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Endocrinology, doi:10.1210/en.2002-0092
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Endocrinology Vol. 144, No. 8 3469-3476
Copyright © 2003 by The Endocrine Society

Thyroid Hormone Is an Inhibitor of Estrogen-Induced Degradation of Estrogen Receptor-{alpha} Protein: Estrogen-Dependent Proteolysis Is Not Essential for Receptor Transactivation Function in the Pituitary

Elaine T. Alarid, Mara T. Preisler-Mashek and Natalia M. Solodin

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

Address all correspondence and requests for reprints to: Elaine T. Alarid, Ph.D., Department of Physiology, University of Wisconsin Madison, 120 Service Memorial Institute, 1300 University Avenue, Madison, Wisconsin 53706. E-mail: alarid{at}physiology.wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proteolysis by the 26S proteasome is an important regulatory mechanism that governs the protein stability of several steroid/nuclear receptors and that has been implicated in the control of receptor transcriptional activation function. Herein, we report that thyroid hormone can prevent estrogen-induced proteolysis of estrogen receptor-{alpha} (ER{alpha}) protein in lactotrope cells of the pituitary. The stabilization of ER{alpha} protein by thyroid hormone represents a selective blockade against estradiol-stimulated degradation, because thyroid hormone (but not glucocorticoid) can protect estrogen-activated ER{alpha}. Moreover, thyroid hormone treatment does not interfere with signal-induced proteolysis of a separate proteasome target, I{kappa}B{alpha} or ER{alpha} proteolysis induced by ICI182780. Using thyroid hormone as a tool to inhibit ER{alpha} proteolysis, we examined the effect of loss of this regulatory function on estrogen-induced transcriptional responses. Consistent with earlier reports, estrogen activation of an idealized estrogen response element reporter gene was inhibited. However, thyroid hormone did not prevent induction of prolactin gene expression or the ability of ER{alpha} to stimulate proliferation. These results demonstrate that estrogen-induced proteolysis of ER{alpha} is not a general requirement for receptor transcriptional activation function, and they demonstrate that proteolytic regulation is a means by which other endocrine factors can indirectly modulate ER{alpha} activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CELLULAR ESTROGEN RECEPTOR-{alpha} (ER{alpha}) levels are dynamically regulated in direct opposition to circulating estrogen, setting up an autoregulatory loop in which receptor concentrations are limited when cells are stimulated with ligand. The feedback regulation of ER{alpha} by estrogen is complex and implements regulatory mechanisms that function at transcriptional, posttranscriptional, and posttranslational levels (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). This complexity not only implies an underlying importance to the regulation of receptor concentration but also provides cells with an increased number of potential mechanisms available for higher-order regulation of estrogen action in a complex cellular environment.

The most acute component of estrogen-induced down-regulation of ER{alpha} is the regulated destruction of receptor protein by the 26S proteasome (1, 5, 7). Proteasome-mediated proteolysis is involved in the regulated turnover of several members of the nuclear receptor superfamily, including receptors for thyroid hormone, androgen, glucocorticoid, progesterone, retinoic acid, 9-cis retinoic acid, and vitamin D (15, 16, 17, 18, 19, 20, 21, 22, 23). The prevalent model describing the function of proteolysis suggests that receptor turnover is essential for efficient receptor transactivation. Pharmacological studies with peptide inhibitors of proteasome and mutagenesis analysis of the coactivator interaction domain of ER{alpha} have demonstrated that blockade of proteolysis decreases the magnitude of ER{alpha}-mediated activation of an estrogen response element (ERE) reporter gene (24). However, this evidence is limited to examination of reporter gene activity in heterologous systems, and the model has not been directly tested in physiologically relevant cell contexts.

We observed previously that diverse ligands, including the short-acting estrogen, estriol, and the cell-type specific agonist, 17{alpha}-estradiol, exhibit variable regulation of proteasome-dependent degradation of ER{alpha} in lactotrope cells of the anterior pituitary (25). These results suggested the possibility that receptor proteolysis might be further regulated by events downstream of ligand binding and possibly by other endocrine factors. Early in vivo studies suggested that thyroid hormone might be just such a modulator. Chronic estrogen treatment of rats results in hyperplastic expansion of the lactotrope population of the anterior pituitary (26, 27, 28). The ability of estrogen to induce pituitary hyperplasia, however, requires that the animal have an intact thyroid gland (29, 30, 31). It has also been demonstrated that thyroid hormone treatment increases estrogen binding in the pituitary and hypothalamus of euthyroid and thyroidectomized animals (32, 33, 34, 35, 36). A similar increase in estrogen binding can be achieved by the inhibition of proteolysis with proteasome inhibitors (1).

In this report, we demonstrate that thyroid hormone can inhibit estrogen-induced proteolysis of ER{alpha} in lactotrope cells. Prevention of ER{alpha} down-regulation by thyroid hormone resulted in differential effects on ER{alpha}-mediated gene activation but did not inhibit estrogen-stimulated proliferation. These results demonstrate that estrogen-induced proteolysis of ER{alpha} is not required for receptor transactivation function, and they identify a novel physiological regulator of the ER{alpha} proteolytic pathway in the pituitary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormone treatments
PR1 cells were maintained under standard CO2 and temperature conditions in high-glucose DMEM medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Techologies, Gaithersburg, MD). Six days before hormone treatment, the cells were moved to phenol-red free DMEM with 10% resin/dextran-coated charcoal stripped serum to deprive them of both estrogen and thyroid hormone. On the day of experimentation, cells were counted with a hemacytometer. Samples containing 1 x 106 cells were then stimulated with the indicated hormone. Treatment proceeded for 2 h unless otherwise indicated. The final concentration of ethanol (EtOH) was 0.1% in all samples. 17ß-Estradiol (E2), T3, and cortisol (F) were purchased from Sigma (St. Louis, MO). ICI182780 (ICI) was a gift from Dr. Jack Gorski.

Experiments with TNF were carried out in phenol-red containing DMEM with 10% fetal bovine serum. As above, equivalent amounts of cells were treated with 10 ng/ml TNF (Calbiochem, La Jolla, CA) for 0, 10, and 20 min. Upon harvest, whole cell lysates were obtained and analyzed by Western blot analysis.

Western blot analysis
Western blot analysis was performed, as previously described (1), on whole-cell lysates that were obtained either by direct dissolution of cell pellet in 2x sample buffer (125 mM Tris-base, 20% glycerol, 4% sodium dodecyl sulfate, 10% ß-mercaptoethanol) or by extraction with Totex buffer (20 mM HEPES, pH 7.9, 350 mM NaCl, 20% glycerol, 1% NP-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, and 0.5 mM dithiothreitol) followed by Bradford assay (37). Immunoblotting was performed using antirat ER{alpha} no. 715 (38) or anti-I{kappa}B{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Blots were reprobed with antiactin (Santa Cruz Biotechnology, Inc.) or anti-{alpha}-tubulin (Calbiochem) to verify equivalent loading of samples. Saturating concentrations of antibody were used to allow for quantitative measurement of receptor levels. Bands were visualized by ECL (Amersham Pharmacia Biotech, Arlington Heights, IL) and quantified by laser densitometry. For each gel, EtOH-treated control samples were used to generate an internal standard curve for linear regression analysis, against which relative ER{alpha} levels were determined. The correlation coefficient for the standard curves ranged from 0.99–1.0, verifying that the measurements of relative receptor levels determined by laser densitometry were within a linear range for the ECL reaction. Statistical differences between groups were determined by one-way ANOVA followed by a paired Student’s t test based on a 95% confidence interval.

Northern blot analysis
Total RNA was isolated by phenol:chloroform extraction and EtOH precipitation (39). Twenty micrograms of RNA were electrophoresed on a 1% agarose gel containing formaldehyde (40). The gels were transferred to nylon membrane and probed with a radiolabeled fragment of cDNA for ER{alpha} (41), prolactin (Prl) (42), or glyceraldehyde 6-phosphate dehydrogenase (GAPDH) (43) using conditions previously described (1). Messenger RNA levels were quantified by phosphoimager analysis using Imagequant software (Molecular Dynamics, Inc., Sunnyvale, CA). ER{alpha} levels were corrected for loading by normalization against GAPDH levels.

Transient transfections
PR1 cells were deprived of steroid and thyroid hormones, for 2 d before transfection, by maintenance in phenol-red free medium containing 10% resin/dextran-coated charcoal-stripped serum. Cells were transfected using Superfect reagent (QIAGEN, Valencia, CA) according to manufacturer’s instructions. To assess ER{alpha} transcriptional activity, two reporter genes were used: an ERE-tk-luc, which is comprised of a multimerized vitellogenin estrogen response element and a thymidine kinase promoter-driving luciferase (44); and a -2.5 Prl-luc comprised of 2.5 kb of the upstream regulatory region of the Prl gene fused to luciferase (45). Thyroid hormone receptor (TR) transcriptional activity was assessed using a reporter construct composed of a palindromic thyroid hormone response element (TRE) upstream of a minimal MTV promoter-driving luciferase expression (46). Glucocorticoid responsiveness was measured using a 4x multimerized glucocorticoid response element (GRE) fused to a tk-Luc reporter gene construct, which was provided by Dr. Chinghai Kao. To control for transfection efficiency, cells were cotransfected with a CMV-ßgal construct. The CMV-ßgal construct used in these experiments did not exhibit any regulation by E2 or T3. Hormone treatment began immediately after removal of transfection reagent. Assays for luciferase (Promega Corp., Madison, WI) and ß-galactosidase (Tropix, Bedford, MA) activity were performed according to manufacturers’ instructions. Statistically significant differences were determined using a paired Student’s t test.

For experiments using proteasome inhibitor, cells were cotransfected, as above, with -2.5 Prl luc and CMV-ßgal. The day after transfection, cells were pretreated for 30 min with either dimethylsulfoxide (DMSO) (solvent) or 100 µM ALLnL (Calbiochem). We previously demonstrated that this dose of ALLnL was sufficient to inhibit estrogen-induced proteolysis in PR1 cells (1). Hormone treatment proceeded for 24 h using 10 nM E2.

Proliferation assay
PR1 cells were placed in resin/dextran-coated charcoal-stripped serum for 3 d to deprive them of steroid and thyroid hormones. On the third day, 1 x 106 cells were replated and treated with the indicated hormone. E2 and T3 were administered at a dose of 0.1 nM. ICI was given at a dose of 100 nM as described (see Ref. 52). Cells were harvested after 24, 48, 72, 96, and 120 h; and total genomic DNA was isolated using the Puregene DNA purification kit (Gentra Systems, Inc., Minneapolis, MN) according to the manufacturer’s protocol. Statistical analysis, using a one-way ANOVA and a paired Student’s t test, was performed on data representing the last day (d 5) of the experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Based on early in vivo evidence, which suggested that thyroid hormone modulated ER{alpha} levels and activity in lactotrope cells of the anterior pituitary, we tested whether estrogen-induced down-regulation of ER{alpha} protein was altered in the presence of thyroid hormone (T3). To address this question, we used the PR1 lactotrope cell line as a model system. PR1 cells are derived from an estrogen-induced pituitary tumor (47) and express ER{alpha}, but not ERß, based on RT-PCR analysis performed in our laboratory (data not shown). Thus, they represent an in vitro correlate to the early in vivo studies documenting thyroid hormone: estrogen interactions in the pituitary. In addition, ER{alpha} protein levels are regulated by induction of proteasome-mediated proteolysis in this system (1). To test the effects of thyroid hormone on estrogen-induced ER{alpha} protein degradation, PR1 cells were treated acutely for 2 h with E2 and T3. During this time frame, changes in steady-state levels of ER{alpha} protein reflect direct regulation of receptor protein by the 26S proteasome (1). Western blot analysis of ER{alpha} protein levels in EtOH-treated vs. E2-treated samples shows that ligand-stimulation induces an approximate 50% reduction in total ER{alpha} protein (Fig 1Go). Treatment with T3 alone did not alter ER{alpha} levels, relative to control samples. However, coadministration of T3 resulted in an inhibition of E2-induced receptor protein down-regulation.



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  		FIG. 1. Thyroid hormone protects ER{alpha} from estrogen-induced down-regulation. A, Representative Western blot analysis, showing ER{alpha} levels (top panel) in PR1 cells treated with 10 nM E2 or 10 nM T3, alone or in combination, for 2 h. For quantitative measure of relative ER{alpha} levels, a standard curve was generated using one fourth and one half of an EtOH-treated control and was run alongside of the experimental samples in each experiment. Equivalent protein loading was confirmed by reprobing blots for actin (bottom panel). B, Measurement of relative ER{alpha} levels in cells treated with 10 nM E2 and increasing concentrations of T3 was based on linear regression analysis of standard curves. Relative ER{alpha} levels in cells treated with E2 alone are represented by {square}. The level of ER{alpha} in EtOH-treated controls was assigned as 1.0. Relative ER{alpha} levels in treated samples are shown as the mean ± SEM for seven independent experiments. Significant differences were determined by one-way ANOVA, followed by paired Student’s t test. *, P <= 0.01.

 
To quantitatively assess changes in ER{alpha} protein levels, Western blots included a standard curve that was generated by dilution of the EtOH-treated control in each experiment. An example is shown in Fig. 1AGo. Relative ER{alpha} levels were then determined by linear regression analysis. The addition of T3, at varying doses ranging from 0.01–1000 nM, resulted in a dose-dependent increase in ER{alpha} levels relative to E2-treated samples (Fig 1BGo). Doses of T3 greater than 1 nM were sufficient to maintain ER{alpha} at control levels (Fig. 1BGo). Based on a sigmoidal dose-response curve, the EC50 for T3 inhibition of receptor down-regulation was determined to be 1.9 nM, which is consistent with T3 activation of TR.

To test whether T3 opposed the actions of estrogen by induction of ER{alpha} gene expression, total RNA was isolated from cells treated with E2 and T3 under identical conditions, as described above. Northern analysis was performed to assess ER{alpha} mRNA levels using radiolabeled probes for ER{alpha} and GAPDH. Examination of the relative receptor expression levels shown in Fig. 2Go reveals that ER{alpha} gene expression is unaffected by treatment with either T3 or E2. These results are consistent with our previous findings and demonstrate that the acute regulation of ER{alpha} protein occurring during a short exposure to hormones is mediated through direct regulation of receptor protein, not receptor mRNA. Further, they demonstrate that T3 does not induce increased synthesis of ER{alpha}.



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  		FIG. 2. Thyroid hormone does not alter ER{alpha} mRNA levels. A, Shown is a representative Northern blot of total RNA isolated from PR1 cells that were treated with 10 nM E2 and/or 10 nM T3 for 2 h. Controls consisted of RNA from cells treated with 0.1% EtOH. Blots were hybridized with 32P-labeled cDNA fragment encoding ER{alpha}. GAPDH served as a loading control. B, Phosphoimager analysis was used to quantify ER{alpha} mRNA levels, and levels were corrected for loading against GAPDH. The data are represented as mean ± SD for six independent experiments.

 
Proteasome activity controls the stability of most short-lived proteins in the cell (48). A classic example of signal-inducible proteolysis by the ubiquitin proteasome pathway is TNF induction of I{kappa}B{alpha} protein degradation (49). To test whether treatment with T3 disrupts proteasome activity, PR1 cells were treated with TNF in the presence and absence of T3, and I{kappa}B{alpha} protein levels were monitored over time. Results shown in Fig. 3Go, A and B, demonstrate that T3 treatment did not alter TNF-stimulated degradation of I{kappa}B{alpha} protein. Similar results were obtained when cells were treated with the pure antiestrogen, ICI, which also stimulates the destruction of ER{alpha} by proteasomes (Fig. 3Go, C and D; and Refs. 22 ,25 , and 50). Pretreatment of cells with T3 for up to 2 h before addition of either TNF or ICI did not prevent inducible degradation of I{kappa}B{alpha} and ICI-bound ER{alpha}, respectively (data not shown). These results suggest that the ability of T3 to protect ER{alpha} protein is not attributable to a generalized inhibition of proteasome activity but is due to a selective disruption of the pathway by which estrogen, but not ICI, targets ER{alpha} protein for degradation.



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  		FIG. 3. Thyroid hormone disruption of proteolysis is limited to estrogen-induced ER{alpha} degradation. A, PR1 cells were treated with 10 ng/ml TNF and 100 nM T3 for the indicated length of time. Whole-cell lysate was analyzed by Western analysis and hybridized with anti-I{kappa}B{alpha}. Equivalent loading is shown based on {alpha}-tubulin protein levels. Shown is a representative Western blot. B, Relative I{kappa}B{alpha} protein levels were determined by laser densitometry and are shown as a percentage of the level in untreated controls. Data are represented as the mean ± SEM for three independent experiments. Error bars are present in the sample group treated with both TNF and T3 but are not visible. C, PR1 cells were treated with either 10 nM E2 or 10 nM ICI along with increasing doses of T3 as indicated. Whole-cell lysates were analyzed by Western analysis for changes in ER{alpha} levels. Blots were reprobed with antiactin antibody to control for loading. Shown is a representative Western blot. D, ER{alpha} levels in cells treated with 10 nM ICI, in the presence and absence of 10 nM T3, were quantified by laser densitometry and are shown as a percentage of those in EtOH-treated controls. Data are represented as the mean ± SEM for five independent experiments. Statistical differences were determined as in Fig. 1Go. *, P <= 0.05, relative to controls.

 
Nuclear receptors can compete with each other for shared regulatory components (51), and it has been suggested that T3 may repress ER{alpha} activity by titrating shared components of the transcriptional machinery (52). To test whether activation of other nuclear receptors can function similarly to T3 and repress ER{alpha} proteolysis, PR1 cells were treated with E2 in combination with either T3 or cortisol. Whereas thyroid hormone was effective at blocking proteolysis of ER{alpha}, identical treatment with cortisol failed to alter estrogen’s ability to induce degradation of receptor protein (Fig. 4AGo). Repeated quantitative measurement of relative ER{alpha} levels in six independent experiments showed that only thyroid hormone significantly increased ER{alpha} steady-state levels in the presence of estrogen, as shown in Fig. 1Go. Cortisol treatment had no effect on estrogen-induced down-regulation of ER{alpha} protein (E2 = 45 ± 6.8%, and E2 + F = 43 ± 7.1%, relative to control ER{alpha} levels). Despite the differences in effects of T3 and cortisol on ER{alpha} protein levels, both hormones were able to induce transcriptional activation of a reporter gene construct (Fig. 4BGo), indicating that both TR and glucocorticoid receptor are active in PR1 cells. These results demonstrate that the ability to inhibit estrogen-induced ER{alpha} proteolysis is specific for T3.



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  		FIG. 4. Specificity of thyroid-hormone-conferred resistance to estrogen-induced receptor proteolysis. A, PR1 cells were treated with 10 nM E2 in combination with 10 nM T3, or 10 nM F. Shown is a representative Western analysis of ER{alpha} protein levels after 2 h of hormone stimulation. Controls consisted of cells treated with EtOH and E2 alone (-). Independent repetitions were carried out six times and yielded similar results. B, PR1 cells were transiently transfected with either a TRE-pal-luc (TRE) or a 4x GRE-tk-luc (GRE) reporter plasmid as indicated. Cells were cotransfected with CMV-ßgal to control for transfection efficiency. After transfection, cells were treated with 100 nM of the indicated hormone for 24 h. Luciferase activity was normalized to ß-gal activity. Fold activation was determined by comparison with solvent-treated controls, which were arbitrarily set at 1.0. Data are shown as mean ± SEM for six independent experiments. Significant differences were determined by paired Student’s t test. *, P < 0.01, relative to control samples.

 
The identification of T3 as a selective inhibitor of estrogen-induced proteolysis provided us with a unique tool to assess the functional consequences of loss of proteolytic regulation on ER{alpha} function without a general inhibition of proteolysis. Moreover, we were afforded the opportunity to examine the impact on both receptor activation of reporter gene activity and endogenous target genes. For comparison with previous studies in HeLa cells (22, 24), an idealized estrogen-response element driven reporter construct (ERE-tk-Luc) was transiently introduced into PR1 cells. In parallel, a separate group of PR1 cells was transfected with a TRE-driven reporter (TRE-pal-Luc) to examine TR transcriptional activity. Cells were treated with E2 and T3, alone and in combination. Figure 5AGo shows that T3 inhibits estrogen induction of an ERE reporter gene in PR1 cells. Reciprocally, E2 inhibits thyroid-hormone-induced TRE-pal-Luc reporter gene activity (Fig. 5BGo). These results are in agreement with the model that inhibition of proteolysis results in decreased ER{alpha} transcriptional activity.



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  		FIG. 5. Transcriptional transrepression of an ERE-driven reporter gene by estrogen and thyroid hormone. PR1 cells were transiently transfected with an idealized ERE-driven reporter gene (A) or a positive palindromic TRE-driven reporter (B) to assess ER{alpha} and TR transcriptional activity, respectively. A CMV-ßgal reporter was cotransfected to control for transfection efficiency. After transfection, cells were treated for 24 h with 10 nM E2 or 10 nM T3, alone or in combination. Luciferase values were normalized against ß-gal activity. Fold activation was determined relative to solvent-treated controls, which were set at 1.0. The data represented the mean ± SEM for four independent experiments. Statistical analysis was performed using a paired Student’s t test. a, P < 0.05, relative to EtOH control; b, P < 0.01, relative to E2-treated sample; c, P < 0.05, relative to T3-treated sample.

 
Transcriptional transrepression by ER{alpha} and TR is consistent with the coupling of ER{alpha} proteolysis and transactivation but can also be explained by competition for a common DNA binding element (52, 53, 54, 55, 56, 57, 58, 59, 60, 61). Therefore, we examined the effects of thyroid hormone on the activation of an endogenous target of ER{alpha} in lactotrope cells, Prl. A Prl reporter gene, consisting of 2.5 kb of the upstream regulatory region of the Prl gene fused to luciferase, was cotransfected into PR1 cells along with a CMV-ßgal reporter gene. As predicted, estrogen treatment stimulated a robust induction of reporter gene activity. T3 alone also induced Prl reporter gene activity. However, in contrast to effects seen on an idealized ERE, the addition of thyroid hormone did not inhibit the activity of ER{alpha} but rather increased it (Fig. 6AGo). To independently confirm this finding, Northern analysis was done on total RNA isolated from PR1 cells treated with E2 and T3. Results shown in Fig. 6BGo illustrate that T3 does not inhibit estrogen induction of endogenous Prl expression. Treatment with estrogen or thyroid hormone alone induced a 2.7 ± 0.3- and 2.2 ± 0.6-fold increase in Prl gene expression, respectively, whereas the combined treatment resulted in a 4.0 ± .9-fold induction. Western analysis of ER{alpha} levels in Fig. 6CGo confirms that T3 inhibits ER{alpha} down-regulation under these conditions.



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  		FIG. 6. Activation of Prl gene expression is enhanced despite a blockade of ER{alpha} proteolysis. A, A luciferase reporter gene, driven by -2.5 kb of the Prl regulatory region, was introduced in PR1 cells along with a CMV-ßgal reporter gene. After transfection, cells were stimulated with 10 nM E2 and/or 100 nM T3 for 24 h. Luciferase values were normalized for transfection efficiency against ß-gal and shown relative to EtOH-treated controls, which were arbitrarily set at 1.0. Data are shown as mean ± SD for three independent experiments (a, P <= 0.02, relative to EtOH control; b, P < 0.02, relative to E2). B, Total RNA was isolated from PR1 cells treated for 24 h with 10 nM E2 or 10 nM T3. Northern analysis was performed using a radiolabeled probe encoding a fragment of rat Prl cDNA. Shown is a representative Northern blot from three independent experiments. C, A representative Western analysis showing ER{alpha} levels in whole-cell lysates treated with E2 in the presence and absence of T3 for 24 h, as in B.

 
To assess the effects of thyroid hormone on a more complex response to estrogen, we examined the impact of loss of down-regulation on proliferation of lactotropes. Cells that were initially deprived of steroid and thyroid hormones were treated, for varying lengths of time, with E2 and/or T3; and total genomic DNA was measured (Fig. 7AGo). Chun et al. (62) previously demonstrated that estrogen directly stimulates the growth of PR1 cells. As expected, estrogen stimulated an increase in DNA content, which was apparent 48 h after initiation of hormone stimulation. Samples treated with EtOH, thyroid hormone, and ICI remained static over the entire period of the experiment. Statistical analyses of DNA content on d 5 of treatment (Fig. 7BGo) show that PR1 cells exhibit a proliferative response to estrogen in the presence and absence of T3. Furthermore, this increased proliferation is mediated through ER{alpha}, as demonstrated by the inhibition by ICI, an antagonist of ER{alpha} but not TR function. These results are similar to the findings with induction of Prl gene expression and indicate that prevention of ER{alpha} proteolysis and blockade of receptor down-regulation does not have a generalized inhibitory effect on ER{alpha}-mediated transcription.



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  		FIG. 7. Thyroid hormone enhances estrogen-stimulated proliferation. A, PR1 cells, maintained in medium supplemented with resin/charcoal-stripped fetal bovine serum, were treated with 0.1% EtOH, 0.1 nM E2, 0.1 nM T3, or 100 nM ICI. Cells were harvested at the indicated times, and total DNA content was determined as described in Materials and Methods. Data shown are a representative growth curve of PR1 cells grown under the indicated conditions. B, Data obtained after 5 d of hormone treatment in five independent experiments were combined to determine the mean ± SEM for each experimental group. Statistical analysis was performed by one-way ANOVA followed by a paired Student’s t test. *, P < 0.05, relative to EtOH control.

 
The actions of thyroid hormone and estrogen in the stimulation of endogenous gene activation are complex and may be independent of thyroid hormone’s inhibition of ER{alpha} proteolysis. Thus, to directly test whether proteolysis is essential in estrogen induction of gene expression in the pituitary, Prl reporter gene activity was measured in PR1 cells in which proteasome function was inhibited by pretreatment with ALLnL. ALLnL has previously been demonstrated to prevent estrogen-induced proteolysis of ER{alpha} in PR1 cells (1). After transfection with -2.5 Prl-luc reporter gene, cells were pretreated with ALLnL for 30 min before stimulation with EtOH or E2 for 24 h. Controls consisted of pretreating cells with DMSO, which is a solvent for ALLnL. Pretreatment of cells with proteasome inhibitor did not disrupt estrogen induction of Prl reporter gene activity (Fig. 8AGo), despite stabilizing ER{alpha} protein (Fig. 8BGo). Thus, inhibition of proteolysis with a pharmacological inhibitor functioned similarly to T3 and did not disrupt ER{alpha} transactivation capacity.



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  		FIG. 8. Proteasome inhibition does not disrupt estrogen induction of Prl expression. A, PR1 cells were cotransfected with -2.5 Prl-Luc and CMV-ßgal reporter constructs as described in Materials and Methods. The day after transfection, parallel samples were pretreated for 30 min with DMSO or 100 µM ALLnL. Treatment with EtOH or 10 nM E2 proceeded for 24 h. Data are shown as mean ± SEM for three independent experiments. Relative Luc/ßgal activity was determined relative to DMSO EtOH-treated sample, which was set at 1.0. Statistical analysis was performed by one-way ANOVA followed by a paired Student’s t test. *, P < 0.01. B, Shown is a representative Western blot of cell lysates from transfection experiment in A, confirming that the proteasome inhibitor prevented ER{alpha} degradation. Blots were probed with anti-ER{alpha}. Actin serves as a loading control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study identifies thyroid hormone as a selective inhibitor of estrogen-induced proteolysis of ER{alpha} by proteasomes. With this endogenous regulator, it was possible to ascertain the functional role(s) of proteolysis in the control of ER{alpha} transcriptional activity in the pituitary without globally inhibiting the activity of proteasomes. Moreover, it allowed the investigation into complex responses to estrogen, including the regulation of an endogenous target gene expression and growth. Thyroid hormone disrupts proteolysis but can modulate ER{alpha} induction of gene expression both positively and negatively in a promoter-specific manner. Whereas ER{alpha} induction of an ERE was diminished by T3, induction of Prl and growth was not inhibited. Thus, ER{alpha} retains the capacity to activate transcriptional processes on certain genes, such as Prl, while escaping destruction by proteasomes. The inference of these findings is that receptor proteolysis is not a general requirement for activation of transcription. Further, these data suggest the possibility that modulation of the proteolytic pathway may contribute to promoter-specific regulation of ER{alpha} function. Elucidation of the mechanism by which thyroid hormone protects ER{alpha} from degradation will likely yield important insight not only into the signaling pathway that targets ER{alpha} to proteasomes but also into a potential underlying mechanism responsible for promoter selectivity.

Neither T3 nor pharmacological inhibition of estrogen-induced proteolysis resulted in a reduction in ER{alpha} activation of Prl gene expression. This is in contrast to other reports that show an inverse relationship between receptor proteolysis and transcriptional efficiency (24, 63). This discrepancy cannot be attributed to compensatory activation of ERß because PR1 cells in our laboratory express only the ER{alpha} isoform (data not shown). However, one possible explanation that could account for this phenomenon in pituitary is that preventing ER{alpha} degradation effectively increases the number of functional receptors available to participate in transcriptional processes. We previously demonstrated that inhibiting receptor proteolysis with proteasome inhibitors increases the estrogen-binding capacity in PR1 cells (1). Thus, receptors that evade proteolysis retain certain ER{alpha} functions, including ligand binding. Similar results have been reported with glucocorticoid receptor in embryonic hippocampal neurons, which show that activated receptors that are not targeted to proteasomes retain transactivation capacity (64). Studies of ER{alpha} in stable CHO (65) and tet-inducible MCF-7 breast cancer cells (Fowler, A. M., N. M. Solodin, M. T. Preisler-Mashek, P. Zhang, A. V. Lee, and E. T. Alarid, submitted) demonstrate that the magnitude of ER{alpha} transcriptional activity is directly related to the concentration of receptor. Thus, any regulatory mechanism that has the potential to elevate the concentration of functional receptor could potentially augment the transcriptional output of ER{alpha}.

The two most efficient ligands at signaling ER{alpha} degradation are E2 and ICI (14, 22, 25). Their shared activity in stimulating proteolysis despite opposing activities on receptor transcriptional function could be explained either by the uncoupling of the proteolytic and transactivation pathways or by multiple ER{alpha} degradation pathways. The finding that thyroid hormone preferentially disrupts estrogen-induced degradation over ICI-induced proteolysis provides evidence in support of the latter hypothesis that estrogen and ICI signal receptor degradation by distinct routes. Our previous studies demonstrate that both are initiated by ligand binding; however, the branch point between these two pathways has not been delineated. Studies by Wijayaratne et al. (22) would argue that activation of transcription distinguishes agonist- vs. antagonist-signaled proteolysis. A conclusive separation, however, awaits the dissection of the events required in estrogen and ICI degradation pathways. Nevertheless, it is intriguing that ER{alpha} may be targeted to proteasomes by more than one mechanism, increasing the number of potential ways through which proteolysis can contribute to the control of ER{alpha} function.

We present evidence that the regulation of proteolysis may serve as an alternative means of controlling ER{alpha} activity. Indeed, a number of conditions and regulatory molecules, such as hypoxia (66), aryl hydrocarbon receptor (67, 68), and calmodulin (69), have been demonstrated to alter ER{alpha} protein stability. The complexity of the ubiquitin proteasome pathway provides multiple entry points where other regulatory pathways can interface with ER{alpha} protein regulation and potentially modulate ER{alpha} function indirectly. Based on the known transcriptional interactions between TR and ER, future investigations will be aimed at identifying the events in ER{alpha} proteolysis that are disrupted by thyroid hormone, with the intent to better understand the control of ER{alpha} activity in complex endocrine environments.


   Acknowledgments
 
The authors would like to thank Jack Gorski for the gifts of ICI and antirat ER{alpha} antibodies and for insightful discussion throughout the undertaking of this project. We thank Dr. Fern Murdoch and Amy Fowler for critical review of the manuscript. We also thank Drs. Dave Furlow, Rich Maurer, and Chinghai Kao for TRE-pal-luc, -2.5 Prl-luc, and pGRE4-luc reporter constructs, respectively.


   Footnotes
 
This work was supported by Research Grant NIH-K01-CA-79090 (to E.T.A.).

Abbreviations: DMSO, Dimethylsulfoxide; E2, 17ß-estradiol; ER{alpha}, estrogen receptor-{alpha}; ERE, estrogen response element; EtOH, ethanol; F, cortisol; GAPDH, glyceraldehyde 6-phosphate dehydrogenase; GRE, glucocorticoid response element; ICI, ICI182780; Prl, prolactin; TR, thyroid hormone receptor; TRE, thyroid hormone response element.

Received December 2, 2002.

Accepted for publication April 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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