The glucocorticoid agonist activities of mifepristone (RU486) and progesterone are dependent on glucocorticoid receptor levels but not on EC50 values

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journal homepage: www.elsevier.com/locate/steroids

The glucocorticoid agonist activities of mifepristone (RU486) and progesterone are dependent on glucocorticoid receptor levels but not on EC50 values Shimin Zhang a,∗ , Jacqueline Jonklaas b , Mark Danielsen c a

Division of Molecular Pathobiology, Department of Environmental and Infectious Disease Sciences, American Registry of Pathology, Armed Forces Institute of Pathology, Washington, DC 20306, United States b Department of Medicine, Division of Endocrinology, Georgetown University Medical Center, Washington, DC 20007, United States c Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, United States

a r t i c l e

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a b s t r a c t

Article history:

Mifepristone is an antagonist of the glucocorticoid receptor (GR) that also has significant

Received 1 December 2006

agonist activity in some cell types. We examined the partial agonist activity of mifepristone

Received in revised form

in COS-7 cells transfected with increasing amounts of a glucocorticoid receptor expression

10 March 2007

vector pmGR. As pmGR levels increased, the response of the reporter, pMTVCAT to dexam-

Accepted 27 March 2007

ethasone increased, consistent with increasing levels of receptor expression; the response

Published on line 7 April 2007

to mifepristone also increased but at a higher rate, resulting in increasing mifepristone agonist and decreasing antagonist activity. In contrast, increasing pMTVCAT levels increased

Keywords:

CAT activity induced by both dexamethasone and mifepristone, but did not change the rel-

Mifepristone

ative agonist activity of mifepristone. We also examined the relationship between agonist

RU486

activity and receptor level in a series of clones of the E8.2.A3 cell line expressing various

Agonist activity

levels of GR. Again, the relative agonist activity of mifepristone increased as GR increased.

Antagonist activity

This increase was not due to changes in the dose response curves to these two ligands

Glucocorticoid receptor

since their EC50 values were independent of receptor levels. These results indicate that the

Transfection

degree of glucocorticoid agonist activity exhibited by mifepristone is dependent on the concentration of GR in the cell. Similar results were obtained with another partial agonist of the GR, progesterone, whereas the complete antagonist ZK98.299 had no agonist activity under any condition. Taken together, these results suggest that the phenomenon of receptor concentration-dependence is a property of partial GR agonists in general. © 2007 Elsevier Inc. All rights reserved.

1.

Introduction

Glucocorticoids are hormones produced in the adrenal cortex, which regulate a diverse set of processes in vertebrates. They are particularly important for glucose homeostasis, for the ability to respond to stress, and for various developmental processes such as lung development. Over a period of many

∗

Corresponding author. Tel.: +1 202 782 1768; fax: +1 202 782 9160. E-mail address: zhangs@afip.osd.mil (S. Zhang). 0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2007.03.012

years, effective synthetic agonists have been developed for clinical use [1]. These synthetic agonists have, for instance, increased selectivity for the GR over the mineralocorticoid receptor. Synthetic antagonists have also been developed, though they tend to have less specificity for the GR and interact with related steroid receptors. One such glucocorticoid antagonist is mifepristone (RU486) that is both a glucocorti-

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coid and progesterone antagonist and can interact with the androgen receptor [2]. Clinically, mifepristone is used mainly to inhibit progesterone action in the first few weeks after conception leading to loss of fetal viability. However, it has other possible uses such as in control of Cushing’s syndrome [3] and in the treatment of depression and neuropsychiatric disorders [4] and has potential for treatment of certain tumors [5]. Steroid receptors are hormone-inducible transcription factors. In the absence of hormone, they are found within cells, bound to a number of chaperone proteins such as HSP90. Hormone-binding brings about a conformational change in the receptor such that the protein complex dissociates and the receptor can then bind to specific DNA response elements [6,7]. The hormone-induced conformational change is also required for interaction with transcriptional coactivators and corepressors [8,9]. It is thought that each steroid receptor forms receptor- and cell-specific complexes with these coregulators and that it is the identity of the coregulators and the ratio between coactivators and corepressors that sets the magnitude of the transcriptional response [8]. Steroid antagonists also bind to steroid receptors, but do not bring about the same conformational changes as agonists. In many cases, the antagonist–receptor complex can bind to DNA, but the altered conformation of the hormone-binding domain of the receptor results the recruitment of a different mix of coactivators and corepressors that results in decreased transcriptional activity [10,11]. Thus, the effects of antagonists can be tissue specific depending on the cell’s complement of transcriptional coregulators [12]. An example of this is the selective estrogen receptor modulator tamoxifen whose estrogen antagonist activity is used to treat breast cancer, while at the same time preserving bone density [13]. Mifepristone is a selective progesterone and glucocorticoid receptor modulator, since the amount of agonist activity it expresses is dependent on cell type and growth conditions of the cells. In an attempt to determine what factors regulate its glucocorticoid agonist activity, we studied its activity in cells expressing various levels of the GR. We show that the level of agonist activity correlates with receptor level in the cells, but not with the levels of target genes. We also showed that these effects were independent of changes in dose response curves. Similar results were obtained with progesterone, which is also a partial agonist of the GR. We also examined the activity of the glucocorticoid antagonist ZK98.299 [14]. Under all conditions ZK98.299 was inactive.

2.

Experimental

2.1.

Materials

COS-7 and CV1 cells were purchased from American Type Culture Collection (ATCC), Manassas, VA. E8.2.A3 cells, a GR-negative cell line derived from L929, were kindly provided by Dr. Paul R. Housley [15]. Dulbecco’s Modified Eagle Medium (DMEM) and trypsin/EDTA solution were purchased from Biofluids Inc. (Rockville, MD). Tissue culture dishes were from Falcon Labware (Lincoln Park, NJ) and multi-well tissue culture plates were from Corning Inc. (Corning, NY). Charcoalstripped bovine calf serum was prepared in our laboratory

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following a procedure described previously [16]. [3 H]-acetyl coenzyme A ([3 H]-acetyl CoA) and [3 H]-triamcinolone acetonide ([3 H]-TA) were from New England Nuclear Life Science Products (Boston, MA). Mifepristone was a gift from Dr. Stony Simons, Jr. (NIH, Bethesda, MD). ZK98.299 was obtained from Schering AG, Berlin, Germany. The Bradford protein assay solution was from Bio-Rad Laboratories (Richmond, CA). 2.5% dextran-coated charcoal was from Wein Laboratories, Inc. (Succasunna, NJ). O-Nitrophenyl-␤-d-galactopyranoside (ONPG) was from Sigma (St. Louis, MO). Chlorophenol red ␤d-galactopyranoside (CPRG) was from Boehringer Mannheim Co. (Indianapolis, IN). General chemicals were from Sigma or J.T. Baker (Phillipsburg, NJ).

2.2.

Plasmids

pmGR is a derivative of pSV2Wrec, which contains the mouse GR under the transcriptional control of SV40 promoter [17]. It was constructed by replacing the pBR322 origin of replication in pSV2Wrec with the origin of replication from pBluescript, which improves plasmid yields. pMTVCAT was made by inserting a glucocorticoid-inducible promoter, mouse mammary tumor virus (MMTV) long terminal repeat into the HindIII site of pSV0CAT that contains the chloramphenicol acetyltransferase (CAT) gene [17]. pBAG contains LacZ gene (ˇ-galactosidase) whose expression is under the control of the Moloney murine leukemia virus long terminal repeat [18]. pSV2neo contains the neomycin-resistance gene under the control of an SV40 promoter [19].

2.3.

Cell culture and transfection

Cells used in this study were cultured in DMEM containing 10% defined/supplemented bovine calf serum. Transfection of cells was performed by a slight modification of the low CO2 calcium phosphate precipitation method [16,20]. Briefly, exponentially growing stock cells were seeded (about 1.25 × 104 cells/cm2 ) into 10- or 15-cm tissue culture plates 16–24 h before transfection and refed with growth medium containing charcoal-stripped serum 4 h before transfection. Transfection mixtures were prepared by mixing stock 1 M CaCl2 and 2× BBS, plasmid DNA, carrier DNA (sonicated salmon sperm DNA) and sterilized distilled water to give a final concentration of 25 mM BES [N-, N-Bis(2 hydroxyethy)-2 amino-ethane-sulfonic acid], 140 mM NaCl, 0.75 mM Na2 HPO4 and 125 mM CaCl2 . The amount of the GR expression vector pmGR used ranged from 10 ng to 3 ␮g per 10-cm dish. In most CAT assay experiments, 10.5 ␮g pMTVCAT and 150 ng pBAG were used per 10-cm dish unless indicated otherwise. Carrier DNA was used to bring the total amount of DNA to 30 ␮g per 10-cm dish. The mixture was incubated for 20 min at 37 ◦ C, and then a 1.5-ml aliquot was added drop wise into each 10cm dish. The dishes were gently swirled several times and incubated for 18 h at 37 ◦ C under 3% CO2 . At the end of the incubation, the medium was removed. The cells were washed twice with 37 ◦ C pre-warmed PBS, refed with medium containing charcoal-stripped serum, and incubated at 37 ◦ C under 10% CO2 overnight. When 15-cm dishes were used, all quantities were increased 2.6-fold.

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For stable transfections, the same calcium phosphate precipitation method described above was used except that the selection vector pSV2neo was included in the transfection mixture at a ratio of 1:20. The transfected cells from one dish were split into 8–10 fresh dishes 15–20 h after the transfection and grown in DEMEM medium containing charcoal stripped serum. The cells were refed 24 h later with the same medium containing 400 ␮g/ml G418 and were grown for a further 3–6 weeks until single cell colonies were large enough to be transferred to separate culture dishes. All experiments were performed using these clones. CAT assay and Western blot analysis (below) were used to screen for positive cell colonies containing both pmGR and pMTVCAT.

2.4.

Hormone induction and CAT assays

The stably or transiently transfected cells were detached with 0.05% trypsin and 0.02% EDTA, pooled together if more than one dish of cells were used, mixed with growth medium, and then distributed into 24-well tissue culture plates (0.5 ml cell suspension per well). Cells from one 10-cm dish were transferred to one 24-well plate. 0.5 ml of growth medium with or without steroids was added into each well, and the cells were then cultured for 2 days before harvest. A simplified single step CAT assay was used to measure the transcriptional activity of the GR [21,22]. This method is based on the phase separation principle [23]. Briefly, the transfected cells in multi-well plates were washed twice with phosphate buffer saline (PBS). Two hundred and fifty microliters of 0.25 M Tris–HCl, pH 7.8 was added to each well. To prepare cell lysates, the plates underwent three cycles of freeze-thaw at −70 ◦ C and room temperature, and then were heated at 65 ◦ C for 10 min. To determine CAT activity, 100 ␮l of cell lysate from each well were mixed with 150 ␮l reaction buffer (0.25 M Tris–HCl, pH 7.8, containing 16.67 nM [3 H] acetyl–CoA and 0.267 mg/ml of chloramphenicol) and 2 ml of non-aqueous scintillation fluid Econofluor-2 (Dupont, Wilmington, DE) in a scintillation vial. CAT activity was determined by continuously counting the vials in a scintillation counter for at least three cycles at regular intervals over a period of 1–4 h. The rate of the CAT catalytic reaction (cpm/min) was obtained by dividing the difference in cpm of two cycles with the time (minutes) between them. Lysates were diluted with reaction buffer when necessary to keep the rate of reaction below 150-cpm/min. To determine ␤-galactosidase activity, 100 ␮l cell lysate were incubated with 900 ␮l of 80 mM sodium phosphate, pH 7.3, containing 9 mM MgCl2 , 102 ␮M 2-mercaptoethanol, and 8 mM of the substrates ONPG or CPRG for 2 h (CPRG) or overnight (ONPG) in a 37 ◦ C water bath. The absorbance at 570 nm (for CPRG) or at 414 nm (for ONPG) was measured on a spectrophotometer [24]. Blank controls were prepared in the same way as experimental samples, except that the transfected cells did not contain LacZ gene. For the whole cell assay of ␤-galactosidase activity [18], transfected cells were rinsed twice with PBS and fixed with cold fixing solution (0.2% glutaraldehyde, 5.4% formaldehyde) for 5 min, followed by three washes with PBS. Cells were stained with X-Gal solution (1 mg/ml X-Gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2 , and 0.01% sodium deoxycholate). Blue-stained cells were counted after overnight

incubation at 37 ◦ C. The ␤-galactosidase assays were within the linear range of the assay. The transfection efficiency was determined by ␤-galactosidase assays. Only experiments in which the transfection efficiencies were within two-fold of each other were quantified. Results of CAT assays were normalized for the transfection efficiency using the amount of ␤-galactosidase activity in the transfected cells when results from two or more transfections of cells were compared.

2.5.

Hormone-binding assay

Cells were transfected as described above. After 3 days, these cells were harvested using a rubber policeman and washed three times with PBS. The stably transfected cells were harvested when the cells reached 90% confluence in exponential growth. The following manipulations were carried out at 4 ◦ C unless indicated otherwise. The cell pellets were suspended in three volumes of binding buffer (20 mM HEPES, pH 7.3, 20 mM molybdate, 5 mM EDTA), placed on ice for 5 min and ruptured by a Dounce homogenizer. The homogenate was centrifuged at 15,000 × g for 30 min. The resulting supernatant was re-centrifuged at 110,000 × g for 1 h. The clear supernatant (cytosol) was used for hormone-binding assays and Western blot analysis. Protein concentration was measured using Bradford reagent [25]. Hormone-binding assays were performed by the charcoal adsorption method [16,26]. Cytosol and [3 H]-TA were incubated in an ice water bath overnight in a total volume of 100 ␮l containing 0.6–1.2 mg protein (total binding). Nonspecific binding was determined by including at least 2000-fold molar excess of non-radioactive steroid in the binding mixture. After incubation, 300 ␮l of 2.5% dextran-coated charcoal were added to each tube and the tubes were then vortexed for 10 s. The tubes were placed at room temperature for 10 min and then spun down in a microcentrifuge for 8 min. [3 H]-TA present in the supernatant was determined in a scintillation counter. Specific hormone-binding was obtained by subtracting nonspecific binding from total binding. The number of GR molecules in the transfected cells was calculated from the maximal specific hormone-binding activities in cell lysate normalized with the cell number.

2.6.

Immunoblotting

The expression of the GR was compared using enhanced chemiluminescence (ECL) Western blot analysis. The GR in approximately 30 ␮g cytosol (above) was separated on a 12% SDS-polyacrylamide gel. Prestained lysozyme (15.3 kDa), ␤-lactoglobulin (18.3 kDa), carbonic anhydrase (27.77 kDa), ovalbumin (44.23 kDa), bovine serum albumin (71.03 kDa), phosphorylase B (105.7 kDa), and H chain of myosin (195.97 kDa) from BRL Life Technologies, Inc. (Gaithersburg, MD) were used as molecular weight markers. After electrophoresis, the gels were immersed in transfer buffer (48 mM Tris, 39 mM glycine, pH 9.2, 20% methanol and 0.00375% SDS) for 20 min. Proteins in gels were transferred from gels to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST (10 mM Tris–HCl, pH 80, 150 mM NaCl and 0.05% Tween 20) for 2 h at room temperature with shaking, followed by washing three times with TBST, each for 15 min. The

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membranes were incubated with primary antibody against the GR (GR49) at room temperature for 1 h with shaking and washed as described above. The membranes were then incubated with a secondary antibody–peroxidase conjugate for 1 h and then washed again. The GR band was visualized by incubating the membranes in enhanced chemiluminescence (ECL) detection solution (Amersham, Arlington Heights, IL) followed by exposing them to X-ray films.

3.

Results

3.1. Mifepristone functions as an antagonist as well as a partial agonist of the We have shown previously that although mifepristone has a high affinity for the GR and competes against glucocorticoid agonists such as dexamethasone (dex) for binding to the GR, inhibition of the GR response is modest even when present at 5000-fold the concentration of dex (10 ␮M mifepristone, 2 nM dex) [27]. This is due to the inherent agonist activity of mifepristone. To further characterize the agonist activity of mifepristone on the GR, we examined its agonist activity in WCL-2 cells. WCL-2 cells are CHO cells that express high levels of GR [28]. In this cell line, both steroids induced CAT activity in a dose-dependent manner; mifepristone had a somewhat higher EC50 than dex (9.2 nM for mifepristone, 3.7 nM for dex). As expected, mifepristone acted as a partial agonist in these cells giving 13.8% of the activity of dex at saturating levels of ligand (Fig. 1A, insert). In Fig. 1B the agonist activity of 5 × 10−8 M mifepristone on the GR in a variety of cell types is presented. Mifepristone had the lowest amount of agonist activity in CV1 cells with only 8% of the activity of 5 × 10−8 M dex and the highest activity in E8.2.A3 cells with 19% as measured by the ability to induce CAT activity.

3.2. The amount of GR agonist activity expressed by mifepristone in transiently transfected COS cells increases as pmGR levels increase COS-7 cells were transiently transfected with increasing amounts of the GR expression vector pmGR, a fixed amount (10.5 ␮g/10 cm dish) of pMTVCAT and carrier DNA to bring the total amount of DNA to 30 ␮g in each transfection (Fig. 2A). A constant amount of DNA was used so that the efficiency of transfection would not vary based on the amount of pmGR used. The level of CAT activity in the absence of steroid did not increase. However, CAT activity in cells treated with 5 × 10−8 M dex increased as GR expression plasmid levels increased. 5 × 10−8 M mifepristone also gave increasing amounts of CAT activity, but the rate of increase was much higher than with dex. This resulted in an increase of mifepristone agonist activity from less than 5% to 25% relative to dex activity. To test whether the effect is specific to mifepristone, or a general feature of agonists–antagonists, we also induced the cells with the partial glucocorticoid agonist progesterone and the glucocorticoid antagonist ZK98.299. The agonist activity of progesterone did correlate with the amount of GR expression vector used in the transfections (Fig. 2) whereas ZK98.299 was inactive under any conditions (not shown). Since the agonist

Fig. 1 – The level of agonist activity of mifepristone is dependent on the cell line. Cells were transfected with pMTVCAT, pBAG with (COS-7 and CV-1) or without (WCL-2 and E8.2.A3 GR) 0.5 ␮g pmGR. The next day, the transfected cells were detached, placed in 24 well plates and induced with dex or mifepristone; CAT and ␤-galactosidase activities were determined 2 days later. Each treatment was performed in triplicate. ␤-Galactosidase activity was used to normalize the CAT activity within each cell line. (A) Dose-response curves of the GR induced by dex or mifepristone in WCL-2 cells. (B) The transcriptional activity of the GR in different cell types induced by 5 × 10−8 Dex or mifepristone.

activity of mifepristone increases as receptor expression vector increases, one would predict that the antagonist activity of mifepristone would decrease with increasing receptor plasmid. To test this, COS-7 cells were transfected with increasing levels of GR expression vector and then treated with a mixture of 5 × 10−8 M dex and 5 × 10−8 M mifepristone. Mifepristone was most effective as an antagonist with low levels of GR expression vector and effectiveness decreased with increasing levels of expression vector as predicted (Fig. 2C). In the experiments described above, cells were transfected with increasing amounts of the expression vector and a constant amount of the reporter plasmid (pMTVCAT). To

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Fig. 2 – The agonist activity of both mifepristone and progesterone increases as the amount of GR expression vector increases. COS-7 cells were transfected with pMTVCAT and pBAG and the indicated amount of pmGR. The next day, cells were detached, seeded into 24 well plates and treated with 5 × 10−8 M dex, 5 × 10−8 M mifepristone, 10−6 M progesterone, or a combination of these ligands as shown. Cells were harvested 2 days later. (A) Induction of the transcriptional activity of the GR with dex, mifepristone, progesterone or vehicle. Vehicle alone values were 2.2 ± 0.6 cpm/min and showed no relationship to the amount of pMGR added (not shown). (B) Agonist activity of mifepristone and progesterone relative to the agonist activity of dex. (C) Antagonist activity of mifepristone. Cells were treated with either 5 × 10−8 dex alone or a mixture of 5 × 10−8 dex and 5 × 10−8 mifepristone. Inhibitory activity of mifepristone was calculated.

determine whether the change in ratio of the reporter to the expression vector led to the results, we transfected increasing amounts of the reporter into COS-7 cells along with a constant amount of pmGR (Fig. 3). Results indicated that increase of pMTVCAT in the transfections increased CAT activity due to treatment with 5 × 10−8 M dex or 5 × 10−8 mifepristone (Fig. 3A). However, the increases were very similar, with little change in the relative agonist or antagonist activity of mifepristone (Fig. 3B). Progesterone again acted in a similar way to mifepristone. These results show that the ratio of expression versus reporter plasmids was not responsible for the increased agonist activity of mifepristone.

3.3. Agonist activity of mifepristone in stable cell lines expressing different levels of GR protein Our interpretation of the results with transfected COS-7 cells is that increasing levels of the GR cause the increased agonist activity of mifepristone and progesterone. To address this directly we turned to the mouse L-cell cell line E8.2.A3. This cell line was developed by Housley and Forsthoefel [15] and does not express the GR. We then stably transfected these cells with pmGR and pMTVCAT and picked clones that stably expressed different levels of the receptor. We chose this method over further analysis of COS-7 cells because, unlike

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Fig. 3 – The agonist activities of both mifepristone and progesterone are not influenced by the amount of reporter plasmid used. COS-7 cells were transfected with 1 ␮g pmGR and 0.5 ␮g pBAG and the amount of pMTVCAT shown. Cells were replated into 24-well tissue culture plates the next day and treated with 5 × 10−8 M ligand or vehicle as shown. Two days later, the cells were harvested for CAT assay. (A) CAT activity induced by the ligands. (B) Agonist and antagonist activity of mifepristone relative to the agonist activity of dex.

COS-7 cells, each L-cell line would be expected to contain cells expressing a relatively homogeneous level of the receptor. In addition, when these cells are stably transfected with constructs containing the MMTV long terminal repeat, MMTV integrates into genomic DNA and the chromatin undergoes GR induced rearrangement when treated with glucocorticoids [29]. An analysis of the GR expression levels in these clones by both hormone-binding assay and Western blot is shown in Fig. 4. Individual clones were treated with dex and or mifepristone (5 × 10−8 M) and CAT activity determined 2 days later (Fig. 5). Similar to COS-7 cells, as cells expressed more GR, CAT activity increased in response to both dex and mifepristone (Fig. 5A). The greater increase in CAT activity in response to mifepristone than to dex resulted in an increase in the relative agonist activity of mifepristone compared to dex (Fig. 5B). Conversely, the antagonist activity of mifepristone decreased with increasing receptor levels (Fig. 5C and D).

Fig. 4 – Expression of mouse GR in stably transfected E82 cells. GR-negative cells, E8.2.A3 were cotransfected with the GR expression vector (pmGR), reporter vector (pMTVCAT) and selection vector (pSV2neo) as described in Section 2. The transfected cells were selected with G418 and cell lysates were prepared from individual clones. Expression of the GR protein was determined by Western blot analysis. The number of GR molecules in individual clones was determined by hormone-binding assay.

Table 1 – Effect of GR levels on EC50 values for dex and mifepristone Cells

EC50 (nM)

COS-7, 100 ng COS-7, 500 ng COS-7, 1000 ng COS-7, 3000 ng E8.2.A3 #19 E8.2.A3 #23 E8.2.A3 #26

Dex

Mifepristone

2 2.3 2.2 1.1 32 28 28

2.2 3.3 2.2 2.2 nd nd nd

COS-7 cells were transfected with pMCAT and the indicated amount of pmGR. Cells were treated with increasing concentrations of dex or mifepristone; CAT activities were determined 2 days later. nd: not determined.

3.4. Dose response curves for dex and mifepristone vary little in response to increasing levels of receptor One possible explanation for our results is that increasing receptor levels change the dose response curve for one or both ligands. To test this, a dose response analysis was performed in COS-7 cells transfected with a constant amount of pMTVCAT and increasing levels of pmGR and in E8.2.A3 cells. Cells were treated with increasing levels of dex or mifepristone, CAT activity determined and EC50 values calculated (Table 1). Increasing GR expression did not lead to a pattern of EC50 change indicating that changes in EC50 were not responsible for the changes in agonist activity of mifepristone.

4.

Discussion

We have shown that raising the level of mouse GR increases the overall transcription rate of MTVCAT in response to both agonists and partial agonists/antagonists. However, the rate of increase for the partial agonists mifepristone and proges-

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Fig. 5 – Partial agonistic activity of RU486 increases with the GR concentration in stably transfected cells. E82 cells were cotransfected with GR expression vectors (pmGR), reporter vector (pMTVCAT) and selection vector (pSV2neo), then selected and screened for GR expressing clones as described in Section 2. The copy number of the GR expressed in individual clones was determined by hormone-binding assays. (A) CAT activities of individual clones induced by 5 × 10−8 M mifepristone. (B) Agonist activity of 5 × 10−8 M mifepristone compared to that of 5 × 10−8 M Dex. (C) Agonist activity of a mixture of 5 × 10−8 M dex and 5 × 10−8 M mifepristone. (D) Antagonist activity of 5 × 10−8 M mifepristone.

terone is greater than for the full agonist dex, resulting in increased agonist activity of these partial agonists relative to dex. In competition experiments with mixtures of agonist and antagonist, the antagonist effect of these ligands decreased as receptor levels rose. These effects were observed within a variety of cell types expressing either transiently or stably expressed receptor. The level of GR within cells in vivo is not only dependent on the cell type but is also affected by growth conditions. There are significant barriers to teasing out the role of receptor levels in partial agonist responses in vivo since the receptor interacts with a whole host of transcriptional regulators whose levels are also dependent on cell type and growth conditions [8]. However, our results suggest that receptor level is at least partly responsible for the different agonist levels of partial agonists–antagonists in different cell types in vivo.

It is not completely understood why some GR ligands are unable to bring about as robust a transcriptional response as other ligands. This lack of understanding is due to the complex effects agonists have on the GR. In the absence of ligand the GR is found in a complex with heat shock proteins and associated factors (reviewed in Ref. [30]). Glucocorticoids bind to the receptor bringing about a conformational change that alters its association with chaperones, allows nuclear localization, DNA binding, and association with a host of other factors including coactivators and corepressors [9,31]. Theoretically partial agonists could act inefficiently at one or more of these processes. There is strong evidence from X-ray crystallography that in general partial agonists of steroid receptors induce a different receptor conformation than agonists. For instance, the coactivator-binding site of estradiol-bound estrogen receptor is very different from that of raloxifene-bound receptor. In the latter case the position of Helix 12 changes such that the original coactivator-binding site is occluded and a new binding site is formed [32]. This has led to the general acceptance that the partial agonist-induced conformations have altered affinity for transcriptional coactivators and corepressors. In this model it is the altered interaction with coregulators that reduces the transcriptional response. This may also occur with mifepristone activation of the GR since this ligand can not only induce a different conformation of the receptor compared to dex [33], but also altered ability to interact with coregulators [10,11,10]. If reduced interaction with coactivators is indeed the main reason for the decreased transcriptional activity of mifepristone-bound receptor, it would be predicted that occupancy rates of DNA binding sites by mifepristone and dex bound receptors would be similar since the decreased efficiency of coactivator interaction would take place subsequent to DNA binding. In a very insightful paper from Gordon Hager’s group, it was shown that this prediction is incorrect [34]. They examined GR-regulated transcription in single cells using an integrated MMTV array, fluorescent GR binding, and FISH analysis of the RNA produced. They found, as expected, that the transcription rate of mifepristone-bound GR is only a fraction of dex-bound receptors. Unexpectedly, mifepristone-treated cells had lower GR binding rates, and there was a correlation between occupancy of the promoter and transcription rate. That is, although mifepristone alters the receptor’s ability to interact with coactivators and corepressors in vitro, when present on the promoter in vivo it has similar transcriptional activity to the dex-bound receptor. A possible explanation for this result is that activation of transcription by the GR is accomplished by a set of reversible processes, one of which would be DNA binding and another binding to transcriptional coactivators. Hager’s group [34] suggests that the formation of an active transcription complex is stochastic in nature. If we assume that GR binding to DNA is stabilized by the formation of a transcriptionally active complex, then decreased affinity with coactivators would lead to decreased occupancy of the DNA (Fig. 6). That is, the probability of forming a productive complex with mifepristone-bound receptor would be decreased. If such a model is correct, why do increased receptor levels have a more dramatic effect on mifepristone-bound GR than on dex-bound receptor? One possibility is that increasing mifepristone-bound receptor levels

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Acknowledgments MD was supported by grants R01 DK42552 and K04 DK02105 from the National Institutes of Health.

references

Fig. 6 – Model to explain the effects of increasing receptor levels on the agonist activity of mifepristone. (A) Dex (D) allows efficient formation of a receptor:DNA:coactivator complex that is converted to a transcriptionally active complex via poorly defined mechanisms. Increased receptor levels have little effect on complex formation, as the underlying process is so efficient. (B) Mifepristone (M) has reduced ability to interact with coactivators making the formation of receptor:DNA:coactivator complexes inefficient. The low level of these complexes limits the formation of transcriptionally active complexes. Increased receptor levels drive formation of receptor:DNA:coactivator complexes that can then be converted to transcriptionally active complexes.

increases levels of receptor occupancy of the promoter, which in turn overcomes the altered binding activity such complexes have with coactivators compared to dex-bound complexes (Fig. 6). Perhaps one way to test these hypotheses would be to repeat Hager’s group’s experiment discussed above [34] but transfecting in increasing amounts of wild type (i.e. nonfluorescent) GR. If our model is correct, prompter occupancy should increase more rapidly with mifepristone-bound GR than with dex-bound GR. We examined the possibility that left shifts in dose response curves were responsible for the increase in relative agonist activity of mifepristone by constructing dose response curves and calculating EC50 values (Table 1). Although there was some variation in EC50 values obtained from these curves, there was no direct correlation between receptor level and the relative agonist activity of mifepristone (Table 1). This was to be expected since increasing receptor levels increased dex and mifepristone activity with no discernible plateau effect at high concentrations (Fig. 5). We also used relatively high concentrations of the ligands, which should decrease the effect of any left shift in the dose response curve. That is, the increased relative agonist activity of mifepristone and progesterone we observed is independent of changes in the dose response curves to these ligands. This is in contrast to previous work in which changes in agonist activity of partial agonists was linked to left shifts in the dose response curve [35,36].

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