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Molecular Endocrinology 11 (1): 87-96
Copyright © 1997 by The Endocrine Society

Repression of cAMP-Induced Expression of The Mouse P450 17{alpha}-Hydroxylase/C17-20 Lyase Gene (Cyp17) by Androgens

María Burgos-Trinidad1, Geri L. Youngblood2, Medardo R. Maroto, Arno Scheller, Diane M. Robins and Anita H. Payne

Department of Obstetrics and Gynecology (M.B.-T., G.L.Y., M.R.M., A.H.P.) and The Reproductive Sciences Program (M.B.-T., D.M.R., A.H.P.) Department of Human Genetics (A.S., D.M.R.) Department of Biological Chemistry (G.L.Y., A.H.P.) The University of Michigan Ann Arbor, Michigan 48109


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17{alpha}-hydroxylase/C17-20 lyase (P450c17) protein and the accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated mechanism. To examine the mechanism by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene, constructs containing 5'-flanking sequences of the mouse Cyp17 linked to the chloramphenicol acetyltransferase (CAT) reporter gene were cotransfected into MA-10 tumor Leydig cells with a mouse AR expression plasmid. In the presence of dihydrotestosterone, the cAMP-induced expression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestosterone was observed when the -1021 bp Cyp17-CAT construct was cotransfected with a human AR expression plasmid missing the second zinc finger of the DNA-binding domain, indicating that DNA binding is involved in AR-mediated repression of Cyp17 expression. Analysis of deletions of the -1021 bp fragment demonstrated that -346 bp of 5'-flanking region of the mouse Cyp17 promoter are sufficient to confer androgen repression of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footprinting analysis indicated that the AR interacts with sequences between -330 and -278 bp of the Cyp17 promoter. This region overlaps with the previously identified cAMP-responsive region located between -346 and -245 bp of the Cyp17 promoter. These results suggest that AR-mediated repression involves binding of the AR to sequences in the cAMP-responsive region of the Cyp17 promoter, possibly interfering with the binding of the protein(s) that mediate cAMP induction of Cyp17.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The biosynthesis of testosterone in Leydig cells requires the activities of the cytochrome P450 enzyme, 17{alpha}-hydroxylase/C17-20 lyase (P450c17). This enzyme is a single polypeptide catalyzing two sequential reactions, the hydroxylation of the C21 steroid progesterone at carbon 17, followed by the cleavage of the C17-20 bond to produce androstenedione, the immediate precursor of testosterone (1). Studies from this laboratory, using primary cultures of mouse Leydig cells, demonstrated that cAMP is essential for the induction of P450c17 enzyme activity (2), de novo synthesis of P450c17 protein (3, 4), and the expression of P450c17 mRNA (5). The synthesis of P450c17 ceases in the absence of cAMP (3). The cAMP-mediated induction of P450c17 mRNA requires newly synthesized proteins (5). Furthermore, testosterone, which is produced by the Leydig cells upon stimulation of the cells with cAMP, negatively regulates cAMP induction of P450c17 activity (6), de novo synthesis (4), and levels of mRNA (5). When testosterone production is inhibited by the addition of aminoglutethimide (AG), an inhibitor of cholesterol metabolism, the cAMP-induced increase in P450c17 enzyme activity, de novo synthesis (4), and the expression of P450c17 mRNA are enhanced (5). The repression caused by testosterone can be mimicked by the addition of the androgen agonist, mibolerone, and prevented by the addition of the androgen antagonist, hydroxyflutamide (4). The synthetic glucocorticoid, dexamethasone, and estradiol have no effect on cAMP induction of P450c17 mRNA levels (5). These results indicate that testosterone represses cAMP-induced synthesis and mRNA accumulation of mouse P450c17 via an androgen receptor (AR)-mediated mechanism.

To investigate the molecular mechanisms involved in the regulation of mouse P450c17 by cAMP and steroid hormones in Leydig cells, the structural gene encoding P450c17 was previously isolated and characterized (7). In transient transfections of MA-10 tumor Leydig cells with Cyp17 promoter-driven reporter constructs, a region necessary for cAMP induction of the gene was localized to between -346 and -245 bp relative to the transcription initiation site and is referred to as the cAMP-responsive region or CRR (8). No functional cAMP response element was identified in this fragment, indicating that cAMP-induction of Cyp17 is not mediated by the cAMP-response element-binding protein (8). Gel mobility shift assays using nuclear extracts from cAMP-treated MA-10 cells demonstrated that cAMP induced a protein or proteins that binds specifically to the CRR of Cyp17 (9). The present study was designed to examine the mechanism(s) by which androgens, via the AR, negatively regulate the cAMP-induced expression of the Cyp17 gene.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To examine whether mouse MA-10 tumor Leydig cells contain endogenous AR, cells were transiently transfected with the 2X(HRE)TATACAT reporter plasmid (see Materials and Methods) and treated for 12 h with the androgen, dihydrotestosterone (DHT). Treatment of the transfected cells with DHT did not increase chloramphenicol acetyltransferase (CAT) activity relative to control, indicating that MA-10 cells do not contain endogenous AR (Fig. 1Go). In contrast, cotransfection of 2X(HRE)TATACAT with the mouse AR (mAR) expression vector resulted in a minor increase in the basal expression of 2X(HRE) TATACAT, which was markedly increased by treatment with DHT. Similar results were obtained using a different androgen-responsive CAT reporter construct, the 2X(HRE)tkCAT, containing two copies of a hormone response element (HRE) from the sex-limited protein (Slp) gene, in front of the thymidine kinase (tk) promoter. In addition, a hormone-binding assay was used to examine for the presence of endogenous AR in MA-10 cells, by measuring specific binding of the androgen agonist [3H]R1881 (Table 1Go). MA-10 cells exhibited specific binding of [3H]R1881 in the absence of transfected AR expression plasmid, indicating that MA-10 cells do contain endogenous AR. However, from the functional data presented above (Fig. 1Go), endogenous AR appears nonfunctional or alternatively, the concentration is too low to mediate induction of androgen-responsive reporter constructs, or androgen repression of the cAMP-induced expression of the Cyp17-CAT constructs.



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  		Figure 1. MA-10 Tumor Leydig Cells Do Not Contain Functional ARs

MA-10 cells were transfected with 5 µg of the 3X(HRE)tkCAT or 5 µg of the 2X(HRE)TATACAT reporter plasmids in the presence or absence of the mAR expression plasmid (0.5 µg). All plates received the same total amount of DNA by adding appropriate amounts of the parent vector pCMV5, lacking the mAR sequences. After 24 h of incubation, cells were untreated or treated with 0.1 µM DHT for 12 h as indicated, and the relative amount of CAT activity was determined as described in Materials and Methods.

 

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  		Table 1. Androgen-Specific Binding of hAR and hAR-DBDm Expressed in MA-10 Tumor Leydig Cells

 
Repression of cAMP Induction of Cyp17 Gene Expression by Androgens
To examine the effect of androgens on the cAMP-induced expression of Cyp17, a construct containing -1021 bp of 5'-flanking sequences of the mouse Cyp17 promoter in front of the CAT reporter gene (-1021 bp Cyp17-CAT) was cotransfected into MA-10 cells with increasing amounts of mAR expression vector, and cells were treated with cAMP or cAMP plus DHT. In the absence of mAR, DHT had no significant effect on the cAMP-induced CAT expression. Increasing amounts of the mAR caused a dose-dependent repression of the cAMP-induced expression of the -1021 bp Cyp17-CAT construct in the absence of DHT (Fig. 2Go). Addition of DHT to cAMP-treated cells resulted in a further decrease in CAT expression. Similar results were obtained when the -1021 bp Cyp17-CAT construct was cotransfected with increasing amounts of a rat androgen receptor (rAR) expression vector (10). These results demonstrate that repression of cAMP-induced expression of P450c17 mRNA and protein synthesis reflects repression at the level of transcription and is mediated by the androgen receptor and that -1021 bp of 5'-flanking sequences are sufficient to mediate repression of Cyp17-CAT expression by the mAR.



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  		Figure 2. Effect of mAR on cAMP-Induced Expression of the -1021-bp Cyp17-CAT Construct

MA-10 cells were transiently transfected with the -1021-bp Cyp17-CAT construct (5 µg) and increasing amounts of a mAR expression plasmid, as indicated. All plates received the same total amount of DNA as described in Fig. 1Go. After 24 h, all the cells were treated with 100 µM aminoglutethimide (AG) in the absence or presence of 500 µM cAMP or cAMP plus 0.1 µM DHT. The relative CAT activity of -1021 bp Cyp17-CAT in the presence of cAMP and in the absence of mAR was arbitrarily set at 100. Values represent the averages of duplicate plates from a representative experiment.

 
A Functional DNA-Binding Domain (DBD) of the AR Is Required to Mediate Repression
To investigate whether the DBD of the AR is necessary for androgen-mediated repression of cAMP-induced expression of Cyp17, the effect of a human AR (hAR)-DBD mutant expression plasmid on the cAMP-induced expression of Cyp17 was examined. The hAR-DBD mutant (DBDm) is missing the second zinc finger of the DBD (11). This mutant has normal androgen-binding affinity and localizes in the nucleus but has reduced DNA binding and failed to activate transcription of an androgen-responsive reporter gene (11). MA-10 cells were cotransfected with the -1021 bp Cyp17-CAT construct and increasing amounts of the DBDm expression plasmid (Fig. 3Go). The DBDm did not repress the cAMP-induced expression of -1021 bp Cyp17-CAT in the absence or presence of DHT. In contrast, the wild type hAR repressed the cAMP-induced expression of -1021 bp Cyp17-CAT to basal levels of expression when cells were treated with DHT (Fig. 3Go). The DBDm was also tested in cotransfections with the 2X(HRE)TATACAT construct, and no increase in CAT expression of this construct was observed when cells were treated with DHT (data not shown), thus confirming that the DBDm cannot mediate induction of a hormone-responsive reporter gene. Interestingly, in contrast to the mAR or the rAR (10), the hAR did not exhibit nearly the same extent of androgen-independent repression of Cyp17-CAT expression even at 2 µg of hAR expression vector. However, the extent of ligand-independent repression at a given concentration of mAR was found to be variable (compare Fig. 2Go and Fig. 4Go).



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  		Figure 3. DNA Binding Is Involved in AR-Mediated Repression of Cyp17-CAT Expression

MA-10 cells were transiently transfected with the -1021-bp Cyp17-CAT construct (5 µg) and increasing amounts of the hAR expression plasmid or the hAR-DBDm expression plasmid, as indicated. All plates were corrected to the same total amount of DNA with the parent vector pCMV5. After 24 h, cells were treated with 100 µM AG in the absence or presence of 500 µM cAMP or cAMP plus DHT. The relative CAT activity of the -1021-bp Cyp17-CAT in the presence of cAMP and in the absence of receptor expression plasmid was arbitrarily set at 100. Values represent the mean ± SD from duplicate plates in three separate experiments.

 


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  		Figure 4. Localization of Cyp17-Regulatory Regions Mediating Androgen Repression

MA-10 cells were transiently transfected with the indicated Cyp17-CAT constructs (5 µg), in the absence (-) or presence (+) of 0.5 µg of the mAR expression plasmid. All cultures were corrected for the same amount of DNA as described in Fig. 1Go. All cultures were treated for 12 h with 100 µM AG in the absence or presence of 500 µM cAMP or cAMP plus DHT. The relative CAT activity of -1021 bp Cyp17-CAT in the presence of cAMP and in the absence of receptor expression plasmid was arbitrarily set at 100. Values represent the average ± the range of duplicate plates in two separate experiments.

 
To demonstrate that the DBDm was expressed to a similar extent as wild type hAR in transfected cells, the amount of each receptor expressed in MA-10 cells was determined by measuring the specific binding of the androgen agonist [3H]R1881 (Table 1Go). The transiently transfected DBDm was expressed in MA-10 cells, although the level of [3H]R1881 bound is about 50% of that observed with the wild type hAR. It is unlikely that this difference in expression could account for the complete loss of repression since even up to 2 µg DBDm expression plasmid failed to show any effect, compared with 0.25 µg hAR. The hAR has the highest affinity for R1881. Dexamethasone failed to inhibit binding of [3H]R1881. Estradiol competes for [3H]R1881 binding with 50% inhibition at 100-fold molar excess concentration, in agreement with previous reports (12). Taken together, these results indicate that a functional DBD of the AR is required to observe repression and suggest the involvement of DNA binding in AR-mediated repression of Cyp17-CAT expression.

Localization of the DNA Sequences in the Cyp17 Promoter Required for Androgen Repression
To identify sequences in the Cyp17 promoter that mediate androgen repression of cAMP induction of Cyp17, CAT reporter constructs containing different lengths of 5'-flanking sequences of the Cyp17 gene were transiently transfected into MA-10 cells in the absence or presence of a mAR expression plasmid. Cells were treated for 12 h with cAMP or cAMP plus DHT. The cAMP-induced expression of each of the constructs was inhibited by about 30–40% in the presence of mAR (Fig. 4Go). Treatment with DHT resulted in a further decrease in CAT expression of each of the constructs, to near basal levels of expression. These results indicate that the first -346 bp of Cyp17 are sufficient to bring about ligand-dependent repression by the mAR of cAMP-induced Cyp17-CAT expression.

Localization of AR-Binding Sites in the Cyp17 Gene
To determine whether AR can interact directly with sequences within the first -346 bp of the Cyp17 promoter and to identify the specific sequences involved in mediating repression, deoxyribonuclease I (DNase I) footprinting experiments were performed using baculovirus extract containing AR. The results shown in Fig. 5AGo indicate that AR interacts with sequences within the Cyp17 promoter. Two footprints were observed when the Cyp17 promoter fragment (-346 to -72) was incubated with AR (Fig. 5AGo). The first region protected, androgen response element 1 (ARE-1), is found between -330 and -313 and comprises the sequence 5'-AATTATTAACTGTGCAGC-3', and the second androgen-response element (ARE-2) lies between -306 and -278 and has the sequence 5'-GACATTACAGCACGCACTCTGAAACCTTG-3'. A more pronounced protection of these two regions was observed with higher amounts of AR (Fig. 5AGo). Both sequences share little homology with the consensus sequences for typical ARE or negative glucocorticoid response element-like sequences (13). Under identical experimental conditions AR protected a region in the Slp-enhancer fragment (Fig. 5BGo) encompassing tandem HRE-like sequences (14, 15). The protected fragments in both the Cyp17 promoter and the Slp-enhancer fragment were specifically competed by a double-stranded oligonucleotide containing a single copy of HRE-3 from the Slp gene (16). These results indicate that AR interacts with sequences within the -346 and -245-bp CRR region of the Cyp17 promoter (8). Binding of AR to this region may overlap or interfere with the binding of transcription factor(s) essential for cAMP induction of Cyp17 gene expression.



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  		Figure 5. Localization of Potential AR-Binding Sites in the Cyp17 Promoter

A, A 275-bp fragment of the Cyp17 promoter (-346 to -72 bp) was end-labeled with 32P and used to analyze AR binding in DNase I protection assays as described in Materials and Methods. Radiolabeled DNA was incubated in the absence (lane 1) or in the presence of 25 (lane 2), 50 (lanes 3 and 6), 75 (lane 4), or 100 (lane 5) µg of baculovirus extracts containing AR or 100 µg (lane 7) of extract from noninfected Sf9 cells. Lane 6 contains HRE competitor oligonucleotide at 500-fold over the concentration of probe. B, A 270-bp fragment containing 160 bp from the androgen-responsive enhancer of the mouse Slp gene and including several sequences similar to HREs, was end-labeled and incubated in the absence (lane 1) or in the presence of 40 (lane 2) or 50 (lanes 3 and 4) µg of baculovirus extracts containing AR or 50 µg Sf9 cell extract (lane 5). Lane 4 contains HRE competitor at 500-fold over the concentration of probe. Lane M is the A+G sequencing reaction of the radiolabeled DNA fragments. The numbers on the left side of the figures correspond to nucleotide sequence in the Cyp17 gene (A) or the Slp gene (B). Protected regions are indicated by lines on the right side.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The expression of P450c17 is essential for testosterone biosynthesis in Leydig cells. Previous studies have shown that expression of P450c17 is absolutely dependent on cAMP (3), and androgens negatively regulate the cAMP-induced expression of P450c17 via an AR-mediated mechanism (4). In the present study we sought to investigate the molecular mechanism(s) by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene. The steroid-binding data presented in these studies indicates that MA-10 cells contain endogenous AR, consistent with previous results demonstrating the presence of AR in purified rat Leydig cells (17). However, androgens failed to induce CAT activity from androgen-responsive reporter constructs, indicating that the amount of endogenous AR present in MA-10 cells is not sufficient to mediate induction of androgen-reponsive genes or that the receptor is not functional. Therefore, to examine whether AR was involved in mediating androgen repression, MA-10 cells were cotransfected with AR expression vectors and Cyp17 promoter constructs. Herein, we report that in cotransfection studies androgens repress the cAMP-induced expression of Cyp17-CAT reporter constructs. We have shown that -346 bp of the Cyp17 promoter are sufficient to confer androgen repression of cAMP-induced Cyp17 expression and that a functional DBD of the AR is required to observe repression. Evidence is presented indicating that the AR interacts directly with sequences within the region between -346 and -245 bp upstream of the start site of transcription, which has been demonstrated previously to be required for cAMP induction of the Cyp17 gene (8).

Interestingly, androgen-independent repression of cAMP-induced expression of Cyp17 was observed in MA-10 cells transfected with the mAR or the rAR (10), but not with the hAR even at 2 µg hAR expression vector. Ligand-independent repression by the mAR (18) and the hAR (19) has been reported previously and attributed to elevated levels of receptor interacting with DNA in the absence of ligand, combined with competition for basal transcription factors by the cytomegalovirus (CMV) promoter (19). Since the hAR, mAR, and rAR were expressed via the same CMV promoter, competition for transcription factors by the CMV promoter is unlikely to account for any species difference. However, the extent of ligand-independent repression observed may reflect differences in the levels of expression of the different AR plasmids transfected into MA-10 cells, rather than an intrinsic species-type difference between the receptors.

The mechanism(s) by which steroid hormones repress gene transcription appears to be different from the mechanisms involved in activation (13, 20, 21). Several mechanisms have been proposed to explain transcriptional repression based on investigations involving the glucocorticoid receptor (GR), which has been shown to repress expression of a variety of genes. These mechanisms include interactions with negative glucocorticoid response elements (nGRE); these sequences differ from positive GRE sequences, which mediate enhancement of transcription (22, 23, 24), interaction of the GR with promoter elements preventing or interfering with the binding of positive transcription factors (25, 26), and protein-protein interactions between GR and other factors required for transcriptional activation (27, 28, 29, 30). nGREs differ from the positive GREs not only in sequence but between each other, so that no clear consensus exists (13). In the first mechanism, the receptor is thought to bind nGRE with a lower affinity than the positive GREs or in a different conformation, resulting in decreased transcriptional activity (22). In addition, a nGRE may be negative in some tissues and act as a positive response element in others as is the case of the proliferin gene where positive or negative regulation is determined by the relative amounts of c-jun and c-fos (AP-1), which differ in different cell types (31). The second mechanism was first proposed for the human glycoprotein hormone {alpha}-subunit gene (25). The interaction of the GR with sequences adjacent to the cAMP-response element (CRE) blocked the binding of the transcription factor, the cAMP-response element-binding protein, thus preventing transcriptional activation. Further studies demonstrated that this repression requires the DBD of GR (26). However, subsequent studies have shown that DNA binding of GR to the glycoprotein hormone {alpha}-subunit gene may not be necessary for repression (28), even though purified GR interacts with the promoter in vitro (25). As examples of the third mechanism, inhibition of the collagenase I induction by glucocorticoids involves repression of AP-1 activity by GR involving protein-protein interactions between the DBD of GR and the DNA-binding region of the jun monomer (30). In contrast, inhibition of rat PRL by glucocorticoids does not appear to require the DBD of GR but involves protein-protein interactions between GR and other transcription factors resulting in repression (27). Any of these mechanisms could contribute or account for the negative regulation of Cyp17 expression by androgens.

Androgens have been documented to repress mRNA levels of several genes, including the testosterone-repressed prostate message TRPM-2 (32, 33) and transforming growth factor ß (34). In the human prostate tumor cell line LNCaP, AR mRNA expression is down-regulated by androgen treatment (35). However, it is not known whether this repression is mediated by a direct action of the activated AR on the transcription of these genes. With regard to negative gene regulation meditated by AR, there are only three examples in which transfection and DNA-binding data have been presented. The expression of the mAR gene is induced by cAMP and repressed by androgens (18). Clay et al. (19) have shown that repression of the glycoprotein hormone {alpha}-subunit gene by androgen may involve direct binding of AR to the proximal promoter (19). They proposed that binding of AR may block the binding of a transcription factor. In contrast, androgen repression of the low-affinity neurotrophin receptor (p75) does not require a direct binding of the AR with specific DNA elements (36), even though an intact DBD of the receptor was required to observe repression.

Our data demonstrate that binding of the AR to the Cyp17 promoter is essential for mediating androgen repression of cAMP-induction of Cyp17 transcription. Deletion of the second zinc finger of the DBD of the hAR abolished the ability of AR to repress the cAMP-induced Cyp17-CAT expression. However, the possibility that deletion of the second zinc finger of the AR-DBD disrupts some unknown protein-protein interactions that accounts for the loss of repression, rather than disruption of AR-DNA binding, cannot be excluded at this time. To provide additional evidence for the role of DNA binding, we examined whether AR can interact directly with sequences within this region by DNase I footprinting experiments. AR binds in vitro to two distinct DNA sites in the Cyp17 promoter. The interaction of AR with Cyp17 DNA sequences was weak, relative to the high-affinity interaction of AR with the consensus HREs from the Slp gene (14, 15). However, interaction of AR with negative AREs (nAREs) may be weaker than with positive AREs (22). In addition, computer analysis of the first -346 bp of Cyp17 did not identify any putative ARE (37) or nGRE-like motifs in this region (13), and thus the sequences protected by AR share no sequence homology with reported consensus HREs. Together, these results suggest a role of DNA binding in the androgen repression of Cyp17 expression. However, mutagenesis studies are necessary to demonstrate that DNA binding of receptor demonstrated in vitro, is also required in vivo.

The molecular mechanism(s) by which the AR represses the cAMP-induced expression of Cyp17 is not yet clear. cAMP is absolutely essential for Cyp17 expression (38), and previous studies have demonstrated that the CRR of the Cyp17 promoter is located between -346 and -245 bp (8). This 101-bp fragment overlaps with the DNA sequences recognized by AR in DNase I footprinting. The protein(s) required for Cyp17 expression in Leydig cells, as well as the specific sequences within the CRR that mediate the cAMP-induced expression of Cyp17, remain to be identified. Taken together, our results suggest that androgens can inhibit the expression of Cyp17 by a mechanism involving the binding of AR to regions in the Cyp17 promoter in a manner that interferes or prevents the binding of factors required for cAMP activation of Cyp17 gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
DL-Aminoglutethimide was obtained from Aldrich (Milwaukee, WI). Waymouth’s MB752/1 medium and horse serum were obtained from GIBCO-BRL Life Technology (Gaithersburg, MD). 8-Bromo-cAMP, HEPES, testosterone, estradiol, and dexamethasone were obtained from Sigma (St. Louis, MO). 5{alpha}-DHT was obtained from Sigma. Unlabeled R1881 and [3H]methyltrienolone [17{alpha}-methyl-3H]R1881, 80 Ci/mmol) were obtained from DuPont-New England Nuclear (Boston, MA).

Plasmids
CAT reporter plasmids containing different length fragments of the mouse Cyp17 promoter have been described previously (8). The pGRE2CAT reporter plasmid [henceforth referred to as 2X(HRE)TATACAT], which contains two copies of a GRE consensus sequence derived from the aminotransferase gene in front of the adenovirus E1b TATA sequence, was a gift from Dr. John A. Cidlowski (39). The 3X(HRE)tkCAT reporter plasmid contains a trimer of HRE-3 of the mouse Slp gene fused to the herpes simplex virus thymidine kinase (tk) promoter (14). The mAR expression plasmid in the pcDNAI/neo expression vector was obtained from Dr. Don J. Tindall (40) and subcloned into the pCMV5 expression vector. The hAR expression vector containing the full-length AR cDNA in the pCMV5 expression plasmid (41) and the hAR-DBDm missing the second zinc finger of the receptor (11) were a gift from Dr. Frank S. French and Dr. Elizabeth M. Wilson. The rAR recombinant baculovirus was generously provided by Dr. Olli A. Jänne (42). Spodoptera frugiperda (Sf9) insect cells were infected with the recombinant baculovirus, and extracts were prepared as previously described (43).

Cell Culture and Transfections
MA-10 mouse tumor Leydig cells (a generous gift from Dr. Mario Ascoli) were grown in Waymouth’s MB 752/1 medium containing 15% horse serum, 20 mM HEPES, and gentamicin. For transfections, cells were plated in 60- mm dishes at approximately 1 x 106 cells per dish 40–48 h before transfection. Four hours before transfection, cells were fed fresh media and transfected in duplicate with the indicated amounts of DNA plasmids by the calcium phosphate precipitation method (44). Calcium phosphate DNA precipitates to be used for several plates were prepared in one solution and equally distributed over all plates (11 µg DNA/plate). Empty vector was added to maintain the same total amount of pCMV5 expression plasmid per transfection. Four hours after addition of the DNA precipitate, cells were treated with 15% glycerol (2–3 min), the plates were washed with PBS to remove the glycerol, and fresh medium containing 15% charcoal-stripped serum was added to all plates. After 24 h of incubation, cells were treated with fresh media containing charcoal-stripped serum and 100 µM aminoglutethimide (AG) plus or minus the indicated factors for 12 h. AG and steroids were added from methanolic stock solutions, and the final concentration of methanol was 0.4% in all treatment media. AG was added to block the production of progesterone, which is produced by the MA-10 cells upon treatment of cells with cAMP. Progesterone can bind to the AR although with lower affinity than the androgen, DHT. MA-10 cells do not express the Cyp17 gene even after treatment of the cells with cAMP (38) and therefore do not produce testosterone. Cells were treated with 500 µM cAMP, a concentration that results in maximal induction of expression of Cyp17-CAT constructs in MA-10 cells (8). Cell extracts were assayed for CAT activity by measuring the amount of [3H]acetylated chloramphenicol produced during a 2-h incubation as previously described (8). All cultures were cotransfected with 4 µg SV2-ß-gal to correct for transfection efficiency. Results are expressed relative to ß-galactosidase activity.

Androgen-specific binding of hAR and hAR-DBDm was determined in MA-10 cells transiently expressing the recombinant AR using a whole cell-binding assay previously described, with minor modifications (45). Cells were transfected with 5 µg of the indicated AR expression plasmid (hAR or DBDm), or with the parent vector lacking the AR sequences (pCMV5), as previously described. Control plates received no DNA. Four hours after addition of the DNA precipitate, cells were treated with 15% glycerol (2–3 min), the plates were washed with PBS to remove the glycerol, and fresh medium containing 15% charcoal-stripped serum was added to all plates. Forty hours after transfection, the medium was removed and the cells were washed twice with warm PBS and once with serum-free Waymouth’s MB 752/1 media. Cells were incubated for 2 h with 5 nM [3H]methyltrienolone (R1881) in duplicate dishes in the presence or absence of 100-fold molar excess unlabeled steroids. Labeling medium was removed, and the cells were washed three times with cold PBS containing 0.1% BSA. The cells were harvested in 2% SDS, 10% glycerol, and 10 mM Tris, pH 6.8, and radioactivity was determined by scintillation counting. Binding is expressed as a percentage of [3H]R1881 bound in the absence of unlabeled steroid. Values represent the average of duplicate plates.

DNase I Footprinting Assay
A 275-bp fragment of the Cyp17 promoter corresponding to bases -346 to +72 bp was labeled at the 5'-end with T4 polynucleotide kinase and [{gamma}-32P]ATP (7000 Ci/mmol, ICN Pharmaceuticals, Costa Mesa, CA) and purified on a 5% polyacrylamide gel. A 270-bp fragment containing 160 bp from the androgen-responsive enhancer of the mouse Slp gene, and which includes several sequences similar to HREs (14, 15, 16), was labeled as above and used as a positive control. DNAse I footprinting assays were performed as described (15) with the following modifications. Extracts from noninfected SF9 cells or baculovirus extracts containing AR (43) were preincubated with 1 µg poly(deoxyinosinic-deoxycytidylic acid) for 20 min on ice in footprinting buffer containing 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 2 mM dithiothreitol, 0.2 mM EDTA, 0.05% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 50 nM DHT, 5 µM leupeptin, 5 µM pepstatin, and 0.3 µM aprotinin. Where indicated, preincubations contained a specific 30-bp competitor oligonucleotide corresponding to one copy of HRE-3 from the Slp enhancer (16) at 500-fold molar excess over the concentration of probe. Radiolabeled DNA (30,000 cpm, ~0.5-2 ng) was added (50 µl final reaction volume) followed by a 1-h incubation on ice. MgCl2 and CaCl2 were added to a final concentration of 2 mM, before DNase I digestion with 188 ng DNase I (Worthington Biochemicals, Freehold, NJ) for 5 min on ice. Naked DNA was cleaved with 54 ng DNase I. Reactions were terminated by addition of 85 µl DNase I stop solution [100 mM Tris (pH 8.0), 100 mM NaCl, 1% sodium lauroyl sarcosine, 10 mM EDTA, 100 µg/ml proteinase K, and 25 µg/ml sheared salmon sperm DNA] and incubation at 37 C for 30 min and then 80 C for 2 min followed by phenol/chloroform extraction and ethanol precipitation. The digestion products were resolved on 8% polyacrylamide gel containing 7 M urea. The Maxam-Gilbert A+G sequencing reaction of the radiolabeled fragments run in an adjacent lane was used to locate the footprinted regions (46).


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. E. M. Wilson and Dr. F. S. French for generously providing the hAR, rAR, and the hAR-DBDm expression vectors; to Dr. D. J. Tindall for the mAR expression vector; to Dr. O. A. Jänne for the recombinant baculovirus rAR; to Dr. J. A. Cidlowski for the pGRE2CAT plasmid; and to Dr. Mario Ascoli for providing the MA-10 cells. We thank Shelly F. Bender and Christa B. Williams for technical assistance.


   FOOTNOTES
 
Address requests for reprints to: Dr. Anita H. Payne, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, 300 Pasteur Drive, Stanford, California 94305-5317.

This work was supported by NIH Grants HD-08358, HD-17916 (to A.H.P.), and GM-31546 (to D.M.R.) and by the National Research Service Award HD-07672 (to M.B.-T.) and P30-HD-18258 (to the Molecular Biology Core of the Reproductive Sciences Program).

1 Current address: University of Michigan Medical Center, 5562 MSRBII, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0678. Back

2 Current address: Lineberger Cancer Center, CB 7295, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. Back

Received for publication June 25, 1996. Revision received September 12, 1996. Accepted for publication October 7, 1996.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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