Ligand-specific regulation of proteasome-mediated proteolysis of estrogen receptor-alpha

Mara T. Preisler-Mashek, Natalia Solodin, Bethany L. Stark, Michael K. Tyriver, and Elaine T. Alarid

Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteasome-mediated proteolysis modulates the cellular concentration of estrogen receptor-alpha (ERalpha ) and is induced by treatment of cells with 17beta -estradiol. Herein, we show that multiple receptor agonists, including 17alpha -estradiol and estriol as well as the antagonist ICI-182780, stimulate proteasome-dependent proteolysis of ERalpha in a process that requires ligand binding to the receptor. Proteolysis of receptor depends on ligand concentration, and there exists a direct correlation between ligand-binding affinity and the half-maximal dose of ligand required to stimulate receptor degradation. Furthermore, introduction of a point mutation into the receptor ligand-binding pocket yields a stable receptor resistant to proteolysis. Interestingly, although all ligands stimulate receptor degradation, the extent to which overall ER levels are affected varies with each ligand and is not related to ligand-binding affinity or activation of transcription. These results demonstrate ligand-specific regulation of ERalpha proteolysis, and they introduce the concept that cellular receptor concentration is governed not only at the level of induction of proteolysis but also by the efficiency with which the receptor is degraded.

steroid; nuclear receptor; antagonist; estriol; pituitary


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CELLULAR ESTROGEN RECEPTOR LEVELS are dynamic and are particularly sensitive to changes in circulating levels of 17beta -estradiol. It has been demonstrated through a number of studies that the decline in estrogen receptor-alpha (ERalpha ) upon exposure to 17beta -estradiol results from a combination of mechanisms that control both receptor synthesis and degradation through transcriptional, posttranscriptional, and posttranslational mechanisms (20, 27, 32, 33, 35, 36). The most rapid of these regulatory mechanisms is the direct loss of ERalpha protein brought about by the induction of proteasome-mediated proteolysis (1, 21, 28).

Regulated proteolysis by proteasomes accounts for the turnover of most short-lived proteins, including many nuclear receptors (9, 16, 23, 29, 41, 43, 45). Through a series of three enzymatic reactions, ubiquitin moieties are attached to a protein substrate, which targets it to the 26S proteasome. The molecular events that direct ERalpha into this pathway have not been clearly established. However, earlier studies that examine changes in receptor-binding capacity have shown that receptor levels can be controlled by both receptor agonists and antagonists, suggesting the possibility that receptor occupation by ligand may provide specificity (3, 4, 15, 18, 22, 33).

In our original report of ERalpha protein regulation by proteolysis, we utilized a pituitary lactotrope model system, the PR1 cell line. The lactotrope cell population of the anterior pituitary is a major target of estrogen action. Animals that lack ERalpha show a decrease in lactotrope cell density and prolactin expression (37). In contrast, animals treated with estrogen for prolonged periods develop hyperplastic pituitaries comprised primarily of lactotrope cells. The F344 rat model is a classic system for the study of estrogen regulation of lactotrope growth and function (11, 44). The PR1 cell line is an in vitro correlate of this model that was derived from an estrogen-induced pituitary hyperplasia in F344 rats (30). Using this lactotrope cell line, we showed that estrogen shortened the half-life of ERalpha protein in the pituitary from >3 h to 1 h, which translated into a decrease in steady-state levels of ERalpha protein within 2 h of ligand exposure (1). During this time frame, there was no measurable change in ERalpha mRNA levels. We have capitalized on the identification of a time window in which proteolysis alone is responsible for changes in ER protein to further examine the regulation of proteasome-dependent degradation of endogenous ERalpha .

Herein, we demonstrate that proteasome-dependent proteolysis of ERalpha can be induced by multiple estrogens, including short-acting estrogens and the pure antiestrogen ICI-182780 (ICI). This response requires the direct binding of the various ligands to receptor, since introduction of a point mutation in a critical residue of the ERalpha ligand-binding pocket yields a receptor resistant to degradation. In addition, we find that binding affinity of ligand for receptor correlates with the potency with which ligand can induce proteolysis, further supporting a critical role for ligand binding in regulating receptor proteolysis. Interestingly, through quantitative assessment of changes in receptor level at varying concentrations of ligand, we observed that the overall decline in receptor levels achieved by proteolysis varies depending on the ligand that occupies the receptor. These results indicate that ligands can differentially regulate receptor proteolysis, and they suggest the possibility that cellular ER concentration is controlled by not only the induction of proteolysis but also its efficiency.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell culture. PR1 lactotrope and HEK 293 cells were maintained under standard conditions of humidity and temperature in high-glucose DMEM medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD).

Steroid stimulation. For experiments involving steroid treatment, cells were grown in either Optimem medium (Life Technologies) or phenol red-free DMEM with 10% charcoal-stripped serum (34) and antibiotics. Cells were maintained in medium lacking steroid for a minimum of 3 days before treatment. Steroid stimulation proceeded for 2 h as previously described (1) unless otherwise indicated. The final concentration of ethanol (EtOH) in control and treated samples was 0.1%. 17beta -Estradiol and 4-hydroxytamoxifen (4-OHTam) were purchased from Sigma Chemical (St. Louis, MO). 17alpha -Estradiol and estriol were purchased from Steraloids (Newport, RI). The antiestrogen ICI was a gift from Dr. Jack Gorski. In experiments utilizing the proteasome inhibitor ALLnL (Calbiochem, La Jolla, CA), samples were pretreated for 30 min with inhibitor before the addition of steroid.

Western blot analysis. Western blot analysis was performed on whole cell extracts that were obtained by either direct dissolution of cell pellets in 2× sample buffer (125 mM Tris-base, 20% glycerol, 4% SDS, 10% beta -mercaptoethanol, and bromphenol blue, pH 6.8) or by extraction in Totex buffer (19) followed by Bradford assay (5). Proteins were separated on a 7.5% acrylamide gel. Immunoblotting was performed as previously described (1), utilizing anti-rat ER (13) or anti-human ER (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies as appropriate. Saturating concentrations of antibody were used in all cases to allow quantitative analysis of relative protein levels. Equivalent loading of lanes was verified by reprobing blots with antibody against alpha -tubulin (Calbiochem) or beta -actin (Santa Cruz Biotechnology). Secondary antibodies were conjugated either to horseradish peroxidase or 125I for visualization and quantification, respectively. Receptor levels were determined by phosphoimager analysis by use of Imagequant software (Molecular Dynamics, Sunnyvale, CA) or laser densitometry. Where densitometry was employed, an internal standard curve was generated for each blot. The EtOH-treated control was arbitrarily set at 100, and a series of dilutions of this control were run in parallel to generate the standard curve for the determination of receptor levels relative to the EtOH-treated control. Each experiment was repeated a minimum of three times to ensure reproducibility. The EC50 for the downregulation response for each ligand was calculated on the basis of nonlinear regression analysis (Prism 3.0; GraphPad Software, San Diego, CA). Statistical significance of differences between treatment groups was determined by one-way ANOVA followed by t-test analysis, with a 95% confidence interval (Microcal Origin, Microcal Software, Northampton, MA).

Transient transfection. Transfections were performed utilizing Fugene reagent (Roche Molecular Biochemicals, Indianapolis, IN) or calcium phosphate precipitation in pituitary and 293 cells, respectively. To assess transcriptional activity of ERalpha , cells were transfected with either an ERE-tk-Luc reporter, which encodes a multimerized vitellogenin estrogen-response-element (ERE) enhancer fused to a thymidine kinase (tk) promoter (42), or a Prl-luc construct bearing 2.5 kb of the Prl regulatory region. The Prl-Luc construct was obtained from Dr. Richard Maurer. In addition, cells were cotransfected with a beta -galactosidase reporter (CMV-beta gal) to control for transfection efficiency. Cells were treated 24 h after transfection with EtOH or the indicated estrogen and were harvested after an additional 24 h. Assays for beta -galactosidase (Tropix, Bedford, MA) and luciferase activity (Promega, Madison, WI) were performed as directed by the manufacturers. Luciferase activity was normalized to beta -galactosidase activity, and degree of activation was determined relative to the ethanol control.

Generation of cell lines expressing wild-type and mutant estrogen receptors. Stable 293 cell lines were generated using calcium phosphate transfection of retroviral expression vector LHL-CA (26) containing wild-type (WT) and mutant ERs. The G521R-ERalpha was generated by PCR mutagenesis, in which primers encoding the mutation (5'-AGTAACAAACGCATG-GAGCATCTGTACAGC-3') were used in two reactions, generating ER fragments between the XbaI site and the mutation and between the mutation and a region downstream of the ER stop codon that contained a BstEII site. The two reactions were used as a template in a third reaction to create a final fragment that was then digested with XbaI and BstEII and subcloned back into an ER-encoding vector. The resultant mutant was verified by sequencing and then subcloned into the LHL-CA expression vector. This vector contains a Moloney murine leukemia viral LTR driving the expression of a gene-encoding hygromycin resistance. Expression of WT and G521R ER is controlled by a CMV enhancer and actin promoter. The LHL-CA vector contains no viral coding regions. Selection of cell colonies with hygromycin B (200 µg/ml) began 24 h after transfection. Colonies were isolated and screened for ER expression by Western blot analysis by use of anti-human ER. All results obtained after experimental treatment were confirmed in multiple clones.

Binding assay. Whole cell binding assays were performed as previously described (1) by use of 10 pM [3H]estradiol (New England Nuclear/Du Pont, Boston, MA) on the basis of the reported disassociation constant of ERalpha in PR1 cells (7). All assays were performed in the presence of ALLnL to prevent the proteolysis of receptor that occurs during establishment of equilibrium conditions. For competition, cells were incubated with increasing concentrations of 17beta -estradiol, 17alpha -estradiol, estriol, or ICI that ranged from 1 pM to 1 µM in log increments. Steroid binding proceeded for 2 h. Assays were performed with duplicate samples and were repeated twice. (Interassay variation was neglible and is included in Fig. 4 but is hidden by the data symbols.) EC50 values were determined by nonlinear regression analysis based on single-site competition (Prism 3.0; GraphPad Software).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Initial experiments were performed to assess the specificity of inducible proteolysis of ERalpha . PR1 cells were exposed for a brief period of 2 h to various steroids, including testosterone, progesterone, cortisol, and estrogens (17beta -estradiol, 17alpha -estradiol, and estriol). In addition, cells were treated with the receptor antagonists 4-OHTam and ICI. After treatment, changes in ERalpha levels were evaluated by Western blot analysis of whole cell lysates. Shown in Fig. 1, treatment of cells with 17beta -estradiol resulted in a decline in steady-state levels of ERalpha protein (10). The acute loss of receptor protein could also be induced by treatment with other receptor ligands, including 17alpha -estradiol and estriol, but not by steroids such as testosterone, progesterone, and cortisol that do not bind specifically to ERalpha (Fig. 1A and Table 1). Unlike agonists, ERalpha antagonists showed differential activity in regulating receptor protein levels. ICI was similar to estrogens in its ability to decrease receptor levels (Fig. 1B and Table 1). However, the partial antagonist 4-OHTam was without effect. These results indicate that downregulation of ERalpha may be restricted to specific ligands of the receptor (Fig. 1A).


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 1.   Induction of estrogen receptor-alpha (ER) protein degradation by estrogens and antiestrogens. Representative Western blots showing ERalpha levels in whole cell lysates from PR1 cells treated with ERalpha agonists and antagonists. Blots were probed with saturating concentrations of anti-rER for ERalpha and anti-alpha -tubulin or anti-actin for loading controls. A: ERalpha levels after a 2-h treatment with a 10 nM dose of the indicated steroid hormone: testosterone (T), progesterone (P), cortisol (F), estriol (E3), 17alpha -estradiol (17alpha -E2), or 17beta -estradiol (17beta -E2). Ethanol (EtOH) treatment served as a control. B: similarly, ERalpha levels were monitored after a 2-h treatment with a 10 nM dose of antiestrogens ICI-182780 (ICI) or 4-hydroxytamoxifen (4-OHTam).


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Specificity of ERalpha downregulation

The involvement of proteasomes in the degradation of ERalpha induced by 17alpha -estradiol, estriol, and ICI was tested using an inhibitor of the proteasome pathway, ALLnL. Proteasome activity was blocked by a 30-min preincubation of cells with proteasome inhibitor. In parallel, a control group was pretreated with DMSO, the inhibitor solvent. After inactivation of proteasomes, cells were treated with estrogens and ICI as before. As shown previously, treatment of cells with estrogen and ICI resulted in a significant decline in receptor level. The loss of receptor was effectively inhibited by ALLnL (Fig. 2A) and a second inhibitor, lactacystin (data not shown). Examination of the relative receptor levels illustrated in Fig. 2C demonstrates that ALLnL stabilized the receptor level to that of control cells and completely prevented the rapid loss of receptor due to steroid treatment. ALLnL was also effective at blocking the actions of 17beta -estradiol, 17alpha -estradiol, and estriol (Fig. 2B). In Fig. 2B, it can be observed that inclusion of the proteasome inhibitor alone can slightly increase basal levels of the receptor. However, upon quantification of relative ER levels by phosphoimager analysis, this apparent increase was not statistically significant.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Ligand-activated ERalpha degradation is proteasome dependent. A: a representative Western blot of ERalpha levels performed, as described in Fig. 1, on whole cell extracts of PR1 cells pretreated with 50 µM ALLnL or DMSO followed by a 2-h treatment with EtOH, 10 nM 17beta -E2, or 10 nM ICI. B: similarly, pretreated PR1 cells were stimulated with 10 nM E3, 17alpha -E2, 17beta -E2, or EtOH, and Western analysis was performed as in A. C: changes in relative ER levels were quantified by phosphoimager analysis of Western blots that were probed with 125I-conjugated secondary antibody. Relative ER levels were normalized to vehicle-treated controls (DMSO/EtOH). Values are means ± SE for >= 4 trials. Statistical significance between treated and control groups was determined by 1-way ANOVA and t-test. * P < 0.05.

The limitation of this proteolytic response to ligands of ERalpha suggested that ligand binding to receptor is an essential first step in directing the receptor to the proteasome pathway. To directly test this possibility, stable cell lines were generated that express either a WT-ERalpha or an ERalpha ligand-binding point mutant (G521R-ERalpha ). HEK 293 cells were transfected with a retroviral expression vector that encodes hygromycin resistance and either WT-ERalpha or G521R-ERalpha . Stable colonies were selected after maintenance in hygromycin-containing medium and screened for the expression of ERalpha by Western blot. Multiple clones were identified for each line, and a representative line was selected for further analysis. The receptor transcriptional activity in the WT and G521R cell lines was confirmed by transient transfection of an estrogen-responsive reporter construct (ERE-tk-LUC). Shown in Fig. 3A, cells that express WT-ERalpha exhibit increases in reporter gene activity in response to 17beta -estradiol, 17alpha -estradiol, and estriol. In line with its antagonistic activity, ICI inhibited transcription, confirming the specificity of the transcriptional response. In contrast, cells expressing G521R-ERalpha did not exhibit ligand-dependent changes in transcription activity (Fig. 3B). These results are consistent with the original characterizations of this mutation in mouse and human ERalpha (12). To examine the effect of introduction of this mutation on estrogen-induced proteolysis, cells were treated with 17beta -estradiol, 17alpha -estradiol, estriol, or ICI for 2 h and analyzed for changes in ERalpha protein levels. Figure 3C shows that cells expressing WT-ERalpha respond to estrogens like PR1 cells by decreasing steady-state levels of ERalpha protein. Thus estrogen-induced proteolysis is faithfully reconstituted in this heterologous system. These cells, however, show a heightened sensitivity to ICI such that, within 2 h, WT-ERalpha is barely detectable. G521R-ERalpha levels, however, were unchanged by stimulation with either agonists or ICI (Fig. 3). These results demonstrate that ligand binding is essential in the induction of ERalpha proteolysis.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3.   Estrogen-induced ERalpha degradation requires ligand binding. HEK 293 cell lines were stably transfected with a retroviral expression vector encoding wild-type (WT-ERalpha ) or mutant G521R (G521R-ERalpha ) ERalpha . Transcriptional activity of WT-ERalpha (A) and G521R-ERalpha (B) was assessed by transient transfection of an ERE-tk-Luc reporter plasmid followed by a 24-h treatment with the indicated estrogen. Luciferase activity was normalized to beta -galactosidase (beta -gal) activity to control for transfection efficiency. ER-expressing cells were treated with 10 nM ICI, E3, 17alpha -E2, and 17beta -E2 for 2 h, and steady-state levels of the WT-ERalpha (C) or G521R-ERalpha (D) were examined by Western analysis of whole cell lysates, as previously described. Blots were reprobed with anti-alpha -tubulin or anti-actin to control for loading. Results were verified in multiple independent experiments and clones.

17beta -Estradiol, 17alpha -estradiol, estriol, and ICI bind to ERalpha with varying affinities. To further examine the relationship between ligand binding and induction of proteolysis, PR1 cells were treated with different doses of estrogens ranging from 10-12 to 10-6 M (Fig. 4, top). Changes in ER levels were determined by quantification of receptor levels relative to an internal standard curve generated by dilution of extract from ethanol-treated control cells. For direct comparison, competitive binding assays were performed within the identical dose range (Fig. 4, bottom). Nonlinear regression analysis was used to calculate an EC50 for both the degradation response and receptor binding in PR1 cells. The rank order of potency indicates that 17beta -estradiol is the most potent agonist and possesses approximately one order of magnitude greater activity in both binding and ability to induce degradation than estriol, 17alpha -estradiol, and ICI. Comparison of the EC50 values for binding and degradation for individual ligands (Table 2) clearly shows a direct correlation between ligand affinity for receptor and the ability of individual ligands to induce receptor proteolysis. Thus ligands stimulate proteolysis at concentrations consistent with their binding affinity, providing further evidence that ligand binding triggers the events that lead to receptor proteolysis by proteasomes. It also points out that proteolysis can be modulated by hormone concentration.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Potency of ligand to induce degradation correlates with binding affinity. Dose-response and competitive binding assays were performed as described in EXPERIMENTAL PROCEDURES with ligand concentrations ranging from 1 pM to 1 µM in log increments. ERalpha levels in PR1 cells (top) treated with the indicated ligand for 2 h relative to untreated controls. Each point represents laser densitometric analysis of >= 4 independent experiments, which were internally controlled by a standard curve as described in EXPERIMENTAL PROCEDURES. Competitive binding assays (bottom) showing displacement of 10 pM [3H]estradiol by the indicated ligand at various concentrations. Binding assays were performed twice in duplicate. Interassay variation is included but is hidden by data symbols.


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Ligand potency in ERalpha binding and proteolysis

Interestingly, although all ligands induce proteolysis, the maximum change in receptor level affected by these ligands varies. Examination of the dose curves illustrated in Fig. 4A shows that the plateau level of receptor achieved by stimulation with estriol and 17alpha -estradiol is higher than that reached by 17beta -estradiol and ICI. This is also supported by data in Table 1, which shows that at a saturable dose for all ligands (10 nM), significant differences exist between the relative receptor levels. 17beta -Estradiol and ICI are similar in their ability to degrade the receptor. Both induce a loss of ~50% of receptor. 17alpha -Estradiol and estriol, however, affect smaller declines in the receptor that are significantly different from 17beta -estradiol and ICI. This ligand-specific variation cannot be accounted for by differences in binding, since all of the ligands tested are capable of occupying 100% of receptor at the 10 nM dose. Furthermore, 17alpha -estradiol, estriol, and ICI share similar binding affinities for the receptor, yet show different capacities to alter ER levels.

Recent studies have suggested that the transcriptional activation of receptor correlates with receptor proteolysis (24). However, in WT-ERalpha stable cells, 17alpha -estradiol, estriol, and 17beta -estradiol appear to activate transcription equivalently despite exhibiting differences in modulating receptor proteolysis (Fig. 2A). To directly compare the ability of agonists to modulate proteolysis and activate gene expression, ERalpha transcriptional activity was assessed in PR1 cells over a range of doses (Fig. 5). Cells were transfected with a reporter construct representative of a natural estrogen-regulated gene in lactotropes (Prl-Luc) and treated with varying doses of 17beta -estradiol, 17alpha -estradiol, estriol, and ICI. Consistent with its pure antiestrogenic activity, ICI inhibited the transcriptional activity of ERalpha in PR1 cells (data not shown). Examination of the transcriptional response at different doses shows that ligand ability to activate transcription parallels the ligand-binding affinity. The EC50 values for transcriptional activation are ~0.09 nM, 1 nM, and 0.4 nM for 17beta -estradiol, 17alpha -estradiol, and estriol, respectively. Interestingly, however, differences in transactivation capacity between these ligands are abolished at doses of 1 nM and higher. Thus it appears that, although ligand binding is required to induce both receptor transcriptional activity and proteolysis, ligand-specific differences in the proteolytic response are not reflected in ligand capacity to activate ERalpha transcriptional function.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Analysis of ligand-induced ERalpha transcriptional activity in PR1 cells. ERalpha transcriptional activity was measured in PR1 cells by transient transfection assay. Prl-Luc reporter gene was cotransfected along with CMV-beta gal with Fugene reagent. After transfection, cells were treated for an additional 24 h with E3, 17alpha -E2, and 17beta -E2, at the doses indicated. All groups contained 0.1% EtOH, including the control sample (0). Luciferase values were normalized against beta -galactosidase values to control for variation in transfection efficiency. Fold (degree of) activation represents the increase in corrected luciferase values relative to EtOH-treated controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteasome-mediated proteolysis has recently garnered increased attention as a conserved mechanism responsible for the control of steroid receptor protein stability. Because of its potential importance to the regulation of receptor function, much interest has been generated in determining the mechanism through which steroids target the receptor for proteolysis. Although it has been demonstrated that estradiol treatment of cells can stimulate proteolysis, the molecular signals that engage the receptor in the proteasome pathway have not been delineated. In this study, we demonstrate that proteolysis of ERalpha is a specific response that is induced by ligand binding to receptor. ERalpha ligands, however, show varying capacity to limit intracellular ERalpha concentration through this pathway. These results demonstrate the existence of ligand-specific regulation of receptor proteolysis by proteasomes.

Multiple ligands stimulate the destruction of ERalpha , including potent agonists such as 17beta -estradiol as well as the weak or short-acting agonists estriol and 17alpha -estradiol. Ligand binding induces a "transformation" of ERalpha in which the receptor becomes tightly associated with components of the nucleus. Release of ERalpha from this complex(es) requires high salt (0.4 M) extraction. In studies with GFP-ERalpha fusion protein, estradiol stimulation results in a formation of nuclear foci, providing a visual representation of the strong interactions of ERalpha with nuclear factors (39). Weak agonists, like estriol and 17alpha -estradiol, are so called because of their ability to activate some but not all of ERalpha responses, depending on the length of time of exposure. For example, a single administration of estriol can induce short-term estrogenic responses, such as water imbibition of the uterus, but not long-term growth responses (17). This selective regulation of receptor activity by estriol is not correlated with its ability to activate transcription, because estriol and estradiol are equally efficient at stimulating transcription in vitro (25), and under chronic stimulation, estriol functionally mimics estradiol (Fig. 5 and Ref. 2). Rather, it is more closely associated with the inability of estriol-bound receptor to sustain tight nuclear interactions (2, 8). This led to the concept that factors that form complexes with ERalpha in the nucleus may have a role in the selective regulation of the receptor by estriol and other weak agonists. Our studies show that estriol and 17alpha -estradiol are less efficient at lowering receptor levels than 17beta -estradiol or ICI, suggesting that fewer receptors are targeted to the proteasome pathway by these ligands despite 100% receptor occupancy. These data suggest that, like receptor transformation, proteolysis may be sensitive to the weak or short-term interactions between receptor and other nuclear factors. This notion is supported by a recent study showing that coactivator interaction with ERalpha is necessary for 17beta -estradiol-induced downregulation (24). Our data, however, do not agree with the further conclusion suggested in the latter report, that receptor turnover is linked to the efficiency of receptor transcriptional activity. We show instead that estriol and 17alpha -estradiol both activate transcription to comparable levels as 17beta -estradiol (Fig. 5), yet they have diminished capacity to decrease receptor concentration (Table 1). Our data, thus, better support the hypothesis that interactions within the nucleus may have a role in providing specificity to the proteolytic response.

We demonstrate that ICI induces proteasome-dependent degradation of ERalpha in lactotrope cells. Early studies by Gibson et al. (14) demonstrated that ICI-164384 could elicit a rapid decrease in uterine ERalpha , and it was suggested that the mechanism through which ICI operated involved proteolytic processing of the receptor (14). Dauvois et al. (10) also demonstrated that ICI-164384 shortened the half-life of mouse ERalpha , supporting the direct action of ICI on protein stability. We show that ICI targets the receptor to proteasomes in lactotrope cells, in agreement with studies by Stenoien et al. (40). In the PR1 cells, however, 17beta -estradiol and ICI are equally effective, and dose-response studies indicate that higher doses of ICI are required to elicit an effect equivalent to that of 17beta -estradiol. This differs from the response in MCF-7 cells (40), in which ICI appears to induce a complete loss of receptor and is more effective than estradiol when saturating concentrations of ligand are used. Differences in the effectiveness of ICI were also apparent in 293 stable cell lines expressing the wild-type receptor (Fig. 3), in which ICI stimulation resulted in a greater reduction in receptor levels, similar to what is seen in MCF-7 cells. These results suggest the possibility that regulation of proteolysis of ERalpha displays cell-type specificity in addition to ligand dependency.

Demonstration that ligand-induced proteolysis is abolished by a point mutant in the ligand-binding pocket provides direct evidence that ligand binding is required to target receptor to proteasomes. This is also supported by deletion of the entire ligand-binding domain (24), and by the correlation between ligand-binding affinity and potency of ligand to induce proteolysis. Ligand binding alone, however, is insufficient to induce proteolysis, since ligands of the triphenyltheylene class, such as tamoxifen, are incapable of downregulating ERalpha (see Fig. 1 and Ref. 4). Structural studies of the ERalpha ligand-binding domain demonstrate that tamoxifen binding disrupts the organization of critical helix 12 of ERalpha and prevents interaction of the receptor with coactivators (6, 38). It is unlikely, however, that tamoxifen's activity in disrupting coactivator receptor interaction is responsible for its lack of ability to induce proteolysis of receptor, because ICI binding produces a similar structural occlusion to the coactivator interface yet is a potent stimulator of receptor degradation (31). The demonstration that not all receptor ligands can target receptor to proteasomes, however, highlights the observation that, although ligand binding is required to induce proteolysis, signals downstream of binding are also necessary to direct receptor into the proteasome pathway.

Varying doses of ligand were utilized in this study to allow for the quantitative analysis of changes in ERalpha levels due to proteolysis. Only a few studies have directly examined proteasome-dependent proteolysis of ERalpha , and commonly, saturating concentrations of ligand are utilized to induce proteolysis. In addition, changes in receptor level are assessed by qualitative determination of relative receptor protein levels. Although these other studies provide important insight relevant to the induction of proteolysis, they overlook ligand-specific differences in the regulation of ERalpha degradation that might provide insight into underlying regulatory mechanisms. Our results indicate that proteolysis can be modulated by ligand concentration. They also demonstrate that ligands that induce proteolysis can differentially control the numbers of receptors that are degraded. These findings illustrate an important concept, that regulation of receptor stability is governed not only at the level of proteolysis induction but also by the efficiency with which receptor is degraded. As such, small changes in receptor stability may be misinterpreted as an absence of proteasome-dependent proteolysis, when instead, they may simply reflect changes in efficiency of receptor processing. Our results highlight the importance of the quantitative assessment of changes in receptor levels and provide evidence for ligand-specific regulation of proteasome-dependent proteolysis of ERalpha .


    ACKNOWLEDGEMENTS

We thank Drs. Jack Gorski, Jyoti Watters, Fern Murdoch, Mike Fritsch, and Shigeki Miyamoto for helpful discussion throughout the course of this investigation and for critical reading of the manuscript. In addition, we thank Dr. Richard Maurer for the gift of the Prl-Luc reporter gene construct.


    FOOTNOTES

This work was supported by National Cancer Institute Grant K01 CA-79090 to E. T. Alarid.

Address for reprint requests and other correspondence: E. T. Alarid, Dept. of Physiology, 120 Service Memorial Institute, 1300 University Ave., Madison, WI 53706 (E-mail: alarid{at}physiology.wisc.edu).

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

10.1152/ajpendo.00353.2001

Received 3 August 2001; accepted in final form 14 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Alarid, ET, Bakopoulos N, and Solodin N. Proteasome-mediated proteolysis of estrogen receptors: a novel component in autologous down-regulation. Mol Endocrinol 13: 1522-1534, 1999[Abstract/Free Full Text].

2.   Anderson, JN, Peck EJ, and Clark JH. Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei. Endocrinology 96: 160-167, 1975[Abstract].

3.   Borras, M, Hardy L, Lempereur F, El Khissiin AH, Legros N, Gol-Winkler R, and Leclercq G. Estradiol-induced down-regulation of estrogen receptor. Effect of various modulators of protein synthesis and expression. J Steroid Biochem Molec Biol 48: 325-336, 1994[ISI][Medline].

4.   Borras, M, Laios I, El Khissin A, Seo HS, Lempereur F, Legros N, and LeClercq G. Estrogenic and anti-estrogenic regulation of half-life of covalently labeled estrogen receptor in MCF-7 breast cancer cells. J Steroid Biochem Mol Biol 57: 203-216, 1996[ISI][Medline].

5.   Bradford, MA. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

6.   Brzozowski, AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engström O, Öhman L, Greene GL, Gustafsson J-Å, and Carlquist M. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389: 753-758, 1997[ISI][Medline].

7.   Chun, TY, Gregg D, Sarkar DK, and Gorski J. Differential regulation by estrogens of growth and prolactin synthesis in pituitary cells suggests that only a small pool of estrogen receptors is required for growth. Proc Natl Acad Sci USA 95: 2325-2330, 1998[Abstract/Free Full Text].

8.   Clark, JH, Paszko Z, and Peck EJ. Nuclear binding and retention of the receptor estrogen complex; relation to the agonist and antagonistic properties of estriol. Endocrinology 100: 91-96, 1977[Abstract].

9.   Dace, A, Zhao L, Park KS, Furuno T, Takamura N, Nakanishi M, West BL, Hanover JA, and Cheng S. Hormone binding induces rapid proteasome-mediated degradation of thyroid hormone receptors. Proc Natl Acad Sci USA 97: 8985-8990, 2000[Abstract/Free Full Text].

10.   Dauvois, S, Danielian PS, White R, and Parker MG. Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA 89: 4037-4041, 1992[Abstract].

11.   DeNicola, AF, van Lawzewitsch I, Kaplan SE, and Libertun C. Biochemical and ultrastructural studies on estrogen-induced pituitary tumors in F344 rats. J Natl Cancer Inst 61: 753, 1978[ISI][Medline].

12.   Ekena, K, Weis KE, Katzenellenbogen JA, and Katzenellenbogen BS. Identification of amino acids in the hormone binding domain of the human estrogen receptor important in estrogen binding. J Biol Chem 271: 20053-20059, 1996[Abstract/Free Full Text].

13.   Furlow, JD, Ahrens H, Mueller G, and Gorski J. Antisera to a synthetic peptide recognize native and denatured rat estrogen receptors. Endocrinology 127: 1028-1032, 1990[Abstract].

14.   Gibson, MK, Nemmers LA, Beckman WC, Jr, Davis VL, Curtis SW, and Korach KS. The mechanism of ICI 164,384 antiestrogenicity involves rapid loss of estrogen receptor in uterine tissue. Endocrinology 129: 2000-2010, 1991[Abstract].

15.   Guilbaud, NF, Gas N, Dupont MA, and Valette A. Effects of differentiation-inducing agents on maturation of human MCF-7 breast cancer cells. J Cell Physiol 145: 162-172, 1990[ISI][Medline].

16.   Hauser, S, Adelmant G, Sarraf P, Wright HM, Mueller E, and Spiegelman BM. Degradation of the peroxisome proliferator-activated receptor gamma is linked to ligand-dependent activation. J Biol Chem 275: 18527-18533, 2000[Abstract/Free Full Text].

17.   Hisaw, FLJ Comparative effectiveness of oestrogens on fluid imbibation and growth of the rat's uterus. Endocrinology 64: 276-289, 1959[ISI].

18.   Horwitz, KB, and McGuire WL. Nuclear mechanism of estrogen action. Effects of estradiol and anti-estrogens on estrogen receptors and nuclear receptor processing. J Biol Chem 253: 8185-8195, 1978[ISI][Medline].

19.   Huang, TT, Wuerzberger-Davis SM, Seufzer BJ, Shumway SD, Kurama T, Boothman DA, and Miyamoto S. NF-kappa B activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events. J Biol Chem 275: 9501-9509, 2000[Abstract/Free Full Text].

20.   Kaneko, K, Furlow JD, and Gorski J. Involvement of the coding sequence for the estrogen receptor gene in autologous ligand-dependent down-regulation. Mol Endocrinol 7: 879-888, 1993[Abstract].

21.   Khissiin, AE, and Leclercq G. Implication of proteasome in estrogen receptor degradation. FEBS Lett 448: 160-166, 1999[ISI][Medline].

22.   Kiang, DT, Kollander RE, Thomas T, and Kennedy B. Up-regulation of estrogen receptors by nonsteroidal antiestrogens in human breast cancer. Cancer Res 49: 5312-5316, 1989[Abstract].

23.   Lange, CA, Shen T, and Horwitz KB. Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26S proteasome. Proc Natl Acad Sci USA 97: 1032-1037, 2000[Abstract/Free Full Text].

24.   Lonard, DM, Nawaz Z, Smith CL, and O'Malley BW. The 26S proteasome is required for estrogen receptor-alpha and coactivator turnover and for efficient estrogen receptor-alpha transactivation. Mol Cell 5: 939-948, 2000[ISI][Medline].

25.   Melamed, M, Castano E, Notides AC, and Sasson S. Molecular and kinetic basis for the mixed agonist/antagonist activity of estriol. Mol Endocrinol 11: 1868-1878, 1997[Abstract/Free Full Text].

26.   Miyamoto, S, Seufzer BJ, and Shumway S. Novel Ikappa Balpha proteolytic pathway in WEHI231 immature B cells. Mol Cell Biol 18: 19-29, 1998[Abstract/Free Full Text].

27.   Monsma, FJ, Jr, Katzenellenbogen BS, Miller MA, Ziegler YS, and Katzenellenbogen JA. Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently attaching antiestrogen. Endocrinology 115: 143-153, 1984[Abstract].

28.   Nawaz, Z, Lonard DM, Dennis AP, Smith CL, and O'Malley BW. Proteasome-dependent degradation of the human estrogen receptor. Proc Natl Acad Sci USA 96: 1858-1862, 1999[Abstract/Free Full Text].

29.   Nomura, Y, Nagaya T, Hayashi Y, Kambe F, and Seo H. 9-cis-retinoic acid decreases the level of its cognate receptor, retinoic X receptor, through acceleration of the turnover. Biochem Biophys Res Comm 260: 729-733, 1999[ISI][Medline].

30.   Pastorcic, M, De A, Boyadjieva N, Vale W, and Sarkar DK. Reduction in the expression and action of transforming growth factor beta 1 on lactotropes during estrogen-induced tumorigenesis in the anterior pituitary. Cancer Res 55: 4892-4898, 1995[Abstract].

31.   Pike, ACW, Brzozowski AM, Walton J, Hubbard RE, Bonn T, Gustafsson JA, and Carlquist M. Structural aspects of agonism and antagonism in the oestrogen receptor. Biochem Soc Trans 28: 396-400, 2000[ISI][Medline].

32.   Pink, JJ, and Jordan VC. Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancer cell lines. Cancer Res 56: 2321-2330, 1996[Abstract].

33.   Read, LD, Greene GL, and Katzenellenbogen BS. Regulation of estrogen receptor messenger ribonucleic acid and protein levels in human breast cancer cell lines by sex steroid hormones, their antagonists, and growth factors. Mol Endocrinol 3: 295-304, 1989[Abstract].

34.   Reddel, RR, Murphy LC, and Sutherland RL. Factors affecting the sensitivity of T-47D human breast cancer cells to tamoxifen. Cancer Res 44: 2398-2405, 1984[Abstract].

35.   Ree, AH, Landmark BF, Eskild W, Levy FO, Lahooti H, Jahnsen T, Aakvaag A, and Hansson V. Autologous down-regulation of messenger ribonucleic acid and protein levels for estrogen receptors in MCF-7 cells: an inverse correlation to progesterone receptors. Endocrinology 124: 2577-2583, 1989[Abstract].

36.   Saceda, M, Lippman ME, Chambon P, Lindsey RL, Ponglikitmongkol M, Puente M, and Martin MB. Regulation of the estrogen receptor in MCF-7 cells by estradiol. Mol Endocrinol 2: 1157-1162, 1988[Abstract].

37.   Scully, KM, Gleiberman AS, Lindzey J, Lubahn DB, Korach KS, and Rosenfeld MG. Role of estrogen receptor-alpha in the anterior pituitary gland. Mol Endocrinol 11: 674-681, 1997[Abstract/Free Full Text].

38.   Shiau, AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, and Greene GL. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95: 927-937, 1998[ISI][Medline].

39.   Stenoien, DL, Mancini MG, Patel K, Allegretto EA, Smith CL, and Mancini MA. Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1. Mol Endocrinol 14: 518-534, 2000[Abstract/Free Full Text].

40.   Stenoien, DL, Patel K, Mancini MG, Dutertre M, Smith CL, O'Malley BW, and Mancini MA. FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and proteasome-dependent. Nature Cell Biology 3: 15-23, 2001[ISI][Medline].

41.   Syvala, H, Vienonen A, Zhuang YH, Kivineva M, Ylikomi T, and Tuohimaa P. Evidence for enhanced ubiquitin-mediated proteolysis of the chicken progesterone receptor. Life Sci 63: 1505-1512, 1998[ISI][Medline].

42.   Watters, JJ, Campbell JS, Cunningham JJ, Krebs EG, and Dorsa DM. Rapid membrane effects of steroids in neuroblastoma cells: effects of estrogen on mitogen activated protein kinase signalling cascade and c-fos immediate early gene transcription. Endocrinology 138: 4030-4033, 1997[Abstract/Free Full Text].

43.   Whitesell, L, and Cook P. Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells. Mol Endocrinol 10: 705-712, 1996[Abstract].

44.   Wiklund, J, Wertz N, and Gorski J. A comparison of estrogen effects on uterine and pituitary growth and prolactin synthesis in F344 and Holtzman rats. Endocrinology 109: 1700-1707, 1981[ISI][Medline].

45.   Zhu, J, Gianni M, Kopf E, Honore N, Chelbi-Alix M, Koken M, Quignon F, Rochette-Egly C, and de The H. Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor alpha  (RARalpha ) and oncogenic RARalpha fusion proteins. Proc Natl Acad Sci USA 96: 14807-14812, 1999[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 282(4):E891-E898
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society