Opposing Effects of Corepressor and Coactivators in Determining the Dose-Response Curve of Agonists, and Residual Agonist Activity of Antagonists, for Glucocorticoid Receptor-Regulated Gene Expression

Daniele Szapary, Ying Huang and S. Stoney Simons, Jr.

Steroid Hormones Section National Institute of Diabetes and Digestive and Kidney Diseases Laboratory of Molecular and Cellular Biology National Institutes of Health Bethesda, Maryland 20892-0805


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A distinguishing, but unexplained, characteristic of steroid hormone action is the dose-response curve for the regulation of gene expression. We have previously reported that the dose-response curve for glucocorticoid induction of a transfected reporter gene in CV-1 and HeLa cells is repositioned in the presence of increasing concentrations of glucocorticoid receptors (GRs). This behavior is now shown to be independent of the reporter, promoter, or enhancer, consistent with our proposal that a transacting factor(s) was being titrated by added receptors. Candidate factors have been identified by the observation that changes in glucocorticoid induction parameters in CV-1 cells could be reproduced by varying the cellular levels of coactivators [transcriptional intermediary factor 2 (TIF2), steroid receptor coactivator 1 (SRC-1), and amplified in breast cancer 1 (AIB1)], comodulator [CREB-binding protein (CBP)], or corepressor [silencing mediator for retinoid and thyroid-hormone receptors (SMRT)] without concomitant increases in GR. Significantly, the effects of TIF2 and SMRT were mutually antagonistic. Similarly, additional SMRT could reverse the action of increased levels of GRs in HeLa cells, thus indicating that the effects of cofactors on transcription may be general for GR in a variety of cells. These data further indicate that GRs are yet an additional target of the corepressor SMRT. At the same time, these cofactors were found to be capable of regulating the level of residual agonist activity displayed by antiglucocorticoids. Finally, these observations suggest that a novel role for cofactors is to participate in processes that determine the dose-response curve, and partial agonist activity, of GR-steroid complexes. This new activity of cofactors is disconnected from their ability to increase or decrease GR transactivation. An equilibrium model is proposed in which the ratio of coactivator-corepressor bound to either receptor-agonist or -antagonist complexes regulates the final transcriptional properties.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The dose-response curve, showing the fold induction of a biological response with increasing concentration of steroid, summarizes a major physiological consequence of steroid hormones in a responsive cell. Many of the events leading to steroid-regulated gene induction have been elucidated and include steroid binding to the cognate receptor, activation or transformation of the receptor-steroid complex, complex binding to the hormone response element, recruitment of coactivators vs. corepressors, and interaction with auxiliary transcription factors followed by a series of events starting with transcription initiation and culminating in the mature, induced protein (1, 2, 3, 4). Steroid binding to receptors is an obligatory step in such steroid-induced responses. The observance of a dose-response curve that could be superimposed on the curve for steroid binding to a receptor was early evidence for the requirement of that receptor protein in steroid hormone action (5). Generally, the concentrations of circulating steroid in humans and animals are below those required for saturation of the receptor. Therefore, a physiologically relevant action of steroids is to specify the percentage of maximal induction that is elicited from a given responsive gene.

The close correlations that were often noted between steroid binding to receptors and steroid induction of a biological response led to the conclusion that the dose-response curve was dictated by the affinity of steroid for the receptor (6, 7, 8). Thus, all genes responding to a particular steroid were predicted to display the same dose-response curves, at least in the same cell. It was not uncommon for a given steroid to afford different dose-response curves in different cells or organisms, possibly due to phenomena such as unequal amounts of metabolism, serum binding proteins, and nonspecific binding to cells. However, reports of dissimilar dose-response curves for the regulation of two genes by the same receptor-steroid complex in the same cell caused the model to be seriously questioned (9, 10, 11, 12). Additional discrepancies included the findings that the dose-response curve of some, but not all, glucocorticoid-responsive genes could be modified by altering the density of the cells in culture (13), by the presence of a 21-bp cis-acting element of the rat tyrosine aminotransferase (TAT) gene (14, 15, 16, 17), and finally by elevated levels of glucocorticoid receptor (GR) (18). In the face of these unexpected results, no model has yet been able to explain the molecular determinants of dose-response curves for steroid induction, or repression, of gene expression.

Another aspect of steroid hormone action that has resisted descriptions at a molecular level pertains to the residual agonist activity displayed by antisteroids. While it has long been thought that the amount of residual agonist activity of each antisteroid is independent of the gene examined, many exceptions have been noted (9, 10, 12, 14, 18, 19, 20, 21, 22, 23, 24). A prominent feature of the current model is that corepressors are bound to the ligand-free, or antagonist-bound, receptors. Agonist binding is believed to result in a conformational change of the protein, thereby releasing the corepressor and/or recruiting coactivators. However, this has been envisaged as an all-or-none process with corepressor or coactivator binding mediating gene repression or activation, respectively (2, 3, 25, 26, 27, 28, 29) and, therefore, cannot readily account for changes in residual agonist activity. The activity of antisteroids can also be altered by trans-acting factors, such as dopamine (30), EGF (31), and protein kinase A inducers (32, 33). Thus, general models are presently lacking for the molecular determinants of both dose-response curves and the residual agonist activity of antisteroids.

We have recently described a system in which both the dose-response curve of glucocorticoids and the residual agonist activity of antiglucocorticoids were modified by increased levels of GRs (18). The parallel behavior of the two responses intimated that they might be closely linked at the molecular level. These effects were seen when receptors were both limiting and nonlimiting for gene induction, thereby suggesting that another component of the transcriptional machinery could be titrated by added receptors. The purpose of the current study was to further define those components that are proposed to affect the dose-response curve and the partial agonist activity of antisteroids. We now report that the effect of transfected receptors was independent of reporter, promoter, and enhancer. Surprisingly, coactivator and corepressor molecules exerted opposing effects that could modify the induction properties in a manner that was independent of changes in the fold induction or GR protein. A model is advanced in which the ratio of coactivator to corepressor is a defining ingredient for both the dose-response curve for agonist steroids and for the amount of residual agonist activity displayed by antisteroids.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Modified Induction Parameters with Increasing Receptor Are Independent of Reporter, Promoter, and Enhancer
We previously reported that the dose-response curve for GR-regulated transactivation of a transiently transfected reporter construct is progressively shifted to lower concentrations of steroid hormone when the GR concentrations are raised (18). Furthermore, we showed that these changes in the dose-response curve under different conditions can be more easily detected by comparing the induction by a subsaturating, physiological level of agonist (~1 nM Dex) after expressing this value as the percent of maximal induction with 1 µM Dex to normalize for interexperimental variations in absolute response. Any increase in the percent agonist activity for approximately 1 nM Dex is then due to a left shift in the dose-response curve (14, 15, 18). Similarly, an increase in the residual agonist activity of an antiglucocorticoid can be directly determined. In this manner, increased GR in CV-1 and HeLa cells caused both a left shift in the dose-response curve with Dex and increased amounts of agonist activity with antiglucocorticoids from a chloramphenicol acetyltransferase reporter (18). This same behavior was obtained with a luciferase reporter in both CV-1 (Fig. 1AGo) and HeLa cells (data not shown). Thus, the effect of transfected GR is independent of the reporter gene. The absence of a statistically significant increase in the agonist activity of RU 486 in CV-1 cells (P = 0.27, n = 5), compared with HeLa cells (18), presumably reflects the lack of a tissue-specific component and is reminiscent of the report that RU 486 is a better agonist for the B form progesterone receptors (PR) than is R5020 in HeLa cells but not in CV-1 cells (24). Also, RU 486 displayed agonist activity with stably transfected PR (and transiently transfected PRE2tkLUC) in HeLa but not CV-1 cells (34).



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Figure 1. Effect of Reporter, Promoter, and Enhancer on Activity of Agonists and Antagonists with Increased Levels of GR

Triplicate dishes of CV-1 cells were transfected with the indicated amounts of pSVLGR and 2 µg of GREtkLuc (panel A) or 1 µg of MMTVLuc reporter (panel B) (18 ). The fold induction by 1 µM Dex at low and high concentrations of GR was typically 7- and 20-fold for GREtkLuc and 200- and 450-fold for MMTVLuc. The activity of each steroid was expressed as percent of maximal induction as described in Materials and Methods. The data are averages ± SEM for n = 4–5 experiments.

 
The role of the promoter was examined in HeLa cells using the chloramphenicol acetyltransferase (CAT) reporter under the control of either the minimum thymidine kinase promoter (tkmin) or the TAT gene promoter (tat). While the absolute activities were different with the two promoters (Table 1Go), both promoters were equally effective in mediating the effect of increased GR. The mouse mammary tumor virus (MMTV) promoter/enhancer, which contains a complex glucocorticoid response element (GRE) (35), gave results in CV-1 cells (Fig. 1BGo) that were very similar to those with the different promoters in CV-1 and HeLa cells (Table 1Go and Ref. 18). Interestingly, the MMTV promoter/enhancer permitted a significant increase in the agonist activity of RU 486 (P = 0.049, n = 4) that was not apparent with the simple GRE enhancer (Fig. 1AGo; P = 0.27, n = 5). This may be due to a heightened capacity of the MMTV promoter/enhancer to mediate increased levels of agonist activity of antisteroids, as witnessed by the very high levels of agonist activity for the antiglucocorticoid dexamethasone 21-mesylate (Dex-Mes) (Fig. 1BGo vs. A and Table 1Go). Nevertheless, the ability of the increased levels of GR to modify the induction parameters was relatively independent of enhancer, promoter, and reporter.


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Table 1. Effect of Promoter on Ability of Transfected GR to Increase Steroid Activity as Percent of Maximal Induction by 1 µM Dex in HeLa Cells

 
We had previously found that the effects of transfected GR were specific in HeLa cells in that no change was seen with the transfected A form of human PR (PR-A) (18). Similarly, the addition in CV-1 cells of 0.36 µg of PR-A cDNA plasmid with 0.04 µg of GR cDNA had no effect compared with the further addition of 0.36 µg of GR cDNA (percent agonist activity of 0.5 nM Dex: 7 ± 11% for 0.04 µg GR; 2.5 ± 9.1% for 0.04 µg GR plus 0.36 µg PR-A; 68 ± 2% for 0.4 µg GR [± range; n = 2]). PR-A was being expressed as seen by the squelching of GR induction when 10 nM R5020 was added along with Dex (data not shown). Therefore, the above effects of transfected GR are not an artifact of transfection but are selective for GR.

Coactivators Mimic Transfected GR
Given the ubiquitous role of coactivators in steroid hormone action, we inquired whether they might participate in determining the GR dose-response curve. Transcriptional intermediary factor 2 (TIF2)/glucocortiocoid interacting protein 1 (GRIP-1) caused a moderate increase in the total transactivation by saturating concentrations of Dex (100 nM) in the presence of low levels of transiently transfected GR in CV-1 cells (Fig. 2AGo). This is consistent with TIF2 being a coactivator. At the same time, the activity of 1 µM concentrations of Dex-Mes increased about 4-fold. To determine whether or not the dose-response curve of Dex, or the percent residual agonist activity of Dex-Mes, had been altered by the added TIF2, the data were replotted as percent of maximal induction by 100 nM Dex. As shown in Fig. 2BGo, the added TIF2 produced both a clear left shift in the Dex dose-response curve and a marked increase in the percent agonist activity of the antagonist Dex-Mes. Further experiments were then conducted with the simpler, three-point dose-response curve (14, 15, 18), in which an increase in the percent of maximal activity by a subsaturating concentration of Dex, such as with 1 or 3 nM in Fig. 2BGo, was diagnostic of a left shift in the dose-response curve. Using this abbreviated assay, we confirmed the ability of added TIF2 to shift the Dex dose-response curve to the left (Fig. 3AGo). This shift was accompanied by a 2- to 3-fold increase in the fold induction by saturating concentrations of Dex (1 µM), as in Fig. 2AGo, which contrasts with the reports that TIF2 has little effect on the fold induction by GR in HeLa cells (36, 37). At the same time, the residual agonist activity of the antagonists Dex-Mes and progesterone was significantly augmented. The lack of appreciable change in RU 486 activity parallels the inability of transfected GR to alter RU 486 activity (Figs. 1Go and 3AGo), suggesting that increased levels of either GR or TIF2 are acting via the same mechanism. These responses require the C-terminal sequences of TIF2 as a truncated protein (TIF2.0 = amino acids 1–627) lacking the receptor interacting and transactivation domains (4) was inactive (data not shown).



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Figure 2. Dose-Response Curves for Dex Induction by Transfected GR ± Cotransfected TIF2

Triplicate dishes of CV-1 cells were transfected with 1 µg of GREtkLuc reporter and low (0.04 µg) amounts of pSVLGR plus 0.2 µg of TIF2, followed by the indicated concentrations of Dex (18 ). A, Total luciferase activity (relative). The total activity of induced luciferase activity was plotted for each series (circles). At the same time, the total activity seen with 1 µM Dex-Mes (DM) was included at the edge of the graph (bars). B, Normalized dose-response curves. To directly determine whether or not the Dex dose-response curves for GR ± TIF2 were the same, the activity at each point in the curves, or bars, of panel A was expressed as percent of maximal induction by 100 nM Dex in similarly treated cells. In both graphs, the responses with GR alone are indicated by the open symbols while the data for GR + TIF2 are designated by the solid symbols.

 


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Figure 3. Ability of Added Coactivator/Comodulator to Reproduce the Enhanced Activity of Agonists and Antagonists with Increased Levels of GR

Triplicate dishes of CV-1 cells were transfected with 1–2 µg of GREtkLuc reporter and low (0.02–0.04 µg) or high (0.2–0.4 µg) amounts of pSVLGR plus 0.2 µg of TIF2 (panel A), 0.9 µg of SRC-1 (panel B), 0.9 µg of AIB1 (panel C), or 0.8 µg of CBP plasmid (panel D) as in Fig. 2Go. The activity of each steroid was expressed as percent of maximal induction by 1 µM Dex as described in Materials and Methods. The data are averages ± SEM for n = 3–4 experiments, except for the high GR data with AIB1 where the average ± range of two experiments is given.

 
TIF2/GRIP-1 is related to a group of approximately 160-kDa coactivators (p160) that includes steroid receptor coactivator 1 (SRC-1) and amplified in breast cancer 1 (AIB1)/activator of thyroid and retinoic receptors (ACTR)/p300/CBP interacting protein (pCIP)/receptor-associated coactivator 3 (RAC3) (3). Almost all of these factors have been shown to interact with GR (37, 38). We therefore examined the effects of these related factors. SRC-1, which is reported to produce only marginal increases in the total activation by GRs with saturating concentrations of Dex in HeLa cells (39), caused a 1.2- to 3-fold increase in GR transactivation in CV-1 cells (data not shown). This small enhancement of the fold induction could reflect the presence of appreciable quantities of endogenous SRC-1 in CV-1 cells (40). AIB1 (41) caused between no change and a 2-fold increase (data not shown). In both cases, however, added cofactor mimicked the ability of additional GR to cause a left shift in the Dex dose-response curve (i.e. increased activity of 1 nM Dex) and to augment the residual agonist activity of antiglucocorticoids (Fig. 3Go, B and C). Again, the increased activity of the antagonists Dex-Mes and progesterone, but not RU 486, with added SRC-1 (Fig. 2BGo) argues that added coactivator, or GR, are each mediating the same responses.

The ability of increased concentrations of GR, SRC-1, TIF2, and AIB1 to cause the same net result suggested that a common factor is being titrated. One candidate was the adapter protein CREB-binding protein (CBP) (42), which is known to interact with all of the above proteins (2, 3, 4, 43, 44, 45, 46). Therefore, if each coactivator acted by sequestering the available CBP, added CBP should reverse the responses and cause a right shift in the Dex dose-response curve and a decrease in the partial agonist activity of antisteroids. However, increased CBP resulted in the same behavior as added GR instead of counteracting the GR (Fig. 3DGo and data not shown). Thus, the effect of increased GR or coactivator cannot be due to titration of endogenous CBP. Interestingly, the increased activity with added CBP occurred under conditions where the fold induction by saturating concentrations of Dex was reduced by 25 ± 8% (±SEM, n = 4).

TIF2 Effects Occur without Raising GR Levels
Given the ability of each of the coactivators, and CBP, to mimic the effects of high levels of transfected GR in causing increased activities for both subsaturating concentrations of Dex (due to a left shift in the dose-response curve, as in Fig. 2Go) and saturating concentrations of antagonists (Fig. 3Go, A–D), we asked whether the coactivators were acting simply by increasing the amount of GR obtained after transfection with low levels of GR cDNA to those seen with high concentrations of transfected GR cDNA. Detection of transfected GR in CV-1 cells was complicated by the presence of endogenous, biologically inactive GR that specifically bound [3H]Dex but was unable to transactivate the GREtkLUC reporter (data not shown). In fact, the specific Dex binding in CV-1 cells transiently transfected with 0.2 µg of pSVLGR was indistinguishable from that in untransfected cells. However, the ratio of transfected GR to endogenous, biologically inactive GR could be dramatically increased by using magnetic isolation procedures to select for those cells that had taken up the exogenous plasmid DNAs. Western blots of the cytosols from those magnetically isolated cells, which had been transfected with 0.4 µg of pSVLGR, revealed the overexpressed GR as the usual 98-kDa species (Fig. 4Go, species labeled as a) plus two lower molecular mass species (labeled as b and c) arising from alternative translational start sites (47) (lane 9 vs. lanes 2 and 4). Cells transfected with 0.04 µg of GR did not contain noticeably higher levels of GR (lanes 7 vs. 6). Most significant, though, was that the transfection of 0.04 µg of GR plus 0.2 µg of TIF2, conditions affording the same increased activities of 1 nM Dex and 1 µM Dex-Mes as obtained with 0.4 µg of GR (see Fig. 3AGo), did not result in any more GR than that without TIF2 (lanes 8 vs. 7) and much less than that seen with 0.4 µg of GR (lanes 8 vs. 9). Therefore, the presence of added TIF2 with low concentrations of GR protein affords the same biological responses as seen with higher amounts of GR in the absence of TIF2. Thus, the ability of coactivators to modulate GR transactivation properties occurs via a mechanism that is different from simply increasing the GR concentrations.



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Figure 4. Effect of TIF2 on GR Concentration in Transiently Transfected CV-1 Cells

CV-1 cells were transfected with 0.1 µg of IL2R cDNA plus the following DNAs (amounts are listed per 60-mm dish but were scaled up for the larger volumes of cells used): carrier and vector DNAs only (lane 6), 40 ng pSVLGR (lane 7), 40 ng pSVLGR plus 200 ng TIF2 (lane 8), or 400 ng pSVLGR (lane 9). The transfected cells were enriched by adding mouse monoclonal antihuman IL2R antibody, followed by M-450 Dynabeads coated with goat antimouse IgG. The magnetically tagged cells were immobilized by magnetic plates on the walls of the flask and washed to remove untransfected cells. The cytosols from the washed pellets were prepared and the receptor was visualized by ECL as described in Materials and Methods. Lanes 4, 5, and 10 contain the indicated units of GR that was overexpressed from pSVLGR in Cos-7 cells. The heterogeneity of receptors overexpressed in Cos (labeled as a, b, and c, with a being the full length GR) and CV-1 cells is due to downstream translational start sites (47 ). Lanes 1 and 2 are a shorter exposure of the lanes 3 and 4 to show that the smaller translation product of pSVLGR is the most intense, consistent with the pattern seen in lane 9 for 400 ng of pSVLGR in CV-1 cells.

 
Corepressor Antagonizes the Effect of Coactivators
Corepressors have inverse effects from coactivators with the nuclear receptors (48, 49) and are present, along with coactivators, in CV-1 and HeLa cells (40). Therefore, we examined the response in our system of added corepressor, even though they have not been described to interact with GR or to alter their transactivation (49). Transfected silencing mediator for retinoid and thyroid hormone receptors (SMRT) repressed the activity of subsaturating concentrations of Dex (i.e. caused a right shift of the dose-response curve) and decreased the residual agonist activity of the antiglucocorticoid Dex-Mes (Fig. 5Go). Thus, the corepressor SMRT acted in the opposite direction of GR, coactivators, or CBP. At the same time, the added SMRT resulted in a 52 ± 12% (±SEM, n = 3) reduction in the fold induction by saturating concentrations of Dex. Importantly, when SMRT and TIF2 were both added, an intermediate response was obtained (Fig. 5Go). The activity of SMRT plus TIF2 in the presence of low concentrations of GR was significantly less than that with TIF2 only (P <= 0.007) and more than that with SMRT only (P <= 0.05; both P values are for the paired Students t test, n = 5). Therefore, added coactivator and corepressor counteract, or antagonize, the effects of each other in establishing the position of the GR dose-response curve with agonists and the partial agonist activity of antiglucocorticoids.



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Figure 5. Activity of Agonists and Antagonists Responds to the Opposing Effects of the Corepressor SMRT and the Coactivator TIF2

Triplicate dishes of CV-1 cells were transfected with 0.04 µg (low GR) and 0.2 µg (high GR) of pSVLGR and 2 µg of GREtkLuc reporter without or with 0.2 µg of SMRT, 0.2 µg of TIF2, or both as in Fig. 2Go. The activity of each steroid was expressed as percent of maximal induction as described in Materials and Methods. The data are averages ± SEM for n = 5 experiments.

 
Effects of Cofactors in HeLa Cells
Cotransfection of TIF2 into HeLa cells produced little change in the fold induction by 1 µM Dex (1.3- to 1.6-fold) and no significant difference in the Dex dose-response curve or residual agonist activity of Dex-Mes (data not shown). Assuming that this could result from high levels of endogenous TIF2, which might be counterbalanced by added SMRT, we treated HeLa cells with SMRT both without and with additional GR. Little if any effect was seen when SMRT was added to the endogenous GR. However, the changes produced by increased GR could be prevented by the added SMRT (Fig. 6Go). Thus, SMRT could repress the increases in activity caused by both added TIF2 in CV-1 cells and extra GR in HeLa cells. It should be noted that the fold induction by 1 µM Dex in HeLa cells was not decreased, but increased by up to 2-fold, in the presence of added SMRT.



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Figure 6. Reversal of Effects of Added GR by SMRT in HeLa Cells

Triplicate dishes of HeLa cells were transfected with 0.8 µg of SMRT with or without and 0.4 µg of pSVLGR and 1 µg of GREtkLuc reporter as in Fig. 2Go. The activity of each steroid was expressed as percent of maximal induction (± SD of the triplicate values) as described in Materials and Methods. Similar results were obtained in two other experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GR dose-response curve can be modified in a manner that is independent of the gene, the promoter, the enhancer (Fig. 1Go and Table 1Go), and the cell (18). We further find that coactivators (TIF2/GRIP-1, SRC-1, AIB1/ACTR/pCIP/RAC3, and CBP) and corepressors (SMRT) display a previously unsuspected property in being able to oppose each other, and added receptor, in the regulation of the dose-response curve for GR. Thus, when the endogenous ratio of coactivator-corepressor in CV-1 cells was perturbed by the overexpression of one factor, the dose-response curve was repositioned. The effects of coactivators/corepressor were specific as neither a TIF2 mutant lacking all GR-interacting domains nor PR was able to produce the same responses. At the same time, this balance of coactivators and corepressors was seen to modify the residual agonist activity of antiglucocorticoids. These are new activities of coactivators and corepressors, which have been thought to affect only the fold induction of receptors bound with saturating concentrations of ligand (36, 39, 41, 43, 45, 48, 49). Thus, coactivators and corepressors are equally important for the activity of both physiological and pharmacological levels of glucocorticoid steroids.

Given the effect of variations in GR levels on both the dose-response curve and the partial agonist activity of antisteroids, the response to increased coactivators and corepressor could theoretically be indirect by modifying the level of GR. However, Western blots of cytosols of transfected CV-1 cells that had been enriched by magnetic isolation of those cells expressing the transfected DNA revealed that biologically active concentrations of TIF2 (Figs. 2Go and 3Go) caused no discernible increase in GR protein levels (Fig. 4Go). Thus, the modification of GR activity by coactivators occurs via mechanisms other than simple elevation of the level of GR protein. This inability of coactivators to increase steroid receptor levels has also been noted for AIB1 with estrogen receptors (ERs) in tumor cell lines or breast tumor samples (41), for CBP and SRC-1 with GR in COS-7 cells (44), and for a fragment of GRIP-1/TIF2 with truncated mouse GR in yeast (27). Even stronger support for this conclusion derives from an analysis of the biologically active GRs ± cofactors in our experiments. We had previously demonstrated that the total induced reporter activity is directly proportional to GR concentration in CV-1 cells (18) (Y. Huang and S. Simons, submitted). Because the level of reporter activity with low levels of GR did not increase in the presence of additional AIB1 or CBP (±10%, data not shown), we conclude that the cotransfection of coactivators did not uniformly increase the levels of either GR protein or functional GR. Also, there was no consistent relationship between the fold induction by 1 µM Dex ± cofactor in CV-1 cells. The fold induction went down with CBP and up with AIB1, SRC-1, and TIF2. Added CBP and SMRT each decreased the fold induction with saturating concentrations of agonist steroid but had opposite effects on the position of the dose-response curve and the residual agonist activity of Dex-Mes (Figs. 3Go and 5Go). Finally, SMRT exhibited some cell-specific effects in that it decreased the fold induction by 1 µM Dex in CV-1 cells but caused an increase in HeLa cells. Nevertheless, SMRT caused a right shift in the Dex dose-response curve and decreased agonist activity for Dex-Mes in both cell lines (Figs. 5Go and 6Go). Thus, the consequences of added SMRT in our assays were unchanged even in HeLa cells where SMRT acted as a coactivator for GR. These results also indicate that the influences of cofactors on the GR induction parameters of the present study can be dissociated from the more extensively studied effects on total transactivation by saturating concentrations of ligand. Therefore, as no consistent relationship could be found between the overexpression of coactivators (or corepressors) and the absolute activity, or fold induction, of the reporter construct, we conclude that the modulation of GR activity in Figs. 2Go, 3Go, 5Go, and 6Go cannot result simply from changes in the concentration of either GR protein or functional GR that might be caused by overexpressed cofactors. It should also be noted that others have observed dramatic increases in the fold induction by progesterone receptors without any change in the dose-response curve (50).

The alteration of glucocorticoid dose-response curves appears to be a novel property of corepressors and the p160 coactivators in mammalian cells. GRIP-1 causes a left shift in the dose-response curve of full-length GR (and mineralocorticoid and ERs) in yeast (27), but no reports have appeared in mammalian cells, where the effect of TIF2/GRIP-1 has varied from squelching (38) to inactivity (36) to a <=2-fold increase in fold induction (37). In our hands, overexpression of TIF2/GRIP-1 in mammalian cells had multiple effects, i.e. of shifting the dose-response curve to the left and enhancing the residual agonist activity of antagonists in addition to magnifying the fold induction by saturating concentrations of agonist by a factor of 2–3. L7/SPA, which does not appear to be related to the p160 coactivators, enhances the residual agonist activity of RU 486 bound to GR but has no effect on agonist-bound receptors (51). It was initially thought that corepressors did not bind to the steroid receptors, like GR and ER (49). More recently, though, corepressors have been found to decrease the residual agonist activity of antisteroids bound to PR and ER (29, 51, 52, 53). In most cases, the corepressor was also seen to interact with the receptor (29, 51, 52, 54). We therefore suspect that the effects of SMRT on GR action (Figs. 3Go, 5Go, and 6Go), which have not been previously reported, also derive from an interaction of SMRT with GR.

TIF2 counteracted the effects of SMRT and vice versa (Fig. 5Go). This contrasts with the report with ERs that added coactivator (SRC-1) could not reverse the effects of the corepressor SMRT (52). Thus, the interactions of coactivators and corepressors with GR may be more closely balanced than with other receptors, such as ERs.

Previous observations that only full-length, functionally active, GR were able to alter the Dex dose-response curve, and the residual agonist activity of antiglucocorticoids (18) suggested that both amino and carboxyl termini of GR are required for productive interactions with at least the coactivators, as has been observed by others (54, 55, 56, 57, 58, 59). The fact that the induction properties of reporters regulated by either TAT or MMTV enhancers responded to increased GR (Fig. 1Go) lends further support to this conclusion since receptors lacking the amino-terminal AF-1 domain were unable to induce MMTV-regulated reporters (60).

Histone acetylation and release of the repressive effects of DNA-bound nucleosomes has emerged as an attractive control mechanism for steroid-regulated gene expression (61, 62, 63). Several, but not all (4), cofactors have been found to possess histone acetylase or deacetylase activity (2, 26, 28, 64, 65). However, histone acetylation has been reported to cause both increased and decreased induction by receptors (66). Also, histone acetylation does not appear to be required in all cases of steroid-regulated transcription, especially in transiently transfected cells (67), and nucleosome reorganization may precede receptor-induced transactivation (68, 69). Nucleosome formation and phasing are highly ordered over the MMTV GREs (70) and have been found to be greatly reduced, or absent, in transiently transfected reporters (71, 72), which is the usual substrate in studies of coactivators and corepressors. Because we see both a high-fold induction of MMTVLuc expression (200–450 fold) and the typical dose-response curve left shift, and increased residual agonist activity of antisteroids, with elevated GR levels (Fig. 1BGo), we suspect that these effects of added GR and cofactors do not require histone acetylation or deacetylation.

The ability of coactivators and corepressors to alter glucocorticoid dose-response curves, and residual agonist activity of antagonists, cannot be explained by the current model in which one or the other group of cofactors is associated exclusively with a single GR species. Such behavior would be expected to lead to an all-or-nothing response as opposed to the gradual increases that are seen with added GR (18) or cofactors (data not shown). Furthermore, if antagonist-bound GR associated only with corepressors, it is not obvious how added coactivator could exert any effect. We propose that the effects described in this study reflect changes in the equilibrium mixture of oligomeric species containing corepressors, or coactivators, bound to GR complexed with either agonist or antagonists (Fig. 7Go). This is similar to the proposal of an equilibrium population of cofactor complexes with ligand-free nuclear receptors (73). However, we now extend this concept to ligand-bound GR. From our previous work with mutant GRs (18, 74), we conclude that effective interactions of coactivators and corepressors occur predominantly, if not exclusively, with steroid-bound GR. Thus, for agonist-bound GR, the ratio of coactivator-bound GR to corepressor-bound GR would determine the position of the dose-response curve without any change in the dissociation constant (Kd) of steroid binding to GR. Addition of more coactivator or CBP would force the equilibrium to the side of coactivator-bound GR by the principle of mass action, thereby causing a further left shift in the dose-response curve. Similarly, the amount of residual agonist activity displayed when an antiglucocorticoid bound to GR would be controlled by the ratio of the two sets of receptor/cofactor complexes (Fig. 7Go). Support for the generality of this model derives from the recent finding that SRC-3 (= AIB1) associated with ERs bound by either agonists or antagonists (75) and that the antiestrogen ICI 164,384 stimulated the binding of the coactivator RIP140 to ER ligand-binding domain (76). Furthermore, coactivators (TIF2, AIB1, and SRC-1) and comodulators (CBP and p300) increased the percent agonist activity of tamoxifen bound to mutant ERs (G440V) (77). A comparable situation appears to exist with the androgen receptor: the binding of antiandrogens has recently been reported to promote the interaction of receptors with the coactivator ARA70 (78). Finally, this determination of biological activity by a balance of interactions with coactivators vs. corepressors may not be unique to mammalian systems. The activity of the Drosophila transcription factor Dorsal appears to be modulated by the association of coactivators (CBP) and corepressors (Groucho) (79).



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Figure 7. Model for Control of Dose-Response Curve and Residual Agonist Activity by Ratio of GR-Steroid Complexes That Are Associated with Coactivators vs. Corepressors

Cartoon depicts some of the species proposed to be associated with DNA-bound GR-steroid complexes (shaded and cross-hatched symbolsrepresent unspecified, probable additional components). However, not all components may be simultaneously associated with the same molecule of GR. The two shapes representing GR depict presumed conformational differences resulting from the association of either coactivators or corepressors. Either agonist or antagonist steroids can be bound to each GR conformer. Extreme ratios of coactivators/corepressors would afford almost exclusively the higher order complexes on the left or right while intermediate ratios would give a mixture of higher order complexes. See text for further details.

 
A mathematical analysis of our model was performed to understand how variations in GR concentration could affect the final ratio of coactivator- vs. corepressor bound to GR-steroid complexes. We assumed that GR bound to either coactivators (CoA) or corepressors (CoR) as dictated by the following simple equations: GR + CoA {leftrightharpoons} GR-CoA, with [GR-CoA] = K1[GR][CoA], and GR + CoR {leftrightharpoons} GR-CoR, with [GR-CoR] = K2[GR][CoR]. Solving these two equations revealed that, for any cellular concentration of coactivators and corepressors, increasing amounts of receptor would yield higher ratios of coactivator-/corepressor-bound GR (i.e. GR-CoA/GR-CoR) only when the affinity of coactivators for steroid-bound GR (K1) was less than that for corepressors (K2). We therefore predict that GR complexes, regardless of the nature of the bound steroid, will have an intrinsically higher affinity for corepressors than for coactivators and/or CBP. However, the magnitude of preferential association of GR with corepressors vs. coactivators might further be modified by steroid-induced changes in receptor tertiary structure (80, 81, 82, 83) such that more coactivators associate with agonist-bound GR. This balance could be additionally modified by other factors binding to cis-acting elements (14, 15, 74).

Our model of an equilibrium interaction of cofactors with GR suggests that the ratio of cofactors plays a pivotal role in both the dose-response curve of agonists and the residual agonist activity of antagonists. Thus, in addition to the absolute level of cofactors (54), the ratio of cofactors is important. Our model is based predominantly on data from the overexpression of GR and cofactors in CV-1 cells. However, the similar results with added GR (18) and SMRT (Fig. 6Go) in HeLa cells suggest that this effect is not cell-specific. Furthermore, the recent reports that transfected GRIP-1/TIF2 caused a left shift in the dose-response curve of ER-mediated induction of an estrogen-responsive reporter gene in yeast (84), that all three classes of coactivators (and two comodulators) increased the agonist activity of the antiestrogen tamoxifen in HeLa cells (77), and that the coactivator ARA70 associates with antagonist-bound androgen receptors in human prostate cancer cells (78) argue that our model may apply to other steroid receptors in numerous cellular environments. These considerations, coupled with the recent findings of variations in levels of cofactors in different (40, 75, 85) or the same (53) cells provide an attractive mechanism for explaining cellular variations in the dose-response curve and residual agonist activity of specific receptor-steroid complexes. These results further suggest novel controls of receptor activity during differentiation, development, and homeostasis.

The model of Fig. 7Go deviates from that which has evolved for the nuclear receptors, where agonist binding causes a dissociation of corepressor and is required for the association of coactivators (2, 3, 25, 26, 27, 28, 29). However, the interactions of cofactors with GR may be much more loosely regulated than those seen for the nuclear receptors. This is consistent with variations in other properties of the nuclear receptors, such as a predominantly cytoplasmic localization of the steroid-free GR, the requirement of bound hsp90 for steroid binding of GR, the absence of either DNA binding or transcriptional repression by steroid-free GR, the loss of steroid binding activity upon deletion of amino acids beyond the proposed helix 12, different tryptic digestion sites in receptor ligand-binding domains (86), and the lack of heterodimerization of GR with retinoid X receptor (RXR). Conversely, an equilibrium nature of cofactor association with receptors may be more prevalent than previously recognized. For example, the presence of a sequence in retinoic acid receptors that interacts with coactivators prevents gene repression by the ligand-free receptor in CV-1 cells (87). Given the recent report of SRC-1 interaction with nuclear factor-{kappa}B (NF-{kappa}B) (88), it will be interesting to determine whether SRC-1 can also modify the dose-response curve of various NF-{kappa}B inducers in addition to other steroid receptors. Finally, our model suggests that an added consequence of titration/squelching, in which one transcription factor competitively binds a cofactor needed by a second transcription factor (11, 18, 44, 89) and thus alters the ratio of interacting factors, can be to modify the dose-response curve and residual agonist activity of a given receptor-steroid complex.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unless otherwise indicated, all operations were performed at 0 C.

Chemicals, Buffers, and Plasmids
Most of the chemicals, buffers, and plasmids, including rat GR (pSVLGR from Keith Yamamoto, UCSF, San Francisco, CA) are the same as described previously (18). GREtkminCAT and GREtatCAT were described elsewhere (14). MMTVLuc (pLTRLuc) was obtained from Gordon Hager (National Institutes of Health, Bethesda, MD). The cDNA plasmids of SRC-1a (B. W. O’Malley, Baylor College of Medicine, Houston, TX), TIF2 and and the truncated TIF2 (TIF2.0) plus the A-form of human progesterone receptor (Hinrich Gronemeyer, IGBMC, Strasbourg, France), AIB1 (Paul Meltzer, National Institutes of Health, Bethesda, MD), CBP (Richard Goodman, Vollum Institute, Portland, OR), and SMRT (Ron Evans, Salk Institute, La Jolla, CA) were each graciously sent as gifts.

Cell Culture and Transfection
The conditions of both growth and transient transfection (in triplicate) of CV-1 and HeLa cells, with the total transfected DNA brought up to 3 µg/60-mm dish with pBSK+ DNA have been described (18). CAT activity was determined as described previously (18). The luciferase activity was assayed using the Luciferase Assay System from Promega Corp. (Madison, WI) and the methods recommended by the supplier, including one cycle of freeze-thaw lysis of the cells in dry ice followed by centrifugation (15,000 rpm) for 15 min. In experiments with cofactor cDNA plasmid, all cells not transfected with cofactor were cotransfected with an equimolar amount of the same plasmid vector to control for artifacts of the vector DNA.

Western Blotting of Cytosols from Magnetically Isolated, Transfected Cells
CV-1 cells (three semiconfluent T150 flasks per treatment) were transfected by the usual calcium phosphate method (18), with the amounts of DNA (pSVLGR, pSG5, and TIF2 in the pSG5 vector, and pBSK+ as carrier DNA) being scaled up from that used for 60-mm dishes. Those cells that had taken up the added DNA were separated for analysis by the magnetic isolation procedure of Smith et al. (90). The initial transfection included 2.1 µg/T150 flask of pCMVIL2R (from Cathy Smith, National Cancer Institute/National Institutes of Health) (90) for the expression of interleukin 2 receptor (IL2R). The transfected cells were harvested by adding mouse monoclonal antihuman IL2R antibody (50 µg per 1 ml of Dynabeads; Upstate Biotechnology, Inc., Lake Placid, NY) followed by M-450 Dynabeads coated with goat antimouse IgG (Perspective Biosystems, Framingham, MA). Magnetic plates (BioMag Separators; Perspective Biosystems) were used to attract the magnetically tagged cells to the walls of the flask. The cytosols from the washed pellets were prepared as usual (83) and separated on an 8% SDS-PAGE gel. The receptors were visualized by Western blotting with BUGR-2 monoclonal antirat GR antibody, followed by enhanced chemiluminescence (ECL) as described (83).

Analysis of Data
The activity for subsaturating concentrations of agonist, or saturating concentrations of antagonist, was expressed as percent of maximal activity with saturating concentrations of agonist [1 µM dexamethasone (Dex) unless otherwise noted]. The fold induction with 1 µM Dex was calculated as the luciferase activity [relative light units/(mg protein)(sec)] with 1 µM Dex divided by the basal activity obtained with ethanol. Individual values were generally within ± 20% of the average, which was plotted. Unless otherwise noted, all statistical analyses were by two-tailed Student’s t test using the program InStat 2.03 for Macintosh (GraphPad Software, Inc., San Diego, CA).


    ACKNOWLEDGMENTS
 
We thank Ron Evans, Richard Goodman, Hinrich Gronemeyer, Gordon Hager, Paul Meltzer, Bert O’Malley, Cathy Smith, and Keith Yamamoto for their generous donation of reagents; Cathy Smith for help with the magnetic isolation of transiently transfected cells; Allen Minton (NIH) for the mathematical analysis of our model; and Keiko Ozato (NIH) and Paul Meltzer (NIH) for their critical review of the paper.


    FOOTNOTES
 
Address requests for reprints to: Dr. S. Stoney Simons, Jr., Building 8, Room B2A-07, National Institute of Diabetes and Digestive and Kidney Diseases/LMCB, National Institutes of Health, Bethesda, Maryland 20892.

Received for publication May 21, 1999. Revision received July 27, 1999. Accepted for publication August 17, 1999.


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