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Endocrinology, doi:10.1210/en.2007-0407
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Endocrinology Vol. 148, No. 12 6083-6091
Copyright © 2007 by The Endocrine Society

Estrogen Enhances Gonadotropin-Releasing Hormone-Stimulated Transcription of the Luteinizing Hormone Subunit Promoters via Altered Expression of Stimulatory and Suppressive Transcription Factors

Takanori Kowase, Heidi E. Walsh, Douglas S. Darling and Margaret A. Shupnik

Division of Endocrinology (T.K., M.A.S.), Department of Medicine, University of Virginia School of Medicine, Neuroscience Graduate Program (H.E.W.), University of Virginia, Charlottesville, Virginia 22908; and Center for Oral Health and Systemic Disease (D.S.D.), Periodontics, Endodontics and Dental Hygiene, University of Louisville School of Dentistry, Louisville, Kentucky 40292

Address all correspondence and requests for reprints to: Margaret A. Shupnik, Ph.D., Box 800578 HSC, University of Virginia, Charlottesville, Virginia 22908. E-mail: mas3x{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transcription of the LH subunit genes is stimulated by GnRH and may be modulated physiologically by steroids such as 17ß-estradiol (E). We found that E treatment amplified GnRH stimulation of the rat LHß and {alpha}-subunit promoters, and expression of the endogenous mRNA, in LßT2 gonadotrope cells 2- to 5-fold above GnRH alone. We examined gene expression in LßT2 cells after E and/or GnRH treatment, and found that E suppressed expression of transcription factor Zfhx1a, and enhanced GnRH stimulation of Egr-1 mRNA and protein. E effects were abolished in the presence of antiestrogen. Egr-1 is critical for LHß expression; however, the role of Zfhx1a, which binds to E-box sequences, was untested. We found E-box motifs in both the rat LHß (–381, –182, and –15 bp) and {alpha}-subunit (–292, –64, –58 bp) promoters. Zfhx1a overexpression suppressed basal and GnRH-stimulated activity of both promoters. Mutation of the {alpha}-subunit promoter E boxes at either –64 or –58 bp eliminated Zfhx1a suppression, whereas mutation of the –292 bp E box had no effect. Gel shift assays demonstrated that Zfhx1a bound to the –64 and –58, but not –292, bp E-box DNA. Similarly, mutation of LHß promoter E boxes at either –381 or –182, but not –15, bp reduced Zfhx1a suppression, correlating with binding of Zfhx1a. The –381 bp LHß E box overlaps with an Sp1 binding site in the distal GnRH-stimulatory region, and increased Sp1 expression overcame Zfhx1a suppression. Thus, one mechanism by which E may enhance GnRH-stimulated LH subunit promoter activity is through regulation of both activators and suppressors of transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PITUITARY gonadotropins LH and FSH are glycoprotein dimers composed of an {alpha}-subunit that is common for LH, FSH, and TSH, and unique LHß and FSHß subunits. Expression of the gonadotropin genes is primarily and differentially controlled by the pattern of hypothalamic GnRH pulses (1, 2, 3). The sex steroids, including 17ß-estradiol (E), testosterone (T), and progesterone (P), can also modulate transcription, either by acting at the hypothalamus to alter GnRH pulse patterns, or by direct actions at the pituitary to influence basal or GnRH-stimulated expression (4, 5, 6, 7, 8, 9, 10, 11, 12). Steroids act to alter transcription directly at gene promoters or "non-genomically" to alter cytoplasmic signaling pathways (13).

In many species E, T, and dihydrotestosterone (DHT) suppress the castration-induced increase in gonadotropins, and one common pathway for this response may be negative feedback at the hypothalamus to alter GnRH pulses (4, 6, 14). In females, mRNA levels and gene transcription of the gonadotropin subunits also vary during the reproductive cycle. Positive E feedback at diestrus and proestrus generally stimulates LHß and {alpha}-subunit mRNA and gene transcription, with FSHß mRNA and transcription highest at late proestrus and diestrus (12, 15, 16). Studies performed with estrogen receptor (ER) knockout mice and other in vivo and cell culture studies suggest that negative E feedback occurs via the hypothalamus; however, positive E feedback may occur via both the hypothalamus and pituitary (17), and may include actions of several hypothalamic peptides, such as kisspeptin (18).

Direct actions of E and T or DHT at the pituitary level differ among gonadotropin subunits and between species. In vitro treatment with T or DHT increases rodent FSHß mRNA levels and promoter activity by mechanisms including direct androgen receptor (AR) association with the promoter and enhanced MAPK activity (10, 19). In contrast, T or DHT suppresses LHß or {alpha}-subunit promoter GnRH-stimulated activity through AR interactions with transcription factors binding to the GnRH response elements (7, 8). E treatment of pituitary cells suppresses ovine FSHß transcription via AP1 sites (20) but stimulates rat LHß gene transcription and basal or GnRH-stimulated LH synthesis and secretion in several species (11, 12, 21). The rat LHß promoter contains an imperfect estrogen response element (ERE) at –1189 bp, but this motif is not conserved in other species (20, 21). E has also cooperated with pulsatile GnRH to regulate GnRH receptor (GnRH-R) levels (22, 23, 24), and the ability of E to modulate GnRH responsiveness of gonadotropin and GnRH-R gene expression is beginning to be defined (24, 25, 26, 27).

Both LH subunits require the orphan nuclear receptor steroidogenic factor 1 (SF-1) for basal activity and several complex promoter elements for GnRH stimulation (8). For complete GnRH stimulation, the mouse {alpha}-glycoprotein subunit promoter requires a composite GnRH response region that contains an Ets-domain protein binding site and a second region that likely binds LIM homeodomain proteins (28). In contrast, deletion and mutation analysis shows that the rat {alpha}-subunit promoter relies primarily on two Ets domain protein binding sites between –405 and –385 bp (29). The transfected rat LHß promoter requires two composite GnRH response regions: a distal region containing two binding sites for Sp1 and a CArG box; and a proximal region with two composite SF-1 and Egr-1 binding sites, separated by a homeodomain protein binding site (29, 30, 31).

In these studies we examined the ability of E to modulate basal and GnRH-stimulated LH subunit gene expression in clonal gonadotrope LßT2 cells using transfected rat LHß (–617 to +44 bp) and {alpha}-subunit (–411 to +77 bp) promoters. We found that E modestly stimulated basal {alpha}-subunit and LHß promoter activity, and markedly stimulated the response to GnRH. Because neither construct contains an ERE, we examined E regulation of other factors that might influence LH subunit expression. E had no effect on GnRH-R or SF-1 mRNA expression but suppressed expression of Zfhx1a, a zinc finger homeodomain protein that acts as a transcriptional repressor (32), and amplified GnRH stimulation of Egr-1. These results suggest that one way that E may potentiate LH subunit gene expression is by regulation of both activators and suppressors of transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Gonadotropin promoter reporter constructs
The rat {alpha}-glycoprotein subunit and LHß promoter-luciferase reporter plasmids were previously described in detail (29). For E-box mutagenesis, the rat {alpha}-glycoprotein subunit promoter construct (–411 {alpha}-LUC) and LHß promoter constructs (–617 LHß-LUC) were used as a template with the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Mutagenesis was performed strictly following the manufacturer’s protocol with the following oligonucleotide primers and their reverse complements. Underlined lowercase bold letters indicate mutated bases: {alpha}-luciferase (LUC)-E box 292, 5'-CTTAGAATCCTGTTTATTTTTAAAGGccTCAA-CTTTCAGAATGTTTTGTGCAAG-3'; {alpha}-LUC-E box 64, 5'-CCCTGGGCTTAGGccCA-GGTGGGAGCAT-3'; {alpha}-LUC-E box 58, 5'-GGGCTTAGGTGCAGGccGGAGCATGCAA-TTTGTA-3'; rLHß-LUC-E box 381, 5'-GGGGCGGCGCCCAtCTCTGGTTGTATTTAA-3'; rLHß-LUC-E box 182, 5'-TCAGTTAAGCTCAGGggCCTGGGCTGAGTG-TG-3'; and rLHß-LUC-E box 15, 5'-CAGGTATAAAGCCAGGccCCCAAGGTAGGGAAGG-3'.

Cell cultures and transient transfection assays
The clonal mouse gonadotrope cell line, LßT2 (33), which expresses GnRH-R, ER, AR, and {alpha}-glycoprotein and LHß subunits, and secretes LH, was originally obtained from Dr. Pamela Mellon (University of California San Diego, San Diego, CA). These cells contain both ER{alpha} and ERß mRNA and protein, with ER{alpha} expressed as the predominant ER subtype (34). LßT2 cells were maintained in DMEM supplemented with 10% fetal bovine sera and 100 U/ml penicillin, 100 µg/ml streptomycin at 37 C in 95% O2/5% CO2. Serum and media were obtained from Mediatech, Inc. (Herndon, VA). For transfection experiments, cells were plated in 12-well (22 mm) plates at a concentration of 500,000 cells per well with phenol red-free DMEM supplemented with 5% charcoal-stripped newborn calf serum. The next day, media were changed before transfection. LßT2 cells were transfected with 0.5 µg/well promoter luciferase constructs and 1 µg/well pcDNA3.1-Zfhx1a/Zfhep/ZEB (35), unless designated otherwise using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to manufacturer’s instructions. Alternatively, pcDNA3.1-Egr-1 was transfected (1.0 µg/well), along with promoter-luciferase constructs. In some experiments the LHß-luciferase promoter construct was cotransfected with 0.5, 1.0, or 3.0 µg/well cytomegalovirus-Sp1 expression construct (36) (a kind gift of Dr. Randall Urban, University of Texas Medical Branch, Galveston, TX), with or without 0.5 µg/well pcDNA3-Zfhx1a, and 0.5 µg/well CMV-ß-galactosidase. Total DNA was normalized with vector alone. After the 16-h transfection period, media were changed, and cells were treated with or without vehicle or E (10 nM) for 24 h, then with or without GnRH (100 nM), for an additional 6 h. After treatment, cells were washed twice with PBS and collected in 1x lysis buffer (Promega Corp., Madison, WI) for luciferase assays. Luciferase activity in the lysate samples was analyzed with a Turner TD-20E luminometer (Turner Designs, Sunnyvale, CA), and protein content was determined using protein dye (Bio-Rad Laboratories, Inc., Richmond, CA). All experiments were performed a minimum of three times with triplicate samples per group.

Nuclear proteins and EMSAs
The binding of Zfhx1a to the subsets of E-box-like sequences of the rat and mouse LHß promoter, and rat {alpha}-glycoprotein subunit promoter, were assessed by nonradioactive EMSAs (Lightshift Chemiluminescent EMSA Kit; Pierce, Rockford, IL). Double-stranded oligonucleotides were designed and include: rLHß-LUC-E box 381 mut, 5'-GGGGCGG-CGCCCAtCTCTGGTTGTATTTAA-3'; rLHß-LUC-E box 182 mut, 5'-TCAGTTAA-GCTCAGGggCCTGGGCTGAGTGTG-3'; rLHß-LUC-E box 15 mut, 5'-CAGGTATAAAGCCAGGccCCCAAGGTAGGGAAGG-3'; {alpha}-LUC-E box 292 mut, 5'-CTTAGAATCCTGTTTATTTTTAAAGGccTCAACTTTCAGAATGTTTTGTGCAAG-3'; {alpha}-LUC-E box 64 mut, 5'-CCCTGGGCTTAGGccCAGGTGGGAGCAT-3'; and {alpha}-LUC-E box 58 mut, 5'-GGGCTT-AGGTGCAGGccGGAGCATGCAATTTGTA-3'.Underlined lowercase bold letters indicate mutated bases.

Nuclear proteins were isolated from LßT2 cell nuclei by the method of Dignam et al. (37), as described previously (29). 3'-Biotin-labeled DNA oligonucleotides were generated using Biotin 3' End DNA Labeling kit (Pierce). 3'-Biotin-labeled or unlabeled DNA oligonucleotides containing the wild-type mutated E-box sites were synthesized, and the complementary oligos were annealed to obtain the double-stranded probe. Nuclear protein extract (6.0 µg) was incubated with 20 fmol labeled probe alone or with 4 pmol unlabeled probe in the 10x binding buffer plus 1 µg poly(dI-dC) in a 20-µl reaction at room temperature (RT) for 20 min. For supershift EMSA, 1 µl rabbit anti-Zfhep/Zfhx1a/ZEB antibody (38) or preimmune rabbit serum was incubated with 6 µg nuclear extract at RT for 30 min in buffer containing 1 µg poly(dI–dC), before addition of 20 fmol labeled probe and further incubation at RT for 20 min. Both incubation conditions worked for EMSA and supershift EMSA. After adding 5 µl of 5x loading buffer to each 20-µl reaction, 20 µl of each sample was loaded on 6% polyacrylamide gel containing 0.5x Tris-borate EDTA, and run at 4 C in 0.5x Tris-borate EDTA at 100 V for 1.5 h. After electrophoresis, complexes were transferred to positively charged nylon membranes (Roche, Indianapolis, IN) in an XCell SureLock Electrophoresis Cell (Invitrogen) at 0.38 A for 1 h, and cross-linked in the FB-UVXL-1000 Microprocessor-Controlled UV Crosslinker (Fisher Scientific, Hampton, NH) at optimal cross-link mode, which delivers an energy dosage of 120 mJ/cm2. Detection of Biotin-labeled DNA probe was performed with the manufacturer’s protocol.

Quantitative real-time RT-PCR and protein expression
To measure mRNA or protein expression levels with or without steroid treatment, LßT2 cells were plated in six-well (35 mm in diameter) plates at 2.5 million cells per well with phenol red-free DMEM supplemented with 5% charcoal-stripped newborn calf serum and 100 U/ml penicillin, 100 µg/ml streptomycin. The next day, media were changed, and cells were treated with either ethanol vehicle control, E2 (10 nM), DHT (10 nM), and incubated at 37 C for an additional 24 h. In some experiments, cells were incubated with the ER antagonist, ICI 182,780 (1 µM), in the absence or presence of 10 nM E, for 24 h before RNA preparation. All compounds were obtained from Sigma Chemical Co. (St. Louis, MO) and solubilized in ethanol, which was added as the vehicle control. GnRH (100 nM) was added to some cells treated with vehicle or steroids for the final 1, 3, or 6 h. After treatment, total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. Samples of RNA were first analyzed by the Specialized Cooperative Centers Program in Infertility and Reproduction Research microarray core at the University of Washington (Seattle, WA). One microgram of total RNA was used for RT with an iScript cDNA synthesis kit (Bio-Rad), and quantitative real-time RT-PCR was performed using published RT-PCR primers (39). Oligonucleotide primers for Zfhx1 were 5'-CGCAGCCAAGCACAGAAG-3' (forward) and 5'-TTCCATCCGCAGGTT-GAGG-3' (reverse). To account for differences in starting material, we normalized mRNA levels with mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA with the primers 5'-TGCGACTTCAACAGCAACTC-3' (forward) and 5'-CTTGCTCAGTGTCCTTG-CTG-3' (reverse). RT-PCR products were detected by the incorporation of DNA-intercalating SyBr green reagent and the iCycler iQ system (Bio-Rad). Melting curves and agarose gel electrophoresis confirmed the presence of a single PCR product for each primer set. Experimental and GAPDH reactions were performed in separate tubes in triplicate, and the average threshold cycle (CT) for the triplicate was used in all subsequent calculations. Relative differences among treatment groups were determined using the {Delta}{Delta}CT method as outlined in the Applied Biosystems protocol (Foster City, CA) for reverse transcriptase-PCR. A {Delta}CT value was calculated for each sample using the CT value for GAPDH to account for RNA recovery between groups.

In some experiments, cells were solubilized in sodium dodecyl sulfate-gel loading buffer as previously described (34), and protein subjected to electrophoresis on sodium dodecyl sulfate-containing 8% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and detected by specific primary antibodies for Egr-1 (Santa Cruz sc-189; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) incubated at 1:1000 1 h at RT, and for Zfhx1a (38), incubated at 1:1000 overnight at 4 C. Secondary antibody for both proteins was horseradish peroxidase-linked donkey antirabbit antibody (Amersham Biosciences, Piscataway, NJ), incubated at 1:10,000 for 1 h at RT. As a loading control, ß-actin was detected on the same blots with primary antibody (Sigma) and used at 1:100,000 for 1 h RT, and secondary sheep antimouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 1:50,000 for 1 h RT. All proteins were detected on film after incubation with SuperSignal West Pico Chemiluminescence (Pierce).

Statistical analysis
Values are expressed as mean ± SEM. Each experiment was performed a minimum of three times, and treatment groups within each transfection were composed of three to six replicates. Statistical significance was determined by ANOVA or unpaired t test using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA). For ANOVA, Tukey’s post hoc test was used to determine significant differences between the means, with P less than 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E potentiates GnRH stimulation of rat LHß and {alpha}-subunit promoter activity in LßT2 gonadotrope cells
Because E and GnRH cooperate during the ovulatory surge to stimulate LH, and E and GnRH can individually stimulate LH secretion and synthesis in vitro, we tested the ability of E to influence basal and GnRH-stimulated subunit promoter gene activity. Luciferase constructs containing either the rat {alpha}-subunit (–411 to +77 or –287 to +77 bp) and LHß (–617 to +44 or –245 to + 44 bp) promoters were transfected into LßT2 cells, which were treated with E, GnRH, or both hormones. E treatment effects were maximal after 24 h. For the {alpha}-subunit, E treatment alone increased basal activity of both promoter constructs approximately 2-fold (Fig. 1AGo). As previously demonstrated (29), the –287 construct does not contain the two Ets-like motifs between –405 and –385 bp, and is not responsive to GnRH, but E or E plus G increased promoter activity 2-fold. GnRH-stimulated activity of the –411 construct was enhanced with E treatment, increasing from 3.3-fold with GnRH alone to 4.7-fold with GnRH plus E. Basal activity of both the –617 and –245 LHß promoter constructs were stimulated approximately 2-fold in the presence of E (Fig. 1BGo). GnRH stimulation of the –617 construct was also markedly enhanced, from 8.5-fold (GnRH) to 19.6-fold (GnRH plus E). GnRH stimulation of the –245 construct that lacks one of two GnRH-responsive elements was not as robust, which is in agreement with earlier reports (29, 40). GnRH stimulation of this construct was also enhanced in the presence of E, from 4- to 5.8-fold. Expression of the endogenous mRNAs for {alpha}-subunit and LHß mRNA were similarly stimulated by E, GnRH, and GnRH plus E (Fig. 1CGo), with E plus GnRH stimulation approximately 2-fold above GnRH alone. Thus, E enhanced GnRH stimulation of both LH subunit promoters, reflecting changes in endogenous LH subunit mRNA levels.


Figure 1
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  		FIG. 1. Estrogen enhances basal and GnRH-stimulated rat {alpha}-subunit and LHß promoter activity and mRNA levels in LßT2 cells. Cells were transfected overnight with luciferase constructs (0.5 µg/well) containing the rat {alpha}-subunit (–411 to +77 or –287 to +77 bp) promoter (A) or rat LHß (–617 to +44 or –245 to +44 bp) promoter (B) constructs. Cartoons of the promoters indicating locations of known or putative binding sites for transcription factors are shown with bent arrows at the transcriptional start sites. Transfected cells were treated for 24 h with vehicle [Con (control)] or 10 nM E, ± 100 nM GnRH for the last 6 h. Cells were then collected, and luciferase activity was measured. Data are expressed as normalized luciferase activity corrected for protein and cotransfected ß-galactosidase activity, and represent the mean ± SEM for four experiments with triplicate samples per group. *, P < 0.05 GnRH vs. control in the same steroid treatment group. ^, P < 0.05 E treatment vs. vehicle, ± GnRH. Fold GnRH stimulation for each construct and treatment is shown in parentheses. C, Regulation of endogenous mRNA. Cells were treated with E and GnRH and total RNA was isolated. Endogenous LHß and {alpha}-subunit mRNAs were measured by quantitative RT-PCR, normalized for GAPDH mRNA expression. Values are expressed as the mean ± SEM for three experiments with triplicate samples. *, P < 0.05 treatment vs. control. ^, P < 0.05 treatment vs. GnRH alone. ALU, Arbitrary light units; GnRH-RE, GnRH response element; GSE, gonadotrope specific element; PGBE, pituitary glycoprotein hormone basal element.

 
E treatment alters expression of transcription factors Egr1 and Zfhx1 in LßT2 cells
E can exert its effects through both genomic and cytoplasmic signaling pathways, and alteration of cyclic adenosine 3',5'-monophosphate response element binding protein (CREB), protein kinase C, and calcium signaling has been proposed to play a role in E sensitization of gonadotropes (22, 25, 26, 27) To determine if E responses might also occur through genomic pathways to alter expression of important transcription factors or the GnRH-R, we performed mRNA analysis of cells treated with vehicle, E, GnRH, or E plus GnRH, and potential candidate mRNAs were further analyzed by quantitative RT-PCR (Fig. 2AGo). Relevant binding sites for transcription factors shown to modulate basal or GnRH-stimulated promoter expression are depicted in Fig. 1Go. Twenty-four hours of E treatment alone, or in combination with GnRH, had little effect on expression of Sp1 or the GnRH-R, or of SF-1, which binds to both the {alpha}-subunit and LHß promoters. We also saw no effects of E on SRF, which binds to CArG box elements, Ptx-1, Ptx-2, or CREB (data not shown). In contrast, E amplified the stimulatory effect of GnRH on Egr-1, an early response protein that is critical for expression of the LHß gene (41), and suppressed expression of mRNA encoding Zfhx1a, a transcription factor that binds GC-rich sites similar to Sp1 sites (35, 42). The 1-h GnRH time point is maximally effective for Egr-1, and we used this time point to demonstrate effects of GnRH plus E2. Zfhx1a is not stimulated at these time points, nor at 24-h continuous GnRH (data not shown). Similarly, we have tested several time points of E2, and found a significant E2 response after at least 8–12 h, and 24 h was maximally effective, with no additional suppression of Zfhx1a or stimulation of Egr-1 after 48 h. Protein levels of Zfhx1a and Egr-1 were also regulated by E and GnRH similarly to their mRNAs (Fig. 2BGo). Results from three experiments for each protein, normalized for ß-actin expression, demonstrated that Zfhx1a protein levels were suppressed to 0.55 ± 0.1 of control levels by E or to 0.48 + 0.05 by E plus GnRH. Stimulation of Egr-1 protein was approximately 2.0- to 2.4-fold higher in the presence of E plus GnRH vs. GnRH alone.


Figure 2
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  		FIG. 2. E and GnRH treatment selectively alter transcription factor mRNA and protein levels in LßT2 cells. A, mRNA measurements. LßT2 cells were treated for 24 h with vehicle (Con), 10 nM E, ± 100 nM GnRH for the last 1-h incubation. Total mRNA was collected and scanned by microarray analysis and verified by quantitative RT-PCR, normalized for expression of GAPDH mRNA. Values are expressed as percent control values and represent the mean ± SEM for duplicate samples in triplicate experiments. *, P < 0.05 vs. vehicle-treated control for each mRNA. B, Protein levels of Egr-1 and Zfhx1a were measured on immunoblots (30 µg total protein) from cells treated as described previously, using ß-actin in the same samples as a loading control. Representative blot of three experiments with duplicate samples.

 
The effect of E was specific, as DHT did not alter expression of either Egr-1 or Zfhx1a, alone or in combination with GnRH (Fig. 3Go). In addition, the antiestrogen ICI 182,780 completely abolished the effects of E. Thus, E potentiation of GnRH-stimulated Egr-1 and E suppression of Zfhx1a expression is specific, and occurs at both the mRNA and protein levels.


Figure 3
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  		FIG. 3. E and GnRH specifically modulate Egr-1 and Zfhx1a mRNA levels in LßT2 cells. Cells were treated with 10 nM E or DHT, or the antiestrogen ICI 182,780 (1 µM), alone or in combination for 24 h, and 100 nM GnRH (G) was added for the last 1 h as indicated. Total RNA was isolated, and mRNA for Egr-1 (A) or Zfhx1a (B) mRNA quantitated as in Fig. 2Go. Data are calculated as percent control (Con) compared with vehicle-treated cells and are presented as the mean ± SEM for duplicate samples in triplicate experiments. *, P < 0.05 vs. vehicle-treated control for each mRNA. ^, P < 0.05 vs. GnRH alone.

 
Increased Egr-1 and Zfhx1 expression influences LH subunit promoter activity
The role of Egr-1 in modulating LHß expression has been described (29, 30, 31), but the potential role of Zfhx1a was unknown. We next tested how overexpression of either Egr-1 or Zfhx1a could influence {alpha}-subunit and LHß promoter activity (Fig. 4Go). Egr-1 overexpression specifically stimulated LHß promoter activity (Fig. 4Go, right lower panel), increasing the basal activity approximately 7-fold and the overall GnRH simulated promoter activity. There was no significant effect on either basal or GnRH-stimulated {alpha}-subunit promoter activity (Fig. 4Go, right upper panel). Fold-GnRH stimulation for LHß was not as great with Egr-1 overexpression (1.7 vs. 4-fold), probably because a significant component of GnRH stimulation includes induction of Egr-1. In contrast, Zfhx1a overexpression suppressed both {alpha}-subunit and LHß promoter activity (Fig. 4Go, left panels). Basal activity of both promoters was maximally suppressed by 40–50%. Stimulation of {alpha}-subunit promoter activity by GnRH in the presence of Zfhx1a was slightly lower than with GnRH alone (2.6 vs. 3.3-fold). GnRH-stimulated LHß promoter activity was suppressed from 7.7-fold with GnRH alone to 3.6-fold with Zfhx1a. Thus, increased Egr-1 would contribute to stimulated LHß expression, and decreased Zfhx1a would result in stimulated {alpha}-subunit and LHß promoter activity.


Figure 4
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  		FIG. 4. Exogenous Egr-1 specifically stimulates LHß, and Zfhx1a suppresses {alpha}-subunit and LHß promoter activity. LßT2 cells were transfected with 0.5 µg of either {alpha}-subunit (–411 to +77 bp) or LHß (–617 to +44 bp) promoter-luciferase constructs, and cotransfected with indicated microgram amounts of expression vectors for Egr-1 or Zfhx1a, and empty vector to normalize total DNA. After 48 h, cells were treated with 100 nM GnRH for 6 h, and luciferase activity was measured. Data are the mean ± SEM for three experiments with three samples per group. *, P < 0.05 vs. vehicle-treated control (Con) for each group. ^, P < 0.05 vs. cells with no exogenous Egr-1 or Zfhx1a. ALU, Arbitrary light units.

 
Suppression of rat {alpha}-subunit promoter activity by Zfhx1a via two E-box sequences
Zfhx1a acts by binding E-box-like GC-rich sequences in many genes, and there are three such motifs in the rat {alpha}-subunit promoter, at bases –292, –64, and –58 relative to the transcriptional start site. The mechanism by which Zfhx1a suppressed the rat {alpha}-subunit promoter was investigated in transient transfection assays with Zfhx1a overexpression and {alpha}-subunit promoter constructs with specific mutations of each E box (Fig. 5AGo). Transfected cells were treated for 6 h with or without GnRH. With the intact promoter, Zfhx1a decreased basal activity, and GnRH stimulated activity. Mutation of E-box –292 alone slightly decreased activity of the promoter, but Zfhx1a still suppressed both basal and GnRH-stimulated promoter activity to the same extent as the wild-type promoter. Mutation of either E-box –64 or E-box –58 completely prevented Zfhx1a suppression of basal and GnRH-stimulated promoter activity. Mutation of all three E boxes (Mut3Ebox) also prevented Zfhx1a suppression of the promoter and resulted in a significant 70% increase in basal promoter activity compared with the wild-type promoter. Mutation of E-box –64 and E-box –58 together gave identical results to the triple mutant (data not shown).


Figure 5
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  		FIG. 5. Zfhx1a suppresses {alpha}-subunit promoter activity via two E-box sequences at –64 and –58 bp. A, Effects of mutations in E-box sequences in the {alpha}-subunit promoter on basal and GnRH-stimulated promoter activity. Cells were transfected with 0.5 µg of either wild-type {alpha}-subunit ({alpha}, –411 to +77 bp) promoter-luciferase construct, or the same construct mutated (Mut) at individual E boxes, or all three E boxes (Mut3Ebox). Cells were treated as in Fig. 4Go. Values are the mean ± SEM for four experiments with triplicates per group. *, P < 0.05 GnRH vs. vehicle-treated control (Con).^, P < 0.05 compared with no exogenous Zfhx1a. B, Protein binding to DNA representing {alpha}-subunit promoter E-box sequences. Labeled DNA was incubated alone (-) or with 6 µg nuclear proteins from LßT2 cells (LßT2 and all other lanes). Some reactions were incubated with antibody for Zfhx1a (Ab), preimmune rabbit serum (NRS), or 200-fold excess oligonucleotides representing wild-type (wt) or mutated E-box DNA. Specific DNA protein complexes are indicated by closed arrowheads; supershifted complexes with Zfhx1a antibody are indicated with arrows. Data shown are a representative gel shift of at least three separate experiments. C, Effects of mutations in all three E-box sequences in the –411 to +77 {alpha}-subunit promoter, on E-stimulated promoter activity. *, P < 0.05 E vs. vehicle-treated control. Values are the mean ± SEM for three experiments with triplicates per group. ALU, Arbitrary light units; GnRH-RE, GnRH response element; GSE, gonadotrope specific element; PGBE, pituitary glycoprotein hormone basal element.

 
To investigate whether any of these regions bound to Zfhx1a protein, we performed EMSA experiments with labeled DNA, including each E-box region. DNA from all three E boxes forms some complexes with nuclear proteins from LßT2 cells (Fig. 5BGo). However, only E-box –58 and E-box –64 DNA bind Zfhx1a, as indicated by the supershifted band that appears with antibody to Zfhx1a (Ab) and not with preimmune serum (NRS). Protein binding to E-box –292 was diminished by unlabeled oligonucleotides representing either wild-type or E-box mutated DNA, suggesting that bound proteins were not associated specifically with the E box. Protein binding to E-box –64 and E-box –58 DNA was inhibited specifically with an excess of oligonucleotide representing intact, but not mutated E-box sequences. Thus, Zfhx1a binds to the same gene regions, the E-box elements at –64 and –58 bp, which confer Zfhx1a suppression of the {alpha}-subunit promoter.

To determine if Zfhx1a binding contributed to E regulation of the {alpha}-subunit promoter, we transfected LßT2 cells with wild-type and Mut3Ebox {alpha}-subunit constructs, and treated the cells with E (Fig. 5CGo). As expected, the wild-type promoter was stimulated by E. Mutation of the E boxes increased basal expression of the promoter and reduced E- stimulation, suggesting that Zfhx1a binding suppresses expression and that reduction of Zfhx1a levels by E could relieve this suppression.

Suppression of rat LHß promoter activity by Zfhx1a via two E-box sequences and rescue by increased Sp1 expression
We next examined if Zfhx1a suppression of the rat LHß promoter was mediated by any of the three E box motifs at –381, –182, or –15 bp relative to the transcriptional start site, by promoter mutagenesis (Fig. 6Go). The promoter contains both proximal and distal GnRH enhancer elements, and Zfhx1a overexpression suppresses both basal and GnRH stimulation of the wild-type promoter. Mutation of either E-box –381 or E-box –182 eliminates Zfhx1a suppression of basal and GnRH-stimulated promoter activity (Fig. 6AGo). In contrast, mutation of E-box-15 decreased basal promoter activity but had no effect on Zfhx1a suppression. Mutation of all three E boxes increased basal activity 60% and slightly enhanced GnRH stimulated promoter activity. Mutation of E-box –381 and E-box –182 together gave identical results to the triple mutant (data not shown). EMSA analysis showed that DNA including E-boxes –381 or –182 formed complexes with a protein that was supershifted with Zfhx1a antibody, and that these complexes were inhibited by the addition of wild-type, but not E-box mutated oligonucleotides (Fig. 6BGo). These results suggest that two of three DNA regions with E-box homology, at –182 and –381 bp, bind to Zfhx1a and mediate the suppressive effect on the LHß promoter.


Figure 6
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  		FIG. 6. Zfhx1a suppresses LHß-subunit promoter activity via two E-box sequences at –381 and –182 bp; suppression is overcome by Sp1. A, Mutations in E-box sequences in the LHß-subunit promoter affect basal and GnRH-stimulated promoter activity. Cells were transfected with 0.5 µg wild-type LHß (–617 to +44 bp) promoter-luciferase construct (LHb), or the same construct mutated (Mut) at individual E boxes, or all three E boxes (Mut3Ebox). Cells were treated as in Fig. 4Go. Values are the mean ± SEM for four experiments with triplicates per group. *, P < 0.05 GnRH vs. vehicle-treated control (Con).^, P < 0.05 vs. cells with no exogenous Zfhx1a. B, Protein binding to DNA representing LHß promoter E-box sequences. Labeled DNA was incubated alone (-) or with 6 µg nuclear proteins from LßT2 cells (LßT2 and all other lanes). Some reactions were incubated with either Zfhx1a antibody (Ab), preimmune rabbit serum (NRS), or 200-fold excess oligonucleotides representing wild-type (wt) or mutated (Mut) E-box DNA. Specific DNA protein complexes are indicated by closed arrowheads; supershifted complexes with Zfhx1a antibody are indicated with arrows and Shift. Data shown are representative of at least three experiments. C, Cells were transfected with 0.5 µg LHß (–617 to +44 bp) promoter-luciferase and 0 (control) or 1 µg expression vector for Zfhx1a (Zfhx), ± cotransfected Sp1 or empty vector to normalize DNA. After 48 h, cells were treated with 100 nM GnRH for 6 h. Data are the mean ± SEM for three experiments with three samples per group. *, P < 0.05 vs. vehicle-treated control for each mRNA. ^, P < 0.05 vs. cells with no exogenous Zfhx1a. ALU, Arbitrary light units.

 
The Zfhx1a binding site/E-box region at –381 bp in the LHß promoter overlaps the 3' Sp1 binding sites between –381 and –355 bp in the distal GnRH response region (29). Thus, Zfhx1a binding might interfere with Sp1 binding, and influence basal and stimulated activity through that mechanism. Therefore, we tested if increased expression of Sp1 could rescue the promoter from the suppressive effects of Zfhx1a. In the presence of a constant level of Zfhx1a, increasing Sp1 overcame the suppressive effects of Zfhx1a on both basal and stimulated promoter activity (Fig. 6CGo), suggesting that competition of Zfhx1a and Sp1 binding at this site could contribute to Zfhx1a effects on this promoter.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In mammals, normal puberty and reproductive function require proper control of LH synthesis and secretion from gonadotropes, and appropriate expression of the {alpha}-subunit and LHß genes. Transcription of the genes encoding {alpha}-subunit and LHß reflects dynamic integration of a positive signal mediated by GnRH from the hypothalamus, negative sex steroid feedback in males and females from estrogens and androgens, and positive feedback from estrogen in females. Negative and positive feedback might occur via alteration of GnRH pulses, or directly at the pituitary level. In this report we found that E treatment enhances GnRH stimulation of the {alpha}-subunit and LHß promoters transfected into LßT2 cells, as well as the endogenous genes (Fig. 1Go). The effect is specific for E, as thyroid hormone, P, and DHT (7) do not enhance GnRH responses.

Steroids may regulate gene expression through many pathways, including modulation of cytoplasmic signaling. For example, DHT treatment amplifies MAPK signaling in gonadotropes, and this contributes to increased FSHß mRNA (19). E treatment suppresses CREB phosphorylation, and may sensitize gonadotropes to GnRH by amplification of protein kinase C and calcium responses (26, 27). Our data provide evidence that E treatment also increases LH subunit gene expression via modulation of regulatory transcription factors. Neither the {alpha}-subunit nor the LHß promoter constructs used in this study contain EREs. Thus, E stimulation must be mediated through other gene elements or by modulation of other transcription factors.

E stimulation of the {alpha}-subunit promoter does not rely on the GnRH-responsive region (Fig. 1AGo), although E can augment the GnRH response. In contrast, E alone has only modest effects on basal expression of the LHß promoter, and is most effective at potentiating GnRH stimulation of the –617 bp construct containing both distal and proximal GnRH response regions (Fig. 1BGo). We performed mRNA analysis of E-treated LßT2 cells, and found that E regulated expression of two transcription factors, Egr-1 and Zfhx1a, at the mRNA and protein levels (Fig. 2Go) that influence LH subunit gene expression. Both factors are specifically regulated by E, and not DHT, through an ER-directed mechanism that is inhibited by the pure ER antagonist ICI 182,780 (Fig. 3Go).

Egr-1 is an immediate early gene product that is required for LHß mRNA expression, and increased expression specifically stimulates LHß but not {alpha}-subunit promoter activity (Fig. 4Go). Egr-1 is dramatically induced by GnRH (30, 31), and we have found that this is enhanced by E (Figs. 2Go and 3Go). The role of Egr-1 in LHß expression is well known, but Zfhx1a activity in gonadotropes had not been explored.

Zfhx1a is a zinc finger homeodomain protein that influences both LH subunit genes. Zfhx1a is the mouse homolog of proteins known by several names in other vertebrates, including ZEB, Zfhep, AREB6, and {delta}EF1 (32, 35, 42, 43, 44, 45, 46, 47). These proteins have over 90% homology in their DNA binding regions, and bind to subsets of E-box-like motifs, with highest affinity for CACCT and CACCTG sequences (42, 43, 44, 45, 46, 47). Zfhx1a and its homologs repress the transcription of several genes, such as {alpha}7 integrin (44), IL-2 (45), and GATA-3 (46), but may also stimulate some genes such as the vitamin D receptor (47). In the pituitary somatolactotrope GH3 cell line, Zfhx1a can bind the thyroid hormone response element of the rat {alpha}-subunit promoter gene and suppress transcription (35). Recently, Zfhx1 has also suppressed GH promoter activity, and is hypothesized to contribute to silencing GH expression in developing lactotropes (48). Thus, Zfhx1a appears to be a repressor in at least two pituitary cell types, somatolactotropes and gonadotropes. Zfhx1 and homologs can be differentially regulated in different tissues and cells (40, 49). Zfhx1 expression (a and b forms not distinguished) was slightly stimulated by E in total pituitary, and in the MMQ lactotrope cell line (48), but Zfhx1a expression is clearly suppressed in gonadotrope cells (Figs. 2Go and 3Go).

For the {alpha}-subunit promoter, Zfhx1a bound to two closely spaced E-box motifs at –64 and –58 bp, and mutation of the Zfhx1a-binding E boxes within the –411 bp {alpha}-subunit promoter construct resulted in increased basal expression (60–70%) and enhanced GnRH stimulation (Fig. 5Go, A and B). Interestingly, the –287 bp deletion construct containing both Zfhx1a binding sites at –64 and –58 bp continues to be fully stimulated by E (Fig. 1AGo). This correlates with data demonstrating that the –411 bp construct with mutated –64 and –58 E boxes (Fig. 5CGo) is no longer stimulated by E. These findings suggest that Zfhx1a binding normally represses {alpha}-subunit expression and prevents full GnRH stimulatory responses in gonadotropes.

The LHß promoter binds Zfhx1a via E-box elements at –381 and –182 bp (Fig. 6Go, A and B), and overexpression of Zfhx1a suppressed GnRH-stimulated promoter activity. Mutation of all three E boxes increased LHß promoter activity, indicating that Zfhx1a binding is normally repressive. Zfhx1a and related proteins are proposed to act as negative regulators both by active suppression and by competition for DNA binding with activator proteins (42, 50, 51, 52, 53). Active repression is largely due to association of CtBP with Zfhx1 (52). Zfhx1a binds to subsets of E boxes that are also the major binding sites for various basic helix-loop-helix proteins and zinc finger proteins such as Sp1, and, thus, might compete with these proteins for DNA binding (42, 51, 53). In the rat LHß gene promoter, the 3’Sp1 binding site of the distal GnRH response region overlaps with the Zfhx1a binding site at E-box 381 (7) In transient transfection assays, increased expression of Sp1 eliminates the suppressive effect of Zfhx1a (Fig. 6CGo). Although the AR may interact with Sp1 at this site (7, 54), we have not found direct interaction of ER{alpha} with Sp1 on this gene region (unpublished data). Thus, Zfhx1a may act to suppress LHß transcription through two pathways, including competition for binding with activating Sp1 at LHß E-box 381, and perhaps active repression at LHß E-box –182, which does not bind other known activators.

The distal and proximal GnRH-responsive regions within the –617 bp LHß promoter must cooperate for full stimulation (29, 40), and E enhancement of GnRH-stimulated promoter activity is most pronounced with both regions present (Fig. 1Go). Our data suggest that E actions may be exerted on the distal response region by decreased Zfhx1a expression, as Zfhx1a may compete with Sp1 binding to this site. By enhancing GnRH stimulation of Egr-1 levels, E treatment would also increase activity of the proximal response region. Thus, E may stimulate overall promoter activity by decreasing repression of the distal and increasing activation of the proximal cooperating response regions.

In addition to regulating expression of transcription factors binding directly to the gonadotropin genes, E may also modulate transcription through other nuclear mechanisms. Recently, ER{alpha} has been associated with the mouse and Chinook salmon LHß genes through SF-1 and Ptx1 sites; Egr-1 was not tested (55). These studies suggest that GnRH and ER{alpha} signaling could cooperate in the nucleus at the level of the LHß gene via transcription factors that do not bind to EREs. As an additional example of this type of regulation, pituitary progesterone receptor is also induced by E at the LH surge. The progesterone receptor is important for facilitating E and GnRH responses in vivo through both ligand-dependent and ligand-independent pathways that include GnRH priming, which may be of particular importance during the gonadotropin surge (56, 57). Thus, cross talk between E and GnRH stimulation of LH promoter activity may occur at many levels, including modulation of cytoplasmic signaling pathways (24, 25, 26, 27), direct interactions of ER{alpha} with other transcription factors tethered to the gene (20, 55), or by influencing expression of transcription factors that modulate basal and GnRH-stimulated activity, as shown in this study. This includes up-regulation of stimulatory proteins such as Egr-1, which stimulates LHß, and down-regulation of the suppressor Zfhx1a, which inhibits both {alpha}-subunit and LHß subunit promoter activity.


   Acknowledgments
 
We thank the Specialized Cooperative Centers Program in Infertility and Reproduction Research Microarray Facility at the University of Washington (Seattle, WA).


   Footnotes
 
This work was supported by National Institute of Child Health and Human Development/National Institutes of Health Cooperative Agreement U54-HD-289934 as part of the Specialized Cooperative Centers Program in Infertility and Reproduction Research through both an individual research project (to M.A.S.) and Molecular Core at the University of Virginia.

Present address for T.K.: Department of Pediatrics, Showa-Machi, Gumma University, Maebashi 371-8511, Japan.

Disclosure Statement: M.A.S. has received royalties from Upstate Biotechnology (now Millipore). T.K., H.E.W., and D.S.D. have nothing to declare.

First Published Online September 6, 2007

Abbreviations: AR, Androgen receptor; CREB, cyclic adenosine 3',5'-monophosphate response element binding protein; DHT, dihydrotestosterone; E, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GnRH-R, GnRH receptor; LUC, luciferase; NRS, preimmune serum; P, progesterone; RT, room temperature; SF-1, steroidogenic factor 1; T, testosterone.

Received March 29, 2007.

Accepted for publication August 30, 2007.


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 Introduction
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 Discussion
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