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

We have previously shown that early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) β deficiency. Egr-1 activates the LHβ 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 LHβ, we examined the activity of the LHβ promoter in the gonadotrope cell line LβT2. GnRH markedly stimulated the LHβ promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHβ promoter attenuated this stimulation, whereas mutation of both promoter elements abrogated GnRH induction of the LHβ promoter. Furthermore, GnRH stimulated Egr-1 but not SF-1 expression in LβT2 cells. Importantly, overexpression of Egr-1 alone was sufficient to enhance LHβ expression. Although other Egr proteins are expressed in LβT2 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 LHβ promoter. We conclude that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHβ gene and plays a central role in the dynamic regulation of LHβ expression.

We have previously shown that early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) ␤ deficiency. Egr-1 activates the LH␤ 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 LH␤, we examined the activity of the LH␤ promoter in the gonadotrope cell line L␤T2. GnRH markedly stimulated the LH␤ promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LH␤ promoter attenuated this stimulation, whereas mutation of both promoter elements abrogated GnRH induction of the LH␤ promoter. Furthermore, GnRH stimulated Egr-1 but not SF-1 expression in L␤T2 cells. Importantly, overexpression of Egr-1 alone was sufficient to enhance LH␤ expression. Although other Egr proteins are expressed in L␤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 LH␤ promoter. We conclude that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LH␤ gene and plays a central role in the dynamic regulation of LH␤ expression.
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 GnRH 1 and transmitted to gonadotropes via the pituitary portal system (1)(2)(3)(4)(5). Follicle-stimulating hormone and luteinizing hormone (LH) are heterodimeric glycoproteins that consist of a common ␣-subunit and a unique ␤-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 LH␤ is modulated at several levels, including mRNA transcription, polyadenylation, and protein glycosylation (2,6,7). Whereas diverse signaling pathways converge on the modulation of LH␤ 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 LH␤ 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 LH␤ promoter contains similar sites at positions Ϫ50 and Ϫ113.
In addition to Egr-1 elements, the LH␤ proximal promoter also contains evolution-conserved SF-1 responsive elements located at positions Ϫ127 and Ϫ59 (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)(18)(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 LH␤ expression, subsequent analysis of SF-1 Ϫ/Ϫ mice, maintained until maturity by corticosteroid rescue treatment, revealed that these mice do express LH␤ in response to GnRH injections (33).
Like SF-1, Egr-1 is also required for normal reproductive development and function, as Egr-1 Ϫ/Ϫfemale mice exhibit arrested uterine development and infertility, reflecting a specific deficiency in LH␤ 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 LH␤ promoter in vitro, suggesting that SF-1 and Egr-1 may provide a means of directing LH␤ 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 LH␤ expression (1,3,5,6,10,13,39). Therefore we hypothesized that GnRH activation of the LH␤ gene requires the synergistic interaction of Egr-1 and SF-1. We tested our hypothesis utilizing the gonadotrope cell line L␤T2, which expresses Egr-1 and SF-1 and responds to GnRH administration with LH␤ production (40 -42).

EXPERIMENTAL PROCEDURES
Plasmids and Mutagenesis-The wild type Ϫ156 to ϩ7 LH␤ 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 LH␤ promoter into a pBSKS vector (Stratagene) and generated the following mutations: Egr-1 at Ϫ113 from CGC-CCCCAA to TAGTACTCA, Egr-1 at Ϫ50 from CACCCCCAC to GAT-TCTTAT, SF-1 at Ϫ127 from TGACCTTG to TGATCATG, and SF-1 at Ϫ59 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 ϫ 0.5 min, 56°C ϫ 2 min, and 72°C ϫ 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 LH␤ 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-1 1-369 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-L␤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% CO 2 , 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 g/well, which included 0.05 g of CMV-␤-galactosidase plasmid (to normalize for cell viability and transfection efficiency). After 24 h the medium was changed to medium that contained 1% charcoal/dextrantreated 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 Ϯ S.D.) normalized to ␤-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 5Ј Egr-1, 3Ј Egr-1, 5Ј SF-1, or 3Ј SF-1 (detailed above) were end-labeled with 25 Ci of [␥-32 P]ATP using polynucleotide kinase. Nuclear extract from L␤T2 cells was generated as described previously (47). For positive control proteins, we used a bacterially expressed and purified His-Egr-1 29 -536 or glutathione S-transferase-SF-1 1-106 , both previously described (48,49). For each binding reaction we mixed 0.02 g of the proteins or 3 g 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 0.5ϫ 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 L␤T2 cells using Tri-reagent (Molecular Research Center, Cincinnati, OH). RNA samples were extracted, precipitated, and resuspended in water. RNA samples (15 g) 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 [ 32 P]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 0.2-2ϫ sodium chloride/sodium citrate buffer (1ϫ 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 Phospho-rImager screen for 4 h and to Kodak film at Ϫ80°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 L␤T2 cells were infected with 3 ϫ 10 8 plaqueforming 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 LH␤ probe previously described (16).

GnRH-dependent Stimulation of LH␤ Gene Requires the Synergistic Interaction of Egr-1 and SF-1-
We used the L␤T2 gonadotrope line to determine whether or not Egr-1 and SF-1 cooperatively transduce GnRH stimulation of the LH␤ promoter. The L␤T2 cell line was derived from a pituitary tumor in transgenic mice that express the SV40 T antigen driven by the rat LH␤ promoter (40). In addition to the SF-1 and GnRH receptors, these cells express both LH␣ and LH␤ subunits, and LH secretion is enhanced by GnRH stimulation in vitro (40,41,51). To test for GnRH enhancement of LH␤ promoter activity, we transfected the L␤T2 cells with a LH␤ reporter construct, which contains nucleotides Ϫ156 to ϩ7 of the rat LH␤ 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 L␤T2 cells (41). We found that GnRH enhanced the activity of the LH␤ 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 LH␤ reporter gene.
It has been previously shown that the LH␤ 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 LH␤ expression, we added GnRH to L␤T2 cells that were transiently transfected with either the wild type LH␤ promoter reporter or the LH␤ promoter mutated in the binding elements for Egr-1 or SF-1. We found that mutation of the higher affinity Egr-1 site at Ϫ50 (29) reduced both basal-and GnRH-stimulated activity of the LH␤ 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 LH␤ 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 L␤T2 cells (Fig. 1C). These results indicate that Egr-1 and SF-1 sites are essential for GnRH stimulation of LH␤ 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 LH␤ 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 L␤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 construct and stimulated with GnRH (100 nM) 24 and 8 h before harvest. Results (mean Ϯ 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 L␤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-1 29 -536 or glutathione S-transferase-SF-1 1-106 . 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. the same conditions. Because it was previously shown that GnRH stimulates the transcription of the LH␤ gene in L␤T2 cells (41), we sought to recapitulate the response of L␤T2 cells to GnRH by directly assessing the influence of enhanced Egr-1 expression on LH␤ transcription. Using adenovirus-mediated transfection of L␤T2 cells, we found that overexpression of Egr-1 enhanced the transcription of the LH␤ gene (Fig. 2B). Together, these results indicate that Egr-1 is not only required for LH␤ expression (16,24) but is also sufficient to induce transcription of the LH␤ gene in L␤T2 cells.
Other Egr Family Members Synergistically Interact with SF-1 in Regulation of LH␤ 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 Ϫ113 and Ϫ50) within the LH␤ promoter (27). Using Western and Northern blot analyses, we have determined that Egr-2, Egr-3, and Egr-4 are all expressed in the ␤T2 gonadotrope line (Fig. 4) (data not shown).
To determine if Egr proteins are capable of synergizing with SF-1 in the activation of LH␤ 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 LH␤ 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 LH␤ 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 LH␤ promoter, as a mutation of these sites abrogated their induction of a LH␤ promoter.
To assess the role of the cooperativity between SF-1 and the members of the Egr family of proteins that stimulate the LH␤ promoter, we sought to determine the effect of GnRH on the expression of these proteins in L␤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 L␤T2 and can synergize with SF-1, Egr-1 may play the most prominent role in GnRH stimulation of the LH␤ gene.
DAX-1 Represses Basal-and GnRH-stimulated Activity of LH␤ 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 LH␤ reporter, we hypothesized that DAX-1 would diminish the activity of the LH␤ 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 LH␤ promoter in JEG3 cells (Fig. 5A). In contrast, DAX-1 markedly diminished the cooperative interaction between all Egr proteins and SF-1 in FIG. 3. Egr proteins synergistically interact with SF-1 in activation of LH␤ promoter. JEG3 cells were transiently transfected with plasmids that contain either the wild type (WT) rat LH␤ promoter (0.5 g) or the LH␤ 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 g) or CMV-SF-1 (0.1 g) 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 g; Egr-2, 0.3 g; Egr-3, 0.01 g; Egr-4, 0.01 g. Results (mean Ϯ S.D.) are expressed as relative luciferase units (RLU) and represent three independent experiments performed in duplicate.

FIG. 4. The effect of GnRH on expression of Egr transcripts in L␤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. activation of the LH␤ 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 LH␤ promoter is SF-1-dependent, yet the ability of DAX-1 to repress SF-1 activity is context-dependent.
To determine whether or not DAX-1 represses the GnRH-dependent stimulation of the LH␤ gene, we transfected DAX-1 into L␤T2 cells. As shown in Fig. 5B, DAX-1 repressed both basal-and GnRH-dependent stimulation of the LH␤ reporter. As expected, this effect was attenuated when a C-terminally truncated form of DAX-1 (DAX-1 1-369 ), which corresponds to naturally occurring mutations that cause adrenal hypoplasia congenita, was used (35). A similar level of attenuation by DAX-1 1-369 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 LH␤ promoter activity by DAX-1 in L␤T2 cells requires the cooperative interaction of Egr-1 and SF-1.

DISCUSSION
Egr-1 is essential for LH␤ production in vivo, as Egr-1 Ϫ/Ϫ mice exhibit LH␤ deficiency, resulting in abnormal sexual development and infertility (16,24). Similarly, SF-1 Ϫ/Ϫ mice are deficient in LH␤ production (33,38), which can be restored with GnRH administration (33). GnRH is the most important physiologic regulator of LH production and stimulates LH␤ 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 LH␤ in vitro (16,29), our results provide the first link between the GnRH-dependent stimulation of LH␤ and the synergistic interaction of Egr-1 and SF-1. Maximal diminution of the LH␤ 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 LH␤ production. It is also evident that mutations in the Egr-1 binding elements within the LH␤ promoter have a more profound effect on GnRH-stimulated activity of the LH␤ 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 LH␤ 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 LH␤ in L␤T2 cells even in the absence of GnRH stimulation. Nevertheless, it is likely that endogenous SF-1 in L␤T2 cells supports the ability of Egr-1 to activate the LH␤ gene.
Our results are consistent with the finding that changes in the transcription rate of LH␤ in vivo are not associated with a concomitant change in SF-1 expression in sheep pituitary (52), although a weak induction (Ͻ1.65-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 LH␤ 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 LH␤ 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 LH␤ (56). However, unlike Egr-1, the action of Ptx1 is not specific to LH␤, as Ptx1 is also required for expression of the gonadotropin ␣-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 LH␤ 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 L␤T2 cells.
In addition to Egr-1 and SF-1, other DNA binding elements within the LH␤ gene may modulate GnRH stimulation of LH␤ expression. Using the rat somatolactotropic cell line GH3, Kaiser et al. (14) have demonstrated that another zinc finger protein, Sp1, binds to the rat LH␤ promoter at 2 sites located between Ϫ451 and Ϫ386 and may play a role in GnRH-stimulated expression of the LH␤ 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 LH␤ promoter involves an integrated response of multiple transcription factors binding to discrete promoter elements. Indeed estradiol, acting via estrogen receptors, modulates LH␤ expression (8), and estrogen receptors were shown to interact with SF-1 in regulation of the salmon gonadotropin II␤ subunit (57). Other sex hormones also alter LH␤ 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 LH␤ gene (2, 4, 6, 13, 15, 39, 58). Whereas FIG. 5. DAX-1 represses the synergistic interaction of Egr-1 and SF-1 in activation of LH␤ promoter. A, JEG3 cells were transiently transfected with plasmids that contain wild type rat LH␤ promoter (0.5 g) and with either control plasmid (CMV-Neo) or CMV-SF-1 (both at 0.1 g) 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 g/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 LH␤ promoter activity in L␤T2 cells. Cells were stimulated with GnRH (100 nM) 24 and 8 h before harvest. ⌬DAX-1 denotes DAX 1-369 , which is deficient in repressive function (36,37). Results (mean Ϯ S.D.) are expressed as relative luciferase units (RLU) and represent three independent experiments performed in duplicate. 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 LH␤ 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 LH␤ promoter, because these cells do not express an appreciable level of either Egr proteins or SF-1, although they are all expressed in L␤T2 cells. All four Egr proteins synergistically interact with SF-1 in transcriptional activation of the LH␤ 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-1deficient 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 ␣T3 cells, a gonadotrope cell line similar to L␤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 LH␤ promoter in the absence of Egr proteins, it markedly represses the synergistic activation of LH␤ 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 LH␤ 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 LH␤ 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 LH␤ promoter to the human StAR promoter failed to elicit an identical hairpinforming sequence, but it remains possible that a component of DAX-1 repression of LH␤ occurs through direct DNA binding. Nevertheless, the repressive effect of DAX-1 is more dramatic when the LH␤ 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 LH␤ 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 LH␤ expression. A fine modulation of the degree of synergy between these proteins may provide a means of modulating LH␤ expression during the estrous cycle and throughout reproductive life.