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Activin A Augments GnRH-Mediated Transcriptional Activation of the Mouse GnRH Receptor Gene

Departments of Obstetrics, Gynecology, and Reproductive Biology (E.R.N., S.X., L.D.W.) and Medicine (K.-H.J., G.Y.B., U.B.K.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; and Eli Lilly \|[amp ]\| Co., Indianapolis, Indiana 46285 (W.W.C.)

Address all correspondence and requests for reprints to: Errol R. Norwitz, M.D., Ph.D., c/o Division of Maternal-Fetal Medicine, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115. E-mail: . enorwitz{at}partners.org

	Abstract

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The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRH receptors (GnRHRs) on the cell surface, which is mediated in part at the level of GnRHR gene expression. We have previously localized GnRH responsiveness in the mouse GnRHR (mGnRHR) gene promoter to two elements: activating protein-1 and sequence underlying responsiveness to GnRH-1. This study was designed to investigate potential synergy between GnRH and activin A in transcriptional activation of the mGnRHR gene. In functional transfection studies using T3-1 cells, GnRH agonist stimulation of the mGnRHR gene promoter (-765/+62) resulted in a 10.9-fold increase in activity, which was further increased 2-fold (to 21.3-fold) following activin pretreatment. Activin pretreatment alone had no effect. Deletion of region -387/-308 or mutation of a putative SMAD-binding element at -331/-324 (5'-GTCTAG[T]C-3') abrogated the augmented response to GnRH in the presence of activin but not the response to GnRH alone. Overexpression of SMAD2 and SMAD3 along with SMAD4 increased transcriptional activity of the mGnRHR gene, which was further increased by GnRH agonist stimulation. These data demonstrate that activin augments GnRH-mediated transcriptional activation of the mGnRHR gene and suggest that this effect may be mediated through SMAD transcription factors.

	Introduction

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A FUNCTIONAL hypothalamic-pituitary-gonadal axis is critical to mammalian reproductive development and function. Pituitary gonadotropes express G protein-coupled receptors specific for the hypothalamic decapeptide, GnRH (1, 2). Activation of these receptors by GnRH stimulates intracellular signal transduction pathways to increase synthesis and release of the pituitary gonadotropins, LH and FSH (3, 4). These hormones then enter the systemic circulation to regulate gonadal function, including steroid hormone synthesis and gametogenesis.

The biosynthesis and secretion of LH and FSH by pituitary gonadotropes is highly regulated and is dependent primarily on the amplitude and frequency of pulsatile GnRH from the hypothalamus (5). The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRH receptors (GnRHRs) on the cell surface, which is mediated in part at the level of GnRHR gene expression (6). A number of factors are known to affect expression of the GnRHR gene, most notably GnRH itself (7, 8, 9, 10). Previous studies in this laboratory have identified and partially characterized the promoter region of the mouse GnRHR (mGnRHR) gene and demonstrated that the regulatory elements for tissue-specific expression as well as for GnRH regulation are present within a 1.2-kb 5'-flanking region of the mGnRHR gene [designated -1164/+62 relative to the major transcriptional start site (TSS) (11)]. Further studies localized GnRH responsiveness of the mGnRHR gene to two distinct DNA elements: the consensus activating protein-1 (AP-1) binding site (5'-TGAGTCA-3') at position -274/-268 and a novel enhancer element (5'-GCTAATTG-3') at position -292/-285, designated sequence underlying responsiveness to GnRH-1 (SURG-1) (12).

Several factors other than GnRH are known to affect expression of the GnRHR gene, including activin (13, 14), steroid hormones (15), and the pituitary-specific, homeodomain-containing transcription factor, pituitary homeobox-1 (16). Exactly how such factors interact with GnRH at a cellular level to affect transcription of the GnRHR gene, however, has not previously been examined.

Activin refers to a family of nonsteroidal peptide hormones that were originally identified as being produced by the gonads and acting systemically to promote FSH secretion from pituitary gonadotropes. It is now evident, however, that these so-called gonadal peptides are expressed in a wide variety of reproductive and nonreproductive tissues and serve diverse, tissue-specific functions beyond the control of FSH secretion (17). Activin is composed of ß-subunit homodimers or heterodimers to form activin A, activin B, or activin AB (18). On the basis of structural homology, activin belongs to the TGF-ß superfamily. Just as activin structurally resembles TGF-ß, the activin receptor and signaling system demonstrate homology with the TGF-ß receptor system. Activin acts by binding directly to activin receptor II (Act-RII), a serine-threonine kinase, on the cell surface, a process that is inhibited by follistatin (19), thereby increasing association with Act-RI. Formation of this complex leads to phosphorylation of Act-RI, which, in turn, activates a member(s) of the SMAD transcription factor family. The activated SMAD protein(s) translocates to the nucleus, in which it binds to DNA through a defined SMAD-binding element (SBE) (5'-GTCTAG[N]C-3') (20) and acts, either alone or in combination with other factors, to regulate gene transcription (21). The mRNAs that encode the activin ß-subunits as well as the Act-RI and Act-RII receptors have been detected in whole pituitaries from a number of species and in the T3-1 gonadotrope-derived mouse cell line (22). Moreover, pituitary gonadotropes have been shown to contain activin that is biologically active, and treatment with an activin-blocking antibody decreases FSH secretion both in vivo and in vitro (23, 24). Activin A has also been shown to increase the number of GnRHRs on the surface of pituitary gonadotropes (13, 25). This process appears to be mediated primarily at a transcriptional level through a cis-regulatory DNA element (5'-CTAGTCACAACA-3') at position -391/-380 of the mGnRHR gene promoter relative to the major TSS, designated GnRH receptor activating sequence (GRAS) (14, 26).

This study was designed to investigate the interaction between GnRH and activin A on transcriptional activation of the mGnRHR gene. Using transfection studies in T3-1 cells, we have demonstrated that GnRH agonist stimulation of the mGnRHR gene promoter (-765/+62) resulted in a 10.9-fold increase in activity, which was further increased 2-fold (to 21.3-fold) following activin A pretreatment. Activin A pretreatment alone had no effect. The augmented response to GnRH in the presence of activin A (but not the response to GnRH alone) was inhibited by follistatin. Deletion of region -387/-308 of the mGnRHR gene promoter or mutation of the putative SBE at -331/-324 (5'-GTCTAG[T]C-3') abrogated the augmented response to GnRH in the presence of activin A but not the response to GnRH alone, suggesting that the SBE is necessary for this response. Overexpression of SMAD2 or SMAD3 along with SMAD4 significantly increased transcriptional activation of the mGnRHR gene, which was further increased by GnRH stimulation. These data define a possible paracrine/autocrine role for activin A in the regulation of GnRH-mediated transcriptional activation of the mGnRHR gene and suggest that this effect may be mediated through SMAD transcription factors.

	Materials and Methods

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Materials
Des-Gly¹⁰,[D-Ala⁶]-GnRH-ethylamide (GnRH agonist) was obtained from Sigma (St. Louis, MO). Recombinant human activin A and follistatin were a gift of Dr. A. F. Parlow and the National Hormone and Pituitary Program. All oligonucleotides were prepared by Life Technologies, Inc. (Gaithersburg, MD). The T3-1 cells were generously donated by Dr. Pamela Mellon (University of California, San Diego, CA).

Reporter plasmids and expression vectors
A fusion construct was prepared by ligation of the 1.2-kb 5'-flanking region of the mGnRHR gene (designated -1164/+62) into the luciferase reporter plasmid, pXP2, as previously described (11, 12). The nucleotide sequence of the mGnRHR gene promoter used in these studies is based on previous work in this laboratory (11), with -1 assigned to the nucleotide immediately 5' of the major TSS. 5'-Deletions of the 1.2-kb GnRHR gene promoter (designated -765/+62, -387/+62, and -308/+62) were synthesized and inserted upstream of the luciferase reporter in pXP2 as described (12). An expression vector expressing ß-galactosidase driven by the Rous sarcoma virus promoter (RSV-ß-galactosidase) was used as an internal standard and control.

PCR-generated fragments of the region -387/-264 of the mGnRHR gene promoter were synthesized using selected sense/antisense primers with the -1164/+62 construct as a template, placed upstream of the rat GH gene minimal promoter (GH_[-50/+1], designated GH₅₀), and inserted into pXP2 as described (12). The constructs were designated GH₅₀/-387/-264, GH₅₀/-308/-264, and GH₅₀/-387/-308.

Two separate mutants of the putative SBE element in the mGnRHR gene promoter were prepared in both the -765/+62 and GH₅₀/-387/-264 constructs of the mGnRHR gene promoter by synthesizing sense and antisense mutant oligonucleotides with an overlap of approximately 15 bp, self-annealing the oligonucleotides, and reconstituting the DNA double strands using Sequenase 2.0 (United States Biochemical, Cleveland, OH). Replacement of an 8-bp segment (-331/-324) with the NotI restriction enzyme site (5'-GCGGCCGC-3') resulted in the creation of two SBE-NotI mutants, designated -765/+62/SBE-NotI mutant and GH₅₀/-387/-264/SBE-NotI mutant. The NotI restriction enzyme site is novel to the mGnRHR gene, and previous transfection experiments have shown no effect of this sequence on GnRH stimulation (12, 27). Similarly, a 3-bp mutation in this same region led to the creation of two SBE-XhoI mutants, designated -765/+62/SBE-XhoI mutant and GH₅₀/-387/-264/SBE-XhoI mutant. The identity of all reporter constructs was confirmed by sequencing using the dideoxynucleotide chain termination method.

Cell culture and transient transfection
The T3-1 (mouse gonadotrope) cells were maintained in monolayer culture in high-glucose DMEM (Life Technologies, Inc.) supplemented with 10% (vol/vol) FBS (Life Technologies, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate (Life Technologies, Inc.) at 37 C in humidified 5% CO₂/95% air. For transient transfection studies, cells were divided into six-well tissue culture plates and cultured overnight in DMEM in the absence of serum or antibiotics. Under these conditions, cells were 60–80% confluent. Cells were then transfected by calcium phosphate coprecipitation, as previously described (12). Briefly, cells were incubated with the calcium phosphate-DNA precipitates for 4 h in media containing 2% (vol/vol) FBS. In each experiment, luciferase reporter was standardized at 4 µg DNA per well. An expression vector expressing RSV-ß-galactosidase (1 µg/well) was cotransfected in all experiments and used as an internal standard. Following a 4-h transfection, cells were treated with 100 nM GnRH agonist or vehicle in DMEM containing 2% serum (2 ml/well) for 4 h immediately before harvest. These experimental conditions have previously been shown to give maximal response to GnRH agonist stimulation in T3-1 cells (12). Following the final incubation, the medium was aspirated, and cells were washed once with ice-cold PBS (pH 7.4). Cells were lysed in the wells by addition of 200 µl lysis buffer [125 mM Tris (pH 7.6), 0.5% (vol/vol) Triton X-100]. Cellular debris was removed from lysate by microcentrifugation at 14,000 x g for 10 min at 4 C. Supernatants were assayed immediately for luciferase and ß-galactosidase activity by standard protocols. Briefly, luciferase activity was determined by adding 100 µl cell lysate to 200 µl luciferin substrate (Promega Corp., Madison, WI) and measuring luminescence with a LB-953 Autolumat (EG\|[amp ]\|G Berthold, Nashua, NH) luminometer set for a 30-sec integration with no delay. ß-Galactosidase activity was determined by adding 50 µl cell lysate to 297 µl substrate [0.1 M Na₂HPO₄ buffer (pH 7.3), 0.013 M 2-nitrophenyl-ß-D-galactopyranoside, 0.1% (vol/vol) 1.0 M MgCl₂, 0.35% (vol/vol) ß-mercaptoethanol], incubating overnight at 37 C, and measuring colorimetrically at 410 nm in a DU640 spectrophotometer (Beckman Coulter, Inc., Fullerton, CA) after the addition of 100 µl of 1.0 M sodium carbonate. Luciferase activity was normalized to expression of RSV-ß-galactosidase.

Characterization of the interaction between activin A and GnRH on transcriptional activation of the mGnRHR gene promoter
To investigate the effect of activin A and/or follistatin on GnRH agonist-mediated transcriptional activation of the mGnRHR gene, T3-1 cells were cultured in 2% serum-containing DMEM with or without activin A (20 ng/ml or 50 ng/ml) and/or follistatin (100 ng/ml) for 20 h. Thereafter, cells were transiently transfected with deletional and mutational constructs of the mGnRHR gene promoter in pXP2-Luc and response to GnRH agonist stimulation measured. The presence or absence of activin A during the 4-h transfection and 4-h GnRH agonist stimulation did not affect the results (data not shown). As such, activin A was not added during these incubations.

Effect of SMAD transcription factors on GnRH-mediated transcriptional activation of the mGnRHR gene
SMAD2, SMAD3, and SMAD4 expression vectors in pCS2 plasmid and A3-1-pCS2-Luc [3x activin response element (GTCT) in pCS2-Luc] were gifts of Dr. Malcolm Whitman (Harvard Medical School, Boston, MA). To investigate the effect of select SMAD proteins on transcriptional activation of the mGnRHR gene, expression vectors encoding SMAD2, SMAD3, or SMAD4 were transiently transfected into T3-1 cells, either singly or in combinations, along with the GH₅₀/-387/-264 construct, and response to GnRH agonist stimulation was measured. Time- and dose-response experiments were performed to optimize the transfection paradigm (data not shown). In light of these data, all subsequent experiments were carried out in a standardized fashion. The T3-1 cells were transfected with GH₅₀/-387/-264 (4 µg/well) plus select SMAD expression vectors (normalized with pCS2 to give a total of 4 µg/well) for 20 h, followed by GnRH agonist stimulation (100 nM) for 4 h. The identity of the SMAD inserts in each of the pCS2 expression vectors was confirmed by restriction enzyme digestion (data not shown), and all SMAD expression vectors have been shown to produce the designated protein by in vitro translation and protein sequencing (28). To confirm that the SMAD proteins overexpressed in T3-1 cells were able to exert a functional effect, the SMAD expression vectors were each cotransfected with a reporter construct (A3-1-pCS2-Luc) known to be responsive to SMAD2, SMAD3, and SMAD4 in vitro (28).

Because activin acts by promoting phosphorylation of existing SMAD proteins in the cytoplasm of target cells, we speculated that the effect of SMAD overexpression on mGnRHR gene expression may be further augmented by pretreatment with activin A. To investigate further this hypothesis, T3-1 cells were pretreated with activin A (20 ng/ml) or vehicle and transiently transfected with GH₅₀/-387/-264 (4 µg/well) and expression vectors for SMAD3 along with SMAD4 or pCS2 control (4 µg/well) for 20 h, and the response to GnRH agonist stimulation (100 nM for 4 h) was measured.

Preparation of nuclear extracts
The T3-1 cells were grown to 60–80% confluence and treated with 100 nM GnRH agonist or vehicle for 1 h. Thereafter, cells were harvested, and nuclear extracts were prepared by the method of Andrews and Faller (29).

EMSA
Probes were prepared for EMSA by annealing of complementary oligonucleotides representing selected regions of the mGnRHR gene promoter, followed by 5'-end-labeling with [-³²P]ATP (PerkinElmer Corp. Life Sciences, Boston, MA) by T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA). The binding reaction for EMSA was performed by incubating 50,000 cpm DNA probe with 5 µg nuclear extract and 2 µg salmon sperm DNA in reaction buffer [20 mM HEPES (pH 7.9), 60 mM KCl, 5 mM MgCl₂, 10 mM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol, 1 mg/ml BSA, and 5% (vol/vol) glycerol] for 30 min at 4 C. For competition studies, excess unlabeled DNA was added 5 min before the addition of probe. Probes used for competition and supershift EMSA experiments included regions -337/-259 (that contains the putative SBE, SURG-1, and AP-1 elements), -281/-261 (AP-1), -337/-317 (putative SBE), and -335/-312 (putative SBE) of the mGnRHR gene promoter as well as CE3 [(5'-GCCTGCCTCACACCAGGATGCTAAGCCTCTGT CCAG-3' (16)] as an unrelated sequence. Protein-DNA complexes were resolved on 5% low ionic strength nondenaturing PAGE in 0.5x EDTA buffer [45 mM Tris-HCl (pH 8.0), 45 mM boric acid, 1 mM EDTA]. Gels were then dried for 1 h and subjected to autoradiography for 24–48 h. Antibody supershift experiments were performed using an anti-Fos antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), which recognizes all members of the Fos oncoprotein family. Similar experiments were carried out using an anti-Jun blocking antibody (Santa Cruz Biotechnology, Inc.) raised against the common DNA-binding domain of all members of the Jun family. Antibody, either anti-Fos or anti-Jun, was added to the EMSA reaction samples after 30 min and incubated at 4 C for an additional 2 h before gel electrophoresis.

GnRHR binding assays
Buserelin (D-tert-butyl-Ser⁶-des-Gly¹⁰-Pro⁹-ethylamide-GnRH), the GnRH agonist used for binding assays, was provided by Hoechst-Roussel Pharmaceuticals (Somerville, NJ) and was iodinated as previously described (30). The T3-1 cells were plated in six-well tissue culture plates and cultured overnight to 60–80% confluence in DMEM containing 2% serum (2 ml/well). After a 20-h incubation at 37 C with activin A (20 ng/ml) or vehicle, cells were rinsed with DMEM containing 0.1% BSA and further incubated for 90 min with 20,000 cpm [¹²⁵I]buserelin. To determine specific binding, excess noniodinated GnRH was added concurrently to select tissue culture plates throughout the 90-min incubation. Thereafter, cells were rinsed twice with ice-cold PBS and lysed with 2 ml 0.2 M sodium hydroxide/0.1% SDS. The protein concentration in lysates was calculated (Coumassie Plus protein assay reagent, Pierce Chemical Co., Rockford, IL), and total radioactivity was measured in a -counter. Specific binding of [¹²⁵I]buserelin to GnRHR was calculated by subtracting nonspecific binding from total radioactivity, and results were expressed as counts per minute per microgram protein.

Statistical analysis
Transfections were performed in triplicate and repeated multiple times. Data in each experiment were expressed as luciferase/ß-galactosidase activity. Data were combined across experiments and results expressed as mean ± SEM for basal and GnRH agonist- and/or activin-stimulated activities for each construct, and fold stimulation in response to agonist was calculated. One-way ANOVA followed by post hoc comparisons with Fisher’s protected least significant difference test was used to assess whether changes in GnRH and/or activin A responsiveness among different GnRHR promoter-luciferase reporter constructs were significant. Significant differences were designated as P < 0.05. When appropriate, data were analyzed by the t test for independent samples.

	Results

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Activin A augments GnRH-mediated transcriptional activation of the mGnRHR gene
To investigate the effect of activin A on GnRH agonist-mediated transcriptional activation of the mGnRHR gene, transfection studies were carried out in T3-1 cells as described. GnRH agonist stimulation of the mGnRHR gene promoter (-765/+62) resulted in a 10.9 ± 0.6-fold increase in luciferase activity, compared with vector alone (P < 0.01; ANOVA), which was further increased 2-fold (to 21.3 ± 1.3-fold) following activin A pretreatment [P < 0.002, compared with GnRH agonist alone; ANOVA (Fig. 1)]. Using this experimental paradigm, pretreatment with activin A alone had no effect. Similar results were seen with the mGnRHR gene promoter construct, -387/+62 (Fig. 1). No difference was noted between 20 ng/ml and 50 ng/ml activin A pretreatment (data not shown). As such, all subsequent experiments were performed using activin A at a final concentration of 20 ng/ml.

View larger version (15K): [in this window] [in a new window]   Figure 1. Activin A augments GnRH agonist-mediated transcriptional activation of the mGnRHR gene. The T3-1 cells were pretreated with activin A (20 ng/ml) or vehicle for 20 h, transfected for 4 h with deletional constructs of the 5'-flanking region of the mGnRHR gene (-765/+62, -387/+62) or pXP2 control, followed by treatment with GnRH agonist (100 nM) or vehicle for 4 h. Measurements are expressed as luciferase/ß-galactosidase. Results are mean ± SEM from five to seven experiments. Fold augmentation of the GnRH agonist response by activin A is shown at right. *, P < 0.01, compared with pXP2-Luc as well as with control and activin A alone within and between groups. #, P < 0.002, compared with GnRH agonist alone.

View larger version (15K): [in this window] [in a new window]   Figure 2. Effect of follistatin on activin A augmentation of GnRH agonist-mediated transcriptional activation of the mGnRHR gene. The T3-1 cells were pretreated with activin A (20 ng/ml), follistatin (100 ng/ml), and/or vehicle for 20 h, transfected for 4 h with the -765/+62 construct of the 5'-flanking region of the mGnRHR gene or pXP2 control, followed by treatment with GnRH agonist (100 nM) or vehicle for 4 h. Measurements are expressed as luciferase/ß-galactosidase. Results are mean ± SEM from three experiments. Fold augmentation of the GnRH agonist response by activin A is shown at right. *, P < 0.0001, compared with pXP2-Luc as well as with control or activin A alone both within and between groups. #, P < 0.05, compared with GnRH agonist alone.

View larger version (20K): [in this window] [in a new window]   Figure 3. Identification and characterization of the element(s) within the mGnRHR gene promoter that mediates the activin A-augmented response to GnRH agonist stimulation. PCR-generated wild-type and deletional and mutational fragments of the region -387/-264 of the mGnRHR gene promoter were placed upstream of the heterologous minimal promoter, GH50, transfected into T3-1 cells, and stimulated with GnRH agonist (100 nM) or vehicle for 4 h. Cells were pretreated with activin (20 ng/ml) or vehicle for 20 h. Measurements are expressed as luciferase/ß-galactosidase. Results are mean ± SEM from five experiments. *, P < 0.05 compared, with pXP2-GH50-Luc as well as with control and activin A alone both within and between groups. #, P < 0.0001, compared with all other data.

View larger version (18K): [in this window] [in a new window]   Figure 4. SBE mutant constructs. Two separate mutants of the SBE were prepared in both -765/+62 and GH50/-387/-264 constructs of the mGnRHR gene promoter. Replacement of 3 bp in the SBE (-331/-324) led to the creation of SBE-XhoI mutants [so called because of the creation of a new XhoI restriction enzyme site (5'-CTC | GAG-3')]. Similarly, replacement of 6 bp in this same region introduced a NotI restriction enzyme site (5'-GCGG | CCGC-3') and led to the creation of SBE-NotI mutants. Sequences defining the restriction enzyme sites are underlined. Mutated bases are highlighted. GRAS is defined by Duval et al. (26 ).

Confirmation of the functional importance of the putative SBE in the homologous mGnRHR gene promoter
To investigate the importance of the putative SBE in the homologous mGnRHR gene promoter, mutant constructs of this element were prepared in -765/+62 and transfection experiments carried out in T3-1 cells as described. As previously shown in Fig. 1, activin A pretreatment augments GnRH agonist stimulation of -765/+62 construct of the mGnRHR gene promoter (Fig. 5). Similar results were noted using -387/+62 construct of the mGnRHR gene promoter (Fig. 1). Transfection with the 5'-deletion construct, -308/+62 (which does not contain the consensus SBE), on the other hand, resulted in loss of the activin A-augmented response, although the response to GnRH agonist alone was maintained (Fig. 5). Similar results were obtained using mutant constructs of the SBE in -765/+62 (Fig. 5), suggesting that the SBE is necessary for the augmented response to GnRH agonist stimulation following activin A pretreatment. No difference was noted between the -765/+62/SBE-XhoI mutant and -765/+62/SBE-NotI mutant constructs (Fig. 5). These data are similar to those obtained using mutant constructs of the SBE in GH₅₀/-387/-264 (Fig. 3), lending further evidence to the hypothesis that the SBE is necessary for the augmented response to GnRH agonist stimulation following activin A pretreatment.

View larger version (19K): [in this window] [in a new window]   Figure 5. Confirmation of the functional importance of the putative SBE in the homologous mGnRHR gene promoter. 5'-Deletion and mutation fragments of the mGnRHR gene promoter in pXP2-Luc were transfected into T3-1 cells and stimulated with GnRH agonist (100 nM) or vehicle for 4 h as described. Cells were pretreated with activin (20 ng/ml) or vehicle for 20 h. Measurements are expressed as luciferase/ß-galactosidase. Results are mean ± SEM from five to nine experiments. *, P < 0.05, compared with pXP2-Luc as well as with control and activin A alone both within and between groups. #, P < 0.01, compared with all other data.

Overexpression of SMAD2 or SMAD3 along with SMAD4 (but not overexpression of any one of the SMAD proteins alone) significantly increased transcriptional activation of the GH₅₀/-387/-264 construct of the mGnRHR gene promoter, compared with overexpression of pCS2, to 3.9 ± 0.5- and 6.6 ± 0.7-fold, respectively [P < 0.05; ANOVA (Fig. 6)]. Overexpression of SMAD3+SMAD4 resulted in significantly higher activation than SMAD2+SMAD4 [P < 0.05; ANOVA (Fig. 6)]. Stimulation with GnRH agonist resulted in a further increase in mGnRHR gene promoter expression, which was similar for all SMAD expression vectors, either alone or in combination (ranging from 2.6- to 3.3-fold) as well as for the pCS2 control vector (3.1-fold stimulation; Fig. 6). Because of the increased expression noted when SMAD2 or SMAD3 was overexpressed along with SMAD4 (in the absence of GnRH agonist stimulation), stimulation of the SMAD2+SMAD4 and SMAD3+SMAD4 experiments with GnRH agonist resulted in a response that was significantly higher than that for each of the SMAD constructs alone [P < 0.01; ANOVA (Fig. 6)]. For example, compared with unstimulated cells transfected with the pCS2 control vector, GnRH agonist alone increased luciferase activity by 3.1 ± 0.7-fold, SMAD3+SMAD4 alone increased activity by 6.6 ± 1.9-fold, and SMAD3+SMAD4 together with GnRH agonist increased activity by 17.8 ± 4.4-fold (Fig. 6). These data suggest a synergistic interaction between SMAD transcription factors and GnRH agonist stimulation.

View larger version (18K): [in this window] [in a new window]   Figure 6. Effect of SMAD overexpression on GnRH-mediated transcriptional activation of the mGnRHR gene. Expression vectors encoding SMAD2, SMAD 3, SMAD4, or pCS2 control were transfected into T3-1 cells along with GH50/-387/-264 construct of the mGnRHR gene promoter for 20 h followed by stimulation with GnRH agonist (100 nM) or vehicle for 4 h. Measurements are expressed as luciferase/ß-galactosidase. Results are mean ± SEM from eight experiments. *, P < 0.05, compared with control (no GnRH) both within and between groups. #, P < 0.03, compared with pCS2 (no GnRH). ##, P < 0.05, compared with SMAD4+SMAD2 (no GnRH). **, P < 0.01, compared with pCS2, SMAD2, SMAD3, SMAD4 (+ GnRH).

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of activin A treatment and SMAD overexpression on GnRH-mediated transcriptional activation of the mGnRHR gene. The T3-1 cells were treated with activin A (20 ng/ml) or vehicle and transiently transfected with GH50/-387/-264 construct of the mGnRHR gene promoter (4 µg/well) along with expression vectors for SMAD3 and SMAD4 or pCS2 control (4 µg/well) for 20 h, followed by stimulation with GnRH agonist (100 nM) or vehicle for 4 h. Measurements are expressed as luciferase/ß-galactosidase. Results are mean ± SEM from three experiments. Fold response to SMAD overexpression and stimulation with GnRH agonist, activin, and activin + GnRH agonist are shown. Augmented response refers to the fold increase in GnRH agonist-mediated transcriptional activation of the mGnRHR gene promoter following activin A treatment, compared with GnRH agonist treatment alone. *, P < 0.05, compared with like groups in pCS2 experiments. **, P < 0.05, compared with control within each group. #, P < 0.05, compared with control and activin stimulation within each group. ##, P < 0.001, compared with all other data with each group.

View larger version (66K): [in this window] [in a new window]   Figure 8. Identification and characterization of binding factors by EMSA. EMSA was performed using T3-1 nuclear extracts and [32P]-end-labeled -337/-259 of the mGnRHR gene promoter as probe. Three distinct protein-DNA complex bands that were not present in probe alone were identified and are designated by arrows (lane 3). The effects of treatment of T3-1 cells with GnRH agonist (100 nM x 1 h) before preparation of nuclear extract were determined (lane 4). Competition with 500-fold excess unlabeled (cold) -337/-259 probe (lanes 2, 5), -281/-261 (which contains the AP-1 consensus binding sequence) (lane 6), and unlabeled -337/-317 (which contains the putative SBE) (lane 7) are shown.

Further EMSA experiments using nuclear extract from T3-1 cells and -335/-312 region of mGnRHR gene promoter (which contains the putative SBE) as probe identified two complexes that were not present in probe alone (Fig. 9). The intensity of both bands was reduced by unlabeled -335/-312 probe but not by unrelated sequence [CE3 probe (Fig. 9A)]. Of particular interest, competition with unlabeled -281/-261 (AP-1) reduced the upper (but not lower) band. Moreover, addition of anti-Fos antibody (Santa Cruz Biotechnology, Inc.) resulted in supershift of the upper band (Fig. 9B), suggesting that an AP-1 protein(s) is able to bind either directly or indirectly to the -335/-312 region of the mGnRHR gene promoter. Taken together, these data suggest that the same factors may be capable of binding to both SBE and AP-1 regions of the mGnRHR gene promoter or that there may be some protein-protein interaction between the transcription factors binding to these two regions. This is consistent with our transfection data suggesting that the SBE at position -331/-324 is necessary for the observed augmented effect on GnRH-mediated stimulation of the mGnRHR gene promoter in the presence of activin A (Figs. 1–5).

View larger version (33K): [in this window] [in a new window]   Figure 9. Further characterization of factors binding to SBE by EMSA. EMSA was performed using T3-1 nuclear extracts and [32P]-end-labeled -337/-259 of the mGnRHR gene promoter as probe. Two distinct protein-DNA complex bands that were not present in probe alone were identified, and are designated by arrows. A, Competition with 500-fold excess unlabeled (cold) -335/-312 (lane 3), unlabeled -281/-261 (AP-1) (lane 4), or unrelated sequence (CE3) (lane 5) are shown. B, Addition of anti-Fos antibody (Santa Cruz Biotechnology, Inc.) resulted in the formation of a supershifted complex (lane 4), as shown by an asterisk.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we demonstrate that activin A augments GnRH-mediated transcriptional activation of the mGnRHR gene in T3-1 cells and that the putative SBE at position -331/-324 appears to be necessary for this response. We have used T3-1 cells, a well-characterized mouse pituitary gonadotrope cell line, as a model for the analysis of cis-regulatory elements in the mGnRHR gene. This cell line, obtained by targeted tumorigenesis in the mouse pituitary with the simian virus 40 large T antigen driven by the human glycoprotein hormone -subunit promoter (31), has been used to study many aspects of gonadotrope physiology. A number of studies have shown that T3-1 cells constitutively express GnRHR and are capable of binding and responding to exogenous GnRH (10, 31, 32). Characterization of this cell model has demonstrated many similarities in the GnRH response compared with that in mouse primary pituitary cells, including the specific intracellular signal transduction pathways activated and the regulation of GnRHR and -subunit gene promoter activities by GnRH (3, 27, 33). The T3-1 cells thus appear to be a useful model for the study of the regulation of expression of the GnRHR gene by GnRH.

The mGnRHR gene has been previously isolated and characterized (34, 35, 36). The major TSS was shown to be located 62 nucleotides upstream of the translational start site (10). A 1.2-kb 5'-flanking region of the mGnRHR gene (designated -1164/+62) has been characterized and shown to be active in transfection studies (10). This region has also been used in transgenic mice to show that it is sufficient to mediate gonadotrope-specific expression in vivo (37). Functional analysis by transient transfection of T3-1 cells has demonstrated that this region contains an element(s) necessary for tissue-specific basal expression (10, 11, 26, 38) as well as for responsiveness to GnRH (10, 11, 39) and activin A (13). Duval et al. (26) have identified a tripartite enhancer responsible for regulating cell-specific basal expression of the mGnRHR gene. Individual elements of this putative enhancer include binding sites for steroidogenic factor-1, AP-1, and a novel element (5'-CTAGTCACAACA-3') at position -329/-318 relative to the major TSS, designated GRAS. The GRAS element has subsequently been shown to be an activin response element (13). We have similarly localized GnRH responsiveness of the mGnRHR gene to two DNA elements, AP-1 and SURG-1 (12), and demonstrated along with others (39) that a Fos/Jun heterodimer binds to the AP-1 consensus sequence. Exactly how such agonists interact at a cellular level to affect transcription of the GnRHR gene has not previously been examined. In this study, we demonstrate that activin A augments GnRH agonist-mediated transcription of the mGnRHR gene (Fig. 1). The constructs used contain the putative SBE, SURG-1, and the AP-1 consensus-binding site. Further transfection studies using 5'-deletion and mutant constructs (Figs. 3 and 5) suggest that the putative SBE, which overlaps with the GRAS element, is necessary for the augmented response to GnRH agonist following activin A pretreatment.

Follistatin refers to a family of a highly glycosylated polypeptides with two major isoforms resulting from differential splicing of follistatin mRNA (40). Follistatin appears to act primarily as an activin-binding protein, thereby preventing interaction between activin and its receptor on the cell surface (19). There is increasing evidence to suggest that follistatin may play an important role in the regulation of gonadotropin gene expression within pituitary gonadotropes (41, 42). Pituitary gonadotropes and gonadotrope cell lines secrete activin and follistatin (43), and both T3-1 and LßT2 mouse gonadotrope cell lines are known to be responsive to activin and follistatin (12, 13, 44, 45). GnRH stimulation of ovine FSHß and rat LHß gene promoters in LßT2 cells is inhibited by follistatin (45), suggesting that the GnRH response in these cells may depend on endogenously produced activin. Taken together, these observations suggest that activin/follistatin may have a role to play in the regulation of gonadotropin and/or GnRHR gene expression. In the present study, the augmented response of mGnRHR gene expression to GnRH agonist in the presence of activin A (but not the response to GnRH agonist alone) was abrogated by follistatin pretreatment (Fig. 2). There was no response to activin A pretreatment alone (Figs. 1–3). Moreover, follistatin pretreatment alone did not affect the response of the mGnRHR gene promoter to GnRH agonist stimulation (Fig. 2), which might have been expected if endogenously produced activin were important for mGnRHR gene expression. These data are in apparent conflict with prior reports suggesting that pretreatment of gonadotrope cell lines with follistatin represses basal mGnRHR gene promoter expression (45) and leads to an exaggerated response to stimulation with exogenous activin (13). This apparent discrepancy likely results from the use of different cell lines (LßT2 cells, compared with T3-1 cells in the present study), different concentrations of activin (100 ng/ml, compared with 20 ng/ml) and follistatin (250 ng/ml, compared with 100 ng/ml), and/or different transfection paradigms. It should also be noted that follistatin can bind to inhibin (46) and other members of the TGF-ß family [including bone morphogenic protein-4 and -7 (47)], which have been shown to be important regulators of gonadotropin gene expression in the pituitary (48). Although the present study does not exclude the possibility that follistatin may exert its effect in part through inhibition of other transcriptional agonists, it argues strongly for an important role for the interaction between activin and follistatin in the regulation of GnRHR gene expression.

Binding of activin to its cell-surface receptor leads to phosphorylation and activation of a member(s) of the SMAD transcription factor family. The activated SMAD protein(s) translocates to the nucleus in which it acts, either alone or in combination with other factors, to regulate gene transcription (21). Overexpression of SMAD2 or SMAD3 along with SMAD4 in functional transfection studies in T3-1 cells significantly increased transcriptional activation of the mGnRHR gene (Fig. 6). This is likely because of activation of overexpressed SMAD proteins by endogenous activin, which is known to be produced by T3-1 cells. The increase in mGnRHR gene expression following activation of the activin signal transduction pathway by overexpression of SMAD proteins was further increased by stimulation with GnRH agonist (Fig. 6), which is similar to the effect seen in experiments using exogenous activin A to activate the activin signal transduction pathway (Figs. 1–3 and 5). Moreover, the 2.6- to 3.3-fold increase in mGnRHR gene promoter expression following GnRH agonist stimulation is consistent with that previously documented following a 20-h transfection incubation (11, 13). These data are consistent with the hypothesis that the augmented response of the mGnRHR gene to GnRH agonist in the presence of activin A may be mediated through SMAD transcription factors. The response of the mGnRHR gene promoter to GnRH agonist stimulation in the presence of SMAD overexpression can be augmented further by pretreatment with activin A (Fig. 7). These data suggest that activation of SMAD proteins within the cytoplasm of T3-1 cells is important for the augmented response of the mGnRHR gene promoter to GnRH agonist stimulation in the presence of activin A. However, a SMAD-independent effect of activin A on the mGnRHR gene promoter cannot be excluded.

One possible explanation for the augmented response of mGnRHR gene expression to GnRH agonist in the presence of activin A may be an increase in the concentration of GnRHR on the cell surface resulting from activin A pretreatment. Indeed, prior studies in this laboratory have demonstrated that activin is capable of up-regulating GnRHR mRNA in T3-1 cells (12), and activin has been shown to increase the number of GnRHR on the surface of pituitary gonadotropes in vitro (25). GnRH agonist-binding assays were performed in T3-1 cells to examine further this possibility. Treatment with activin A for 20 h increased [¹²⁵I]buserelin binding by 13%, which was not statistically significant. These data, along with the observed abrogation of the augmented response in transfection studies using mGnRHR gene promoter constructs in which the SBE was deleted or mutated (Figs. 3 and 5), suggest that the primary mechanism responsible for this augmented response occurs at the level of gene transcription.

Using nuclear extracts from T3-1 cells, three distinct protein-DNA bands were identified on EMSA (Fig. 8). The intensity of the middle band increased in response to treatment of T3-1 cells with GnRH agonist. Using competition (Fig. 8) and antibody-blocking and -supershift EMSA experiments (data not shown) (12, 39), we have demonstrated that the middle band represents a complex containing a member(s) of the Jun/Fos heterodimer superfamily, also known as the AP-1 protein complex. This is consistent with functional transfection studies demonstrating that the AP-1 binding site is critical for GnRH responsiveness of the mGnRHR gene (12, 39). Further competition using unlabeled SBE consistently reduced the upper and middle bands, suggesting a possible interaction between the factors binding to the SBE and AP-1 cis-regulatory elements in the 5'-flanking region of the mGnRHR gene. This hypothesis is reinforced by the identification by EMSA of two DNA-protein complexes that bind to the -335/-312 region of the mGnRHR gene promoter (which contains the putative SBE), with a demonstrated supershift with anti-Fos antibody (Fig. 9). These data are again consistent with our transfection data suggesting that the SBE at position -331/-324 is necessary for the observed augmented effect on GnRH-mediated stimulation of the mGnRHR gene promoter in the presence of activin A (Figs. 1–3 and 5). Exactly which of the AP-1 and SMAD family members make up this multimer complex has yet to be determined.

There is considerable evidence to suggest that SMAD proteins exert their transcriptional effects primarily after binding to one or more "transcriptional partners" to form a multifactor complex, commonly referred to as the activin-responsive factor (28, 49, 50, 51). Moreover, detailed analysis of the GRAS element in the mGnRHR gene promoter shows that there is a 6-bp sequence (5'-ACAACA-3') at position -323/-318 immediately downstream from the putative SBE (Fig. 4) that is necessary for activin responsiveness (14, 26). It is possible that an additional transcription factor(s) is required that binds to both this cis-regulatory element and the SMAD proteins to effect maximal response of the mGnRHR gene to activin stimulation. Competition and supershift EMSA experiments detailed above suggest that this second transcription factor may be a member of the AP-1 protein family. Cooperation between SMAD transcription factors and Fos/Jun family members has been previously described. Zhang et al. (52) showed that SMAD3 and SMAD4 act together with c-Jun and c-Fos to induce transcription from a synthetic reporter (which contains four tandem AP-1-binding sites from the collagenase I promoter) in response to TGF-ß. This interaction has also been demonstrated in the c-Jun promoter (53). These data suggest that SMAD signaling and MAPK signaling may converge at AP-1-binding promoter sites.

In summary, we have used deletional and mutational constructs of the mGnRHR gene promoter coupled with functional transfection studies in the murine gonadotrope-derived T3-1 cell line to investigate the interaction between GnRH and activin A on transcriptional activation of the mGnRHR gene. We demonstrated that activin A augments GnRH-mediated transcriptional activation of the mGnRHR gene and that the augmented response to GnRH in the presence of activin A (but not the response to GnRH alone) is inhibited by follistatin. Moreover, deletion or mutation of the SBE (5'-GTCTAG[T]C-3') at position -331/-324 abrogated the activin A-augmented response to GnRH, but not the response to GnRH alone, suggesting that the SBE is necessary for this response. Overexpression of SMAD2 or SMAD3 along with SMAD4 significantly increased transcriptional activation of the mGnRHR gene, which was further increased by GnRH stimulation. Using EMSA, we further demonstrated a putative interaction between the transcription factors binding to the SBE and AP-1 cis-regulatory elements in the 5'-flanking region of the mGnRHR gene. These data define a possible paracrine/autocrine role for activin A in the regulation of GnRH-mediated transcriptional activation of the mGnRHR gene and suggest that this effect may be mediated through the interaction of SMAD transcription factors with AP-1.

	Acknowledgments

 
We thank Dr. A. F. Parlow and the National Hormone and Pituitary Program for supplying the activin A and follistatin. We thank Dr. Constance Albarracin for her mGnRHR promoter construct (-1164/+62). We also thank Dr. Malcolm Whitman for the SMAD expression vectors, Dr. Pamela Mellon for her gift of T3-1 cells, and Dr. P. Michael Conn for providing iodinated buserelin.

	Footnotes

 
This work was supported in part by the Reproductive Scientist Development Program through the Association of Professors of Obstetricians and Gynecologists and the NIH (NIH Grant K12-HD00840), the Women’s Reproductive Health Research Award (NIH K12-HD98-004; both to E.R.N.), and NIH R01-HD19938 (to U.B.K.). An award from the George W. Thorn Center further supported U.B.K. L.W. was supported by a Student’s Research Fellowship from the Endocrine Society. K.-H.J. and G.Y.B. were supported by the Lalor Foundation.

Abbreviations: Act-R, Activin receptor; AP-1, activating protein-1; GnRHR, GnRH receptor; GRAS, GnRH receptor activating sequence; mGnRHR, mouse GnRHR; RSV-ß-galactosidase, Rous sarcoma virus promoter; SBE, SMAD-binding element; SURG-1, sequence underlying responsiveness to GnRH-1; TSS, transcriptional start site.

Received August 28, 2001.

Accepted for publication October 31, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Stojilkovic SS, Reinhart J, Catt KJ 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15:462–499[CrossRef][Medline]
2. Clayton RN 1989 Gonadotropin-releasing hormone: its actions and receptors. Endocr Rev 120:11–19
3. Gharib SD, Wierman ME, Shupnik MA, Chin WW 1990 Molecular biology of the pituitary gonadotropins. Endocr Rev 11:177–199[Medline]
4. Mercer JE, Chin WW 1995 Regulation of pituitary gonadotropin gene expression. Hum Reprod Update 1:363–384[Free Full Text]
5. Kaiser UB, Jakubowiak A, Steinberger A, Chin WW 1993 Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro. Endocrinology 133:931–934[Abstract]
6. Stojilkovic SS, Catt KJ 1995 Expression and signal transduction pathways of gonadotropin-releasing hormone receptors. Recent Prog Horm Res 50:161–205
7. Papavasiliou SS, Zmeili S, Khoury S, Landefeld TD, Chin WW, Marshall JC 1986 Gonadotropin-releasing hormone differentially regulates expression of the genes for LH and ß subunits in male rats. Proc Natl Acad Sci USA 83:4026–4029[Abstract/Free Full Text]
8. Marian J, Cooper RL, Conn PM 1981 Regulation of the rat pituitary gonadotropin-releasing hormone receptor. Mol Pharmacol 19:399–405[Abstract/Free Full Text]
9. Katt JA, Duncan JA, Herbon L, Barkan L, Marshall JC 1985 The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors. Endocrinology 116:2113–2115[Abstract]
10. Yasin M, Dalkin AC, Haisenleder DJ, Kerrigan JR, Marshall JC 1995 Gonadotropin-releasing hormone (GnRH) pulse pattern regulates GnRH receptor gene expression: augmentation by estradiol. Endocrinology 136:1559–1564[Abstract]
11. Albarracin CT, Kaiser UB, Chin WW 1994 Isolation and characterization of the 5'-flanking region of the mouse gonadotropin-releasing hormone receptor gene. Endocrinology 135:2300–2306[Abstract]
12. Norwitz ER, Cardona GR, Jeong K-H, Chin WW 1999 Identification and characterization of the gonadotropin-releasing hormone response elements in the mouse gonadotropin-releasing hormone receptor gene. J Biol Chem 274:867–880[Abstract/Free Full Text]
13. Fernandez-Vasquez G, Kaiser UB, Albarracin CT, Chin WW 1996 Transcriptional activation of the gonadotropin-releasing hormone receptor gene by activin A. Mol Endocrinol 10:356–366[Abstract]
14. Duval DL, Ellsworth BS, Clay CM 1999 Is gonadotrope expression of the gonadotropin-releasing hormone receptor gene is mediated by autocrine/paracrine stimulation of an activin response element? Endocrinology 140:1949–1952[Abstract/Free Full Text]
15. Clayton RN, Catt KJ 1981 Regulation of pituitary gonadotropin-releasing hormone receptors by gonadal hormones. Endocrinology 108:887–895[Medline]
16. Drouin J, Lamolet B, Lamonerie T, Lanctot C, Tremblay JJ 1998 The PTX family of homeodomain transcription factors during pituitary developments. Mol Cell Endocrinol 140:31–36[CrossRef][Medline]
17. Meunier H, Rivier C, Evans RM, Vale W 1988 Gonadal and extragonadal expression of inhibin , ßA, and ßB subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247–251[Abstract/Free Full Text]
18. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH release protein from porcine ovarian follicular fluid. Nature 321:776–779[CrossRef][Medline]
19. de Winter JP, ten Dijke P, de Vries CJ, van Achterberg TA, Sugino H, de Waele P, Huylebroeck D, Verschueren K, van den Eijnden-van Raaij AJ 1996 Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors. Mol Cell Endocrinol 116:105–114[CrossRef][Medline]
20. Zawel L, Dai JL, Buckhaults P, Zhou S, Kinzler KW, Vogelstein B, Kern SE 1998 Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell 1:611–617[CrossRef][Medline]
21. Attisano L, Wrana JL, Montalvo E, Massague J 1996 Activation of signaling by the activin receptor complex. Mol Cell Biol 16:1066–1073[Abstract]
22. Halvorson LM, DeCherney AH 1996 Inhibin, activin, and follistatin in reproductive medicine. Fertil Steril 65:459–469[Medline]
23. Corrigan AZ, Bilezikjian LM, Carroll RS, Bald LN, Schmelzer CH, Fendly BM, Mason AJ, Chin WW, Schwall RH, Vale W 1991 Evidence for an autocrine role of activin B with rat anterior pituitary cultures. Endocrinology 128:1682–1684[Abstract]
24. DePaolo LV, Bald LN, Fendly BM 1992 Passive immunoneutralization with a monoclonal antibody reveals a role for endogenous activin-B in mediating FSH hypersecretion during estrus and following ovariectomy of hypophysectomized pituitary-grafted rats. Endocrinology 130:1741–1743[Abstract]
25. Braden TD, Conn PM 1992 Activin-A stimulates the synthesis of gonadotropin-releasing hormone receptors. Endocrinology 130:2101–2105[Abstract]
26. Duval DL, Nelson SE, Clay CM 1997 The tripartite basal enhancer of the gonadotropin-releasing hormone (GnRH) receptor gene promoter regulates cell-specific expression through a novel GnRH receptor activating sequence. Mol Endocrinol 11:1814–1821[Abstract/Free Full Text]
27. Schoderbek WE, Roberson MS, Maurer RA 1993 Two different DNA elements mediate gonadotropin releasing hormone effects on expression of the glycoprotein hormone -subunit gene. J Biol Chem 268:3903–3910[Abstract/Free Full Text]
28. Yeo CY, Chen X, Whitman M 1999 The role of FAST-1 and Smads in transcriptional regulation by activin during early Xenopus embryogenesis. J Biol Chem 274:26584–26590[Abstract/Free Full Text]
29. Andrews NC, Faller DV 1991 A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 19:2499[Free Full Text]
30. Marian J, Conn PM 1980 The calcium requirement in GnRH-stimulated LH release is not mediated through a specific action on receptor binding. Life Sci 27:87–92[CrossRef][Medline]
31. Windle JJ, Weiner RI, Mellon PL 1990 Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol 4:597–603[Abstract]
32. Costa EMF, Bédécarrats GY, Mendonca BB, Arnhold IJP, Kaiser UB, Latronico AC 2001 Two novel mutations in the gonadotropin-releasing hormone receptor gene in Brazilian patients with hypogonadotropic hypogonadism and normal olfaction. J Clin Endocrinol Metab 86:2680–2686[Abstract/Free Full Text]
33. Horn F, Bilezikjian LM, Perrin MH, Bosma MM, Windle JJ, Huber KS, Blount AL, Hille B, Vale W, Mellon PL 1991 Intracellular responses to gonadotropin-releasing hormone in a clonal cell line of the gonadotrope lineage. Mol Endocrinol 5:347–355[Abstract]
34. Kaiser UB, Zhao D, Cardona GR, Chin WW 1992 Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor. Biochem Biophys Res Commun 189:1645–1652[CrossRef][Medline]
35. Perrin MH, Bilezikjian LM, Hoeger D, Donaldson CJ, Rivier J, Haas Y, Vale WW 1993 Molecular and functional characterization of GnRH receptors cloned from rat pituitary and a mouse pituitary cell line. Biochem Biophys Res Commun 191:1139–1144[CrossRef][Medline]
36. Zhou W, Sealfon SC 1994 Structure of the mouse gonadotropin-releasing hormone receptor gene: variant transcripts generated by alternative processing. DNA Cell Biol 13:605–614[Medline]
37. McCue JM, Quirk CC, Nelson SE, Bowen RA, Clay CM 1997 Expression of a murine gonadotropin-releasing hormone receptor-luciferase fusion gene in transgenic mice is diminished by immunoneutralization of gonadotropin-releasing hormone. Endocrinology 138:3154–3160[Abstract/Free Full Text]
38. Clay CM, Nelson SE, DiGregorio GB, Campion CE, Wiedemann AL, Nett RJ 1995 Cell-specific expression of the mouse gonadotropin-releasing hormone (GnRH) receptor gene is conferred by elements residing within 500 bp of the proximal 5'-flanking region. Endocrine 3:615–622
39. White BR, Duval DL, Mulvaney M, Roberson MS, Clay CM 1999 Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element. Mol Endocrinol 13:566–577[Abstract/Free Full Text]
40. Michel U, Farnworth P, Findlay JK 1993 Follistatins: more than follicle-stimulating hormone suppressing proteins. Mol Cell Endocrinol 91:1–11[CrossRef][Medline]
41. Khoury RH, Wang QF, Crowley Jr WF, Hall JE, Schneyer AL, Toth T, Midgley AR Jr, Sluss PM 1995 Serum follistatin levels in women: evidence against an endocrine function of ovarian follistatin. J Clin Endocrinol Metab 80:1361–1368[Abstract]
42. Kettel LM, DePaolo LV, Morales AJ, Apter D, Ling N, Yen SS 1996 Circulating levels of follistatin from puberty to menopause. Fertil Steril 65:472–476[Medline]
43. Kaiser UB, Lee BL, Carroll RS, Unabia G, Chin WW, Childs GV 1992 Follistatin gene expression in the pituitary: localization in gonadotrophs and folliculostellate cells in diestrous rats. Endocrinology 130:3048–3056[Abstract]
44. Attardi B, Klatt B, Little G 1995 Repression of glycoprotein hormone -subunit gene expression and secretion by activin in T3-1 cells. Mol Endocrinol 9:1737–1749[Abstract]
45. Pernasetti F, Vasilyev VV, Rosenberg SB, Bailey JS, Huang HJ, Miller WL, Mellon PL 2001 Cell-specific transcriptional regulation of follicle-stimulating hormone-ß by activin and gonadotropin-releasing hormone in the LßT2 pituitary gonadotrope cell model. Endocrinology 142:2284–2295[Abstract/Free Full Text]
46. Shimonaka M, Inouye S, Shimasaki S, Ling N 1991 Follistatin binds to both activin and inhibin through the common subunit. Endocrinology 128:3313–3315[Abstract]
47. Iemura S-I, Yamamoto TS, Takagi C, Uchiyama H, Natsume T, Shimasaki S, Sugino H, Ueno N 1998 Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo. Proc Natl Acad Sci USA 95:9337–9342[Abstract/Free Full Text]
48. Huang H-J, Wu JC, Su P, Zhirnov O, Miller WL 2001 A novel role for bone morphoge