Androgen Suppression of GnRH-Stimulated Rat LHß Gene Transcription Occurs Through Sp1 Sites in the Distal GnRH-Responsive Promoter Region

Denis Curtin, Shannon Jenkins, Nicole Farmer, Alice C. Anderson, Daniel J. Haisenleder, Emilie Rissman, Elizabeth M. Wilson and Margaret A. Shupnik

Departments of Pharmacology (D.C.), Internal Medicine (S.J., N.F., A.C.A., D.J.H., M.A.S.), and Biology (E.R.), University of Virginia, Charlottesville, Virginia 22908; and Department of Pediatrics and Department of Biochemistry and Biophysics (E.M.W.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroids may regulate LH subunit gene transcription by modulating hypothalamic GnRH pulse patterns or by acting at the pituitary gonadotrope to alter promoter activity. We tested direct pituitary effects of the androgen dihydrotestosterone (DHT) to modulate the rat LHß promoter in transfected LßT2 clonal gonadotrope cells and in pituitaries of transgenic mice expressing LHß-luciferase. The LHß promoter (-617 to +44 bp)-luciferase construct was stimulated in LßT2 cells 7- to 10-fold by GnRH. Androgen treatment had little effect on basal promoter activity but suppressed GnRH stimulation by approximately 75%. GnRH stimulation of LHß was also suppressed by DHT in isolated pituitary cells from male or female mice with functional nuclear ARs, but not in male littermates with mutant AR. GnRH stimulation of the LHß promoter requires interactions between a complex distal response element containing two specificity protein-1 (Sp1) binding sites and a CArG box, and a proximal element with two bipartite binding sites for steroidogenic factor-1 and early growth response protein-1 (Egr-1). DHT effectively suppressed promoter constructs with an intact distal response element. The distal response element does not bind AR, but AR reduces Sp1 binding to this region. Glutathione-S-transferase pull-down studies demonstrated direct interactions of AR with Sp1, which requires the DNA-binding domain of AR, and weaker interactions with Egr-1. We conclude that androgen suppression of the rat LHß promoter occurs primarily through direct interaction of AR with Sp1, with some possible role through binding to Egr-1. These interactions result in interference with GnRH-stimulated gene transcription by reducing cooperation between the distal and proximal GnRH response elements.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EXPRESSION OF THE pituitary gonadotropin subunit genes are regulated by several physiological signals. These include pulsatile release of GnRH from the hypothalamus, which regulates the three gonadotropin genes ({alpha}, LHß, and FSHß) in a subunit-specific, frequency-dependent manner, as well as gonadal steroids and peptide hormones (1, 2, 3, 4, 5, 6). The sex steroids, including E, progesterone, and T, stimulate or inhibit gonadotropin gene transcription by acting on the hypothalamus or the pituitary (5, 6, 7, 8). At the hypothalamic level, steroids may alter GnRH pulse patterns and thus indirectly regulate gonadotropin gene transcription (9, 10). Alternatively, steroids may act directly on the pituitary gonadotrope to modulate either basal or GnRH-stimulated gene transcription rates (8, 11).

In rats, both E and T suppress the castration-induced rise in gonadotropin gene transcription (7, 12), although the mechanisms for these steroid effects have not been completely defined. Data from transgenic mice bearing promoter-reporter transgenes for the human glycoprotein {alpha}-subunit, rat LHß subunit, and bovine LHß subunit promoters suggest that E suppresses gene activity of these constructs primarily by feedback at the hypothalamus to alter GnRH pulse patterns, rather than acting directly at the gonadotrope (13, 14, 15). In contrast, androgens appear to act at least partially at the level of the gonadotrope to regulate both basal gene transcription and the responses to GnRH. Human {alpha}-subunit gene promoter activity was directly suppressed by androgens in transient transfection studies (16). Although the human {alpha}-subunit promoter can bind AR directly, androgen suppression is mediated through gene elements distinct from the receptor binding site (17). Transcription suppression is proposed to occur through protein-protein interactions between transcription factors binding to the promoter in these regions, and the DNA binding and ligand-binding domains of the AR (17).

The rat LHß promoter is stimulated by GnRH through complex distal and proximal response elements that interact functionally for full responsiveness (18, 19, 20, 21, 22, 23). In vivo, the interplay between androgens and GnRH stimulation is complex. Low physiological (pM) levels of androgens are required for GnRH stimulation of rat LHß mRNA levels in female rats treated with phenoxybenzamine to clamp endogenous GnRH pulses, whereas higher (nM) androgen levels invariably suppress stimulation by exogenous GnRH (24). In these studies, we examined the direct effects of the nonaromatizable androgen, dihydrotestosterone (DHT), to modulate the basal or GnRH-stimulated transcription of the rat LHß promoter in cultured pituitary cells from transgenic animals and clonal gonadotrope cells. Transient transfection studies were performed in LßT2 cells, a clonal gonadotrope line that expresses the endogenous LHß and {alpha}-subunit genes and the GnRH receptor (25). Androgen treatment directly suppressed the response of the LHß promoter to GnRH in pituitary cells, and this effect required both the AR and upstream GnRH-responsive regions in the LHß gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Direct Suppression of the Transfected LHß Promoter by DHT
The effects of steroid hormones on the activation of the LHß promoter were investigated using a luciferase reporter construct containing the GnRH-responsive LHß promoter region between -617 and +44 bp. This construct contains both the proximal and distal GnRH-responsive elements described by several investigators (18, 19, 20, 21, 22, 23). Transfected cells were treated for 24 h with 1 nM concentrations of steroid followed by 6 h of treatment with 10 nM GnRH. GnRH alone stimulated the LHß promoter approximately 10-fold over control cells. E treatment increased the GnRH response somewhat to 14-fold, while DHT decreased the GnRH stimulation to approximately 3-fold (Fig. 1AGo). Suppression of the GnRH response was specific for androgens as it was observed only with DHT, or with T in separate experiments (not shown), but not with thyroid hormone, E, or progesterone. Little effect on basal promoter activity was seen with any hormone. Suppression of the GnRH effect was observed with DHT concentrations of 10 pM and higher, and was maximally effective at 1 nM, in reasonable agreement with the affinity of the nuclear androgen receptor for DHT (Fig. 1BGo).



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Figure 1. Suppression of LHß Promoter Activity by DHT

A, LßT2 cells were transfected with 3 µg of -617 LHß-luciferase reporter construct and treated with 1 nM E (E2), DHT, T3, or progesterone (P) for 24 h. Some wells in each steroid treatment group were then treated with 10 nM GnRH for 6 h. Luciferase activity was measured in cell extracts, and normalized activity (ALU) is represented as the mean ± SEM for three experiments each with three wells per group. *, P < 0.05; **, P < 0.01 GnRH vs. untreated or steroid controls. B, LßT2 cells were treated with 10 nM GnRH (horizontal arrow) and varying concentrations of DHT (1 pM to100 nM) for 24 h. Data are expressed as in panel A for three experiments with three wells per group in each experiment. *, P < 0.05; **, P < 0.01 vs. GnRH alone.

 
GnRH Receptor mRNA Levels
One potential reason for androgen suppression of GnRH effects could be a reduction in GnRH receptors. Treatment of LßT2 cells with either DHT, E, or GnRH under the same conditions used for transfection assays was followed by measurement of GnRH receptor (GnRH-R) mRNA (Fig. 2Go). None of the steroid treatments resulted in a decrease in GnRH-R mRNA levels when compared with control. The level of GnRH-R mRNA was not suppressed but was slightly greater with DHT treatment when compared with control (Fig. 2Go).



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Figure 2. GnRH-R mRNA Levels in LßT2 Cells After Steroid Treatment

LßT2 cells were treated with 1 nM DHT or E (E2) for 24 h or 10 nM GnRH for 6 h. Total RNA was isolated and GnRH-R mRNA was quantified by dot blot analysis and by calculations from an RNA standard curve spotted on the same filter. Results are presented as the mean ± SEM for three experiments, with six to seven samples per group. *, P < 0.05 vs. untreated controls.

 
Requirement for AR
We next tested whether androgen suppression of GnRH-stimulated LHß promoter activity could be observed in normal gonadotropes, and whether the androgen receptor was required for the suppression of the GnRH stimulation response by DHT. To perform these studies, we used previously characterized transgenic animals that express the LHß-luciferase reporter transgene in their pituitaries (14). The LHß promoter driving luciferase activity contains both distal and proximal GnRH responsive regions. Pituitary luciferase activity, driven by the LHß promoter, was stimulated when the animals were castrated and was suppressed when animals were treated in vivo with steroids or a GnRH antagonist (14). Thus, the transgene responded identically to the endogenous LHß gene in rat pituitaries as measured by transcription run-off assays.

Male mice bearing the LHß-luciferase transgene were bred to heterozygous testicular feminization (Tfm) female mice, which have a frameshift mutation in the AR gene on one of their X chromosomes (38). This breeding resulted in male progeny which all expressed LHß-luciferase in their pituitaries, but which had either a functional wild-type or mutant AR. Female littermates also expressed LHß-luciferase in their pituitaries, but were either homozygous for wild-type AR or heterozygous for the AR mutation. Pituitary cells from both groups of male mice and from female mice homozygous for wild-type AR were cultured and then treated in vitro with GnRH in the absence and presence of DHT.

As shown in Fig. 3Go, GnRH stimulated LHß promoter activity in cultured pituitary cells from male mice with wild-type AR (WT-M) or mutant AR (Tfm-M), or in pituitary cells from female mice with wild-type AR (WT-F). DHT treatment suppressed the GnRH-stimulatory response in pituitary cells containing wild-type AR from males or females. In contrast, DHT treatment had no effect on the GnRH response in pituitary cell cultures from Tfm males (Fig. 3Go, middle panel). Thus, DHT can exert a suppressive effect on the GnRH response in normal gonadotropes, and this effect requires wild-type AR.



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Figure 3. DHT Suppressive Effects in LHß-Luciferase Transgene Activity in Transgenic Mouse Pituitary Cells

Male transgenic mice bearing the LHß-luciferase transgene were crossed with heterozygous female Tfm mice bearing one mutant AR gene. Male and female offspring of this cross were genotyped for the presence of wild-type (WT) or mutant (Tfm) AR. Pituitary cells isolated from both groups of males (WT-M and Tfm-M) and from females with only wild-type AR (WT-F) were treated in vitro with media alone (Con), 1 nM DHT, 10 nM GnRH, or both. Normalized luciferase activity is expressed as the mean ± SEM for 5 experiments, with 9–14 determinations per group. **, P < 0.01 vs. untreated controls.

 
The Upstream GnRH-Responsive Element Is Required for Complete Androgen Suppression
We next investigated which part of the LHß promoter was necessary for the DHT suppression of GnRH-stimulated promoter activity. The rat LHß promoter contains two composite GnRH-responsive regions that cooperate to confer GnRH stimulation (18, 19, 20, 21, 22, 23). The distal region (-456 to -350 bp) contains two specificity protein-1 (Sp1) sites, including an overlapping Sp1/CArG box site at the 5'-end, while the proximal region (-112 to -50 bp) contains two bipartite binding sites for SF1 and Egr-1, separated by a Ptx-1 binding site (Fig. 4Go). Mutation of individual elements (5'Sp1, CArG box, and 3'Sp1) in the distal GnRH-responsive region interfere severely with GnRH stimulation, but multiple mutations or deletion of this region permits the proximal sites to function effectively (18, 19). The ability of DHT to suppress the GnRH stimulation of three constructs, including the entire -617 bp promoter (-617), the same promoter with mutated CArG and 3'Sp1 sites (CG/Spmut), and a deletion construct in which all distal sites (-245) were removed was tested.



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Figure 4. Response of Transfected LHß-Luciferase Constructs to GnRH and DHT

The LHß promoter contains two GnRH responsive elements including a distal region (-456 to -350 bp) comprised of two Sp1 binding sites and an overlapping Sp1/CArG box and a proximal region (-112 to -50 bp) including one Ptx-1, two SF1, and two Egr-1 binding sites. Transfected constructs include the full-length (-617 to + 44 bp) LHß-luciferase promoter, the -617 promoter construct with mutations in Sp1 and CArG as shown (CG/Spmut), and a construct (-245) with the upstream region deleted. LßT2 cells were transfected and treated with 1 nM DHT for 24 h alone or in combination with 10 nM GnRH for 6 h. Normalized luciferase activity is expressed as the mean of four experiments with three wells/group in each experiment. *, P < 0.05; **, P < 0.01 vs. untreated controls. a, **, P < 0.01 for GnRH vs. GnRH plus DHT.

 
The intact -617 to +44-bp promoter construct was stimulated 8-fold by GnRH, and this stimulation was severely diminished by DHT (Fig. 4Go). In contrast, the CArG/Sp1 mutant and the -245-bp promoter were stimulated by GnRH approximately 4.4- and 3.5-fold, respectively, and this response was unaffected or only slightly suppressed by DHT. This indicates that the distal response region plays a critical role in mediating DHT suppression of the GnRH response.

AR Does Not Bind Directly to the LHß Promoter
The sequence of the distal regulatory region of LHß does not contain any consensus steroid receptor binding sites or any obvious androgen response elements. We investigated whether AR could bind directly to this region using EMSAs. Two oligonucleotides representing the 5'Sp1/CArG region or the 3'Sp1 region of the distal GnRH-responsive region were used as probes with nuclear proteins from LßT2 cells treated with DHT (Fig. 5Go). Both gene regions bind several proteins. The most slowly migrating complex (at arrow) for each oligonucleotide probe contains Sp1, as previously demonstrated by a change in the mobility of these complexes by the addition of Sp1 antibody, and by the formation of identical complexes with recombinant Sp1 (18). AR protein does not directly bind to these gene regions, as the addition of anti-AR antibody did not supershift or eliminate any of the observed DNA-protein complexes from LßT2 cells. Similarly, recombinant AR did not bind to the LHß gene regions (as in Fig. 8Go), but did bind to an androgen response element (ARE) (not shown).



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Figure 5. AR Does Not Bind to the Upstream Response Region in the LHß Promoter

Oligonucleotides representing the upstream GnRH response element containing the overlapping Sp1/CArG binding sites (5'Sp1CArG) or the second Sp1 element (3'Sp1) were end-labeled with [{gamma}-32P]ATP. Probes were incubated with nuclear proteins isolated from LßT2 cells treated with 1 nM DHT (LßT2 in figure) and with antibody to the N terminus of the AR (AR-Ab) in some reactions, and then separated on an acrylamide gel. Lanes with no protein added are labeled minus (-). The position of complexes previously demonstrated to contain Sp1 are also indicated. A representative gel is shown from one of seven separate experiments.

 


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Figure 8. AR Reduces Sp1 Binding to the Distal GnRH-Responsive Region

The oligonucleotide representing the 3'Sp1 region of the LHß promoter was end-labeled with [{gamma}-32P]ATP and incubated with recombinant AR protein (lanes 2–4), DHT-treated LßT2 nuclear protein (lane 5), or AR in increasing concentrations with DHT-treated LßT2 proteins (lanes 6–8). The samples were electrophoresed for 4 h and subjected to autoradiography. AR concentrations used were as follows: lanes 2–4, 0.607 µg/µl; lane 6, 0.103 µg/µl; lane 7, 0.207 µg/µl; and lane 8, 1.035 µg/µl. Lane 1 contains no protein and is labeled minus (-), and lanes 3 and 4 contain anti-AR antibody and anti-Sp1 antibody, respectively. A representative gel is shown from one of five independent experiments.

 
AR Binds to Sp1
Because functional studies indicated that DHT effects were mediated via the distal GnRH response region, and AR did not bind directly to this DNA, we investigated the potential for AR interactions with transcription factors binding to the GnRH-responsive LHß promoter regions. Given that there are two Sp1 binding sites in the distal GnRH-responsive region of the LHß promoter required for full GnRH activation, we first examined the potential role of Sp1 in DHT suppression of GnRH stimulation by performing glutathione-S-transferase (GST) pull-down experiments with a GST-Sp1 fusion protein. Full-length AR, steroidogenic factor 1 (SF-1), and Pit-1 protein were in vitro translated with [35S]-labeled methionine and used in pull-down experiments with GST-Sp1. Labeled AR specifically bound to GST-Sp1 (Fig. 6AGo), on average 20- to 30-fold greater that to GST alone in eight separate experiments. In contrast, neither SF-1 nor Pit-1 bound significantly to GST-Sp1 (Fig. 6BGo).



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Figure 6. AR Specifically Interacts with Sp1 in GST Pull-Down Experiments

A, In vitro translated [35S]methionine-labeled AR was incubated with recombinant proteins (1 µg) of GST alone (GST), GST-Sp1 (Sp1), or GST-Egr-1 (Egr-1). Bound proteins are shown on the autoradiogram. The migration of labeled AR alone (0.5 µl of lysate) and AR binding in the absence (-) or presence (+) of 1 nM DHT to recombinant proteins (2 µl lysate) was detected by autoradiography. The arrow indicates migration of AR. B and C, Pull-down results using GST alone and GST-Sp1 (B) or GST-Egr-1 (C) constructs incubated with in vitro translated [35S]methionine-labeled SF-1 and Pit-1. In vitro translated proteins and GST were in identical quantities as in panel A, and the migration position of each translated protein is indicated by an arrow. Representative gels are shown from one of eight independent experiments.

 
Because there was a trend for DHT suppression of the GnRH response in transfection studies with the -245 LHß-luciferase construct containing only the proximal GnRH-responsive element (Fig. 4Go) and because Egr-1 is clearly critical in basal and GnRH-stimulated promoter activity (19, 20, 21, 22, 23), we tested the potential for AR to interact with Egr-1. Labeled AR consistently bound to GST-Egr-1 less strongly than to GST-Sp1 (Fig. 6AGo) in eight separate experiments, with average binding to GST-Egr-1 approximately 25% (ranging from 12 to 39%) that of binding to GST-Sp1 under the same film exposure conditions. This correlated with the milder suppression of the LHß-luciferase construct containing Egr-1, but not Sp1, sites (Fig. 4Go). As with GST-Sp1, neither SF1 nor Pit-1 bound specifically to GST-Egr-1 (Fig. 6CGo).

The AR DBD Is Required for Interactions with Sp1 and Egr-1
To determine which region of the AR was required for interactions with Sp1 and Egr-1, several different AR constructs were tested in pull-down experiments with GST-Sp1 and GST-Egr-1 (Fig. 7Go). Labeled constructs containing the entire AR (AR), the N-terminal region and the DNA binding domain or DBD (AR-N), or the DBD and C-terminal region of AR (AR-C), all bound to both GST-Sp1 and GST-Egr-1. In contrast, an AR protein containing the C-terminal and N-terminal regions, but no DBD (AR{Delta}DBD), failed to bind to GST-Sp1 or GST-Egr-1. Thus, the DBD region of the receptor is critical for binding of the AR protein to these transcription factors, and this was observed in four independent experiments.



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Figure 7. The DBD of AR Is Required for Interactions with Sp1 and Egr-1

Top panel, Schematic representation of the four AR proteins that were in vitro translated and labeled with [35S]methionine for use in GST pull-down experiments. AR is the full-length receptor, AR-N includes aa 1–660 of the amino terminus, AR-C includes aa 507–919 of the carboxyl terminus, and AR{Delta}DBD is the full-length receptor with aa 538–614 deleted. Middle and bottom panels, Autoradiograms of proteins bound in GST pull-down experiments with GST-Sp1 and GST-Egr-1, respectively. The arrows designate the migration position of each translated AR protein in the input lanes. Representative gels are shown from one of four separate experiments.

 
AR Reduces Sp1 Binding to the LHß Distal GnRH-Responsive Region
To determine whether interactions between AR and transcription factors binding to the LHß promoter could have functional consequences in the context of the promoter, we performed gel shift studies with LßT2 nuclear proteins and recombinant AR. We concentrated on the 3'Sp1 binding region of the LHß distal GnRH-responsive region, because the mutation of this Sp1 site largely eliminates the androgen suppression of the -617 LHß-luciferase construct (Fig. 4Go). As shown, recombinant AR did not bind to this DNA (Fig. 8Go, lanes 2–4). Nuclear proteins from LßT2 cells bound to this DNA with characteristic Sp1-containing bands (lane 5). Addition of recombinant AR at increasing concentrations reduced Sp1 binding to probe in a concentration-dependent manner (lanes 6–8).

Sp1 Cotransfection Abrogates DHT Suppression of GnRH Stimulation
Cotransfection studies were performed to investigate the effects of additional Sp1 on DHT suppression of the GnRH-stimulated –617 bp LHß-luciferase promoter in LßT2 cells. Cotransfection of Sp1 overcame the suppressive effects of DHT in the presence of GnRH, and promoter activity was restored to levels approaching that of GnRH alone (Fig. 9Go).



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Figure 9. Sp1 Cotransfection Restores GnRH Stimulation of LHß-Luciferase Promoter

LßT2 cells were transfected with 3 µg of the -617 LHß-luciferase promoter construct and with 3 µg CMV-Sp1 as indicated and treated with 1 nM DHT for 24 h alone or in combination with 10 nM GnRH for 6 h. Normalized luciferase activity from cell extracts was plotted as a function of percentage of control for three separate experiments, with three wells per group in each experiment. **, P < 0.01 vs. control.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have found that androgens specifically suppress GnRH stimulation of the rat LHß gene promoter. This response does not occur through a consensus binding site for AR in the promoter but does require AR in normal gonadotropes. Complete suppression requires an intact distal GnRH response region, containing the Sp1 and CArG binding sites. A promoter construct in which the distal response region is mutated is not suppressed by DHT, while a deletion construct containing only the proximal GnRH response region with binding sites for Egr-1 and SF-1 is only partially suppressed. We postulate that the androgen response occurs primarily via protein-protein interactions between the AR and proteins binding directly to the distal response region, or proteins that otherwise interact with this site. Our studies suggest that Sp1 plays a primary role in this regard. This does not preclude some physiological role for Egr-1 in this response, and AR interactions with both proteins may be required for complete suppression in vivo.

Both androgens and estrogens have been shown to modulate the response of the gonadotrope to GnRH; however, both the direction and the mechanism of these steroid responses appear to be different. For example, in vivo E can act on the hypothalamus to alter GnRH pulses (9), but also enhances pituitary responses to GnRH (26, 27, 28). This may be at least partially due to increases in GnRH receptor mRNA and protein noted after chronic E treatment in vivo, a stimulation that is amplified by the stimulatory feedback of GnRH on its own receptor levels (26, 27). However, other investigators have reported enhanced GnRH transcriptional responses and lower basal transcription for the human {alpha}-subunit promoter in transfection studies with pituitary cells from female rats treated with E (11). This enhancement resulted from decreased phosphorylation of CREB in E-treated rats, presumably via altered GnRH pulse patterns, and subsequent restoration of phosphorylation and enhanced transcriptional responses when GnRH was restored (28). Thus, the majority of E-mediated responses on the gonadotrope genes appear to result from effects mediated through the hypothalamic effects on GnRH. We did not observe significant effects of E on either GnRH-R mRNA, possibly due to our fairly short (24 h) treatment period, or on the LHß promoter stimulation to an acute GnRH challenge.

In contrast, androgens have a direct effect on gonadotrope gene transcription at the pituitary level. We have shown that androgen suppression of LHß occurs in both clonal cell lines and normal pituitary gonadotropes and requires an intact AR. There is little effect of androgen treatment on GnRH-R mRNA, either in our studies or in vivo (26). However, suppressive effects of androgens on human {alpha}-subunit, as well as the rat and bovine LHß, promoter activity have been observed in transient transfection studies (16, 17, 29). All of these genes appear to be suppressed by interactions of the AR with other transcription factors, rather than by direct binding to DNA. The human {alpha}-subunit promoter does contain an ARE, and binding of the AR to the DNA has been demonstrated (16). However, transcriptional suppression by androgens does not occur through this DNA region, but rather via the {alpha} basal element and tandem cAMP response element sites contained elsewhere in the human {alpha}-subunit promoter (17). The suppression requires the DNA and ligand binding regions of the AR, although the DBD alone was sufficient to suppress transcription in transfection studies. Suppression of the {alpha}-subunit gene is proposed to occur by AR interactions with proteins binding to the cAMP response element and {alpha} basal element sites. The specific proteins involved on the {alpha}-subunit gene have yet to be defined, but are unlikely to be Sp1 and Egr-1, and androgen suppression of the two LH subunit genes thus occurs through divergent gene elements. In at least one other gene, GnRH itself, androgen appears to suppress gene transcription not by AR binding to DNA but via protein-protein interactions through as many as three promoter sites that bind to potential neuronal-specific transcription factors (29).

Promoter deletion/mutation studies from our laboratory (18) and that of Kaiser et al. (19) demonstrated that full GnRH stimulation of the rat LHß gene promoter requires functional cooperation between the distal Sp1/CArG region (-456 to -350 bp) and the proximal tripartite enhancer region (-112 to -50 bp) containing binding sites for Egr-1, SF-1, and Ptx-1. The mechanism for this cooperation is at present unknown but may include direct physical communication between the distal and proximal response regions, possibly through a looping mechanism bringing the two response regions into proximity (19). Physical contact could then occur either directly between transcription factors or via a cofactor that binds both regions. In either scenario, binding of the AR to Sp1 or to Sp1 and Egr-1, could interfere with functional cooperation between the regions, either by preventing binding of the proposed cofactor or by disrupting direct interactions between transcription factors.

Stimulation of the bovine LHß gene by GnRH occurs primarily via a proximal promoter element containing SF-1 and Egr-1 sites, rather than by cooperation between multiple response elements (30). Androgen suppression requires the Egr-1 and SF-1 binding region of the promoter, suggesting that AR binding to proteins such as Egr-1 or SF-1 may play a critical role in this context. In contrast, androgen suppression through a similar region in the rat LHß gene is not predominant, although a partial decrease of transcription occurs with a short construct (-245 bp) containing only these sites. These functional data correlate with the weaker although significant association of Egr-1 and AR in our GST pull-down assays. We have found that the greatest androgen repression of the rat LHß gene occurs via the distal GnRH-responsive region of the gene containing the Sp1 sites, and that cotransfection of Sp1 can abrogate the suppressive response. These data correlate with our biochemical findings that Sp1 and AR directly interact in vitro, and that this interaction reduces Sp1 binding to the distal GnRH-responsive region of the promoter. AR reduction of Sp1 binding to DNA, in addition to AR binding to Egr-1, would then cooperate to reduce or abolish the functional cooperativity of the two GnRH-responsive regions in the rat LHß promoter.

Functional interaction between AR and Sp1 has also been documented in the cyclin-dependent kinase inhibitor p21 gene (31), which is stimulated rather than suppressed by androgen treatment. The p21 promoter contains an ARE and six Sp1 sites. Deletion of the ARE does not eliminate androgen stimulation, which only occurs upon mutation of specific Sp1 sites. Direct AR-Sp1 interactions were demonstrated by mammalian one-hybrid analysis and by coimmunoprecipitation (31). Direct interactions of AR and Sp1 on this gene promoter were not demonstrated, but it is unlikely that AR would reduce Sp1 binding in this context. Thus, AR may modulate Sp1-mediated transcriptional activation by several mechanisms, functionally requiring direct binding of Sp1, but not AR, to DNA. Interestingly, E stimulation of several genes is conferred by Sp1 sites and occurs by binding of ER to Sp1 rather than to DNA. In those studies, EMSAs do not detect a unique ER{alpha}-Sp1 complex, and antibodies to ER{alpha} do not eliminate or supershift DNA-protein complexes; rather, Sp1 binding intensity to DNA is enhanced (32, 33).

Overall, these studies demonstrate a direct effect of DHT on the pituitary gonadotrope to suppress LHß promoter stimulation by GnRH. This mechanism might play an important role in modulating gonadotropin gene responses to hypothalamic stimulation when androgen levels are high and could be an important aspect of steroid feedback on this axis. In light of recent studies demonstrating similar actions of androgens on the human {alpha}-subunit, bovine LHß, and GnRH genes (16, 17, 29, 30), this mechanism appears to be a common and significant means of regulation of hypothalamic-pituitary function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transfection Studies
The clonal gonadotrope cell line, LßT2 cells (24), which express GnRH, ERs, ARs, and both LH subunits and secrete LH, were originally obtained from Dr. Pamela Mellon (University of California San Diego). Immunoblots performed with mouse and rat pituitary and LßT2 cell extracts suggest that AR levels are similar. Cells were grown in phenol red-free DMEM with resin-stripped 10% FBS and antibiotics and were plated in 35-mm wells at a density of 1–1.5 x 106 cells per well 16–20 h before CaPO4 transfection. Each well was transfected with 3 µg of reporter vector as indicated for 16 h, washed, and treated with 10 nM GnRH for 6 h before collection and measurement of luciferase activity. Steroids as indicated were included during the transfection period and the GnRH treatment period, for a total treatment of 24 h. In several experiments, a cytomegalovirus (CMV)-ß-galactosidase vector (0.3 µg/well) was included to normalize for luciferase activity, and in all cases luciferase activity was normalized for protein. After transfection, cells were washed with PBS and lysates were collected in 250 µl lysis buffer (Promega Corp., Madison, WI), vortexed, centrifuged for 1 min, and assayed in a Turner 20e luminometer (Turner Designs, Mountain View, CA). Protein concentrations were determined by the colorimetric assay from Bio-Rad Laboratories, Inc. (Hercules, CA). Hormones were obtained from Sigma (St. Louis, MO). Data for normalized luciferase activity are presented as the mean ± SEM for six wells per group, compared with untreated controls, and each experiment was performed between 3 and 6 times with equivalent results.

LHß-luciferase reporter vectors have been described elsewhere (18). For most studies, the construct containing the promoter region from -617 to +44 bp relative to the transcriptional start site, and both GnRH-responsive elements, was used. A related mutant construct (CGm3'Sp1 m), in which point mutations in the CArG box and 3'Sp1 site within the distal GnRH response element (-456 to -350 bp) were introduced into the -617 to +44 promoter region, was used to determine the effects of androgen on the distal element in the context of the entire LHß promoter. In some experiments the deletion construct from -245 to +44 bp, containing only the proximal GnRH response element, was also tested. In additional experiments, the -617 to +44 LHß-luciferase promoter construct was cotransfected with a CMV-Sp1 expression construct (a generous gift of Dr. Randall Urban, University of Texas Medical Branch, Galveston, TX), as indicated, to determine whether additional Sp1 could rescue the suppressive effect of androgens on the promoter. Total DNA was normalized with vector alone.

GnRH-R mRNA Measurements
LßT2 cells were grown in T75 flasks treated for 24 h with DHT or E (both at 1 nM concentrations) in the presence or absence of 10 nM GnRH for 6 h, as for transfection studies. After treatment, cells were washed with PBS and collected in guanidinium isothiocyanate, and total RNA was isolated as previously described (35). GnRH-R mRNA was measured by a quantitative dot blot procedure previously described (27). GnRH-R sense strand RNA was synthesized by in vitro transcription with Riboprobe Gemini kit (Promega Corp.). A standard curve of sense RNA (50–1000 pg/dot) was spotted on each nitrocellulose filter along with cellular RNA samples (10 µg/sample), and a sample of pooled rat pituitary RNA was included as a positive control and as a measure of filter-filter variability. Hybridization was performed with a saturating amount (1 ng cDNA/µg RNA) of labeled cDNA. GnRH-R mRNA levels were calculated by linear regression using the sense RNA standard curve. Results were expressed as picograms of GnRH-R mRNA per 100 µg cellular DNA.

Transgenic Animal Studies
Transgenic mice bearing the LHß-luciferase transgene have been described previously, and the isolated pituitary cells from these animals have been demonstrated to respond to GnRH in culture (36). LHß-luciferase mice were bred in the C57/B6J background, the same background as the Tfm carrier females heterozygous for the AR mutation on the X chromosome (37, 38). The female Tfm heterozygotes were purchased from The Jackson Laboratory (Bar Harbor, ME), and bred to male LHß-luciferase males. Tail DNA of these progeny were tested by PCR analysis for AR receptor status, in addition to phenotypic gonadal laparotomy. Two groups of male siblings, all expressing LHß-luciferase, and expressing either wild-type or mutant AR, were selected for further study. In addition, female mice homozygous for wild-type AR were also selected for comparison with the males. Pituitaries from mice were collected after 8 wk of age, and the isolated cells were cultured and treated with 1 nM DHT and/or 10 nM GnRH, as for transfected cells. Cells representing one pituitary equivalent were incubated in one 35-mm well. After treatment, luciferase activity was measured as for transfected cells, and results are expressed as luciferase activity/mg protein. Data are plotted as the mean ± SEM for 5 experiments, and each group contains 9–14 individual wells.

Nuclear Proteins and EMSAs
Nuclear proteins were isolated from LßT2 cell nuclei by the method of Dignam et al. (39). The extraction buffer [20 mM HEPES, pH 7.3, 0.6 M KCl, 20 µg/ml ZnCl2, 0.2 mM EGTA, 0.5 mM dithiothreitol (DTT)] contained protease inhibitors (10 µg/ml each aprotinin, antipain, chymostatin, leupeptin, and 1 µg/ml pepstatin). After ultracentrifugation at 100,000 x g, supernatant proteins were subjected to chromatography on a Sephadex G column with buffer (20 mM HEPES, pH 7.3, 1.5 mM MgCl2, 0.15 M KCl, 0.5 mM DTT, and protease inhibitors). For EMSAs, nuclear protein (4–6 µg) was incubated with labeled DNA (50,000–100,000 cpm) and buffer containing final concentrations of 10 mM Tris-HCl, pH 7.5, 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, to mM NaCl, and 1 µg poly(dI-dC). Final volumes were 15–20 µl, and final salt concentrations were adjusted to 100–125 mM KCl. Samples were incubated on ice for 45 min and subjected to electrophoresis on a 5% acrylamide 1x Tris-borate EDTA gel for 1.5 h (18).

Recombinant AR used in EMSA studies was isolated from Sf9 cells (40). A sample of Sf9 cells was mixed in lysis buffer (20 mM Tris, pH 8, 350 nM NaCl, 10% glycerol, 10 mM imidazole, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/µl aprotinin, 1 mM phenylmethylsulfonylfluoride, 1 µM DHT, and 1 mM DTT), freeze-thawed three times, and spun at 100,000 x g for 30 min and supernatant was collected. Total protein was quantified using BCA protein assay (Pierce, Rockford, IL), and AR content was compared against AR that had been column purified using a metal affinity resin (Talon from CLONTECH Laboratories, Inc., Palo Alto, CA). AR content was assessed by immunoblot analysis using AR (N-20) antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Complimentary oligonucleotides representing rat LHß gene sequences for EMSA included wild-type sequences representing the 5'Sp1/CArG box region and 3'Sp1 site. The wild-type sense strand sequence for 5'Sp1/CArG was (5'-GCTAAACCACACCCATTTTTGGACCCAATCCAGGCATCC-3'). The oligonucleotide representing the wild-type 3'Sp1 site was (5'-GCTGGGCGAGGGGCGGCGCCCACCTC-3'). Double-stranded DNA containing one copy of the LHß promoter was end-labeled with [{gamma}-32P]ATP and purified from a 6% acrylamide gel. The wild-type sense strand sequence that was used as a representative androgen response element was ARE (5'-GAAGTCTGGTACAGGGTGTTCTTTTTG-3'). This oligonucleotide was labeled by the same method as above. EMSA experiments using the LHß promoter oligonucleotides and AR were run on a 5% acrylamide 1x Tris-borate EDTA gel for 3 h. Antibodies used were AR (N-20) and Sp1 from Santa Cruz Biotechnology, Inc. Recombinant AR was incubated with DNA under the same conditions as for nuclear proteins and was added simultaneously with nuclear proteins in some experiments.

GST Pull-Down Experiments
BL21 bacterial cells were transformed with constructs expressing GST, GST-Sp1, or GST-Egr-1. Luria Broth (100 ml) containing 50 µg/ml ampicillin was inoculated with 1 ml of bacteria and incubated in an orbital shaker at 37 C. Bacteria were grown to A600 = 0.5, induced with 0.1 mM isopropyl ß-thiogalactopyranoside, and shaken overnight at room temperature. The bacterial pellet was resuspended in 5 ml of buffer containing 50 mM Tris, pH 7.5, 0.5 mM EDTA, 300 mM NaCl, 10 mg/ml lysozyme, and 1 mM DTT. One hundred microliters of 10% Nonidet P-40 were added, and after 10 min, the lysate was frozen at -70 C in an ethanol bath. Lysate was thawed at room temperature and then incubated for 1 h in 5 ml of buffer containing 1.5 M NaCl, 12 mM MgCl2, 5 µg deoxyribonuclease I, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, and 0.1 mM phenylmethylsulfonylfluoride. Lysates were passed through a 20-gauge needle and centrifuged for 30 min at 7,500 x g. Soluble lysate was conjugated with glutathione beads (Sigma) overnight at 4 C. Beads were washed with PBS, and protein concentrations were assessed after electrophoresis on 12% polyacrylamide denaturing gels by Coomassie Blue stain and immunoblotting (GST-Sp1 and GST-Egr-1) with Sp1 and Egr-1 antibody from Santa Cruz Biotechnology, Inc. For pull-down experiments, approximately 1 µg of GST fusion protein was used in each sample incubation. In steroid treatment groups, 1 nM DHT was added and equivalent volumes of ethanol were added to untreated samples. BSA (20 µg/ml) was added to each incubation containing [35S]methionine-labeled (0.04 mCi/50-µl reaction) in vitro translated proteins (TNT Rabbit Reticulocyte Transcription/Translation Kit; Promega Corp.). Labeled proteins included full-length AR (amino acids 1–919), amino-terminal AR (AR-N, aa 1–660), carboxy-terminal AR (AR-C, aa 507–919), and the DBD-deleted AR (AR{Delta}DBD, aa 1–919, {Delta} aa 538–614). Total volume was adjusted to 150 µl with GST wash buffer (10 mM MgCl2, 150 mM KCl, 20 mM HEPES, 10% glycerol, and 0.12% Nonidet P-40). Beads and proteins were incubated for 1.5 h at 4 C and then centrifuged and washed four times in GST wash buffer. Beads were resuspended in 10 µl of SDS loading buffer and boiled for 5 min. Proteins were electrophoresed on SDS containing 10% acrylamide gels at 150 V, along with standard molecular weight markers (Benchmark, Life Technologies, Inc., Gaithersburg, MD). Gels containing [35S]methionine-labeled proteins were dried and exposed to film for 24–72 h at -70 C.


    ACKNOWLEDGMENTS
 
We thank Ms. Savera Shetty for technical assistance with the transgenic mice.


    FOOTNOTES
 
Address all correspondence and requests for reprints to: Margaret A. Shupnik, Ph.D., Box 8800578, 7141 Multistory Building OMS, Department of Internal Medicine/Endocrinology, University of Virginia Medical Center, Charlottesville, Virginia 22908.

This work was supported by National Institute of Child Health and Human Development/National Institutes of Health through cooperative agreement (U54-HD-28934) as part of the Specialized Cooperative Centers Program in Reproduction Research though both an individual research project (M.A.S. and D.J.H.) and the Molecular Core at the University of Virginia, and at the University of North Carolina at Chapel Hill (U54-HD-35041). We also acknowledge additional support from the National Institutes of Health (R01 MH01349).

Abbreviations: ARE, Androgen response element; CMV, cytomegalovirus; DBD, DNA-binding domain; DHT, dihydrotestosterone; DTT, dithiothreitol; Egr-1, early growth response protein-1; GnRH-R, GnRH receptor; GST, glutathione-S-transferase; SF-1, steroidogenic factor 1; Sp1, specificity protein-1.

Received for publication December 14, 2000. Accepted for publication July 20, 2001.


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 DISCUSSION
 MATERIALS AND METHODS
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