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Molecular Endocrinology 12 (2): 278-289
Copyright © 1998 by The Endocrine Society

Synergistic Enhancement of PRB-Mediated RU486 and R5020 Agonist Activities through Cyclic Adenosine 3',5'-Monophosphate Represents a Delayed Primary Response

Sabine Kahmann, Lothar Vaßen and Ludger Klein-Hitpass

Institut für Zellbiologie (Tumorforschung) Universitätsklinikum D-45122 Essen, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activators of protein kinase A have been shown to affect the transactivation potential of progestins and antiprogestins. To analyze the mechanisms and factors involved, we have created HeLa and CV1 cell clones stably expressing isoform B of progesterone receptor. In the HeLa cell background, the progesterone antagonist RU486 significantly induces progesterone-regulatable reporter genes, and this agonistic effect is synergistically enhanced by elevating cAMP or through overexpression of protein kinase A catalytic subunit. In contrast, in CV1 cells containing functional progesterone receptors no agonist activity of RU486 could be detected, suggesting the involvement of cell specifically expressed factors. In a PRB-positive HeLa cell clone containing stably integrated copies of a thymidine kinase-luciferase reporter gene with two progesterone response elements, we observed a complete loss of RU486 antagonist potential upon cotreatment with cAMP for 25 h while partial antagonist potential was maintained in a 5-h experiment. This result shows that, particularly in the presence of protein kinase A activators, the duration of hormone treatment is a crucial parameter in the evaluation of antagonist properties of antiprogestins. A detailed analysis of the kinetics of the hormone effects on transcription revealed that the onset of cAMP/RU486 synergism is delayed relative to the responses induced by RU486 or R5020 alone. Moreover, partial inhibition of protein synthesis by cycloheximide completely abolished cAMP/RU486 synergism while R5020 and RU486 responses were not inhibited. Together, these data indicate that cAMP/RU486 synergism is a delayed primary response requiring the intermediate induction of an essential factor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The progesterone receptor (PR) is a member of the family of ligand-inducible nuclear receptors that regulate hormone-responsive genes by altering the rate of transcription initiation. These proteins share common functional domains for ligand binding, dimerization, DNA binding, and transactivation (1). The binding of ligand to the hormone-binding domain of PRs triggers a cascade of events that includes dissociation of associated heat shock proteins, conformational changes, phosphorylation, and dimerization (1, 2, 3). Ligand-activated receptors bind with high affinity to specific DNA sequences, termed progesterone response elements (PREs), which can also mediate glucocorticoid induction by binding of glucocorticoid receptors (4, 5). After binding to DNA, PRs are thought to interact with components of the basal transcription machinery, either directly or through coactivator proteins (6, 7), to facilitate formation of RNA polymerase II initiation complexes (8).

In all tissues and cell lines examined, human PR is expressed in two isoforms (PRA and PRB) of 94 and 116 kDa in size, which arise from different messages transcribed from two promoters in the human PR gene (9). PRB differs from PRA only in that it contains an additional 164 amino acids at the N terminus. The two PR isoforms display indistinguishable hormone- and DNA-binding properties and can heterodimerize with each other (9). However, several studies have shown that, depending on the cell- and promoter context, PRA and PRB display remarkably different transcriptional activities, suggesting that they may have distinct physiological functions (9, 10, 11, 12, 13, 14, 15). Possibly, an additional activation function that has been identified in the PRB-unique N-terminal region is responsible for the different transactivation potential of PRA and PRB (14). Together, these data raise the possibility that alterations of the PRA/PRB ratio, as have been detected in human breast tumors (16), might profoundly affect the progesterone responsiveness of target cells.

As progesterone is implicated in a variety of hormone-dependent cancers, much effort has been dedicated to the development of ligands, which display relatively little or no agonist activity at all. By competing with progesterone for binding to the receptor, such compounds can partially (partial antagonists) or completely (pure antagonists) prevent induction of progesterone-inducible genes. Based on their effects on the DNA binding of PR in the gel retardation assay, two different types of antiprogestins have been distinguished (17). Antiprogestins, such as ZK98299, which were classified as type I antiprogestins, do not induce stable DNA binding of PR in vitro, suggesting that they inhibit receptor activity at a step before DNA binding. However, analysis of the mechanism of action in in vivo systems has led to conflicting data (18, 19). Type II antiprogestins, including RU486, induce stable binding of PRs to DNA in vitro and in vivo but normally generate a nonproductive receptor conformation that is unable to stimulate transcription of progesterone-inducible genes (17, 20, 21, 22). Consistently, RU486 fails to induce the interaction of the PR ligand-binding domain with coactivators, such as steroid receptor coactivator-1 (SRC-1) and transcriptional intermediary factor-2 (TIF2), which have been shown to play an important role in mediating the effects of agonist-loaded receptors (23, 24). Deletion of the 42 C-terminal amino acids or mutations within a 12-amino acid region in the C terminus rendered PR transcriptionally active in response to RU486, indicating that the very C-terminal end plays an important role in preventing transcriptional activity of RU486-loaded PR (25, 26). As overexpression of the 12-amino acid region stimulated the transcription activation potential of RU486-liganded full-length PR, it has been postulated that suppression of RU486 activity involves a titrable corepressor associating with the C terminus of PR (26). Occasionally observed agonistic effects of RU486 on transcription of specific reporter genes have been postulated to be due to the action of DNA-bound PRB that activates transcription via the N-terminal activation function (20). However, a study from K. Horwitz’s group (11) presented data suggesting that PRB-mediated activation of reporter genes by RU486 may also occur through a yet poorly defined mechanism that does not require binding of RU486-loaded PR to PREs.

The transactivation potential of steroid receptors can be modulated by simultaneous activation of various signal transduction pathways, which transmit signals from cell surface receptors to the nucleus. Examples of such cross-talks are the enhancement of the transactivation potential of dexamethasone-induced glucocorticoid receptor (GR) by 12-O-tetradecanoylphorbol 13-acetate (TPA) (27), an inducer of the protein kinase C pathway, or the potentiation of agonist-loaded steroid receptors by activators of the protein kinase A (PKA) pathway (28, 29, 30, 31). Interestingly, the modulating effect of cAMP is not restricted to receptors bound to natural agonists, as cotreatment with activators of PKA can also elicit strong transcriptional effects of antagonist-loaded receptors (32, 33, 34, 35, 36, 37). Synergistic effects of progesterone antagonist RU486 and cAMP on transfected reporter genes have been shown to be mediated by GR and PRB, but not PRA (36, 37). This phenomenon, which has also been designated as antagonist/agonist switching, is dependent on the promoter used for the analysis (36) and specific for type II antiprogestins (32, 33, 34, 36). cAMP has been shown to have little effect on the phosphorylation state of PR (32, 38). Moreover, mutations abolishing all putative phosphorylation sites within the N-terminal 164 amino acids specific for PRB did not interfere with cAMP/RU486 synergism (38). Thus, it appears unlikely that cAMP-mediated phosphorylation is responsible for the increased activity of RU486-loaded PRs (38).

For the analysis of the mechanism(s) responsible for the enhancement of agonist and antagonist activities through activators of PKA, we have created cell lines stably expressing PRB as well as PRE2-TK-luc and mouse mammary tumor virus (MMTV)-luc reporter genes, respectively. Using these model systems, we demonstrate a role of PKA in the mechanism. Analyzing the kinetics of agonist/antagonist induction both in the presence and absence of cAMP, we find that the synergistic induction of reporter genes by RU486 or R5020 and cAMP is a delayed response. Furthermore, we show that cAMP/RU486 synergism is abolished by cycloheximide, a protein synthesis inhibitor. Our data suggest that the synergism between cAMP-dependent PKA and liganded PRs involves the induction of an additional factor, which plays a crucial role in this mechanism.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stable Cell Lines Expressing PRB
As a model for the analysis of the mechanism of cross-talk between PKA and steroid receptor signaling pathways, we chose a HeLa cell clone (3B2), which had been stably transfected with a SV40 early promoter-driven expression plasmid encoding a His- and myc-tagged human PRB (see Materials and Methods). Using transient transfections, we previously confirmed that the introduction of the tags, which allow Ni-NTA- and myc-antibody affinity purification, has no influence on the transactivation properties of PRB (data not shown). Immunoblotting with antibodies against PR showed that the transfected HeLa cell clone 3B2 (Fig. 1AGo, lane 3) expresses a protein that comigrates with the PRB in T47D cells (lane 1) but is absent in the parental HeLa cells (lane 2). As detected by immunoblotting, the level of PRB in HeLa3B2 cells is about half as high as in the T47D cell line. The DNA-binding ability of PRB expressed in the HeLa3B2 cells was analyzed in gel retardation experiments using whole cell extracts and a PRE as a probe (Fig. 1BGo). Extracts from HeLa3B2 cells pretreated with progesterone formed a specific protein-PRE complex (lane 4), which was not detected in HeLa cell extract (lane 8). Addition of PR antibodies resulted in a further retardation of this protein-PRE complex, demonstrating the presence of PRB (lane 6). Without hormone, no PRB-PRE complex was formed (lane 3), indicating that DNA binding of PRB expressed in HeLa3B2 cells is hormone dependent. As shown in Fig. 2Go, the introduced PRB proved to be capable of mediating a significant R5020 induction of a transiently transfected reporter gene containing two PREs in front of the TK promoter and the luciferase gene (PRE2-TK-luc).



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  		Figure 1. Expression and DNA Binding Activity of PRB in Stably Transfected HeLa and CV-1 Cell Clones

A, Whole cell extracts from parental HeLa cells (165 µg protein, lane 2), HeLa3B2 (165 µg protein, lane 3), CV-1 (42 µg, lane 4), or CV-1.1B (42 µg, lane 5) were separated by SDS-PAGE and blotted to nitrocellulose. Receptors were detected with a PR-specific monoclonal antibody (Medac). Protein (165 µg) from T47D cell extract was separated for comparison (lane 1). The positions of both isoforms of PR detected in T47D cells are indicated. B, Whole cell extracts from the indicated cells were preincubated in a 10 µl standard binding reaction containing a radioactively labeled PRE probe and 1 µM progesterone (+) or control vehicle (-) for 20 min on ice. To some of the binding reactions 1 µl of a PR-specific monoclonal antibody was added as indicated. Specific PR-PRE complexes and a nonspecific protein-PRE complex are noted.

 


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  		Figure 2. Effect of cAMP and Overexpression of PKA Catalytic Subunit on RU486 Agonist Activity in PRB-Positive HeLa3B2 Cells

HeLa3B2 cells were transiently transfected with PRE2-TK-luc reporter plasmid and expression vectors encoding wild-type PKA (wt) or mutated PKA (mut) catalytic subunit. Cells were incubated for 25 h with 10 nM R5020 or RU486 in the presence or absence of 0.2 mM cAMP as indicated. Luciferase activities were normalized to the untreated control. Bars represent the mean of four independent experiments ± SD. Extracts from untreated controls showed 2656 ± 73 luciferase units per 25 µl.

 
Similarly, we stably transfected the PR-negative monkey kidney cell line CV-1 with an expression vector encoding PRB. Immunoblotting indicated that clone CV-1.1B expressed authentic PRB, while no PR could be detected in the parental CV-1 cells (Fig. 1AGo, lanes 4 and 5). On a protein basis CV-1.1B cells expressed about 4-fold higher amounts of PRB than the HeLa3B2 clone (Fig. 1AGo). Gel retardation analysis and supershifting with PR antibodies confirmed that the PR expressed in CV-1.1B cells bound to PREs in a hormone-dependent manner (Fig. 1BGo, lanes 9–13). Transfection studies confirmed that the expressed PRB is able to mediate R5020 induction of PRE2- and PRE4-TK-luc reporters while no response was seen in the parental CV-1 cells (Fig. 3Go and data not shown). Thus, we have created two model cell lines that allow us to specifically address the influence of PKA activation on ligand-activated PRB in two different cellular backgrounds.



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  		Figure 3. Lack of RU486 Agonist Activity in PR-Positive CV-1.1B Cells

CV1.1B were transfected with a PRE4-TK-luc reporter and PKA expression vectors and treated with hormones as described in the legend of Fig. 2Go. Bars represent the mean of two independent experiments. Controls showed 480 luciferase units after substraction of background activity.

 
PRB-Mediated cAMP/RU486 Synergism Is a Cell-Specific Phenomenon and Involves PKA
First we analyzed the effects of the antiprogestin RU486 and 8-Br-cAMP (cAMP), a membrane-permeable activator of PKA, on transcription of a PR target promoter in HeLa clone 3B2. For this purpose HeLa3B2 cells were transiently transfected with the PRE2-TK-luc reporter plasmid. Cells were incubated in the absence or presence of ligands with or without cAMP for 25 h. Treatment with R5020 induced reporter gene activity about 9-fold. cAMP alone stimulated luciferase activity about 3-fold while the antiprogestin RU486 induced luciferase expression about 16-fold (Fig. 2Go). Cotreatment with cAMP and RU486 resulted in 130-fold induction. Similar results were obtained using a PRE4-TK-luc reporter or the MMTV promoter (data not shown). Thus, in this transient model RU486 displays substantial agonist activity even in the absence of exogenous cAMP, and this agonist activity is synergistically enhanced by addition of cAMP.

Since cAMP is an activator of PKA, we asked whether directly increasing the intracellular level of PKA catalytic subunits (isoform ß) might also bring about enhancement of RU486 agonist activity (Fig. 2Go). In the absence of RU486, transient transfection of an expression vector encoding wild-type catalytic subunit (wt) of PKA stimulated PRE2-TK-luc activity about 12-fold. However, in the presence of RU486, overexpression of catalytic subunit led to about 15-fold potentiation of the RU486 activity that was even higher in magnitude than the potentiation evoked by cAMP (8-fold). In contrast, coexpression of a mutated catalytic subunit (mut), which lacks kinase activity due to a point mutation within the catalytic site, did not significantly enhance the RU486 response, proving that the catalytic activity of PKA is essential for the effect. Together, these results clearly suggest that the stimulatory effect of cAMP on the RU486 agonist activity is mediated via activation of PKA.

A similar transfection experiment was performed with a TK-luc reporter containing four PREs (PRE4-TK-luc) in the PRB-positive clone (CV-1.1B) derived from the monkey kidney cell line CV-1. As shown in Fig. 3Go, treatment with R5020 for 25 h strongly induced luciferase activity in CV-1.1B cells, indicating that the introduced receptor is functional. cAMP alone had no detectable effect on the relative low basal promoter activity. In contrast to the results observed in HeLa3B2 cells, both in the absence and presence of cAMP, RU486 displayed no agonist activity at all. Like cAMP, cotransfection of the expression vector encoding the wild-type catalytic subunit of PKA (isoform ß) did not elicit any agonist activity of RU486 (Fig. 3Go). Therefore, it seems unlikely that the failure to observe cAMP/RU486 synergism in CV-1.1B cells is due to insufficient amounts of endogenous PKA. The remarkable difference in the ability of PRB-positive HeLa and CV-1 cells to mediate a synergistic response of cAMP or PKA and RU486 suggests that a cell specifically expressed factor(s), possibly acting downstream of PKA, might play an essential role in the mechanism.

HeLa3B2 Derivatives Containing Chromosomally Integrated Copies of PRE2-TK-luc or MMTV-luc Reporter Genes
Most studies analyzing cross-talk between steroids and PKA activators employed transiently transfected receptor expression vectors and reporter genes. However, transient transfections have several important disadvantages: 1) they are laborious and expensive; 2) levels of transiently transfected reporter genes as well as receptor levels can vary from experiment to experiment; 3) they may not correctly reflect the regulation of endogenous genes due to the lack of chromatin structure on the promoter of interest; and 4) they do not allow precise kinetic analysis in long-term experiments, as the amount of available expression vector and reporter gene may decrease with time. To avoid these possible drawbacks, we stably transfected HeLa3B2 cells either with a reporter plasmid containing two PREs in front of a TK promoter and the luciferase gene (PRE2-TK-luc) or with a luciferase reporter containing the MMTV promoter (MMTV-luc). For further analysis we selected representative PRE2-TK-luc (3B2.TK) and MMTV-luc (3B2.M) clones expressing luciferase activities clearly above background level, ensuring an accurate estimation of fold induction values. Regulatory phenomenons observed in the transient transfections were reproduced in several independent stable transformants analyzed (Figs. 2Go and 4AGo). However, fold stimulation values as determined in cell lines containing stably integrated copies of the reporter genes were generally lower than those observed with transiently transfected reporters. The reason for the decreased responsiveness of stably integrated reporter genes remains unclear.



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  		Figure 4. Effect of cAMP Cotreatment on the Time Courses of R5020 and RU486 Responses of a PRE2-TK-luc Reporter Gene

A, HeLa3B2.TK cells stably expressing PRB and containing stably integrated copies of the PRE2-TK-luc reporter plasmid were incubated with 10 nM R5020 or 10 nM RU486 in the presence or absence of 0.2 mM cAMP. At different times, cells were harvested and assayed for luciferase activity. Luciferase activities were normalized to untreated controls. Points represent the mean of two independent experiments with less than 10% deviation. Control showed 25,884 luciferase units per 25 µl of extract. B, HeLa.TK cells (PRB-negative) containing stably integrated copies of PRE2-TK-luc were cultured in medium containing 10 nM dexamethasone, 10 nM RU486, or 0.2 mM cAMP as indicated and analyzed as described in panel A. Controls showed 3,258 luciferase units per 25 µl of extract.

 
cAMP/RU486 Synergism Is Delayed with Respect to the Onset of the R5020 and RU486 Responses on PRE2-TK-luc
Using the HeLa3B2.TK clone containing chromosomally integrated copies of PRE2-TK-luc, we next analyzed the time course of the responses to R5020 and RU486 in the presence and absence of cAMP in detail (Fig. 4AGo). Treatment with cAMP alone had little effect on transcription at all times investigated. Transcriptional stimulation by R5020 was clearly detectable after 5 h, reached a maximum of 11-fold at 10 h, and decreased to about 5-fold induction values at 25 h. As already observed in the transient transfection experiment (Fig. 2Go), RU486 alone led to an induction (5-fold) of reporter gene activity. The RU486 induction occurred as rapidly as the R5020 response, but remained more or less constant up to 35 h of incubation. Importantly, cotreatment with cAMP for 5 h had no significant effect on the activity of RU486 while longer treatments resulted in increasing synergistic enhancement. This delayed onset of the cAMP/RU486 synergism relative to the RU486 response suggests that de novo protein synthesis of an involved factor is required for the mechanism. Interestingly, cAMP slightly increased the R5020 response only at earlier time points. Thus, cAMP modulates extent and kinetics of R5020 and RU486 responses on PRE2-TK-luc in a distinct manner.

Synergistic Induction of PRE2-TK-luc by cAMP and RU486 Is Mediated by PRB
As HeLa cells contain functional GRs, which possess high affinity for RU486 and bind to the same response elements as PRB (4, 5), it was important to determine whether the RU486 effects observed in HeLa3B2 cells were indeed mediated by the introduced PRB. We thus created HeLa cell clones containing chromosomally integrated copies of the PRE2-TK-luc reporter and determined the effect of RU486 in the presence and absence of cAMP in a representative clone (HeLa.TK). In this PRB-negative clone, dexamethasone treatment caused about 6-fold induction of luciferase activity, proving the presence of functional GRs (Fig. 4BGo). Compared to HeLa3B2 cells, HeLa.TK cells showed a relatively strong response to cAMP, which increased up to 6-fold at 35 h. Possibly, this increased cAMP responsiveness reflects a clonal effect. However, in contrast to the results observed in HeLa3B2 cells (Fig. 4AGo), RU486 alone exhibited no agonist activity at all and no potentiation of RU486 activity was observed upon cotreatment with cAMP, suggesting that the RU486-loaded GRs are not capable of mediating agonistic effects on PRE2-TK-luc in HeLa.TK cells. Similar results were obtained in transient transfections using the parental HeLa cell line (data not shown), excluding the possibility that the lack of GR-mediated cAMP/RU486 synergism in the HeLa.TK clone might represent a clone-specific defect. We conclude that the RU486 agonist effects on PRE2-TK-luc transcription, which we observed in HeLa3B2 cells in the absence or presence of cAMP, are both mediated exclusively by the introduced PRB.

Length of Treatment Largely Affects the Ability of RU486 to Antagonize R5020 Induction of PRE2-TK-luc
As shown in Fig. 4AGo, upon cotreatment with cAMP R5020 is a more potent inducer of PRE2-TK-luc transcription than RU486 at 5- and 10-h time points while the order is reversed at 25- and 35-h time points. Thus one would predict that, in the presence of cAMP, RU486 might still be able to partially antagonize R5020 induction of PRE2-TK-luc in a competition experiment employing 5–10 h of treatment, but completely lack antagonist potential in 25- to 35-h experiments. To test this prediction directly, we performed R5020/RU486 competition experiments with 5 and 25 h of treatment in the presence of cAMP (Fig. 5Go). A saturating concentration of R5020 (10-8 M) and a 100-fold molar excess of RU486 were used. After 5 h of treatment, luciferase activity was higher with R5020 (10-fold, left panel) than with RU486 (4-fold). In the competition experiment (R5020 + RU486) excess of RU486 blocked the R5020 stimulation down to the value observed with RU486 alone. Thus, despite the presence of cAMP, in this short-term experiment RU486 still displays partial antagonist activity. As already shown in Fig. 4AGo, cells exposed for 25 h showed a R5020 response (4-fold) that was lower than that observed with RU486 (15-fold). Addition of excess RU486 did not result in inhibition of the R5020 response, but rather an increase in luciferase activity up to the level observed with RU486 alone was observed. Thus, in a 25-h experiment RU486 no longer exhibits any antagonist activity. These data illustrate that cotreatment with an activator of PKA can indeed result in a complete loss of antagonist activity of RU486. Moreover, our experiments reveal that the duration of hormone treatment is a crucial parameter in the evaluation of antagonist properties, as complete loss of antagonist potential of RU486 is only observed in the experiment involving 25 h of incubation.



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  		Figure 5. Time-Dependent Reversal of Antagonist/Agonist Properties of RU486 in the Presence of cAMP

HeLa3B2.TK cells stably expressing PRB and containing stably integrated copies of the PRE2-TK-luc reporter plasmid were incubated for 5 and 25 h with 0.2 mM cAMP and 10 nM R5020 or 1 µM RU486. Cells were harvested and assayed as described in Fig. 2Go.

 
cAMP Acts Synergistically Both with Progesterone Agonists and Antagonists on Transcription of an MMTV-promoter
In the studies described above, we have analyzed the hormonal effects on transcription using the artificial PRE2-TK-luc reporter gene. To examine a different and more complex promoter, we determined the kinetics of cAMP/ligand responses using a HeLa3B2-derived clone (3B2.M) stably transfected with a reporter containing MMTV-long terminal repeat (LTR) sequences from position -235 to +122 in front of the luciferase gene (MMTV-luc). cAMP by itself gave a modest induction of MMTV-luc transcription (Fig. 6Go). RU486 induced luciferase activity about 3-fold at maximum. This RU486 response was already detectable after 5 h of incubation and remained constant over the time period investigated. Cotreatment with cAMP and RU486 for 25 and 35 h resulted in a strong and synergistic enhancement of MMTV-luc transcription (18- and 53-fold induction, respectively) while synergism was not evident at 5, 10, and 15 h. Thus, cAMP/RU486 synergism on MMTV-luc is a delayed response, confirming the results obtained with the PRE2-TK-luc reporter (Fig. 4AGo). Furthermore, our data demonstrate that cAMP/RU486 synergism is not a PRE2-TK-luc-specific phenomenon. Induction of the MMTV reporter by the progestin R5020 became detectable at the earliest time point (5 h) analyzed and increased continuously up to 85-fold at 35 h. At 5 to 15 h of incubation, cotreatment with R5020 and cAMP had just an additive effect on MMTV-luc activity while at 25 and 35 h synergism became evident. Thus, our experiments show that cAMP/R5020 as well as cAMP/RU486 synergism occur with a pronounced delay relative to the onset of R5020 and RU486 responses. This similarity suggests that enhancement of R5020 and RU486 agonist activities through activation of PKA involves related mechanisms.



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  		Figure 6. Effect of cAMP on the Time Courses of R5020 and RU486 Responses of a Stably Integrated MMTV-luc Reporter Gene

HeLa3B2.M cells stably expressing PRB and containing stably integrated copies of the MMTV-luc reporter plasmid were incubated with 10 nM R5020 or 10 nM RU486 in the presence or absence of 0.2 mM cAMP. At the indicated times, cells were harvested and assayed for luciferase activity. Luciferase activities were normalized to untreated controls and represent the mean of two independent experiments. Controls showed 33487 luciferase units in 25 µl of extract.

 
cAMP Does Not Alter the Subcellular Distribution or Increase Expression of PRB
A possible mechanism that could account for the stimulating effect of cAMP on transcription of PR-dependent reporter genes would be for cAMP to promote nuclear localization of the receptor or to increase the PR content in the cell. To investigate an effect on subcellular localization, immunofluorescence studies with HeLa3B2.TK cells were performed. Using antibody 9E10 directed against the myc-tag present in the transfected PRB, we observed a strong nuclear staining with HeLa3B2.TK cells in untreated controls (Fig. 7Go, lower panel). In contrast, no such signal was observed with the parental HeLa cells, confirming the specificity of the antibody (Fig. 7Go, upper panel). Clearly, treatment with R5020, RU486, and cAMP, alone or in combinations, for up to 25 h did not alter the subcellular distribution significantly. Thus, the cAMP effects on PRB-mediated transcription are not due to an effect on PR localization. However, prolonged treatment with R5020 significantly decreased the nuclear PR staining. To investigate the effect of hormones on the PR content more precisely, extracts from HeLa3B2.TK cells treated with the various hormones for 5 to 35 h were subjected to Western blotting with a PR-specific antibody. As shown in Fig. 8Go, R5020 mediated a strong down-regulation of PR levels, which became clearly evident after 15 h of incubation. In contrast, RU486 alone had relatively little effect on the PR content of the cells while cAMP treatment alone resulted in a modest reduction of PR. Cotreatment with cAMP seemed to enhance down-regulation observed by both R5020 and RU486. Because the effect of cAMP on PR levels is contrary to its effect on RU486- or R5020-dependent transcription, we conclude that the transcriptional enhancement resulting from cotreatment with cAMP cannot be ascribed to changes in PR content.



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  		Figure 7. Lack of an Effect of cAMP on the Subcellular Localization of PRB in HeLa3B2.TK Cells

HeLa3B2.TK cells (lower panel) were treated with hormones (0.2 mM cAMP, 10 nM RU486, 10 nM R5020) as indicated and analyzed by immunofluorescence using antibody 9E10. The immunofluorescence picture of untreated control cells (HeLa3B2.TK) is shown together with the corresponding phase contrast picture. PRB-negative HeLa cells are shown for comparison (upper panel).

 


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  		Figure 8. Effect of cAMP and Steroids on PRB Levels

HeLa3B2.TK cells were treated with cAMP (0.2 mM), RU486 (10 nM), R5020 (10 nM), or cycloheximide (chx, 40 µM) as indicated. Cycloheximide treatment was started 40 min before the addition of hormones. Whole cell extracts were assayed for PR content by Western blotting with a PR-specific antibody (B30).

 
Interestingly, concomitant treatment of cells with cycloheximide, an inhibitor of protein synthesis, completely prevented down-regulation of PR under all conditions (Fig. 8Go). This could indicate the hormone-mediated down-regulation of PR involves a factor that is subject to a fast turnover.

cAMP/RU486 Synergism Requires Protein de Novo Synthesis
Our analysis of the cAMP-mediated modulation of RU486 and R5020 responses revealed that the synergistic increase of ligand induction through cAMP represents a delayed response relative to the effects of the ligands alone (Figs. 4AGo and 6Go). This delay could indicate that de novo synthesis of an essential factor(s) is required for this synergism. To study the requirement of ongoing protein synthesis, we investigated the effect of cycloheximide, an inhibitor of protein synthesis, on the regulation of luciferase mRNA in HeLa3B2.TK cells by RNase protection analysis (39). A cycloheximide concentration was used that caused about 70% inhibition of the luciferase enzyme activity in untreated controls but had little effect on cell viability (data not shown). Luciferase RNA was determined using an in vitro synthesized antisense RNA of 331 nucleotides in length that yielded a protected RNA of 276 nucleotides, as expected (Fig. 9Go). As internal control, {gamma}-actin antisense RNA resulting in three major signals of 125 to 130 nucleotides was used. Although fold induction values as measured by RNA analysis were generally lower than those obtained in luciferase assays, RNA analysis results qualitatively confirmed the effects of steroids and cAMP/RU486 synergism (Fig. 9Go). Consistent with a direct PRB-mediated mechanism, luciferase mRNA induction elicited by R5020 was not decreased, but elevated by cycloheximide (compare lanes 1 and 6 with 5 and 10, respectively). Similarly, the RU486 response proved to be insensitive to cycloheximide (lanes 3 and 8), suggesting that it also represents a primary PRB-mediated transcriptional response. Importantly, the cAMP/RU486 synergism that was evident in the absence of protein synthesis inhibitor (lanes 2–4) was completely abolished by cycloheximide treatment (lanes 7–9). This result, which was consistently observed in three independent experiments, indicates that cAMP/RU486 synergism requires de novo synthesis of an involved factor.



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  		Figure 9. Effect of Partial Inhibition of Protein Synthesis on the cAMP/RU486 Synergism

HeLa3B2.TK cells stably expressing PRB and containing stably integrated copies of the PRE2-TK-luc reporter plasmid were incubated with hormones (10 nM R5020 and RU486, 0.2 mM cAMP) in the absence (-) or presence (+) of cycloheximide (chx, 40 µM) as indicated above the lanes. Cellular RNA was harvested after 15 h of incubation and luciferase (upper panel) and {gamma}-actin (lower panel) mRNA were analyzed by RNase protection assay. On the right hand, the undigested luciferase (upper panel) and {gamma}-actin (lower panel) probes are shown. Signals representing luciferase (Luc, 276 nucleotides) and {gamma}-actin transcripts (125–130 nucleotides) are indicated. The autoradiogram showing luciferase mRNA signals was exposed 20 times longer than the one showing the {gamma}-actin signals. In the bar diagram below, normalized fold activation values are given for each lane. Controls in lane 1 (-chx, open bars) and lane 6 (+chx, filled bars) were set to be 1. The loss of cAMP/RU486 synergism by cycloheximide treatment was consistently observed in three independent RNase protection experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Several studies using different cellular models have demonstrated that cAMP can dramatically increase the agonist potential of progesterone antagonists (32, 33, 34, 36, 37). By stable transfection of a PRB cDNA expression vector, we have created novel cell culture models that allowed us to specifically address the regulation of PRB through ligands and activators of PKA in the HeLa and CV-1 cell background. In a second step, we stably transfected HeLa3B2 cells with two different hormone-responsive reporter genes, namely PRE2-TK-luc and MMTV-luc, to create model systems that combine the advantages of stable transfectants with the superior sensitivity of the luciferase reporter system. As shown by immunoblotting and gel retardation, HeLa3B2 cells express slightly lower levels of PRB as compared to T47D cells (Fig. 1AGo), which have been the established cell culture model for the analysis of the mechanism of progestin and antiprogestin action. It is important to note that the PRB-positive HeLa cell clone expresses functional endogenous GRs, which bind RU486 with high affinity and can recognize the PREs present in the PRE2-TK- and MMTV-luc reporters. Using the PRE2-TK-luc reporter, we observed significant cAMP-independent agonist activity of RU486 in GR/PRB-positive, but not GR-only cells, indicating that this is a PRB-specific phenomenon (Fig. 4Go). This finding confirms a previous study from K. Horwitz’s group (11) in which a TK-chloramphenicol acetyltransferase (CAT) reporter with a single PRE was used. A synergistic stimulation of PRE2-TK-luc transcription by RU486 and an activator of PKA was also detected exclusively with the PRB-positive HeLa cell clone, suggesting that only RU486-loaded PRB, but not RU486-occupied GR, is sensitive to activation of PKA. This conclusion apparently contradicts results of Nordeen and co-workers (36), who reported a GR-mediated synergism of cAMP and RU486 on GRE2E1b- and MMTV-CAT reporter genes in two different GR-positive fibroblast cell lines. Whether this discrepancy is due to the different reporter genes used remains to be established.

Previous reports analyzing modulation of RU486 and R5020 agonist activities already indicated that PKA activators preferentially increase RU486 agonist activity and therefore partially decrease the antagonist potential (33, 34, 36). Here, in HeLa3B2 cells containing chromosomally integrated copies of PRE2-TK-luc, we observed that upon incubation with cAMP for 25 h, RU486 can acquire much stronger agonist potential than R5020, resulting in a complete loss of its antagonist potential (Fig. 5Go). To our knowledge such a complete reversal of the agonist/antagonist roles of RU486 and R5020, leading to the paradoxical situation in which the agonist R5020 has the potential to act as a partial antagonist of the antagonist RU486, has not been observed in previous studies. The complete loss of RU486 antagonist potential, as observed with the PRE2-TK-luc reporter gene, is partly due to the cAMP-mediated enhancement of the RU486 agonist activity beginning after 10 h of incubation and partly due to the concomitant decrease of the R5020 response (Fig. 4AGo). As the decrease of the R5020 response of PRE2-TK-luc observed during incubation periods exceeding 10 h is also observed in the absence of the PKA activator, we conclude that it is not an effect of cAMP (Fig. 4AGo). Rather, we believe that this decrease of R5020 responsiveness is due to the R5020-mediated down-regulation of PRB levels (Figs. 7Go and 8Go). With the MMTV-luc reporter we also observed a strong enhancement of RU486 agonist activity upon cotreatment with cAMP (Fig. 6Go). However, partial RU486 antagonist potential was maintained at all time points investigated, since the R5020 response of this promoter did not decrease during long incubation periods. This is quite astonishing as the PRB levels in the stable HeLa cell clone analyzed were down-regulated through R5020 treatment to a similar extent as in the PRE2-TK-luc expressing HeLa cell clone showing a dramatic loss of responsiveness to R5020. We speculate that the ability of MMTV-luc to maintain high responsiveness at lower PRB concentrations could be due to the extreme cooperativity of the PREs of the MMTV promoter, which may ensure sufficient PRE occupancy even at low receptor levels.

Another novel aspect of our studies that has not been elaborated in previous papers is that, in the presence of cAMP, the ratio of the agonist activities of R5020 to RU486 and consequently also the antagonist potential of RU486 vary considerably with the duration of the hormone treatment (Figs. 4AGo, 5Go, and 6Go). As can be deduced from the time courses of agonist responses on PRE2-TK-luc and MMTV-luc transcription, RU486 (as the weaker agonist) maintains relatively high antagonist potential during short incubation periods; with longer incubation time, an increasing loss of antagonist potential of RU486 can be observed. This time-dependent variation of the RU486 antagonist potential was evident both with the PRE2-TK-luc and MMTV-luc reporter and thus appears not to represent a promoter-specific phenomenon.

In agreement with studies on T47D cells (40), immunocytofluorescence studies indicated that the unliganded PRB of HeLa3B2 cells is predominantly located in the nucleus and that R5020, RU486, and cAMP, alone or in combinations, do not alter the intracellular distribution (Fig. 7Go). By immunoblotting we show that PRB levels in HeLa3B2.TK cells are substantially down-regulated by R5020 treatment (Fig. 8Go), confirming similar results of a study that used a stable transfectant expressing PRB in the T47D background (37). Compared to R5020, both RU486 and cAMP had relatively little effect on PR expression. Clearly, cotreatment with cAMP did not prevent RU486- and R5020-mediated down-regulation of PR (Fig. 8Go), but rather an enhancement of ligand-induced downregulation was observed. Together, these data indicate that the enhancement of PRB-mediated agonist activities of RU486 and R5020 by activators of PKA cannot be ascribed to effects on PRB levels or subcellular localization.

We demonstrated that in PRB expressing HeLa3B2 cells the effect of cAMP could be mimicked by overexpression of PKA catalytic subunit (Fig. 2Go). This result indicates that the cAMP effect is not due to some nonspecific effects of this compound, but attributable to activation of PKA via an intracellular rise in cAMP. A PKA mutant without the catalytic activity had no effect on RU486 agonist activity (Fig. 2Go). This result proves directly that PKA-mediated phosphorylation represents an essential step in the mechanism. Since the PR is a phosphoprotein, it has been assumed that PKA-mediated alteration of the phosphorylation state of the PR itself might play an important role in the mechanism. However, several studies attempting to address this question did not provide direct evidence for this concept (32, 38). Interestingly, in a CV1 cell clone stably expressing functional PRB, RU486 showed no agonist activity at all, even when a PKA catalytic subunit was cotransfected (Fig. 3Go). Our finding that overexpression of the catalytic subunit of PKA does not suffice to elicit RU486 agonist activity in CV-1.1B cells also argues against a simple model, in which direct phosphorylation of PRB through PKA triggers the increased transcriptional activity of RU486-liganded receptors. Furthermore, these data suggest that in addition to catalytically active PKA and RU486-loaded PRB, the mechanism requires another cellular factor(s), which appear(s) to be expressed in a cell-specific manner.

The perhaps most important contribution of this study to the understanding of cAMP/RU486 synergism is our finding that the mechanism requires the intermediate induction of an essential factor and therefore represents a delayed primary response (41). Two different experiments support this notion. First, we demonstrated that synergistic induction of PRE2-TK-luc by cAMP and RU486 is sensitive to partial inhibition of protein synthesis by cycloheximide (Fig. 9Go). The effect of cycloheximide on the synergism was specific, since neither R5020 induction nor the cAMP-independent RU486 induction of PRE2-TK-luc were decreased by the inhibitor. Second, our analysis of the time courses of the hormonal responses on both PRE2-TK-luc and MMTV-luc promoters revealed that the onset of synergism is delayed by 5 to 10 h relative to the cAMP-independent RU486 response (Figs. 4AGo and 6Go). Together, these data are consistent with a model in which cAMP/RU486 treatment induces a factor(s) that is necessary for synergism. Interestingly, the synergism between the agonist R5020 and cAMP, which was specific for the MMTV-luc reporter gene, also required at least 10 h to develop (Fig. 6Go). This similarity suggests that PKA-mediated enhancement of both R5020 and RU486 agonist activities occur through the same mechanism involving the intermediate synthesis of an essential factor.

The functional role of the postulated intermediately induced factor enabling synergism between PKA and RU486-loaded PRB on PRE-containing reporter genes remains completely unclear at present. However, as originally proposed by Nordeen et al. (36), this factor could be a protein that mediates protein-protein interactions between RU486-loaded PR and the initiation complex and thereby enhances transcription initiation by RNA-polymerase II. Alternatively, the induced factor could be a kinase that modifies a preexisting transcriptional coactivator of PRB, e.g. SRC-1 or TIF-2 (23, 24), in a way that allows it to interact with antagonist-loaded PRB. Recently, Jackson and co-workers (42) provided evidence that the partial agonist activity of RU486-loaded PR is inhibited by nuclear receptor corepressor (N-CoR) and silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), two related nuclear proteins previously characterized as corepressors of unliganded thyroid and retinoid acid receptors, but stimulated through L7/SPA, a factor that interacts with the hinge region of PR. Importantly, both corepressors were able to reverse the stimulatory effect of L7/SPA on RU486 agonist activity, suggesting that the ratio of coactivators and corepressors is a crucial parameter in regulating the activity of RU486-loaded PR. Thus, it is an attractive possibility that cAMP exerts its stimulatory effect on RU486 agonist activity through altering the balance between positively and negatively acting cofactors. Consistent with the requirement for intermediate protein synthesis, this could be achieved either through increased synthesis of a positive cofactor facilitating agonist activity or through a more indirect mechanism that involves the induction of a factor, e.g. a protein kinase or specific protease, that ultimately reduces the expression or inactivates corepressors, thereby leading to increased transcriptional activity of RU486-loaded PR bound to DNA. It has been postulated that corepressor could also be associated with unliganded PRs, but dissociate from the receptors upon binding of progesterone (26). Assuming that free and corepressor-bound PRs might be in equilibrium in the agonist-loaded state, our model could also account for the stimulating effect of cAMP on R5020-loaded PRs, as cAMP-mediated down-regulation of corepressors would shift the equilibrium toward the free and active form.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
pSGN-His6-myc-hPR0 is a eukaryotic SV40 early promoter-driven expression vector encoding human PR isoform B with N-terminal his- and myc-tags (further information is available upon inquiry). The luciferase reporter plasmid PRE2-TK-luc contains two PRE consensus sequences inserted into the SacI site of pT81luc (43) in front of the TK kinase promoter (-81 to +52) upstream of the luciferase gene. The MMTV-luc reporter plasmid (pHCwtluc, Ref.44) contains MMTV-LTR sequences from position -235 to +122 linked to the luciferase cassette of pXP1 (43) at the XhoI site. RSV-CHO-PKA-Cß and RSV-CHO-PKA-Cßmut are mammalian expression vectors for the catalytic subunit ß or the mutant subunit ßmut of the cAMP-dependent protein kinase of chinese hamster ovary cells (45).

Cell Culture and Transfection
HeLa and CV1 cells were grown in DMEM supplemented with 1 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 10% FBS (Cytogen). Puromycin (0.5 µg/ml) or G418 (0.5 mg/ml) was added to the medium for stably transfected cells containing pPUR or pSV2neo selection markers, respectively. HeLa cells were cotransfected with pSGN-His6-myc-hPR0 and pSV2neo and selected for G418 resistance. Similarly, CV1 cells were cotransfected with a PRB expression vector and pPUR (CLONTECH, Palo Alto, CA) and selected for puromycin resistance. PRB expressing clones (HeLa3B2 and CV1.1B) were identified by immunocytofluorescence with PR antibodies (Medac, Hamburg, Germany) and further characterized by Western blot analysis. HeLa3B2.TK cells were created by stable transfection of HeLa3B2 cells with PRE2-TK-luc and pPUR, followed by selection in medium containing G418 and puromycin. Similarly, HeLa3B2.M transformants containing an MMTV-luc reporter (pHCwt-luc) were generated by cotransfection with pPUR. HeLa.TK (PR-) cells were obtained by cotransfection of HeLa cells with PRE2-TK-luc and pPUR and puromycin selection. For stable transfection of CV1 cells, Lipofectamin (GIBCO BRL, Gaithersburg, MD) was used while HeLa were transfected by lipofection (GIBCO BRL).

For transient transfection, 6 x 105 HeLa3B2 or 4 x 105 CV1.1B cells were plated per well of a six-well dish (Falcon, Becton-Dickinson, Bedford, MA) and grown for 1 day. Opti-MEM (200 µl) containing 1 µg PRE2-TK-luc, 100 ng RSV-CHO-PKA-Cß or RSV-CHO-Cßmut, 900 ng pBluescript, and 5 µg Lipofectin were incubated for 30 min at 20 C. After addition of 1.3 ml Opti-MEM, the DNA/lipofectin mixture was added to the cells, which had previously been washed two times with Opti-MEM. After 5 h, cells were transferred to DMEM medium containing 10% FCS and incubated overnight. Hormone treatment was started about 5 h after the addition of DNA. After a further incubation for 25 h, cells were harvested and analyzed for luciferase expression.

HeLa cell clones containing stably integrated luciferase reporter genes were plated in six-well dishes at a density of 6 x 105 cells per well and grown for 1 day. Then cells were incubated in medium containing hormones as given in the figure legends. Control samples received appropriate amounts of vehicle. Harvested cells were washed twice in PBS and lysed by the addition of 100 µl of lysis buffer (25 mM Tris-phosphate pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-cyclohexanediamine NNN'N'-tetraacetic acid, 10% glycerol, 1% Triton X-100). After removal of cell debris by centrifugation, luciferase assays were performed on 25 µl of extracts using a Lumat LB9501 luminometer (Berthold). In each experiment cell culture treatment groups were in duplicate.

SDS-PAGE, Western Blot, and Gel Retardation Assay
For the preparation of whole cell extracts, cells were washed with PBS, centrifuged and resuspended in whole cell extract buffer (10% glycerol, 20 mM HEPES, pH 7.9, 1 mM EDTA, 600 mM NaCl, 1 mM dithiothreitol, and protease inhibitors). Cells were lysed by three cycles of freeze-thawing. After centrifugation, supernatants were saved as extracts. Protein concentrations were determined with the Bio-Rad protein assay. SDS-PAGE and transfer to nitrocellulose was performed as described by Sambrook et al. (46). Blots were incubated with monoclonal antibodies against human PR (Medac) and rabbit anti-mouse IgG antibodies linked to peroxidase. PR immunoreactivity was revealed using a chemiluminescence method (ECL, Amersham, Arlington Heights, IL).

The DNA-binding activity of PR was determined in a gel-shift assay using a 32P-labeled PRE-oligonucleotide and preincubated with whole cell extract 1 µM progesterone or ethanol in a 10 µl standard binding reaction for 20 min on ice (17). Monoclonal PR antibodies (B30) were added to the binding reaction as indicated. Samples were subjected to electrophoresis on a nondenaturing 4% polyacrylamide gel. After fixing and drying of the gel, protein PRE-complexes were visualized by autoradiography.

Immunocytofluorescence
Cells were cultured on cover slides in 24-well dishes for 5–25 h in the presence or absence of the indicated hormones. Samples were fixed and permeabilized with ice-cold methanol for 10 min. After blocking in PBS containing 0.5% BSA for 10 min, cells were incubated with antibody 9E10 against the myc-tag (47) for 90 min at 37 C, washed three times with blocking buffer, and incubated with FITC-conjugated antimouse antibody (Dianova, Hamburg, Germany) for 60 min at 37 C.

RNase Protection Analysis
To generate an RNase protection probe for luciferase RNA, a 276-bp EcoRI/RsaI fragment of the luciferase gene of pTK81luc (43) was cloned between the EcoRI and EcoRV sites of pBluescript SK+. Radiolabeled antisense RNA of 331 nucleotides in length was synthesized from this plasmid (EcoRI linearized) by T7 polymerase in vitro. As an internal control, a {gamma}-actin antisense RNA synthesized by SP6 polymerase from a HinfI digested plasmid (kindly provided by Ian Kerr, London) was included in all hybridization reactions. Specific activity of {gamma}-actin antisense RNA was reduced by including unlabeled UTP in the SP6 polymerase reaction.

For isolation of total RNA, HeLa3B2.TK cells were treated for 15 h in medium containing the indicated hormones or vehicles. Hormone treatment of cells receiving cycloheximide (40 µM) was initiated 40 min after addition of the protein synthesis inhibitor. Total RNA was isolated using Trizol reagent (Gibco BRL). Hybridization of 40 µg cellular RNA with luciferase (200,000 cpm) and {gamma}-actin (50,000 cpm) antisense RNAs and RNase digestion was performed as described (48). Protected transcripts were separated on sequencing gels. After fixing and drying, protected bands were quantified on a bio-imaging analyser (BAS1500, Fuji).


   ACKNOWLEDGMENTS
 
We are grateful to W. Schulz (Düsseldorf, Germany), A.C.B. Cato (Karlsruhe, Germany), R. A. Maurer (Iowa City, IA), Ian Kerr (London, U.K.), and H. Gronemeyer (Strasbourg, France) for providing plasmids. We thank D. P. Edwards (Denver, CO) for providing the monoclonal antibody B30 and Roussel-Uclaf (France) for providing RU486. We also thank V. Ulber for excellent technical assistance and C. Schwerk and D. Michels for a critical reading of the manuscript.


   FOOTNOTES
 
Address requests for reprints to: Dr. Ludger Klein-Hitpass, Institut für Zellbiologie (Tumorforschung), Universitätsklinikum, Virchowstr. 173, D-45122 Essen, Germany.

This work was supported by a joint grant of Schering AG and BMBF (to L. K.-H.).

Received for publication June 5, 1997. Revision received October 30, 1997. Accepted for publication November 14, 1997.


   REFERENCES
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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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