AR Suppresses Transcription of the LHß Subunit by Interacting with Steroidogenic Factor-1

Joan S. Jorgensen and John H. Nilson

Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106-4965


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Synthesis of LH is suppressed by feedback from gonadal steroids. Previously, we demonstrated that 779 bp of the bovine LHß promoter was sufficient to target expression of a chloramphenicol acetyltransferase reporter gene specifically to the pituitary in transgenic mice, and found that it was appropriately suppressed after administration of T or E2. In this study, we report that ligand-bound AR, but not ligand-bound ER, directly suppressed activity of the bovine LHß promoter when examined in a gonadotrope-derived cell line. Additional studies with mutated bovine LHß promoter constructs focused on the proximal 5'-flanking region because of the presence of several cis-acting elements that are highly conserved across all mammals. These include regulatory elements that bind steroidogenic factor 1 (SF-1), Egr-1, and Pitx1. When tested by cotransfection with AR, overexpression of Egr-1, Pitx1, and constitutively active steroidogenic factor 1 (SF-1{Delta}LBD) each individually rescued androgen-mediated suppression of the bovine LHß promoter. This suggested a functional interaction between each of these transcription proteins and AR. In contrast, overexpression of full-length SF-1 was incapable of relieving the bovine LHß promoter from the suppressive effect imposed by AR. This suggested that the ligand-binding domain of SF-1 plays an important role in functional interactions that occur between this protein and AR. This notion was further supported by binding assays performed with glutathione-S-transferase-AR: these identified SF-1 as a key interactive partner and localized this interaction to the ligand-binding domain of the protein. Additional binding studies indicated that protein interactions between SF-1, Pitx1, and Egr-1 interfere with formation of a binary complex that contains AR and SF-1. Thus, we conclude that AR suppresses activity of the bovine LHß promoter through protein-protein interactions with SF-1 and that the degree of this interaction can be modified by the presence of Egr-1 and Pitx1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SEX STEROIDS PLAY a critical role in regulating the synthesis of LH by exerting feedback at both the level of the pituitary and hypothalamus. For example, regular pulses of GnRH, released from hypothalamic neurons, stimulate expression of the two genes that encode the heterodimeric subunits of LH, the common {alpha}-glycoprotein subunit ({alpha}GSU) and LHß, as well as trigger pulsatile secretion of the mature hormone from its intracellular stores (1, 2). This increase in circulating LH results in greater occupancy of its cognate receptor in both the ovaries and testes, causing elevated production of both estrogens and androgens (2). These gonadal sex steroids then circulate back to the hypothalamus and pituitary gland to regulate synthesis of GnRH and LH (3, 4), respectively. Here, we will focus on gonadal steroids and determine their mechanism of action specifically at the level of the pituitary.

The {alpha}GSU and LHß genes that encode the subunits of LH reside on different chromosomes and yet respond in the same temporal and directional manner to the stimulatory signal provided by GnRH, and to the negative feedback conferred by estrogens and androgens. Increasing evidence suggests that overall control of {alpha}GSU and LHß gene expression occurs through selected use of specific regulatory elements from a larger complex array located in the 5'-flanking region of both genes (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Thus, it makes intuitive sense that some of these will also be targets of steroid-mediated negative regulation.

In the preceding paper (23) we demonstrated that the bZip proteins cJun and ATF2 act as preferential binding partners of the tandem cAMP-response elements located in the {alpha}GSU promoter-regulatory region that mediates the negative transcriptional effect of androgens. Furthermore, we demonstrated that AR confers its negative effect by binding directly to cJun and ATF2. In this paper, we consider how steroids suppress activity of the LHß promoter.

The proximal promoter (-779/+10 bp) of the bovine (b) LHß subunit gene has been shown to be responsive to both E and T in a transgenic mouse model (24). Nevertheless, it lacks high-affinity binding sites for either ligand-occupied ER or AR (24). Therefore, if steroids act directly at the pituitary to suppress activity of the LHß promoter, the mechanism most likely involves protein-protein interactions that occur between steroid nuclear receptors and specific DNA-binding proteins that regulate transcriptional activity of the LHß gene.

Steadily emerging evidence indicates that three different regulatory elements reside in the proximal 140 bp of the LHß promoter and are remarkably conserved across species (10). These include two elements that bind steroidogenic factor 1 (SF-1), an orphan nuclear receptor family member (9, 12, 16, 18); an element that binds Pitx1, a bicoid-related homeodomain protein (11); and two elements that bind Egr-1, an immediate early response protein (10, 13, 17). All three of these DNA-binding proteins are essential for LHß promoter activity as the absence of any single one abrogates activity of the promoter (10, 13, 17, 18, 20). Egr-1 plays an additional regulatory role since its concentration appears to be regulated directly by GnRH (10, 13, 17). While SF-1 and Pitx1 levels are unaffected by GnRH, they interact synergistically with each other and with Egr-1 to increase LHß gene activity (9, 10, 17, 21). Although regulatory elements distal to -140 bp have been characterized in the rat and bovine LHß promoters, these are much less conserved and display considerable species-specific variation (15, 22, 25). Therefore, we have restricted our analysis of the mechanism of steroid negative regulation to the more highly conserved promoter proximal region.

Herein we report that AR suppressed activity of the LHß promoter whereas ER had no direct effect. Although AR negatively regulates activity of the LHß promoter, it does so without binding directly to DNA. Instead, AR binds specifically to one of the three transcription factors that occupy sites in the regulatory region of the proximal promoter and disrupts the functional synergy that occurs when all the proteins that bind to this region interact. Finally, we compare and contrast how a single nuclear receptor, namely AR, uses two different mechanisms to choreograph the negative regulation of two genes that encode a single glycoprotein hormone.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AR Suppresses Both {alpha}GSU And LHß Promoter Activity While ER Has No Ligand-Dependent Effect
Androgens and estrogens suppressed bLHß reporter activity in transgenic mice (24). Even though the promoter regulatory region lacks specific sequences that have a high affinity for either steroid receptor (24), we decided to explore whether either steroid could exert a negative effect on the bLHß promoter directly at the level of the pituitary.

Transient transfection assays in gonadotrope- derived cell lines were used to determine whether ligand-bound AR or ER{alpha} had an effect on {alpha}GSU or LHß promoter activity. The {alpha}GSU promoter served as a positive control for androgen-dependent suppression. As previously reported, {alpha}GSU was suppressed by ligand-bound AR, but ER{alpha} had no effect (26, 27). Similarly, activity of the -7791 bp bLHß promoter was also suppressed by AR, with ER{alpha} lacking a ligand-dependent effect (Fig. 1Go). Overexpression studies with ERß also indicated that this nuclear receptor was incapable of regulating either the {alpha}GSU or LHß promoter (data not shown). Together, these data suggest that only ligand-bound AR suppresses activity of both {alpha}GSU and LHß promoters directly in the gonadotrope, while the mechanism of ER action occurs elsewhere.



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Figure 1. AR Suppresses Both {alpha}GSU and LHß Promoter Activity While ER Has No Effect

The luciferase gene driven by either {alpha}GSU or LHß promoters was assayed for responsiveness to 60 ng/well cotransfected control (CMVGH), AR (CMVhAR), or ER{alpha} (CMVhER{alpha}), and normalized to ß-galactosidase activity (0.25 µg/well rous sarcoma virus (RSV)-ß-galactosidase). The (-1,500 to +45 bp) {alpha}GSU promoter (1.25 µg/well) was cotransfected with each steroid receptor in {alpha}T3–1 cells. LßT2 cells were used for transfections harboring the (-779/+10 bp) bLHß reporter vector (1.25 µg/well). All cells were maintained in charcoal-stripped media with phenol red-free media used for E2-treated cells. Cells cotransfected with AR were treated with 100 nM DHT while 100 nM estradiol was used for cells harboring ER expression vectors. Control data (CMVGH) represent results from both E2- and DHT-treated cells. Data represent luciferase/ß-galactosidase activity of steroid-treated normalized to vehicle-treated cells. Transfections were done a minimum of three times and the error bars represent SEM.

 
AR Suppresses Wild-Type And Mutant LHß Promoter Constructs
In an attempt to isolate important elements involved in AR-mediated suppression, we cotransfected a variety of mutant LHß promoter constructs with either a control expression vector that encodes GH (CMVGH) or one that encodes AR. All experiments were carried out in the presence of GnRH to optimize the ability to detect suppression, and in the presence of dihydrotestosterone (DHT) due to the necessity of ligand for AR suppression.

As expected, most of the constructs that harbored a mutated element known to be required for bLHß transcription displayed reduced transcriptional activity compared with the parent LHß reporter (Fig. 2Go). The only exception occurred with the bLHß promoter that carried the double nuclear factor Y (NF-Y) (µ5'/3' NF-Y) block mutations. Interestingly, while activity of LHß promoter constructs with a block mutation in Pitx1, gonadotrope specific element (GSE), or, most notably, Egr-1 was attenuated, AR-mediated suppression was still evident. These data suggest that no single element may be responsible for AR-dependent suppression because the remaining elements may be providing compensatory targets for the nuclear receptor.



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Figure 2. AR Suppresses Response Element Block Mutations of the LHß Promoter in LßT2 Cells

-779/+10 bp bLHß promoter constructs carrying mutations through various response elements were cotransfected with either CMVGH (control, black bars) or CMVhAR (AR, white bars). All cells were maintained in charcoal-stripped media with 100 nM DHT. Relative luciferase activity represents the luciferase/ß-galactosidase activity of each promoter. The relative activity, AR divided by control activity of each construct, was used to calculate the percent activity (inset).

 
In an attempt to isolate a region on the bLHß promoter that may be involved in AR-mediated repression, we cotransfected LßT2 cells with a series of progressively truncated promoters. We were surprised to find that AR suppressed activity of all truncated constructs, including the smallest that harbors one GSE and one Egr-1 binding element (-83 bp, Fig. 3Go). In contrast, AR did not suppress a promoterless luciferase control. While these data precluded firm identification of a region required for hormone responsiveness, the presence of two known regulatory elements within the androgen-responsive -83 bp bLHß promoter suggested that their cognate DNA-binding proteins may be targets for AR action.



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Figure 3. AR Suppresses Truncated LHß Promoter Constructs

AR suppresses truncated LHß promoter constructs in LßT2 cells. The -779/+10 bp bLHß promoter construct was truncated to -603 bp, -425 bp, -190 bp, and -83 bp and cotransfected with either CMVGH (control, black bars) or CMVhAR (AR, white bars). All cells were maintained in charcoal-stripped media with 100 nM DHT. Relative luciferase activity represents the luciferase/ß-galactosidase activity of each promoter. The relative activity, AR divided by control activity of each construct, was used to calculate the percent activity (inset).

 
Pitx1, Egr-1, and SF-1{Delta}LBD, but Not SF-1, Rescue AR-Mediated LHß Promoter Suppression
Because identification of single elements and a defined region was problematic, we decided to explore whether transcription factors known to bind the LHß promoter had the capacity to rescue it from AR- mediated suppression. We reasoned that if AR attenuates activity of the LHß promoter by interfering with the action of known transcription factors, then their overexpression should rescue the promoter from the negative effect of androgens. We tested for this possibility by cotransfecting increasing amounts of expression vectors encoding either Pitx1, SF-1, or Egr-1 with a constant amount of an AR expression vector in LßT2 cells and then determined activity of bLHß reporter vector after 24 h of culture. The effect of each transcription factor was examined independently in separate experiments.

In the absence of AR, overexpression of either Pitx1 or SF-1 alone had no effect on activity of the LHß promoter. In contrast, Egr-1 stimulated activity of the reporter vector approximately 20-fold (Fig. 4AGo). As expected, inclusion of an AR expression vector diminished bLHß promoter activity. Indeed, the LHß promoter was still suppressed by approximately 70% in the presence of both Egr-1 and AR, when compared with activity in the presence of Egr-1 alone. Addition of increasing amounts of expression vectors encoding either Pitx1 or Egr-1 rescued the LHß promoter fromthe suppressive effects of AR. While Pitx1 overexpression overcame AR suppression in a dose-responsive manner, Egr-1 rescue from AR suppression required a 10-fold increase in transfected cDNA. In contrast, addition of increasing amounts of expression vectors encoding either full-length SF-1 (Fig. 4AGo), cJun, or CRE binding protein (CREB) (data not shown) had no effect on bLHß promoter activity in the absence of AR and provided no relief from suppression upon the inclusion of AR. Importantly, in the absence of AR, no significant increase in promoter activity was found when cDNA from individual expression vectors was increased from 60 to 600 ng/well. This suggests that overexpression of Pitx1 or Egr-1, in the presence of AR, prevents LHß suppression by specific interactions with AR, and not by increased expression of transcription factor cDNA. In short, of the transcription proteins that regulate activity of the LHß promoter, only Egr-1 or Pitx1 appeared to provide relief from AR-mediated suppression when tested as full-length proteins.



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Figure 4. Egr-1, Pitx1, and SF-1{Delta}LBD, but Not SF-1, Rescues AR-Mediated LHß Promoter Suppression

A, The (-779/+10 bp, inset) bLHß promoter linked to luciferase was cotransfected with a control expression plasmid (CMVGH, speckled bar), AR (hatched bar), or the indicated transcription factors. Pitx1 (CMVPitx1, gray bars), SF-1 (CMVSF-1, white bars), and Egr-1 (CMVEgr-1, black bars) expression vectors were also added in the indicated increasing DNA concentrations in the presence of a constant amount of AR plasmid DNA. Transfections were performed in LßT2 cells maintained in charcoal-stripped media with 100 nM DHT. Luciferase activity of each experiment was normalized to RSV-ß-galactosidase activity. Data represent the luciferase/ß-galactosidase activity of each assay normalized to luciferase/ß-galactosidase of the -779 bp bLHß promoter in the presence of CMVGH. B, Transfections were performed as in panel A. The full-length bLHß promoter and SF-1{Delta}LBD (CMVSF-1{Delta}LBD, dotted bars) expression vector were cotransfected and luciferase/ß-galactosidase activity was measured.

 
Given the classification of SF-1 as an orphan nuclear receptor (28), we considered whether the failure of overexpressed SF-1 to rescue the LHß promoter from the suppressive effect of AR could be due to limiting concentrations of its endogenous ligand. Therefore, we tested this possibility indirectly by repeating the cotransfection assays with a construct that encodes a constitutively active form of SF-1 due to truncation of its putative ligand-binding domain (SF-1{Delta}LBD) (29). Indeed, this construct stimulated activity of the bLHß reporter by approximately 2.5-fold in the absence of AR and suppressed reporter activity by approximately 50% in the presence of AR when compared with activity in the presence of SF-1{Delta}LBD alone. Finally, AR-mediated suppression was prevented in a dose-responsive manner upon the addition of increasing amounts of SF-1{Delta}LBD cDNA (Fig. 4BGo). Importantly, overexpression of SF-1{Delta}LBD did not increase activity of the {alpha}GSU promoter, nor did it prevent AR-mediated suppression (data not shown). Variations in the percent suppression by AR (Fig. 4Go) were likely due to a combination of factors that may include the use of different aliquots of LßT2 cells or different preparations of plasmid DNA. Together, these data provide evidence that Pitx1, Egr-1, and activated SF-1 are functional targets of AR-mediated suppression of LHß promoter activity. Furthermore, the difference in rescue activity displayed by the vector that encodes full-length vs. truncated SF-1 protein suggests that its LBD may be playing a critical role.

AR-DBD Is Necessary and Sufficient for LHß Promoter Suppression
Previously, we demonstrated that the DNA-binding domain (DBD) of AR was both necessary and sufficient for suppression of the {alpha}GSU promoter (26). To determine whether this was also true for the LHß promoter, we cotransfected expression vectors encoding various AR mutants along with the LHß reporter vector in LßT2 cells. Two AR-DBD mutants, a point mutation of the cysteine in the first zinc finger (C576A) and one lacking the entire DBD ({Delta}538–614), were incapable of suppressing the LHß promoter, indicating that the DBD may be essential (Fig. 5Go). In fact, the {Delta}538–614 mutant increased LHß promoter activity approximately 2-fold, suggesting a change in AR conformation that promotes transactivation rather than repression, even in the absence of a DBD. Like its effect on {alpha}GSU, the construct expressing the AR-DBD (554–644) alone resulted in suppressed activity of LHß promoter, although not as completely as that conferred by the vector encoding full-length AR (Fig. 5Go). As expected, the suppressive effect of the AR-DBD construct occurred in the absence of DHT (data not shown). Neither of the vectors encoding the AR-DBD mutants was able to activate a promoter harboring an androgen response element (data not shown and Ref. 26), suggesting fundamental differences in the mechanisms underlying repression vs. activation. Together, these experiments suggest that the DBD plays a critical role in AR suppression of the LHß promoter as it does for the {alpha}GSU promoter (26). In addition, AR exerts its suppressive effect on the LHß promoter without binding directly to DNA, another property also shared by the {alpha}GSU promoter. Thus, we propose that the AR-DBD provides an interactive surface for protein-protein interactions with specific DNA-binding proteins that are required for activity of both promoters.



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Figure 5. AR-DBD Is Necessary and Sufficient for LHß Promoter Suppression

The -779 bp bLHß promoter construct was cotransfected with expression vectors for GH (control), AR, an AR mutant containing the DBD and hinge regions (AR-DBD, aa 554–644), full-length AR with a point mutation in the first zinc finger (C576A), or an AR mutant lacking the entire DBD ({Delta}538–614). Luciferase activity of each experiment was normalized to RSV-ß-galactosidase activity. Data represent the luciferase/ß-galactosidase activity of each assay normalized to luciferase/ß-galactosidase of the proximal 779 bp bLHß promoter in the presence of CMVGH. (*, P < 0.006 when compared with control).

 
AR-DBD Interacts With Full-Length SF-1, but Not In The Presence Of Pitx1 and Egr-1
The promoter-specific rescue activity associated with overexpression of Pitx1, Egr-1, or SF-1{Delta}LBD implicated each as a direct target for interaction with AR. Consequently, we used glutathione-S-transferase (GST)-AR fusion proteins in binding assays to test for direct interactions with these DNA-binding proteins, either alone or in combination. Since AR-DBD was found to be sufficient for bLHß suppression, we restricted potential interaction sites specifically to this domain (GST-AR-DBD). No ligand was included in the reaction. We also took advantage of the C576A point mutation in the first zinc finger of the AR-DBD that eliminates its repressor activity and used it as a control for binding specificity (GST-AR-DBD C576A).

When the binding activities of Pitx1, Egr-1, full-length SF-1, and SF-1{Delta}LBD were examined individually, only full-length SF-1 interacted specifically with AR-DBD (Fig. 6Go). Although SF-1{Delta}LBD interacted weakly with the mutant AR-DBD devoid of repressor activity, it failed to interact with the wild-type AR-DBD. These data suggest that the LBD is necessary for selective binding of SF-1 to the DBD of AR.



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Figure 6. AR DBD Interacts with SF-1 Alone, but Not in the Presence of Egr-1 and Pitx1

Pull-down assays were performed with bacterially produced GST fusion proteins (GST-AR-DBD, GST-AR-DBD C576A, GST control) and in vitro transcribed and translated [35S]methionine-labeled Egr-1, Pitx1, SF-1, and SF-1{Delta}LBD. Combined products were made by adding equal amounts of cDNA to the transcription/translation reaction. Interacting proteins were separated on a 10% SDS-PAGE gel, transferred to 3M paper (Whatman, Clifton, NJ) and visualized by autoradiography. Interactions are compared with 20% labeled input for each protein. Binding assays were repeated a minimum of four times.

 
The observation that only full-length SF-1 displays a direct interaction with the DBD of AR seems paradoxical since this is the only form of the three different DNA-binding proteins that cannot rescue the LHß promoter from androgen-negative regulation. Thus, we considered the possibility that in isolation, full-length SF-1 adopts an inactive conformation that readily binds the DBD of AR. Upon addition of its protein partners, Egr-1 and Pitx1, conformation of SF-1 is changed to a transcriptionally active form (mimicked by SF-1{Delta}LBD) and no longer binds AR. To examine this notion further, we carried out additional GST-AR binding assays, incubating SF-1 with Pitx1, Egr-1, or both. The combination of SF-1 and Pitx1 did not appear to affect the ability of SF-1 to interact with AR-DBD. In contrast, coincubation of SF-1 with Egr-1 promoted alternative binding such that previously undetectable interactions with the mutant AR-DBD occurred to the same extent as those with wild-type AR-DBD. The addition of all three proteins, SF-1, Egr-1, and Pitx1, destroyed any interactions between SF-1 and AR-DBD (Fig. 6Go). Together, these data suggest that sufficient concentrations of Egr-1 and Pitx1 can effectively disrupt the specific interaction that can occur between SF-1 and the DBD of AR. Below, we will present a physiological model whereby changes in the concentration of Egr-1 may set a critical threshold that rescues the LHß promoter from the otherwise suppressive effects of androgen and its receptor.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of LHß gene expression requires an intricate balance between stimulation by GnRH and suppression by gonadal steroids. Concerted action between SF-1, Pitx1, and Egr-1 has been shown to be required for GnRH stimulation (9, 10, 11). In particular, work from several laboratories has established that synergistic interactions between these three proteins and their DNA binding elements are the essential component required for GnRH activation of LHß gene activity (10, 21). Furthermore, a specific interaction between Pitx1 and SF-1 has been shown to alter the dynamics of SF-1 activity (9). Here, we report evidence that suggests these same factors are also important for AR-mediated suppression of LHß transcription.

While the proximal region of the LHß promoter clearly mediated the repressive effect of AR (Fig. 3Go), studies with block replacement mutations failed to isolate any single element (Fig. 2Go). In addition, increasing amounts of Egr-1, Pitx1, or SF-1{Delta}LBD all rescued AR-mediated suppression of the bLHß promoter (Fig. 4Go). Together, these data imply that no single element or transcription factor is responsible for mediating androgen suppression of the LHß promoter. Instead, all three conserved elements and their binding partners appear to play functional roles in mediating the suppressive effects conferred by AR.

Although our findings emphasize the role of the promoter-proximal region of the LHß gene in conferring responsiveness to ligand-occupied AR, they contrast to preliminary results reported by Shupnik et al. for the rat LHß promoter (30). Their studies indicated that AR-mediated suppression of the rat LHß promoter was lost when elements that occupy the distal region of the LHß promoter, namely Sp1 and CArG box sites, were deleted (30). Importantly, this region of the LHß promoter is highly divergent when compared across mammalian species. Indeed, the bLHß promoter lacks comparable elements (15, 19, 22) that prevents us from carrying out a parallel series of studies. Nevertheless, since we were unable to observe a loss in AR-mediated suppression upon individual deletion of regulatory elements within the proximal region of the bLHß promoter, it is possible that elements in the distal region of this promoter are responsible for the compensation and perhaps even necessary, but not sufficient, for mediating the negative response to androgens. Whatever the final explanation, our data clearly support a steroid-responsive role for the regulatory elements within the proximal region of the bLHß promoter. That this region is strongly conserved in the flanking region of the LHß gene in all mammals further underscores the likelihood that it plays a central role in mediating the negative transcriptional effects of androgens.

As we reported in the companion paper regarding {alpha}GSU repression by AR (23), we have considered whether other mechanisms account for nuclear receptor suppression of the LHß transcriptional activity. Indeed, we have tested for changes in critical protein expression, dependence on phosphorylation status of the AR-DBD, and reliance on alterations in histone acetyl transferase activity. We found no change in expression of either SF-1 or Egr-1 as a result of DHT treatment in LßT2 cells (data not shown). In addition, studies by Shupnik et al. (30) determined that pretreatment with DHT resulted in no change in GnRH receptor mRNA expression. Similar to the {alpha}GSU data (23), overexpression studies with a vector encoding a phosphorylation mutant of AR, S650A, indicated no difference in the degree of AR-dependent suppression of bLHß promoter activity when compared with the effects observed with a vector that encodes wild-type AR (data not shown). Although this finding stands in contrast to a study demonstrating that this mutant AR had a reduced capacity to transactivate the MMTV promoter (31), it suggests that activation and repression properties of AR have a differential requirement for phosphorylation. Finally, we analyzed whether AR represses activity of the bLHß promoter by recruiting histone deacetylase and nuclear corepressors. While increasing doses of the histone deacetylase inhibitor, trichostatin A, resulted in an increase in thymidine kinase promoter activity (32, 33), there was little change in bLHß promoter activity, and no difference in the ability of AR to suppress transcription (data not shown). Together, these data suggest that these alternative mechanisms of nuclear receptor suppression are not involved in AR suppression of bLHß gene activity.

As noted earlier, our overexpression studies with vectors that encode Egr-1, Pitx1, and SF-1{Delta}LBD indicated that all of these DNA-binding proteins have the potential to rescue the bLHß promoter from AR- mediated suppression. The construct that encodes SF-1{Delta}LBD is missing half of the putative LBD. Overexpression of this construct increases transcriptional activity of the promoter from the Müllerian inhibiting substance gene (29) as well as bLHß promoter (Fig. 4BGo and Ref. 9). Thus, this truncated form of SF-1 can be viewed as having ligand-independent, constitutive activity. Although the endogenous ligand for SF-1 awaits clear definition, Tremblay and colleagues (9) suggest that Pitx1 can act as a surrogate ligand through direct interaction with SF-1 and convert the orphan receptor into a transcriptionally active form. Since our data suggest that interaction between AR and SF-1 requires the LBD of the orphan receptor, then bound AR may prevent Pitx1 from establishing a productive interaction with SF-1, especially if concentrations of Pitx1 are limiting. If the foregoing is true, then overexpression of Pitx1 would be expected to rescue the LHß promoter from the suppressive effects of AR, which is what we observed. This is also consistent with the known synergistic increase in activity of the LHß promoter that occurs when both SF-1 and Pitx1 are overexpressed (10, 11).

Although we view Pitx1 as playing an important role in activating SF-1, this interaction alone may not be sufficient. Tremblay and Drouin (10) have provided data indicating that direct interactions with functional synergy can also occur between Egr-1 and SF-1 and between Egr-1 and Pitx1. In this regard, our observation that the binding of the AR-DBD to SF-1 is lost when both Pitx1 and Egr-1 are included in the assay (Fig. 6Go) provides another important clue for explaining how dynamic changes in concentrations of Egr-1 may be an important key for explaining how AR negatively regulates the LHß promoter. This is summarized through the model depicted in Fig. 7Go. In the absence of GnRH, little Egr-1 is present in gonadotropes (10, 17, 21). Because of the synergism normally contributed by Egr-1, interactions between Pitx1 and SF-1 should be weak. Thus, ligand-bound AR probably binds strongly to SF-1 and prevents it from assuming a transcriptionally active state. However, upon GnRH stimulation, Egr-1 synthesis markedly increases, with the change in its concentration having a synergistic impact on the formation of a ternary complex that includes Pitx1 and SF-1 and results in the release of AR. In fact, given the degree of reported synergism between Egr-1 and its partners, small changes in its concentration would be expected to have substantial impact on the equilibrium between two transcription complexes that form on the LHß promoter: a ternary complex composed of Egr-1, Pitx-1, and SF-1 that stimulates transcription, and a binary complex composed of SF-1 and AR that represses transcription. Alternatively, because no single element or region of the bLHß promoter was determined to be responsible for mediating androgen suppression, AR need not bind SF-1 directly at the promoter sequences. It is possible that AR may interact with SF-1 and prevent its binding to LHß promoter elements. In addition, other coactivators or adaptor proteins, currently uncharacterized, may play a role in mediating AR suppression of LHß gene activity.



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Figure 7. AR Binds SF-1 to Block Communication between Pitx1, SF-1 and the Initiation Complex and Thus Suppresses LHß Promoter Activity

AR can interact with SF-1 in the presence of Pitx1. A pulse of GnRH then stimulates Egr-1 production that synergistically impacts the microenvironment of protein interactions between Egr-1, Pitx1, and SF-1 at the LHß promoter, and consequently disrupts the AR/SF-1 binary complex.

 
Dynamic alterations in conformation need not be limited to SF-1. Indeed, work by several groups has demonstrated that the addition of ligand induces an interaction between the amino and carboxy termini of AR that is critical for transactivation (34, 35, 36). Previously, we found that AR-DBD alone, and a construct that contained both DBD and LBD, had the ability to suppress {alpha}GSU transcription (26). In contrast, an AR construct missing the LBD failed to repress {alpha}GSU gene activity. Thus, in the context of the entire receptor, both the DBD and LBD are required for AR-mediated suppression of the {alpha}GSU gene (26). Based on these data, we predict that the addition of ligand induces a conformational change that exposes the AR-DBD and enables it to interact directly with SF-1 bound to the LHß promoter.

Although the two subunits that make up the LH heterodimer reside on completely different chromosomes (37), gonadal steroids regulate expression of each gene in the same temporal and directional manner. With the studies presented in this and the companion paper (23), we have a unique opportunity to compare and contrast the mechanisms that androgens use to suppress transcription of each gene. In this regard, we find that transcription of both the {alpha}GSU and LHß genes is directly regulated by ligand-bound AR while ER has no effect. AR also exerts its repressive effect without binding directly to the promoter-regulatory region of either gene. In addition, the DBD of AR and the adjoining hinge domain are both necessary and sufficient for transcriptional repression of each gene. Despite these similarities, however, AR exerts its repressive effect by interacting with different DNA-binding proteins that ultimately contribute to the transcriptional activity of each promoter. For the {alpha}GSU promoter, the bZip proteins, cJun and ATF2, act as preferential binding partners of the tandem cAMP response elements and mediate the negative transcriptional effect of androgens by binding directly to AR. In contrast, the bLHß promoter depends on an entirely different set of DNA-binding proteins, as explained above, with SF-1 serving as a direct target of AR, whereas Egr-1 and Pitx1 appear to modulate the ability of AR to interact with its direct target. In short, we feel these two manuscripts together explain how AR uses different mechanisms to coordinately suppress transcription of the two genes that encode LH. These studies also reveal additional new protein targets of AR, namely ATF2 and SF-1, and therefore increase our understanding of the array of transcriptional proteins used to confer responsiveness to nuclear receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Dihydrotestosterone (DHT), E2, isopropyl ß-D-thiogalactopyranoside, GnRH, Igepal, phenylmethyl sulfonylfluoride, pepstatin A, leupeptin, and aprotinin were purchased from Sigma (St. Louis, MO). Radiolabeled methionine was purchased from Dupont-NEN Life Science Products (Boston, MA). DNA-modifying enzymes and restriction enzymes were purchased from either Roche Molecular Biochemicals (Indianapolis, IN) or Life Technologies, Inc. (Gaithersburg, MD).

DNA
All plasmid DNAs were prepared from overnight bacterial cultures using QIAGEN DNA plasmid columns according to manufacturer’s protocol (QIAGEN, Chatsworth, CA). The (-1,500/+45) human {alpha}GSU and (-779/+10) bLHß promoter constructs have been described previously (7, 8, 24). Wild-type human AR expression vector consists of the full-length AR cDNA fused to the cytomegalovirus (CMV) promoter (38). CMVGH (26) and AR mutants hAR(C576A) and {Delta}538–614 (39) were described previously. AR-DBD (CMVAR-DBD) was made by inserting the PCR fragment containing residues 554–644 of wild-type hAR (39) into pCMV5 (PCR primers 5' with BamHI linker 5'-GCGCGGATCCTTTCCACCCCAGAAGACCTGC-3', 3' with EcoRI linker 5'-GCGCGAATTCCTCTCCTTCCTCCTGTAGTTTCAG-3'). CMV5-ERß was generously provided by Benita Katzenellenbogen (University of Illinois, Champaign/Urbana, IL); CMV5-ER{alpha} was described previously (27). The SF-1 (CMVSF-1) expression vector was kindly donated by Keith Parker (University of Texas Southwestern Medical Center, Dallas, TX) (40); Pitx1 expression construct (CMV-P-OTX) was considerately provided by M.G. Rosenfeld (University of California at San Diego, La Jolla, CA) (41); Egr-1 (CMVNGF1A) expression vector was generously provided by Jeffrey Milbrandt (Washington University Medical School, St. Louis, MO) (42). SF1-{Delta}LBD (CMVSF-1{Delta}LBD) was made by digesting CMVSF-1 with SalI and religating at the SalI site in the pCMV5 polylinker. cDNAs from expression vectors containing Pitx1, Egr-1, or SF-1{Delta}LBD were isolated from EcoRI and XbaI digests; and SF-1 was isolated from EcoRI digests and religated into pcDNA3 (Invitrogen, Carlsbad, CA) for TnT reactions. GST-hAR-DBD encoding amino acids 554–644 of hAR inserted into pGEX-5X-1 (Amersham Pharmacia Biotech, Uppsala, Sweden) was kindly provided by Drs. Olli Janne and Jorma Palvimo (43). GST-hAR DBD-C576A was made by inserting the PCR fragment containing residues 554–644 of full-length mutant AR-C576A (39) (PCR primers 5' with BamHI linker 5'-GCGCGGATCCTTTCCACCCCAGAAGACCTGC-3', 3' with EcoRI linker 5'-GCGCGAATTCCTCTCCTTCCTCCTGTAGTTTCAG-3') into pGEX-2T (Amersham Pharmacia Biotech).

The (-779/+10) bLHß pGL2 construct (7, 8, 24) was used to make all LHß promoter mutants. µPitx1 bLHß pGL2 has been described (20). µ5'/3' NF-Y, µ5'/3' GSE, and µ5'/3' Egr-1 were made using traditional methods of incorporating block mutations by PCR and QuikChange Site-Directed Mutagenesis (Stratagene Cloning Systems, La Jolla, CA). In making the µ5'/3' NF-Y, the 5'-mutation was described (19); PCR primers used for the 3'-mutation are as follows: 5' primer, 5'-AGCCTAGTACTTTGAAAAGACGTCGCTTGCTCTTATATGGACACCTTACCTATTAACTGCTGAGGGCCTCC-AATA-3', and 3'-primer, 5'-TAATAGGTAAGGTGTCCATATAAGAGCAAGCGACGTCTTTTCAAAGTACTAGGCT-GCAGCACCGCCCCTC-3'. Similarly, the 3'-mutation in µ5'/3' GSE has been reported (18); we used 5'-GTCTTATACCTGCAGGCTGTGGGGGCGATCCATGGACCGGGGGTGGCA-3' for the 5'-mutation PCR primer. µ5'/3' Egr-1 was made with 5'-mutant primers, 5'-GTCTGCCTCTTATTCTAGAGGAGATTAGTGTCC-3' and 5'-GGACACTAATCTCCTCTAGAATAAGAGGCAGAC-3', and the 3'- mutant primer, 5'-TTATACCTGCAGGCTCTAGAAATAGCAAGGCCGGG-3'. 5'-Truncation mutants of bLHß pGL2 were also made using traditional cloning techniques. The -603 bp mutant was made by engineering a SalI site at -603 bp using PCR primer 5'-TTTTGTCGACAATCATGCTCTTTGCTGGGTTTGGTTCCG-3'. The -425 bp mutant was made by digesting with EaeI, -190 bp with a RsaI digestion, and the -83 bp mutant was made with a BstEII digestion.

Cell Culture and Transient Transfections
{alpha}T3–1 cells were maintained in high-glucose DMEM supplemented with 5% FBS, 5% horse serum, penicillin, and streptomycin. LßT2 cells were maintained in DMEM supplemented with 10% FBS, penicillin, and streptomycin (Life Technologies, Inc.). Twenty-four hours before transfection, 180,000 cells were plated per 35-mm well in six-well plates. Cells were transfected with the indicated DNAs using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer’s guidelines. Reporter constructs (luciferase, 1.25 µg/well) were cotransfected with expression vectors (as indicated). The amount of transfected cDNAs was kept constant in the dose-response transfections by adding empty CMV expression vector (CMV5). The lipofectamine/DNA solution was replaced with complete medium containing charcoal-stripped serum along with various treatments after 12–16 h. Treatments included 100 nM DHT, 100% EtOH vehicle, and GnRH (100 nM). Cells were harvested 24 h later using 150 µl of reporter lysis buffer (Promega Corp., Madison, WI). Luciferase activity was quantified by luminescence using 15 µl lysate and 100 µl luciferase assay reagent (Promega Corp.). ß-Galactosidase activity was quantified also by luminescence using the Galacto-light assay system (Tropix, Bedford, MA). Luciferase/ß-galactosidase activity of each construct was normalized to the luciferase/ß-galactosidase activity of the wild-type promoter in the presence of CMVGH as described (26). Data in Fig. 1Go reported luciferase/ß-galactosidase activity of each construct in the presence of steroid normalized to luciferase/ß-galactosidase activity of each construct in the presence of ethanol. The values were then averaged over a minimum of three independent experiments.

Purification of GST-Fusion Constructs and GST-Pull-Down Assays
Empty GST construct (pGEX-5X-1), GST-hAR-DBD, or GST-hAR-DBD-C576A were transformed into the DH5{alpha} strain of Escherichia coli. A single colony was inoculated into 2 ml LB + ampicillin (100 µg/ml) and incubated in a 37 C shaker for 5 h. This inoculation was then diluted 1:15 in fresh LB-amp broth and incubated at 37 C overnight. Sixteen hours later, a further dilution (1:100) was incubated for 3 h at 37 C. Isopropyl ß-D-thiogalactopyranoside was added to a final concentration of 1 mM and culture was grown for another 4 h at 37 C. Bacteria were pelleted at 7,500 x g for 10 min at 4 C and then frozen overnight at -80 C. The pellet was resuspended in 3 ml NET (150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 5 mM EDTA, phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and aprotinin protease inhibitor solution) with 1 mg/ml lysozyme added. The pellet was vortexed frequently during the 15- to 30-min incubation. The cells were disrupted by one freeze/thaw cycle, a 30-sec sonication, and two additional freeze/thaw cycles. The suspension was centrifuged at 35,000 rpm for 1 h at 4 C, and the supernatant was aliquoted and stored at -80 C.

GST-protein extracts (75 µl) were incubated with 25 µl glutathione Sepharose Beads (Amersham Pharmacia Biotech) for at least 2.5 h at 4 C and then washed twice with 1 ml NENT buffer (20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, 0.5% dry milk, and the protease inhibitor solution) and twice with 1 ml binding buffer (20 mM HEPES, pH 7.9, 10% glycerol, 60 mM NaCl, 1 mM dithiothreitol, 6 mM MgCl2, 1 mM EDTA, and the protease inhibitor solution). The beads were resuspended in 200 µl binding buffer and incubated with 4–6 µl in vitro translated products at 4 C overnight. Indicated expression vectors were used to make [35S] methionine-labeled in vitro translated products with TnT Coupled Reticulocyte Lysate reaction system according to manufacturer’s instructions (Promega Corp.). The matrix was washed three times with 1 ml NENT buffer, and then twice with binding buffer and resuspended in 20 µl elution buffer (3 mg/ml glutathione in 50 mM Tris-Cl, pH 7.5). After a 10-min incubation at room temperature, the suspension was centrifuged, and 18 µl of the eluant were loaded onto 10% SDS-PAGE for analysis. GST-hAR-DBD, GST-hAR-DBD-C576A, or GST-bound radiolabeled protein products were visualized after 5 d film exposure (Biomax MR, Eastman Kodak Co., Rochester, NY).

Statistical Analysis
Luciferase activity was analyzed by one-way ANOVA (Fig. 5Go) and Tukey’s Honestly Significant Difference determined differences between treatments.


    ACKNOWLEDGMENTS
 
The authors wish to thank Drs. Monica Montano and Paul MacDonald for critical evaluation of this manuscript. We are also grateful to Drs. Ruth Keri and Christine Quirk for mutant LHß promoter constructs.


    FOOTNOTES
 
Address requests for reprints to: Dr. John Nilson, Department of Pharmacology, Case Western Reserve University School of Medicine, W319, 2109 Adelbert Road, Cleveland, Ohio 44106-4965. E-mail: jhn{at}po.cwru.edu

This work was supported by NIH Grants R01-DK-28559 (J.H.N.) and K08-DK-02600 (J.S.J.).

Abbreviations: bLH, bovine LH; CMV, cytomegalovirus; DBD, DNA binding domain; DHT, dihydrotestosterone; GSE, gonadotrope specific element; GST, glutathione-S-transferase; GSU, glycoprotein subunit; LBD, ligand binding domain; NF-Y, nuclear factor Y; RSV, rous sarcoma virus; SF-1, steroidogenic factor 1.

Received for publication March 13, 2001. Accepted for publication May 14, 2001.


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