24 h. In the type II experiments (Fig. 5Go), RU was added just after transfection, when cells were plated, and the kinetics of reversal were analyzed 24 h later for times varying from 1–24 h. For each experiment, the control is represented by the analysis of the cells 24 h after transfection and pretreatment (Fig. 4Go). At least three experiments were performed for all the studied conditions, and for each condition, 200 transfected cells were counted and classified. The subcellular receptor distribution followed four major patterns and was defined as N, when the staining was exclusively nuclear; N>C, when nuclear staining was stronger than the cytoplasmic one; N<C, when the cytoplasmic staining was stronger than the nuclear one; and C, when the staining was exclusively or almost exclusively cytoplasmic. The cytoplasmic ER immunofluorescence due to RU was always speckled and clustered. The N = C category was not considered because it represented approximately 3% of the transfected cells, and it did not significantly vary in different experimental conditions. Some speckled nuclear fluorescence of ER was also detectable at intermediary times of RU treatment (6–16 h).



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Figure 4. Kinetics of Appearance of the RU Effect in Type I Experiments

Subcellular distribution of ER was classified as N, N>C, N<C, and C. The receptor was transfected in Cos-7 cells and detected by H222 antibody. The kinetics started 24 h after pretreatment with medium: A, RU; B, RU + CHX; or after pretreatment with E2: C, RU; D, RU + CHX. For panels A and B, initial conditions were control (C) 24 h, for panels C and D, initial conditions were E2 24 h. One tick on the Y axis represents 20%.

 


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Figure 5. Kinetics of Reversal of the RU Effect in Type II Experiments

Subcellular distribution of ER was classified as N, N>C, N<C, and C. The receptor was transfected in Cos-7 cells and detected by H222 antibody. The kinetics started 24 h after pretreatment with RU. Reversal of the RU effect by : A, change of medium; B, CHX; C, RU + CHX; D, E2; E, E2 + CHX. The initial conditions were RU 24 h. One tick on the Y axis represents 20%.

 
Appearance of RU Effect
RU or RU plus CHX was added to transfected control (Fig. 4Go, A and C) or E2-pretreated (Fig. 4Go, B and D) cells. Some cytoplasmic distribution of ER appeared after 1 h of RU treatment, this effect being evident between 6 and 16 h. At 24 h, 65% of the RU-treated cells were devoid of nuclear staining. Cytoplasmic clustering of ER by RU was less efficient in cells pretreated with E2 than in control cells (only 40% of the cells were devoid of nuclear staining by 24 h) (Fig. 4CGo), due to tight binding of ER to DNA (27) and/or to ER stabilization upon E2 treatment (see below).

Both in control and E2-pretreated cells, the presence of CHX inhibited the RU-dependent cytoplasmic mislocalization of ER, which remained nuclear, indicating that the RU-induced cytoplasmic clustering of ER is strictly dependent on protein synthesis.

Reversal of RU Effect
After 24 h of RU treatment, different conditions for reversal of RU effect were used (Fig. 5Go). The RU clustering effect established during the first 24 h after transfection was found to be reversible in the presence of fresh medium, CHX, RU + CHX, E2, and E2 + CHX (Fig. 5Go, A–E). ER cytoplasmic clustering disappeared progressively during the time course of the treatment substituting the RU. The recovery to N or N>C ER localization was more efficient in short time courses in the absence of CHX, probably on account of the neosynthesis of the receptor. The experiments with E2 (Fig. 5DGo) showed a more efficient reversal to exclusive nuclear staining after 24 h (75%), even in the presence of CHX (Fig. 5EGo). The reversal of the RU effect progressively consisted of more diffuse cytoplasmic ER immunofluorescence and nuclear localization.

These results prove that the RU-dependent formation of cytoplasmic ER clusters is not an irreversible phenomenon, the receptor being able to recover, at least in part, during the RU withdrawal, the constitutive nuclear localization function (pNLS), N and N>C categories representing 20% and 60%, respectively, of the cells at 24 h in Fig. 5Go, A, B, and C.

In the presence of E2, the N category reached 80% at 24 h (Fig. 5Go, D and E), compared with 20–25% in the absence of agonist ligand (Fig. 5Go, A, B, and C). CHX did not interfere with the recovery of the normal nuclear localization of ER in the absence or presence of E2 (Fig. 5Go, B and E), even though it inhibited the establishment of RU-dependent ER cytoplasmic mislocalization (Fig. 4Go, B and D). More interestingly, in the presence of CHX, RU was unable to maintain the cytoplasmic clustering of ER synthesized in the presence of RU alone (Fig. 5CGo). Therefore, we favor the hypothesis that clustering and mislocalization of ER by RU require the synthesis of other protein(s): possibly RU provokes an abnormal receptor conformation maintained and clustered in the cytoplasm via other protein(s).

ER clusters under RU treatment, detected with the monoclonal H222 antibody directed against the LBD (28), was also recognized in fixed cells by the JS32/34 antibody (23), and by the H226 antibody (28), directed against the N-terminal region of the protein (data not shown), indicating that clustering of ER by RU did not modify at least three epitopes.

In the ERC3 cell line, the kinetics of appearance and reversal of RU-dependent cytoplasmic ER clusters were faster, the 8-h time lapse of each treatment being comparable to 16 h in Cos-7 cells. This was possibly due to a faster turnover of the receptor and/or to its expression at a lower level. Indeed, we noted that, in the presence of RU, the level of expression of ER decreased in ERC3 cells and not in Cos-7 cells on the basis of immunofluorescence studies. Western blot analyses and quantification of ER expression level under control, E2, and RU treatment demonstrated an increase of ER after E2 (Cos-7 cells: 2.7- and 1.8-fold at 16 and 24 h treatment, respectively; ERC3 cells: 2.1- and 1.2-fold at 8 and 16 h treatment, respectively, compared with the control), while a decreased expression after RU was evident only in ERC3 cells (2.2-fold decrease at 16 h treatment) (Fig. 6Go). This result, although ER is not controlled by its promoter in ERC3 and Cos-7 cells, is in agreement with previous findings showing different effects of ligands on ER stability, according to cell line (29).



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Figure 6. ER Level after E2 and RU Treatment

ER level was monitored by Western blot analysis using H222 antibody in total cellular extracts of ERC3 and Cos-7 cells (24 h after transfection) in the absence of hormone or after the indicated times of E2 (10 nM) or RU (100 nM) treatment.

 
Subcellular Partitioning of ER in the Presence of RU
The in vivo demonstration of cytoplasmic clustering of ER in the presence of RU led us to investigate its distribution in different subcellular fractions and its possible association with a particular cytoplasmic subcompartment. Cellular extracts of Cos-7 cells expressing ER were analyzed after 16 h treatment with medium alone, E2 (10 nM), and RU (100 nM) by Western blot analysis. The quality of subcellular fractions was suggested by the different pattern of Coomassie blue-stained proteins (not shown) and assessed by measuring the lactate dehydrogenase (LDH) activity that, in all experiments, was as follows: 70% in cytosol, 20% in nuclear extract, and 10% in cytosolic pellet. Cellular proteins were reproducibly partitioned: 33% in the cytosol, 46% in the nuclear extract, and 21% in the cytosolic pellet.

The scanning of the ER signal in Fig. 7Go revealed that in control cells, cytosolic ER signal was greater [integrated optical density (IOD) = 0.93] than in E2- (IOD = 0.09) or RU- (IOD = 0.17) treated cells, due to leakage from the nucleus during cell fractionation (28). Demonstration of ER nuclear retention in control cells was the result of rapid preparation of nuclear extract, the nuclear retention in E2-treated cells being due to the tight interaction of the receptor within the nucleus (27). After RU treatment, the ER signal of nuclear extract was lower (IOD = 2.33) than in control (IOD = 5.81) and E2-treated (IOD = 10.74) cells: some nuclear receptor after 16 h of RU treatment (see Fig. 4AGo) and the possible cofractionation of some ER clusters may account for this signal. The presence of ER in cytosolic pellets of control (IOD = 2.49) and E2-treated (IOD = 1.94) cells was possibly a result of overexpression; the most important signal (IOD =6.83) was found in this fraction after RU treatment, indicating that ER did lose its solubility. This fraction generally contains particulate cytoplasmic material such as plasma membrane, microsomes, lysosomes, peroxisomes, and mitochondria. Moreover, we did not notice any preferential subcellular copartitioning of the receptor with Hsp90 and Hsp70 by Western blot analysis (data not shown).



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Figure 7. In Vitro Subcellular Localization of ER in Transfected Cos-7 Cells

Cytosols, nuclear extracts, and cytosolic pellets were analyzed by Western blot and ER was revealed by the H222 antibody. E2 or RU was added 24 h after transfection. Control experiments (C), E2 treatment 16 h (E2) and RU treatment 16 h (RU).

 
To determine whether ER clustered in the presence of RU was localized in specific cytoplasmic structures, we used antibodies directed against proteins or enzymes specific for different organelles and cellular compartment for indirect immunofluorescence. RU treatment was performed at 100 nM as usual, and at 10 nM, to avoid the formation of large clusters of ER that could mask the internal structure of the cell.

Coimmunolocalization of RU-dependent clusters and Grp78/94, specifically localized in endoplasmic reticulum and Golgi apparatus, or catalase, localized in peroxisomes, or Grp75, a specific mitochondrial marker, or cathepsin D, which is found in lysosomes, was investigated by double indirect immunofluorescence. A low level of colocalization was found with endoplasmic reticulum or peroxisome markers using scatter diagram analysis (data not shown), but it was only observed in a few percent of transfected RU-treated cells and not considered as significant. Lysosomal and mitochondrial fluorescence was completely independent from ER clusters fluorescence (data not shown). In addition, we tested the possible association between clustered ER and Hsp70. As with Hsp90, we did not find any preferential colocalization of the two proteins using the scatter diagrams (data not shown).

In parallel, using immunogold labeling, electron microscopy studies were carried out on transfected control Cos-7 cells and after 16 h of E2 or RU treatment. In control experiments, immunogold decoration was found predominantly distributed in the nucleus (Fig. 8aGo), as after E2 treatment (Fig. 8bGo). The treatment with RU during 16 h led to immunogold labeling of cytoplasmic clusters found near the cytoplasmic side of nuclear pores (Fig. 8Go, c and d) and in cytoplasmic regions rich in ribosomes and polyribosomes (Fig. 8Go, e and f). Despite the dramatic decrease of nuclear decoration, some clustering of ER by RU was also found in the nucleus.



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Figure 8. Electron Microscopic Analysis of the Subcellular Localization of ER in Transfected Cos-7 Cells

The receptor was revealed by the H222 antibody and a gold-conjugated secondary antibody. a, Control; b, E2 treatment 16 h; c–f, RU treatment 16 h. Magnification, 24 x 103.

 
Using biochemical and immunological approaches, RU-dependent ER clustering was not found associated to a specific subcellular compartment and was interpreted as an aggregation-prone insoluble ER conformation.

The LBD of ER Is Sufficient for RU-Dependent Cytoplasmic Clustering
In transfected Cos-7 cells or in the ERC3 cell line, the treatment with the antiestrogen RU elicited the mislocalization and the clustering of ER in the cytoplasm (Fig. 1Go, f and h, and Fig. 2Go f), indicating a loss of constitutive and ligand-dependent nuclear localization functions.

To analyze the involvement of different domains of the ER in the cytoplasmic clustering due to RU, we transfected Cos-7 cells with two ER mutants. HE255G, a mutant deleted from amino acids (aa) 1–274, contains part of the hinge region and the LBD with the p-NLS1 located between the two regions (aa 299–303). HE241G is a mutant deleted from aa 250–303, a region containing pNLS1, pNLS2, and pNLS3. Both mutants are unable to be efficiently nuclear localized, in the absence and presence of E2 (5). As shown in Fig. 9Go, in the absence of hormone, HE255G (Fig. 9aGo) and HE241G (Fig. 9bGo) were localized in the nucleus and in the cytoplasm, showing a homogeneous staining. For both mutants, the addition of RU (100 nM) led to the formation of cytoplasmic clusters and to a disappearance of the nuclear staining (Fig. 9Go, c and d), indicating that the LBD alone bound to RU was sufficient for the observed clustering while the N-terminal portion of the receptor encompassing the AF-1 region plus the entire DNA-binding domain was dispensable. The constitutive nuclear localization function contained in the aa 250–303 region, although completely repressed by RU binding, was not necessary to determine ER mislocalization.



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Figure 9. The RU Effect on ER Mutants

Subcellular localization of HE255G (a and c) and HE241G (b and d). Mutants were transfected in Cos-7 cells and detected by H222 antibody. RU was added 24 h after transfection. a and b, Control; c and d, RU 24 h.

 
RU Inhibits the E2-Induced Transcriptional Activation of a Reporter Gene
Finally, the antiestrogenic properties of RU were tested on the reporter gene ERE-TK-LUC (30) transfected in CHO cells together with the ER expression plasmid HEGO (5). As shown in Fig. 10Go, RU alone, up to 100 nM, was unable to elicite transcriptional activation and, when associated with E2, completely inhibited the E2-induced response.



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Figure 10. The RU Effect on the Estrogen-Induced Transcriptional Activation

CHO cells were transfected with the reporter gene ERE-TK-LUC in the presence of HEGO and immediatly treated with medium, E2, RU, or E2 + RU during 48 h. The figure represents the mean of four independent experiments. The activation by E2 was considered as 100% of transcriptional activation.

 
Because in the reported experiment, virtually no ER is present in the nucleus due to the addition of RU from the beginning of the transfection, it is worth noting that the same lack of agonistic effect and the same extent of antiestrogenic effect were found when RU, E2, or both were given 24 h after transfection (data not shown). In particular, the subcellular localization of ER in the presence of 10 nM E2 plus 100 nM RU was similar to that observed with RU alone.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have shown at the whole cell level that the interaction between Hsp90 and ER was disrupted in the presence of agonistic and antagonistic ligands and that a new pure antiestrogen, RU 58668, inhibited ER nuclear import by clustering the receptor into the cytoplasm in a protein synthesis-dependent manner.

ER Ligands Abolish ER-Hsp90 Interaction
Here we have extended a previous description of the in vivo Hsp90-ER interaction, based on nuclear cotranslocation assay of the two proteins overexpressed in Cos-7 cells (15), to a CHO-derived cell line, ERC3, stably expressing ER.

Moreover, we found that the Hsp90 nuclear localization, due to its interaction with the karyophilic ER, decreased in the presence of the agonist ligand, E2, as well as in the presence of tamoxifen, a mixed agonist-antagonist, and RU, a fully antiestrogenic compound. All ER ligands tested allowed a diffuse cytoplasmic relocalization of transfected or endogenous Hsp90.

Both the ER-dependent nuclear translocation of Hsp90 in the absence of hormone and Hsp90 exit from the nucleus when ER is liganded and tightly bound to the nuclear structure are insensitive to CHX. It is worth noting that the hormone-independent, NLS-based, and the hormone-dependent nuclear localization functions of nuclear steroid receptors, as well as their nucleo-cytoplasmic shuttling, are also insensitive to CHX (6, 31, 32).

With respect to Hsp90 nuclear cotranslocation with unliganded ER, a piggy-back transport by the karyophilic interacting protein is the most plausible explanation, even though Hsp90 itself may possess a weak or regulated nuclear localization function (15). Exit of Hsp90 from the nucleus as a consequence of its dissociation from liganded ER seems not to be a rapid phenomenon (8–12 h are needed to visualize a decrease in the nuclear fluorescence in 80% of the cells). By contrast, the exit of nuclear receptors from the nucleus, in the presence or absence of hormone, needs only 3–4 h in order to be documented in experiments with heterokaryons (32).

The NLS-based mechanism of nuclear ER localization, involving {alpha}- and ß-importins and Ran GTP/GDP cycle, is, to a large degree, established and may account for some coimport of Hsp90. However, steps intervening in the exit of ER from the nucleus, a prerequisite for nucleo-cytoplasmic shuttling, are largely unknown (32, 33, 34).

We suggest therefore that in the presence of ligand, the dynamic association between Hsp90 and ER being shifted toward a dissociated status, the export of Hsp90 from the nucleus, is mostly independent of that of ER. Whether Hsp90 possesses nuclear export signals (33) remains to be determined.

RU Induces Cytoplasmic Clustering of ER
When the pure antiestrogen RU was used, the subcellular distribution of ER was profoundly modified in a dose- and time-dependent manner. ER immunolocalization was found to be cytoplasmic and speckled, clusters of specific immunofluorescence being found in large perinuclear regions without significant colocalization with Hsp90 and Hsp70. The localization of ER under RU treatment was similar to that described after treatment with two pure antiestrogens, ICI 164384 and ICI 182780 (6, 19, 35). Probably due to a lack of time course experiments, the predominantly cytoplasmic clustered localization of ER was observed only in a few percent of cells (4.5%) after 1 h of ICI 184780 treatment (6). Similarly, a short time and a low dose of antiestrogen treatment also failed to provide evidence for the ICI 164384 effect on the subcellular mislocalization of ER (5). The overall effect of RU was a dose- and time-dependent accumulation of ER in cytoplasmic clusters concomitant with a progressive nuclear depletion of ER, indicating the inefficiency of the constitutive and ligand-dependent nuclear localization functions of ER under RU treatment.

The kinetics of nuclear depletion of ER under RU seems to be the result of a blocked nuclear import, which may be concomitant with a diminished ER steady state level according to cell line. It will be of interest to perform experiments to look for a lack of functional interaction between the NLS of ER and importin {alpha} under RU treatment and to elucidate how the hormone-dependent nuclear localization function located in the LBD of ER (5) is disrupted. The finding that some transcription intermediary factors (TIFs) or coactivators interacting with the AF-2 domain of the LBD in an agonist-dependent or -enhanced manner, possess functional NLSs and are able, according to their intracellular levels, to bring mutated "cytoplasmic" receptors to the nucleus, gave some explanation for the hormone-dependent nuclear localization function of the LBD and for the inactivation of this function by RU (36, 37, 38, 39).

The ER mislocalization was reversed by RU withdrawal, and, more efficiently, by 10 nM E2 treatment. Surprisingly, the cytoplasmic clustering and mislocalization of ER by RU was inhibited in the presence of CHX, suggesting that this RU effect was dependent on ongoing protein synthesis, possibly the synthesis of ER itself. However and most importantly, once the mislocalization of ER was established by RU, its reversal by CHX, even in the continuous presence of RU, strongly indicates the participation of protein(s) turning over faster than ER, in the maintenance of RU-dependent ER mislocalization. Such protein(s) may interact with ER in a RU-dependent or -enhanced manner.

Because a similar subcellular mislocalization of ER under ICI 164384 and 184780 treatment has been reported (6, 19), we suspect that RU and both ICI antiestrogens act in a similar way. Indeed, the ICI and RU antiestrogens possess a long side chain at the 7{alpha} and 11ß positions, respectively, that confers a chiral symmetry between the two steroidal structures (6, 21, 22). However, in contrast to the indication of an impeded nucleo-cytoplasmic shuttling of ER in heterokarions in the presence of ICI 184780 plus CHX (6), our experiments clearly demonstrated that the nuclear import is restored in the presence of RU plus CHX, pointing to a protein synthesis-dependent step as a crucial event in the RU mechanism.

By testing for the ER domain responsible for the RU-dependent mislocalization, we found that the LBD alone was sufficient to observe ER cytoplasmic clustering and that the region bearing the constitutive NLS was dispensable.

The fact that a certain mutation in the AF-2 domain (H12 helix of ER) reverses the antiestrogenic properties of ICI 164384 to estrogenic properties (19, 40) is in agreement with the importance of this region in the functional and physical interaction with coactivators (37). The binding of antiestrogens seems to preclude these interactions (37, 38, 39, 41, 42). RU, in particular, may promote or enhance the interaction of the LBD, possibly the H12 helix, with a rapidly turning over protein that sequesters ER out of the nucleus.

Protein(s) Responsible for ER Clustering: a New Step in Pure Antiestrogen Action
We propose that conformational changes in the LBD of ER provoked by RU binding may include 1) incorrect positioning of the H12 helix of the LBD, precluding interaction with transcription intermediary factors that may also be involved in the hormone-dependent nuclear localization of the receptor; 2) disruption of hormone-dependent as well as constitutive nuclear localization functions of ER, thus impeding the access of the receptor to specific ERE sequences; 3) interaction of the LBD with protein(s) responsible for ER clustering. It is not excluded that such a protein may interact transiently with ER in the nucleus, in particular when RU was reversing the natural nuclear localization of ER.

The sequence and the interdependency of these events cannot be inferred on the basis of the results presented here, opening the way to further investigations and to a new hypothesis to be tested in addition to the already proposed mechanisms for antiestrogens based on accelerated receptor degradation (35), inhibition of dimerization (43), and of interaction with coactivators (36, 37, 38). Interestingly, ER clustering by RU, concomitant to a decreased immunoreactivity, can be detected in target tissues such as uterus or MCF-7 derived tumors (F. Delahaye, unpublished observations).

Most importantly, because ER clustering seems to be the major determinant of the biocharacter of the antagonist, this may suggest rapid screening strategies for antiestrogenic and antitumoral properties of synthetic and natural compounds relevant for an overall long-term antiestrogenic effect required in cancer therapy. Such a screening may be complementary to testing for inhibition of ER AF-1 and AF-2 activity by antiestrogens.

We can indeed predict that antiestrogens inducing ER clustering, in addition to being pure antiestrogens, will also be effective in inhibiting the estrogen-independent transcription activation and growth promotion by ER (44) and, more generally, all effects of ER at nuclear level.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estradiol, tamoxifen, and RU58668 were kindly provided by Roussel UCLAF (Romainville, France) and ICI 164384 was from ICI Pharmaceuticals (Macclesfield, U.K.).

Plasmids
Wild-type ER (HEGO), and mutants HE255G and HE241G cDNA cloned in the expression vector pSG5 (Stratagene, La Jolla, CA) have been previously described (5).

To obtain the highest coexpression of ER and Hsp90, the SalI-SalI fragment from pSG5 HEGO containing ER cDNA was subcloned into the plasmid pSVK3 90 containing the Hsp90 cDNA (17) to give the plasmid pSVK3 90-HEGO. In this construct, the cDNAs of Hsp90 and ER are under the control of separate promoters.

Plasmid ERE-TK-LUC containing ERE with the reporter gene luciferase has been described previously (30).

Cell Culture and DNA Transfection
Cos-7 cells were grown in DMEM (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FCS (Life Technologies, Gaithersburg, MD). For transient transfection, DMEM without phenol red (Sigma) was supplemented with 10% of twice charcoal-stripped FCS.

DNA transfections were performed using an Easy Ject Plus electroporator (Eurogentec, Seraing, Belgium) under the following conditions: voltage = 230 V and capacity = 1650 µFarads. The resulting pulse times varied between 30 and 35 msec.

The ERC3 cell line was a generous gift from P. Kushner (24). Cells were cultured in Ham’s F-12/DMEM (1:1) without phenol red (Sigma). The medium was supplemented with 10% of iron-supplemented newborn calf serum (Sigma).

Transactivation Assays
CHO cells were grown in Ham’s F12/DMEM (1:1) (Sigma). Transfections were performed in the same medium, without phenol red and with 10% of twice charcoal-stripped FCS (Life Technologies), by electroporation using 320 V and 1800 µFarads. The resulting pulse times varied between 35 and 40 msec. For each dish (6 cm diameter), 150 ng HEGO, 1 µg ERE-TK-LUC, 3 µg pCH110 (ß-Gal internal standard plasmid), and 10 µg salmon sperm DNA were transfected. Each experimental point was done in triplicate. Hormone or anti-hormone was added when cells were plated just after electroporation and for 48 h. Cells were harvested and lysed to perform ß-Gal and luciferase assays as previously described (45).

Antibodies and Indirect Immunofluorescence
Indirect simple and double immunofluorescence studies were performed as described (15, 17). Monoclonal rat BF4 antiavian Hsp90 antibody (7), which did not recognize endogenous Cos-7 cells Hsp90, was used. The anti-Hsp90 polyclonal antibody used to detect endogenous Hsp90 in ERC3 cell line was a generous gift from Y. Miyata (26).

Human ER and the mutants HE255G and HE241G were revealed with rat H222 or H226 monoclonal antibodies (25) or with mouse JS 34–32 monoclonal antibody (23).

Confocal Laser Scanning Microscopy
Analyses were performed at Service de Microscopie Confocale de l’IFR 21 (Hormones et Génétique, Kremlin-Bicêtre, France) using a Zeiss Axiovert 135 M with 63/1.4 and 100/1.4 Oil plan-Apochromat objectives (Carl Zeiss, Iena, Germany). Selected images were treated by the Zeiss LSM 410 confocal microscopy system with software version 3.92 beta. An AR laser (exitation wave length 488 nm) was used for fluorescein isothiocyanate (FITC) detection and a HeNe laser (excitation wave length, 543 nm) for tetramethyl rhodamine isothiocyonate (TRITC) detection. Double staining microfluorometry analysis was performed as previously described (46).

Preparation of Cytosols and Nuclear Extracts, Electrophoresis, and Western Blot Analysis
Cytosols and nuclear extracts of transfected Cos-7 cells were prepared as follows: cells were harvested 48 h after transfection and centrifuged, and the pellet was resuspended in a hypotonic buffer (20 mM Tris-HCl, pH 7.4; 15% glycerol; 1 mM EDTA; 30 mM natrium molybdate) containing protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride; 5 µg/ml leupeptin; 5 µg/ml antipaïn; 5 µg/ml aprotinin; and 1 mM dithioerythritol). The cells were transferred to a glass Dounce homogenizer and homogenized with 40 up-and-down strokes using a type B pestle. The homogenate (total cell extract) was centrifuged 10 min at 800 x g. The supernatant was centrifuged 45 min at 100,000 x g to obtain the cytosolic fraction. The 100,000 x g pellet was resuspended in the cytosol buffer. The 800 x g nuclear pellet was washed and resuspended in high ionic strength buffer (20 mM HEPES, pH 7.9; 20% glycerol; 0.55 M KCl; 1.5 mM MgCl2; 0.2 mM EDTA; 2 mM dithiothreitol) containing protease inhibitors as described above. Nuclei were lysed in a glass Dounce homogenizer with 10 up-and-down strokes using a type B pestle, and then centrifuged 30 min at 15,000 x g. The supernatant was kept as nuclear extract.

LDH was assayed in all fractions using the kit Enzyline LDH/HBDH (bio Mérieux, Marcy l’Etoile, France).

The cytosols, the resuspended 100,000 x g pellets, and the nuclear extracts (40 µg of each fraction) were directly submitted to 10% SDS-PAGE analysis followed by Western blot.

Human ER was then detected with the monoclonal rat H222 antibody and avidin-biotin Vectastain reagents kit (Vector Laboratories Burlingame, CA), with revelation by enhanced chemiluminescence (ECL) reactions (Amersham, Arlington Heights, IL). The signal corresponding to ER was analyzed utilzing BioImage Software on BioImage Station (Millipore, Bedford, MA) and was expressed as IOD. The range of IODs between 0.01 and 10 was linear when using increasing amounts of loaded proteins.

Preparation of Total Extracts
After transfection cells were harvested and collected by centrifugation. The pellet was frozen at -80 C and then resuspended in high salt buffer (25 mM Tris/HPO4, pH 7.8; 0.55 M KCl; 10 mM MgCl2; 1% Triton X-100; 15% glycerol; 1 mM EDTA; 1 mM dithiothreitol), vortexed, and passed five times through a needle. The suspensions were then centrifuged 15 min at 15,000 x g to remove debris. The supernatants, called total extracts, were submitted to 10% SDS-PAGE (50 µg of proteins), and the signals were analyzed as described above.

Electron Microscopy
Cells were fixed in situ sequentially by glutaraldehyde and osmic acid (47). Thin sections of 50 nM were cut tangently to the cell layer with an Ultratome III (LKB) and mounted on 200-mesh nickel grids coated with parlodion film. They were pretreated with saturated sodium metaperiodate (Merck) to restore antigenicity of osmium fixed tissues (47) and washed thoroughly in distilled water before processing for immunological labeling. Sections were first incubated with 3% normal goat serum in PBS to reduce background. The grids were floated on a drop of monoclonal rat H222 antibody at a final concentration of 5 µg/ml, then incubated with gold-conjugated secondary antibody (Biocell), washed in PBS containing 2% Tween, floated on 2% glutaraldehyde and finally stained with alcoholic uranyl acetate and Reynolds reagent. A control test was made by substitution of the primary antibody against ER by an irrelevant one or by PBS. Grids were analyzed on a Siemens Elmiskop 101 at 80 KV.


    ACKNOWLEDGMENTS
 
We thank G. Geraud (Service de Microscopie Confocale, Institut Jacques Monod, Université de Paris VII, France) for assistance in preliminary experiments; M.C. Lebeau, K. Rajkowski, D. Philibert, P. Van de Velde and J. Bremaud for helpful suggestions and discussion ; L. Outin and J.C. Lambert for figures preparation. We also thank G. Greene and B. Moncharmont for providing ER monoclonal antibodies, P.J. Kushner for ERC3 cell line, the laboratory of P. Chambon for plasmids containing ER and ER mutants cDNA and Roussel UCLAF for providing estradiol and antiestrogens.


    FOOTNOTES
 
Address requests for reprints to: Maria-Grazia Catelli, Lab Hormones, Institut National de la Sante et de la Recherche Medicale U33, 80 rue du General Leclerc, 94276 Le Kremlin Bicetre, Cedex France.

This work was supported by Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche sur le Cancer, Ligue contre le Cancer, Fondation pour la Recherche Médicale and Ministère de la Défense (Direction de la Recherche et de la Technologie). X. Meng is a fellow of the Lalor Foundation.

Received for publication August 13, 1997. Revision received February 18, 1998. Accepted for publication February 20, 1998.


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