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Institut National de la Santé et de la Recherche Médicale (J.-M.L., N.P., B.T., V.G., S.L,. C.S)., INSERM U439, Pathologie Moléculaire des Récepteurs Nucléaires, 34090 Montpellier, France; Unité Biochimie Endocrinienne du Développement et de la Reproduction, Hôpital Lapeyronie (N.P., S.L., C.S.), 34295 Montpellier, France; Physiologie Comparée et Endocrinologie (S.F.), CNRS-ERS63, Les Cézeaux, 63177 Aubières, France; Unité Endocrinologie Pédiatrique (C.S.), Hôpital A. de Villeneuve, 34295 Montpellier, France
Address all correspondence and requests for reprints to: Prof. Charles Sultan, INSERM Unité 439, 70 rue de Navacelles, 34090 Montpellier, France. E-mail: chsultan{at}u439.montp.inserm.fr
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Introduction |
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Although the AR structure has been studied with the use of wild-type and mutant ARs from patients with androgen resistance, the action of the androgen-AR complex in target cells is only partially understood. This is mainly because AR is believed to be part of a large, multicomponent transcription regulation complex, the precise components of which are incompletely defined. Moreover, it is well known that nuclear receptor action can also be modulated by transcription factors. One of these factors is the composite transcription factor activating protein 1 (AP-1), composed either of a c-Fos/c-Jun heterodimer or a c-Jun homodimer.
Hence, AP-1 has been demonstrated to have a positive (6) or a negative (7) effect on the AR-mediated transcription activity. There are also controversial data regarding the mechanism involved in these transcriptional interferences based on gel retardation assays. Bubulaya et al. (6) suggested only a direct interaction between AR and AP-1. Conversely, Aarnisalo et al. (8) reported that the intracellular CREB-binding-protein (CBP) could mediate the interactions in part through competition, as has been demonstrated for other nuclear receptors (9).
Although the transcriptional interactions between AR and AP-1 have been studied, few of the regions in the AR involved in these interactions have been defined. To this end, we studied several engineered and naturally occurring AR mutations. After transient transfections in CV-1 cells, we investigated the capacity of these mutants to interfere with AP-1 on an androgen (transactivation) or an AP-1 (transrepression) modulated gene. In addition, we used staurosporine, a microbial alkaloid that blocks phosphorylation by membrane-translocated protein kinase C (10) in presence of the AP-1 activator 12-O-tetradecanoyl phorbol-13-acetate (TPA). Calyculin-A and okadaic acid, specific inhibitors of protein phosphatases (11), were also used to study the reversible phosphorylation of proteins involved in AP-1/androgen pathway interactions. Mutants of the three functional domains of the AR showed that the hormone-binding domain, part of the DNA binding domain, and the first part of the N-terminal domain are all necessary to establish the fully competent three-dimensional structure of the interface with the AP-1 pathway.
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Materials and Methods |
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) were constructed from pCMV-AR (12).
Mutants Gly568Trp, Val581Phe, and Arg608Lys were previously described
by our group in patients with complete or partial androgen
insensitivity syndromes (13, 14, 15). Mutants Cys579Tyr and Arg585Lys were
found in two patients with complete androgen insensitivity syndrome
(manuscript in preparation), whereas Arg607Gln was detected in two
unrelated families with partial androgen insensitivity syndrome (16).
Two truncated ARs were constructed by cloning the
AocI-AgeI fragment containing the
ligand and DNA binding domains and part of the amino terminal domain of
the AR complementary DNA (cDNA) into the
KpnI/BamHI-cleaved pUC19. Deletions were then
constructed using single restriction fragment sites and ligations: the
KpnI-EspI fragment was recloned into the
KpnI/EspI-cleaved pCMVAR. AR
661–919, deleted
from amino acid 661, was obtained with the
Tth-111I/SmaI and
Tth-111I/StuI restriction enzymes. AR1–188,
starting with a methionine at position 189, was obtained by excision of
the BglII/AflII fragment of pCMV-AR. This mutant
corresponds to the A form of the AR described by Wilson and McPhaul
(17). All of the recombinant plasmids were verified by sequencing.
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The mouse vas deferens protein (MVDP)-Luc reporter gene was constructed from the promoterless basic plasmid pGL3 (Promega Corp., Lyon, France) expressing luciferase under the control of the androgen-regulated promoter of the 0.8-kb fragment of the MVDP (19), which did not contain any functional TPA-response element (TRE) (20). The TPA-regulated reporter gene TRE3-tk-Luc described by Astruc et al. (21) is under control of the AP-1 complex.
Transfection of COS-7 cells and immunoblot analysis
COS-7 cells were plated in 100-mm dishes (1.2 x
10-6 cells), and 8 h later they were transiently
transfected with 10 µg of expression plasmid of wild-type or mutants
using the calcium phosphate precipitate, as previously described (22).
The suspension was added to the culture media for 16 h. Cells were
cultured in DMEM with 3% FCS and harvested 2 days later in 100 µl of
160 mM Tris, pH 6.8, 200 mM dithiothreitol,
20% glycerol, 4% SDS, and 0.004% bromophenol blue in the presence of
protease inhibitors (1 mM PMSF, 0.05 mM
leupeptin, 0.01 mM pepstatin). Samples were boiled for 5
min and centrifuged at 13,000 x g for 10 min. Analysis
of COS-7 cell extracts was done by immunoblotting with the polyclonal
antibody SpO61 (23) directed against the N-terminal domain of the human
AR, using the ECL detection system (Amersham, Cergy, France). For
binding assays, COS-7 cells were transfected in 12-well tissue culture
dishes with 0.1 µg of different expression vectors (mutants and
wild-type AR) and 0.1 µg of pCMV-ß galactosidase. Whole steroid
binding assays were performed as described by Georget et al.
(24).
Cotransfections of CV-1 cells and luciferase assays
CV-1 cells were grown in DMEM supplemented with 10% FCS
(Gibco BRL, Cergy, France) and cultivated in 6-well
plates. The transfections were performed using the calcium-phosphate
technique as previously described (22). Briefly, cells were
cotransfected with various amounts (10–100 ng) of wild-type or mutant
AR, or c-Fos or c-Jun expression vectors, 500 ng of
pCMV5-ß-galactosidase as an internal control for transfection
efficiency, and 1.5 µg of the reporter gene.
Transactivation has been defined as the capacity of the AR to induce the expression of the androgen-regulated gene in the presence or absence of TPA, which stimulates PKC by mimicking the action of the diacylglycerol and ultimately activates AP-1. Conversely, transrepression has been defined as the capacity of the AR to repress the expression of the TPA-regulated gene in the presence or absence of androgen.
For the transactivation assays, cells transfected with the androgen target gene MVDP-Luc were incubated for one day with various concentrations of the androgen agonist methyltrienolone (R1881, NEN Life Science Products, Paris, France) or ethanol, in the presence or absence of TPA. For the transrepression assays, cells transfected with the TPA target gene (TRE3-tk-Luc) were incubated in the presence or absence of R1881 for 30 h and in the presence of TPA. CV-1 cells were chosen for the luciferase assays because they give a higher induction factor with TRE3-tk-Luc than COS-7 cells. Cells were harvested after incubations and assayed for luciferase and ß galactosidase activities and protein content. The fold increase in luciferase activity was determined relative to basal activity in the absence of androgen or TPA and was corrected for transfection efficiency using the ß-galactosidase activity. In each experiment, the mock pCMV5 was used as control (22).
For the study of phosphorylation processes, staurosporine (30 nM), calyculin-A (1 nM) or okadaic acid (1 nM) was added 10 min before TPA (50 nM). It should be noted that at more than 10 nM, both calyculin-A and okadaic acid presented significant toxicity, as shown by the cells that became rapidly (within 2 h) detached from the culture dish.
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). The
discrepancy between the predicted molecular mass based on the AR
sequence and the AR protein size determined by SDS-PAGE, except for the
mutant AR1–188, has already been reported by Jenster et
al. (25). The N-terminal region harboring most of the
phosphorylation sites is believed to be responsible for this mobility
retardation. In addition, the different AR mutants have also been
studied for their androgen-binding capacity by saturation curves and
Scatchard analyzes. As already described, only AR mutant AR661–919
(3) was unable to bind androgen. The other ARs had Kd
values within the same range (0.6 ± 0.2 nM) as the
wild-type (data not shown).
In CV-1 cells, TPA and androgen have a reciprocal antagonistic
action on both MVDP- and TRE3-tk-Luc reporter genes
Transcriptional cross-modulation between AR and AP-1 factor was
studied in CV-1 cells lacking endogenous AR. Transient transfections of
MVDP-Luc reporter gene in these cells have already been proved to give
a suitable system for studying the effects of androgen (19). Different
amounts of expression vector (20, 50, 100 ng) and two R1881
concentrations (0.1 nM and 10 nM) were used to
study the production of luciferase (Fig. 2A). Whatever the amount of expression
vector, TPA (50 nM) clearly antagonized the effect of R1881
on the luciferase transactivation by the wild-type AR. We observed up
to a 50% decrease in luciferase activity. When CV-1 cells were
cotransfected with the empty pCMV vector, no increase or decrease in
basal luciferase activity was observed after TPA incubation. A
reciprocal antagonistic effect between TPA and androgens was observed
on the TRE3-tk-Luc gene, demonstrating a transrepression
activity of AR (Fig. 2B): R1881 inhibited the transcription of the
TPA-regulated luciferase by 43%, reducing the fold induction from 5.4
to 2.3. No decrease in reporter gene activity occurred if the empty
pCMV vector was used in lieu of pCMV-AR.
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, the antagonistic action of the TPA on
the androgen induced MVDP-Luc activation was time dependent. The
inhibition was approximately in the same range between 10–12 h of TPA
treatment. At 24 h, no luciferase activity was detected. The AP-1
regulated luciferase transactivation increased after 6 h to
10 h of TPA incubation. Prolonged treatment with TPA caused a
decrease in AP-1 activity. Based on these results, we chose to study
the transrepression/transactivation mechanisms after 10 h of TPA
treatment. Figure 3B shows that the TPA concentration had to be equal
to 1 nM to induce an AP-1 transactivation with the
TRE3-tk-Luc reporter gene in CV-1 cells, and higher than 1
nM to trigger the inhibition of MVDP-Luc
transactivation.
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).
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).
Effects of staurosporine, calyculin-A and okadaic acid on
TPA/androgen interactions on MVDP-Luc
To identify the biological processes that control the reversible
phosphorylation of proteins involved in the TPA/androgen interactions,
staurosporine was used as an inhibitor of the PKC. The inhibition of
serine/threonine-specific protein phosphatase-1 and/or -2A (PP-1 and
PP-2A) was obtained by the specific inhibitors, calyculin-A and okadaic
acid, respectively, which induced a potentiation of the TPA effects. At
a concentration of 1 nM, okadaic acid inhibits PP-2A but
does not inhibit the protein tyrosine phosphatases, other phosphatases
or the kinases that have been tested (26). Conversely, calyculin-A, 20-
to 300-fold more potent than okadaic acid, has been used as a specific
PP-1 class phosphatase inhibitor (11).
In CV-1 cells transfected by the androgen-regulated reporter gene,
staurosporine (30 nM) administered along with TPA slightly
blocked TPA action on the wild-type AR (66 ± 2% vs.
54 ± 2% for TPA alone, P = 0.01) (Fig. 5). This partial effect may be due to the
low dose of staurosporine used. Indeed, Young et al. (27)
only obtained 70% of the initial signal after incubation of LNCaP
cells with 10 nM TPA and 200 nM staurosporine.
Our data further support the hypothesis that protein kinase C
activation is involved in the TPA-mediated suppression of androgen
action in the transfected CV-1 cells.
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).
Androgen receptor mutants present various interaction profiles in
CV-1 cells
Based on the previous experiments, 100 ng of various AR expression
vector, 10 nM of R1881 and 50 nM of TPA were
used. Treatments with TPA were performed for 10 h. As previously
shown (Fig. 2B), the mock vector did not inhibit or increase induction
of luciferase.
According to the results summarized in Fig. 6, A and B, mutants have been classified
in three types: a) mutants that were unable to transactivate and to
transrepress AP-1 activity, b) mutants that only had a transrepressive
activity, and c) mutants that did not highly differ from the wild-type
AR in either transactivation or transrepression function. The case of
mutants Gly568Trp and Cys579Tyr will be reported in the following
section.
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661–919, which does not bind androgen but is
constitutively active, did not repress AP-1 activity. An absence of
AP-1 repression was also observed with the mutant Leu707Arg (data not
shown), which lacks androgen binding and causes complete androgen
insensitivity (28). This indicates that a functional and intact ligand
binding domain is critical for the modulation of androgen/AP-1 pathway
interactions. b) Mutants that cause complete androgen insensitivity syndrome and lack any transcriptional activity, such as Val581Phe or Arg585Lys, had a capacity to transrepress similar to that of the wild-type. So, mutations that dramatically affect the DNA binding capacity of the AR do not necessarily alter the interactions with the AP-1 pathway. This suggests that a different structural organization of the DBD of the AR is involved in these processes.
c) The transcriptional activity of mutants Arg607Gln and Arg608Lys, described in men with partial androgen insensitivity syndrome (15, 16, 29), was inhibited by TPA as well as the wild-type receptor from 14.8 to 6.3 and from 14 to 5.6, respectively. However, the Arg608Lys mutation was more efficient in transrepression than the wild-type receptor (P < 0.02), while Arg607 slightly impaired the AP-1 activity.
The 1–188 mutant gave a slight transactivation with MVDP-Luc and 10 nM R1881. This transactivation was little affected by TPA and the AP-1 activity was weakly repressed.
Amino acids in the DNA binding domain discriminate between
transactivation and transrepression
Among the different mutants, Gly568Trp presents an interesting
profile. Indeed, the MVDP-dependent luciferase activity induced by this
mutant is less inhibited by TPA incubation (75 ± 6% of the
reference signal) than the wild-type AR or the other mutants (Fig. 6A),
and the TPA-induced luciferase activity is not impaired by this mutant
(110 ± 2% of the initial signal vs. 44 ± 7%
for the wild-type AR, P < 0.001) (Fig. 6B). These
results suggest that the glycine residue at position 568 is more
important for transrepression than transactivation. Cys579Tyr shows an
intermediary effect between the wild-type and the Gly568Trp, with a
lower level of transrepression (72 ± 8% of the initial
signal).
The striking activation of the TRE-regulated gene by Gly568Trp in absence of TPA cannot be explained at this time. For the mutant Gly568Trp, the action of transfected c-Fos and c-Jun resulted in an inhibition in the same range as that observed with the wild-type receptor, and it mimicked the effect of extended TPA incubations (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we provide evidence that TPA and androgen have a reciprocal antagonistic action in CV-1 cells: TPA decreases AR transcriptional activity measured with MVDP-Luc, whereas AP-1 activity is inhibited by androgens. We also report a dose-dependent inhibition by exogenous c-Jun on AR transcriptional activity. When c-Fos was transfected into CV-1 cells, the degree of inhibition was lower. Such a mechanism might depend in part on the ratio of AR to c-Jun and c-Fos; however, one cannot exclude other co-regulator proteins. Bubulya et al. (6) have demonstrated that c-Jun mediates an androgen-induced transactivation in COS-7 cells and that c-Fos is able to block AR activity in the presence or absence of transfected c-Jun. Sato et al. (7) have found that mutual repression of DNA binding activities is due to a direct interaction between AR and c-Jun in the LNCaP cell line. These authors found no difference in the inhibition of the luciferase activities in CV-1 cells transfected by c-Jun or c-Fos. These results, however, cannot be compared with our data because the authors only tested one concentration for the AP-1 components (500 ng/well) and rat AR cDNA (500 ng/well), i.e. higher than our conditions of transient transfection. The discrepancy of the agonistic and antagonistic effects between both the AR and AP-1 pathways could be relevant to the cell type and/or the availability of CBP concentration (36, 37, 38).
To provide insight into the mechanism of cross-modulation between the
androgen and the AP-1 pathways, we used AR mutations to identify the
subregions of the regions involved in these interactions. The results
from the amino-terminal deletion (1–188) showed a transactivation
equal to 30% of the AR and a slight inhibition of the AP-1 activity.
Similar data have been reported by Kallio et al. (36):
deletion of amino acid residues 40–107 in the rat AR (40–126 in the
human AR) did not significantly modify the androgen/AP-1 cross-talk,
while a more extensive deletion in the N-terminal domain, from amino
acid 38 to 296 (38–298 in the human AR), abolished the repressive
activity of AR. It is interesting to note that this last deletion
overlaps the N-terminal portion that we deleted (from amino acid 1 to
188). Similarly, Chamberlain et al. (39) have shown that
amino-terminal deletion (40–218) induced an activation and repression
equal to the 50% of the wild-type. One of the key subregions may thus
be located within amino acids 126–218. Recently, an important helical
region (189–201) was described in the 
1-core transactivation domain
of the human GR (40). We propose that the homologous helical region in
human AR starts at Ser 187. The deletion 1–188 could therefore prevent
this 
-helix formation by suppression of the two initial amino acid
residues conserved in AR and GR (Ser187 and Thr188). Further studies
are underway to delineate this region precisely, to determine its
participation in the interferences with AP-1 and to analyze its
coactivator recruitment.
The data obtained from the mutants of the ligand binding domain led us
to hypothesize that a liganded and active AR is necessary and
sufficient for interaction with AP-1. Transrepression is impaired by
mutant AR661–919, which is constitutively active because it lacks
an important part of the ligand binding domain encompassing the
HSP-binding region. This suggests that the nuclear localization of the
AR alone is not sufficient to repress AP-1 activity. Thus, in addition
to an androgen binding, transrepression requires the integrity of the
regions conformationally modified by this ligand binding.
Concerning the mutants within the DNA binding domain, we showed that Val581Phe and Arg585Lys, which do not bind DNA and are thus totally inactive on an androgen-regulated gene, have the same capacity of inhibition as the wild-type receptor, even though transrepression by the Arg585Lys mutant is lower. A functional DNA binding capacity, i.e. the ability to bind a target sequence, is thus not required to repress the AP-1 activity, as described for the AR-Ets interaction (41) and in contrast to the AR-RelA interaction (42).
It seems that the spatial position of the substituted amino acids in the DNA binding domain is more important than their contribution to the overall structure of the receptor. Our three-dimensional model suggests that valine 581 and arginine 585 are located in the protein-DNA interface and are unable to interact with other factors, whereas glycine at position 568 is located on the opposite surface, possibly interacting with proteins involved in the control of target gene transcription or part of the AR itself. Even though this model has been defined with the AR bound to DNA, we assume that in solution and liganded, the overall structure of the receptor is maintained. The substitution of this glycine, a small and polar amino acid, by a tryptophane, a more cumbersome and hydrophobic residue, would potentially modify or disrupt any protein-to-protein interactions, but it appears to be more important for transrepression than for transactivation. Similar dissociation of repression activity and transcriptional activity has been described for GR (43). Mutant Cys579Tyr presents similarly interesting results. Cysteine at position 579 is one of the four residues that coordinate the zinc ion to maintain the correct geometry of the first zinc finger. One might thus assume that substitution of this residue would definitively alter the functionality of the AR. However, mutant Cys579Tyr is half as active in transcription as the wild-type receptor. The functionality of this mutant could be explained by the fact that a tyrosine residue may also coordinate a zinc ion but with lower energy (44). Even though the first zinc finger is not totally destabilized, the structure of this domain is modified. This would explain why this mutant partially inhibits TPA-induced luciferase activity even though this difference is less clear cut than for mutant Gly568Trp. The importance of the spatial location of the substitution is also established by the study of mutants Arg607Gln and Arg608Lys. Arginine at position 608 interacts with residues of the first and second zinc fingers and is conserved among all members of the nuclear receptor superfamily. Substitution of this arginine with a lysine is predicted to produce a significant conformational change (19). Molecular modeling revealed that both arginines are partially surface-exposed, and may participate in a region of protein-protein interaction, in agreement with the hypothesis proposed for GR by Scheena et al. (45). Because of this modification in the second zinc finger structure, one may speculate that amino acid displacements are responsible for the increased transrepression capacity of the mutant Arg608Lys. In summary, the transrepression function appears to be mediated by a structure insensitive to some of the mutations that impair the major DNA recognition function of the receptor. Wise et al. (46) have similarly shown that a c-Jun mutant deficient in transactivation is able to interact with AR activity. These data demonstrate that transactivation and transrepression activity may also be separated for c-Jun. Conversely, mutations affecting amino acids located on the exposed surface of the DNA binding domain, including positions 568, 579, 608, and 562 (36), negatively or positively modulate interactions with AP-1 factor.
In addition to defining the regions and/or amino acids involved in AR functioning, analysis of the interactions between natural AR mutants and the AP-1 pathway may help us to understand the mechanisms by which phenotypes are determined, or at least to raise hypotheses. Among the natural mutants, two present puzzling transactivation functions. Gly568Trp and Arg608Lys, which have been described in severe forms of partial androgen insensitivity syndrome, present normal transactivation capacity. These data are paradoxical, and one may speculate that a patient’s phenotype is due not only to a direct action of androgens on androgen-regulated genes (transactivation function) but also to the cross-talk between androgen-mediated signaling systems and growth factor pathways, as suggested by Reinikainen et al. (35).
In conclusion, our data demonstrate that the complex interaction between the androgen and AP-1 pathways involves several subregions of the AR, presumably jointly establishing the fully competent three-dimensional structure of the repression interface. These findings open up a new field of investigation into the identification and characterization of new AR subregions involved in the complex processes that modulate androgen action in target cells.
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Acknowledgments |
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Footnotes |
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2 Present address: Howard Hughes Medical Institute (HHMI) and
Department of Pharmacology, The University of Texas Southwestern
Medical Center at Dallas, Dallas, Texas, 75235-9050.
Received April 29, 1998.
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