Activation of Luteinizing Hormone beta  Gene by Gonadotropin-releasing Hormone Requires the Synergy of Early Growth Response-1 and Steroidogenic Factor-1*

Christoph DornDagger , Qinglin OuDagger , John Svaren§, Peter A. Crawford§, and Yoel SadovskyDagger parallel

From the Dagger  Department of Obstetrics and Gynecology, the § Department of Pathology, and the  Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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We have previously shown that early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) beta  deficiency. Egr-1 activates the LHbeta gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for gonadotropin-releasing hormone (GnRH) stimulation of LHbeta , we examined the activity of the LHbeta promoter in the gonadotrope cell line Lbeta T2. GnRH markedly stimulated the LHbeta promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHbeta promoter attenuated this stimulation, whereas mutation of both promoter elements abrogated GnRH induction of the LHbeta promoter. Furthermore, GnRH stimulated Egr-1 but not SF-1 expression in Lbeta T2 cells. Importantly, overexpression of Egr-1 alone was sufficient to enhance LHbeta expression. Although other Egr proteins are expressed in Lbeta T2 cells and are capable of interacting with SF-1, GnRH stimulation of Egr-1 was the most robust. We also found that the nuclear receptor DAX-1, a repressor of SF-1 activity, reduced Egr-1-SF-1 synergy and diminished GnRH stimulation of the LHbeta promoter. We conclude that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHbeta gene and plays a central role in the dynamic regulation of LHbeta expression.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Endocrine signals within the female hypothalamus-pituitary-gonad axis regulate ovarian follicle development, ovulation, and steroidogenesis. Broadly, these changes in the hormonal milieu not only regulate sex organ development and reproductive function but also influence bone formation and cardiovascular homeostasis. Gonadotropins play a central role in orchestrating gonadal function. Gonadotropin production is directed by pulsatile secretion of hypothalamic GnRH1 and transmitted to gonadotropes via the pituitary portal system (1-5). Follicle-stimulating hormone and luteinizing hormone (LH) are heterodimeric glycoproteins that consist of a common alpha -subunit and a unique beta -subunit (4). Follicle-stimulating hormone and LH are secreted in a cyclic, fluctuating manner. Whereas follicle-stimulating hormone plays a major role in oocyte development and estrogen production during the follicular phase of the cycle, mid-cycle LH surge promotes luteinization of the dominant follicle and progesterone production (4).

The production of LHbeta is modulated at several levels, including mRNA transcription, polyadenylation, and protein glycosylation (2, 6, 7). Whereas diverse signaling pathways converge on the modulation of LHbeta gene expression (4, 8-15), recent analysis of mice bearing loss of function mutations in either early growth response-1 (Egr-1, also known as NGFI-A, Zif268, or Krox24) or steroidogenic factor-1 (SF-1) demonstrated that these two transcription factors play a critical role in directing LHbeta expression (16-19). Egr-1, a zinc finger transcription factor, is the prototype of a family of egr genes (20, 21). These genes are induced in response to a variety of extracellular stimuli that lead to proliferation, differentiation, or apoptosis (21). The expression of Egr-1 is widespread (22, 23). In the pituitary it is expressed in the anterior lobe, primarily in gonadotropes and somatotropes (24). The DNA binding domain of Egr-1 is highly homologous among three other members of this family, including Egr-2 (Krox20), Egr-3, and Egr-4 (nerve growth factor induced-C) (21, 25, 26). These Egr proteins bind to sites that resemble the consensus site TGCG(T/G)(G/A)GG(C/A/T)G(G/T) (27). The LHbeta promoter contains similar sites at positions -50 and -113.

In addition to Egr-1 elements, the LHbeta proximal promoter also contains evolution-conserved SF-1 responsive elements located at positions -127 and -59 (12, 16, 28, 29). SF-1 is an orphan member of the nuclear receptor superfamily of proteins (30), which binds to its cognate DNA element as a monomer (31, 32). SF-1 regulates the expression of most steroidogenic enzymes in both female and male gonads and adrenal cortex as well as other proteins relevant to reproductive development and function (31, 32). Importantly, SF-1 is essential for reproductive development, because mice deficient in this transcription factor lack gonads and adrenal glands, which consequently leads to persistence of Mullerian structures even in male embryos as well as early neonatal death from adrenal insufficiency (17-19). SF-1-deficient mice also have impaired development of the hypothalamic ventromedial nucleus (33). Interestingly, the transcriptional activity of SF-1 is repressed by DAX-1, an orphan nuclear receptor implicated in the pathogenesis of adrenal hypoplasia congenita and hypogonadotropic hypogonadism (34-37). Although several studies (12, 38) indicated that SF-1 is absolutely required for LHbeta expression, subsequent analysis of SF-1 -/- mice, maintained until maturity by corticosteroid rescue treatment, revealed that these mice do express LHbeta in response to GnRH injections (33).

Like SF-1, Egr-1 is also required for normal reproductive development and function, as Egr-1 -/-female mice exhibit arrested uterine development and infertility, reflecting a specific deficiency in LHbeta expression in pituitary gonadotropes (16, 24). Importantly, we and others (16, 29) have recently demonstrated that a synergistic interaction between Egr-1 and SF-1 is essential for the activation of the LHbeta promoter in vitro, suggesting that SF-1 and Egr-1 may provide a means of directing LHbeta expression in response to physiological stimuli that orchestrate gonadotrope function. Pulsatile release of GnRH acting through GnRH receptors plays a central role in dynamic regulation of LHbeta expression (1, 3, 5, 6, 10, 13, 39). Therefore we hypothesized that GnRH activation of the LHbeta gene requires the synergistic interaction of Egr-1 and SF-1. We tested our hypothesis utilizing the gonadotrope cell line Lbeta T2, which expresses Egr-1 and SF-1 and responds to GnRH administration with LHbeta production (40-42).

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Plasmids and Mutagenesis-- The wild type -156 to +7 LHbeta promoter construct, cloned upstream of luciferase in PL(KS)b-Luc vector (a gift from Stuart Adler, Washington University, St. Louis, MO), was described previously (16). To generate mutations of either Egr-1 or SF-1 sites, we subcloned the LHbeta promoter into a pBSKS vector (Stratagene) and generated the following mutations: Egr-1 at -113 from CGCCCCCAA to TAGTACTCA, Egr-1 at -50 from CACCCCCAC to GATTCTTAT, SF-1 at -127 from TGACCTTG to TGATCATG, and SF-1 at -59 from TGGCCTTG to TGGAATTC. For mutagenesis, overlapping mutagenic oligonucleotides were synthesized and used in PCR performed with Klentaq (43) for high fidelity and processivity (PCR parameters were 25 cycles of 94 °C × 0.5 min, 56 °C × 2 min, and 72 °C × 4 min). The PCR products were phenol/chloroform, which was extracted and digested with DpnI (NEB) to select for mutated, PCR-generated plasmid. The plasmid mixture was amplified in XL-1 blue bacteria (Stratagene), isolated, and sequenced using the dideoxy method in an automatic sequencer (Applied Biosystems). All LHbeta promoter mutants were subcloned back into the PL(KS)b-Luc vector at the SpeI/XhoI sites. Expression vectors for the Egr-1, SF-1, DAX-1, and DAX-11-369 deletion mutants were previously described (37, 44, 45). Expression vectors for Egr-2, Egr-3, and Egr-4, all cloned in a CMV-Neo expression vector, were kindly provided by Jeffrey Milbrandt (Washington University, St. Louis, MO).

Cell Culture and Transfection-- Lbeta T2 cells (40) were generously provided by P. Mellon (La Jolla, CA) and maintained in monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics in humidified 10% CO2, 90% air at 37 °C. For transfection experiments the cells were plated in 6-well plates at a density of 350,000 cells/well. We have determined that optimal results were obtained when transfections were conducted 24 h after plating. The growth medium was routinely replaced with standard fresh medium 4 h before transfection. Cells were transfected using the modified calcium-phosphate method described previously (46) using a total of 2.5 µg/well, which included 0.05 µg of CMV-beta -galactosidase plasmid (to normalize for cell viability and transfection efficiency). After 24 h the medium was changed to medium that contained 1% charcoal/dextran-treated serum (Hyclone). GnRH (Sigma) was added to the medium at a concentration range of 0.1-100 nM. After 15 min the medium was removed and replaced by the 1% serum containing medium. In some of the experiments, this cycle was repeated every 90 min for a total of four cycles following a previously published protocol (41). Standard luciferase assays were performed 48 h after transfection as described previously (45). All experiments were performed in duplicate and repeated at least three times. Results (mean ± S.D.) normalized to beta -galactosidase activity were expressed as relative luciferase units. The human choriocarcinoma cell line JEG3 was maintained and transfected as described previously (45).

Electromobility Shift Assay-- Double-stranded oligonucleotides (100 ng) that contained the wild type or mutated binding elements of either 5' Egr-1, 3' Egr-1, 5' SF-1, or 3' SF-1 (detailed above) were end-labeled with 25 µCi of [gamma -32P]ATP using polynucleotide kinase. Nuclear extract from Lbeta T2 cells was generated as described previously (47). For positive control proteins, we used a bacterially expressed and purified His-Egr-129-536 or glutathione S-transferase-SF-11-106, both previously described (48, 49). For each binding reaction we mixed 0.02 µg of the proteins or 3 µg of the extracts with 1 ng of a labeled probe in a binding buffer that contained 50 mM NaCl, 1 mM EDTA, and 5% glycerol in 10 mM Tris (pH 7.5) and incubated for 30 min at 25 °C. Each binding reaction was loaded onto a 5% polyacrylamide gel and run in 0.5× Trisborate/EDTA buffer at 150 V for 2 h. The gel was then dried and exposed to a PhosphorImager screen or film.

Expression Analysis-- Total RNA was isolated from Lbeta T2 cells using Tri-reagent (Molecular Research Center, Cincinnati, OH). RNA samples were extracted, precipitated, and resuspended in water. RNA samples (15 µg) were resolved by electrophoresis using a 1% agarose, 1.5% formaldehyde gel. Specific probes for Northern blot analysis of each murine Egr transcript as well as SF-1 were generated by PCR using standard techniques and labeled with [32P]dCTP using a Prime-It II (Stratagene) labeling kit. RNA was transferred to nylon membranes (Zeta-Probe, Bio-Rad) and hybridized overnight at 42 °C. The blots were washed three times in 0.2-2× sodium chloride/sodium citrate buffer (1× buffer is 0.15 M sodium chloride and 0.015 M sodium citrate) with 0.1% sodium dodecyl sulfate at 65 °C. Blots were exposed to a PhosphorImager screen for 4 h and to Kodak film at -80 °C overnight. The ImageQuant software for the PhosphorImager was used for quantitative analysis of the expression of Egr proteins or SF-1 corrected to glyceraldehyde-3-phosphate dehydrogenase expression.

A recombinant adenovirus expressing rat Egr-1 from the CMV promoter in the Ad5PacIGFP vector was provided by Markus Ehrengruber (Brain Research Institute, University of Zurich, Switzerland) (50).2 Approximately 100,000 Lbeta T2 cells were infected with 3 × 108 plaque-forming units/ml of adenovirus for 2 h, and the medium was changed twice. Cells were harvested 24 h after infection, and total RNA was analyzed by Northern blotting using a mouse LHbeta probe previously described (16).

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ABSTRACT
INTRODUCTION
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GnRH-dependent Stimulation of LHbeta Gene Requires the Synergistic Interaction of Egr-1 and SF-1-- We used the Lbeta T2 gonadotrope line to determine whether or not Egr-1 and SF-1 cooperatively transduce GnRH stimulation of the LHbeta promoter. The Lbeta T2 cell line was derived from a pituitary tumor in transgenic mice that express the SV40 T antigen driven by the rat LHbeta promoter (40). In addition to the SF-1 and GnRH receptors, these cells express both LHalpha and LHbeta subunits, and LH secretion is enhanced by GnRH stimulation in vitro (40, 41, 51). To test for GnRH enhancement of LHbeta promoter activity, we transfected the Lbeta T2 cells with a LHbeta reporter construct, which contains nucleotides -156 to +7 of the rat LHbeta promoter, upstream of luciferase (16). As shown in Fig. 1A, we exposed the cells to either single or multiple pulses of GnRH, which were previously shown to stimulate LH secretion in Lbeta T2 cells (41). We found that GnRH enhanced the activity of the LHbeta reporter in a time- and concentration-dependent fashion, and two GnRH pulses (100 nM each) given for 24 and 8 h resulted in maximum enhancement (15-fold) of the LHbeta reporter gene.


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Fig. 1.   GnRH-dependent stimulation of the LHbeta promoter requires the synergistic interaction of Egr-1 and SF-1. A, Lbeta T2 cells were transiently transfected with the rat -156 to +7 LHbeta -luciferase construct and stimulated with GnRH 24 h after transfection. In two paradigms (depicted at the bottom of the figure) GnRH was administered in four 90 min cycles 24 h before harvest and one additional administration 1 h before harvest as described previously under "Experimental Procedures" (41). Results are expressed as -fold luciferase activity over baseline (mean ± S.D.) and represent three independent experiments performed in duplicate. B, Lbeta T2 cells were transiently transfected with wild type or mutated forms of the rat LHbeta -luciferase construct and stimulated with GnRH (100 nM) 24 and 8 h before harvest. Results (mean ± S.D.) are expressed as relative luciferase units (RLU) and represent three independent experiments performed in duplicates. TATA, TATA box. C, mutated Egr-1 or SF-1 elements cannot bind their respective proteins present in an Lbeta T2 nuclear extract. Electromobility shift assay was performed as described under "Experimental Procedures." For positive control we used a bacterially expressed and purified His-Egr-129-536 or glutathione S-transferase-SF-11-106. The SF-1 fusion protein is truncated at the ligand binding domain and therefore migrates slightly faster than wild type SF-1. Results represent two independent experiments.

It has been previously shown that the LHbeta promoter fragment utilized in our studies contains two binding elements for Egr-1 and two for SF-1, which are essential for basal LH production in vivo (16, 29). To analyze the role of these transcription factors in GnRH-dependent stimulation of LHbeta expression, we added GnRH to Lbeta T2 cells that were transiently transfected with either the wild type LHbeta promoter reporter or the LHbeta promoter mutated in the binding elements for Egr-1 or SF-1. We found that mutation of the higher affinity Egr-1 site at -50 (29) reduced both basal- and GnRH-stimulated activity of the LHbeta promoter (Fig. 1B). Promoter activity was further diminished when both Egr-1 sites were mutated. Similarly, we found that mutation of both SF-1 sites reduced the basal- and GnRH-induced activity of the LHbeta promoter, albeit to a lower extent. As expected, mutation of all four Egr-1 and SF-1 sites abolished basal- as well as GnRH-stimulated promoter activity. We used an electromobility shift assay to confirm that the mutated promoter elements for either Egr-1 or SF-1 could not bind their respective protein when expressed in a nuclear extract from Lbeta T2 cells (Fig. 1C). These results indicate that Egr-1 and SF-1 sites are essential for GnRH stimulation of LHbeta gene expression and suggest that between the two proteins, the influence of Egr-1 is stronger than that of SF-1 on the GnRH-stimulated LHbeta promoter.

To test whether or not GnRH regulates the expression of either Egr-1 or SF-1, we used Northern blot analysis to determine their expression in GnRH-stimulated Lbeta T2 cells. As shown in Fig. 2A, we found that GnRH stimulated the expression of Egr-1 in a concentration-dependent manner. In contrast, GnRH had no significant effect on SF-1 expression under the same conditions. Because it was previously shown that GnRH stimulates the transcription of the LHbeta gene in Lbeta T2 cells (41), we sought to recapitulate the response of Lbeta T2 cells to GnRH by directly assessing the influence of enhanced Egr-1 expression on LHbeta transcription. Using adenovirus-mediated transfection of Lbeta T2 cells, we found that overexpression of Egr-1 enhanced the transcription of the LHbeta gene (Fig. 2B). Together, these results indicate that Egr-1 is not only required for LHbeta expression (16, 24) but is also sufficient to induce transcription of the LHbeta gene in Lbeta T2 cells.


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Fig. 2.   GnRH enhances Egr-1 expression in Lbeta T2 cells. A, GnRH (0-30 nM) was added to the culture medium 24 and 8 h before harvest. Total RNA was isolated from the cells and hybridized with the indicated probes as described under "Experimental Procedures." Results represent three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, quantitative analysis of the data presented in Fig. 2A presented for each protein as -fold over control and corrected to glyceraldehyde-3-phosphate dehydrogenase expression. C, control. C, enhanced expression of LHbeta in Lbeta T2 cells that overexpress Egr-1. Lbeta T2 cells were infected with a recombinant adenovirus expressing Egr1. Total RNA was isolated 24 h after infection and analyzed by Northern blotting using a mouse LHbeta probe. The 18 S ribosomal RNA from these samples demonstrated equal loading of the samples. The control lane is derived from uninfected cells. Infection with a control adenovirus demonstrated that adenovirus infection itself does not up-regulate the LHbeta gene (data not shown).

Other Egr Family Members Synergistically Interact with SF-1 in Regulation of LHbeta Promoter in Vitro-- All four members of the Egr family of proteins share a similar binding specificity and were previously shown to bind promoter elements that resemble Egr-1 sites (at -113 and -50) within the LHbeta promoter (27). Using Western and Northern blot analyses, we have determined that Egr-2, Egr-3, and Egr-4 are all expressed in the beta T2 gonadotrope line (Fig. 4) (data not shown). To determine if Egr proteins are capable of synergizing with SF-1 in the activation of LHbeta promoter, we expressed each Egr protein in JEG3 cells, which express a low level of these proteins (data not shown), and tested for activation of the LHbeta reporter in the presence or absence of SF-1. As shown in Fig. 3, transcriptional activity of either Egr-3 or Egr-4 was similar to that exhibited by Egr-1, yet the activity of Egr-2 was higher than that of the other Egr proteins. Nevertheless, all four members of the Egr family were capable of synergistically interacting with SF-1 in activation of the LHbeta promoter. Importantly, the transcriptional activity of all four Egr proteins as well as their synergy with SF-1 required intact Egr-1 sites in the LHbeta promoter, as a mutation of these sites abrogated their induction of a LHbeta promoter.


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Fig. 3.   Egr proteins synergistically interact with SF-1 in activation of LHbeta promoter. JEG3 cells were transiently transfected with plasmids that contain either the wild type (WT) rat LHbeta promoter (0.5 µg) or the LHbeta promoter that harbors mutations in the two Egr elements or in the two Egr elements as well as the two SF-1 elements (see Fig. 1B for promoter structure). Cells were co-transfected with either control plasmid (CMV-Neo, 0.1 µg) or CMV-SF-1 (0.1 µg) along with CMV-driven expression vectors for Egr proteins. The following amounts of plasmid (previously optimized in similar experiments not shown) were used: Egr-1, 0.1 µg; Egr-2, 0.3 µg; Egr-3, 0.01 µg; Egr-4, 0.01 µg. Results (mean ± S.D.) are expressed as relative luciferase units (RLU) and represent three independent experiments performed in duplicate.

To assess the role of the cooperativity between SF-1 and the members of the Egr family of proteins that stimulate the LHbeta promoter, we sought to determine the effect of GnRH on the expression of these proteins in Lbeta T2 cells. We found that GnRH administration 24 and 8 h before harvest enhanced the expression of Egr-1 to a greater extent than its effect on Egr-2, Egr-3, and Egr-4. A similar result was obtained when the expression of Egr proteins was measured 1 h after GnRH administration (Fig. 4). Together, these results suggest that although all four Egr proteins are expressed in Lbeta T2 and can synergize with SF-1, Egr-1 may play the most prominent role in GnRH stimulation of the LHbeta gene.


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Fig. 4.   The effect of GnRH on expression of Egr transcripts in Lbeta T2 cells. A, GnRH was added to the culture medium either 24 and 8 h prior to harvest at a concentration range of 0-30 nM or 1 h prior to harvest at a concentration of 30 nM. Total RNA was isolated from the cells and hybridized with the indicated probes as described under "Experimental Procedures." Results represent two independent experiments. B, quantitative analysis of the data presented in Fig. 4A presented for each protein as -fold over control (C) and corrected to glyceraldehyde-3-phosphate dehydrogenase expression.

DAX-1 Represses Basal- and GnRH-stimulated Activity of LHbeta Promoter-- We and others (36, 37) have previously utilized a synthetic SF-1 reporter gene to demonstrate that the nuclear receptor DAX-1 represses the transcriptional activation of SF-1. Because GnRH enhances Egr-1 expression, and Egr-1 synergistically interacts with SF-1 to induce the activity of LHbeta reporter, we hypothesized that DAX-1 would diminish the activity of the LHbeta promoter. To test our hypothesis, we first determined the effect of DAX-1 on the synergy between Egr-1 and SF-1 in JEG3 cells. We found that unlike its effect on a synthetic promoter, DAX-1 had a minimal repressive activity on SF-1-dependent activation of the LHbeta promoter in JEG3 cells (Fig. 5A). In contrast, DAX-1 markedly diminished the cooperative interaction between all Egr proteins and SF-1 in activation of the LHbeta promoter. As expected, DAX-1 had no effect when the Egr proteins were expressed alone. These results indicate that the repressive effect of DAX-1 on the LHbeta promoter is SF-1-dependent, yet the ability of DAX-1 to repress SF-1 activity is context-dependent.


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Fig. 5.   DAX-1 represses the synergistic interaction of Egr-1 and SF-1 in activation of LHbeta promoter. A, JEG3 cells were transiently transfected with plasmids that contain wild type rat LHbeta promoter (0.5 µg) and with either control plasmid (CMV-Neo) or CMV-SF-1 (both at 0.1 µg) along with CMV-driven expression vectors for Egr proteins (see legend to Fig. 3 for plasmid concentrations). Cells were co-transfected with CMV-DAX-1 vector at 0.01 µg/well previously found optimal for DAX-1 expression in JEG-3 cells (not shown). B, the effect of increasing concentration of DAX-1 on basal- or GnRH-stimulated LHbeta promoter activity in Lbeta T2 cells. Cells were stimulated with GnRH (100 nM) 24 and 8 h before harvest. Delta DAX-1 denotes DAX1-369, which is deficient in repressive function (36, 37). Results (mean ± S.D.) are expressed as relative luciferase units (RLU) and represent three independent experiments performed in duplicate.

To determine whether or not DAX-1 represses the GnRH-dependent stimulation of the LHbeta gene, we transfected DAX-1 into Lbeta T2 cells. As shown in Fig. 5B, DAX-1 repressed both basal- and GnRH-dependent stimulation of the LHbeta reporter. As expected, this effect was attenuated when a C-terminally truncated form of DAX-1 (DAX-11-369), which corresponds to naturally occurring mutations that cause adrenal hypoplasia congenita, was used (35). A similar level of attenuation by DAX-11-369 was previously observed using a synthetic SF-1 reporter (37). The repressive effect of DAX-1 was diminished when Egr-1 and SF-1 sites were mutated (not shown). Together, these results indicate that optimal repression of the LHbeta promoter activity by DAX-1 in Lbeta T2 cells requires the cooperative interaction of Egr-1 and SF-1.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Egr-1 is essential for LHbeta production in vivo, as Egr-1 -/- mice exhibit LHbeta deficiency, resulting in abnormal sexual development and infertility (16, 24). Similarly, SF-1 -/- mice are deficient in LHbeta production (33, 38), which can be restored with GnRH administration (33). GnRH is the most important physiologic regulator of LH production and stimulates LHbeta production and secretion in vitro (41). Whereas it is known that the synergy between Egr-1 and SF-1 is required for basal expression of LHbeta in vitro (16, 29), our results provide the first link between the GnRH-dependent stimulation of LHbeta and the synergistic interaction of Egr-1 and SF-1. Maximal diminution of the LHbeta promoter activity is observed only when both Egr-1 and SF-1 elements are mutated, providing further support to the role of Egr-1-SF-1 synergy in directing LHbeta production. It is also evident that mutations in the Egr-1 binding elements within the LHbeta promoter have a more profound effect on GnRH-stimulated activity of the LHbeta promoter when compared with analogous mutations in the SF-1 elements, suggesting that Egr-1 plays a more important role in the dynamic regulation of the LHbeta gene by GnRH. This conclusion is supported by the finding that GnRH regulates the expression of Egr-1 and not SF-1. In addition, we confirmed that overexpression of Egr-1 alone was sufficient to activate expression of endogenous LHbeta in Lbeta T2 cells even in the absence of GnRH stimulation. Nevertheless, it is likely that endogenous SF-1 in Lbeta T2 cells supports the ability of Egr-1 to activate the LHbeta gene.

Our results are consistent with the finding that changes in the transcription rate of LHbeta in vivo are not associated with a concomitant change in SF-1 expression in sheep pituitary (52), although a weak induction (<1.65-fold) of SF-1 expression by pulsed GnRH treatment of GnRH-deficient female rats has been reported (53). Although Keri and Nilson (12) suggested that SF-1 sites are required for GnRH stimulation of the bovine LHbeta promoter in transgenic mice, Ikeda et al. (33) have demonstrated that SF-1 is not obligatory for GnRH effect, because GnRH can at least partially restore LHbeta expression even in SF-1-deficient mice. These inconsistencies may be partly explained by a different promoter or cellular contexts or by compensation by other nuclear proteins for the absence of functional SF-1 in the knock-out mouse model. Interestingly, a cooperative interaction has also been demonstrated between the pituitary transcription factors Ptx1 and Pit1 in stimulation of the prl gene (54, 55) as well as between Ptx1 and SF-1 in regulation of LHbeta (56). However, unlike Egr-1, the action of Ptx1 is not specific to LHbeta , as Ptx1 is also required for expression of the gonadotropin alpha -subunit (56).

The mechanism of synergy between Egr-1 and SF-1 is currently unknown. Our results, as well as the results of others (29), indicate that both proteins must be DNA-bound to synergistically activate LHbeta gene expression. It is possible that in addition to enhancement of Egr-1 expression, GnRH-dependent signals modulate the functional interaction between Egr-1 and SF-1. Previous studies utilizing a protein-protein interaction assay in vitro (29) suggest that Egr-1 and SF-1 physically interact with each other, and this interaction requires the zinc finger domain of Egr-1. Current studies are underway to test for the presence and significance of this interaction in Lbeta T2 cells.

In addition to Egr-1 and SF-1, other DNA binding elements within the LHbeta gene may modulate GnRH stimulation of LHbeta expression. Using the rat somatolactotropic cell line GH3, Kaiser et al. (14) have demonstrated that another zinc finger protein, Sp1, binds to the rat LHbeta promoter at 2 sites located between -451 and -386 and may play a role in GnRH-stimulated expression of the LHbeta gene. Although the Sp1-dependent GnRH effect was markedly weaker (2-3-fold) when compared with the Egr-1-SF-1-dependent effect in our experiments, these results suggest that GnRH stimulation of the LHbeta promoter involves an integrated response of multiple transcription factors binding to discrete promoter elements. Indeed estradiol, acting via estrogen receptors, modulates LHbeta expression (8), and estrogen receptors were shown to interact with SF-1 in regulation of the salmon gonadotropin IIbeta subunit (57). Other sex hormones also alter LHbeta expression (9, 11). It is likely that signaling by these promoter-bound transcription factors is modulated by diverse signal transduction pathways that participate in the transmission of GnRH stimuli from its surface receptors to the LHbeta gene (2, 4, 6, 13, 15, 39, 58). Whereas potential cross-talk between these second messenger pathways and Egr-1-SF-1 synergy remains to be established, such coordinated interaction is likely to contribute to the fine tuned LHbeta response to GnRH pulses under diverse physiological conditions.

Unlike Egr-1, little is known at the present time about the role of other Egr proteins in reproductive physiology or pathophysiology. Mice homozygous for mutant Egr-2 exhibit abnormal hindbrain development and die within 2 weeks after birth (59, 60). Egr-3-deficient mice have muscle spindle agenesis, which results in sensory ataxia, and are not known to have gonadotropin deficiency (61). We used JEG3 cells to assess cooperativity between Egr proteins and SF-1 in activation of the LHbeta promoter, because these cells do not express an appreciable level of either Egr proteins or SF-1, although they are all expressed in Lbeta T2 cells. All four Egr proteins synergistically interact with SF-1 in transcriptional activation of the LHbeta promoter. Interestingly, whereas GnRH stimulates the expression of all Egr proteins to some extent, Egr-1 exhibits the largest and most prolonged response to GnRH stimulation. This selective effect of GnRH may provide at least a partial explanation for the fact that the profound phenotype of Egr-1-deficient mice was not compensated by any of the other proteins from the Egr family. Studies that focus on the expression of Egr proteins in wild type and Egr-1-deficient mice are required to further elucidate these findings.

DAX-1 is a potent repressor of SF-1 activity (36, 37), and this repression is achieved, at least in part, through recruitment of the co-repressor N-CoR to DNA-bound SF-1. DAX-1 is expressed in the anterior pituitary and in alpha T3 cells, a gonadotrope cell line similar to Lbeta T2 (62). It is also expressed in LH-expressing human pituitary adenomas (63). Interestingly, whereas DAX-1 has only a marginal effect on SF-1-mediated activation of the LHbeta promoter in the absence of Egr proteins, it markedly represses the synergistic activation of LHbeta by Egr-1 and SF-1. These data provide more insight into the range of the repressive function of DAX-1 and suggest that the ability of DAX-1 to repress SF-1-mediated regulation of LHbeta expression depends on the interaction with the synergistic factor Egr-1. A similar effect was recently observed by Nachtigal et al. (64) who found that WT1, an Egr-1-related zinc finger protein, synergistically interacts with SF-1 to promote Mullerian inhibitory substance expression. DAX-1 antagonizes this synergy, but only when both WT1 and SF-1 are co-expressed (64). Whereas our results demonstrate an analogous repressive effect of DAX-1 on SF-1 and Egr-1 in the context of the LHbeta promoter, versions of the promoter harboring mutations to the SF-1 and/or Egr-1 binding sites remain partially repressible by DAX-1 (data not shown). SF-1-independent repression by DAX-1 also occurs on the human steroidogenic acute regulatory protein (StAR) promoter via direct DNA binding by DAX-1 to hairpin structures (65). Comparison of the rat LHbeta promoter to the human StAR promoter failed to elicit an identical hairpin-forming sequence, but it remains possible that a component of DAX-1 repression of LHbeta occurs through direct DNA binding. Nevertheless, the repressive effect of DAX-1 is more dramatic when the LHbeta promoter is stimulated by GnRH and is diminished when Egr-1 and SF-1 sites are mutated. These results support the notion that optimal repression of the LHbeta promoter activity by DAX-1 relies heavily upon the synergistic interaction of Egr-1 and SF-1.

Our findings highlight the significance of the synergy between the immediate early gene product Egr-1 and the gonadotrope-specific SF-1 in GnRH stimulation of LHbeta expression. A fine modulation of the degree of synergy between these proteins may provide a means of modulating LHbeta expression during the estrous cycle and throughout reproductive life.

    ACKNOWLEDGEMENTS

We are grateful to Pamela Mellon for generously providing the Lbeta T2 cells, Stuart Adler for providing the PL(KS)b-Luc plasmid, Markus Ehrengruber for the Ad5PacIGFP adenovirus vector, and Brad Sevetson for the recombinant Egr-1 protein. We are also grateful to Jeffrey Milbrandt for providing the expression vectors for the Egr family members and for helpful discussions. We thank Elena Sadovsky and Lori Rideout for technical assistance.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant DO-653/1 (to C. D.), National Institutes of Health Grant HD-34110, and the Howard Hughes Medical Institute Pilot Research Projects Award (to Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Washington University School of Medicine, P. O. Box 8064, 4911 Barnes-Jewish Hospital Plaza, St. Louis, MO 63110. Tel.: 314-747-0937; Fax: 314-362-8580; E-mail: sadovskyy{at}msnotes.wustl.edu.

2 J. Svaren, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; Egr, early growth response; SF-1, steroidogenic factor-1; DAX-1, dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on chromosome X, gene 1; PCR, polymerase chain reaction; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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