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NEUROENDOCRINOLOGY |
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
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Abstract |
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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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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.
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Materials and Methods |
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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.
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Results |
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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.
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). Follistatin alone had no effect. Basal luciferase activity was not significantly different between the various constructs. Similar results were obtained using the -387/+62 construct of the mGnRHR gene promoter (data not shown). These data suggest that the augmented response of the mGnRHR gene promoter to GnRH agonist stimulation in the presence of activin A is mediated by activin A-activin receptor binding, a process that can be inhibited by follistatin.
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T3-1 cells, and the response to GnRH agonist stimulation measured with or without activin A pretreatment. Wild-type expression vector (GH50/-387/-264) showed a 12.0 ± 1.3-fold increase in luciferase activity in response to GnRH agonist stimulation, compared with control (P < 0.0001; ANOVA), which was further increased 3.1-fold (to 37.3 ± 4.1-fold) following activin A pretreatment [P < 0.0001, compared with GnRH agonist stimulation; ANOVA (Fig. 3)]. GH50-pXP2 plasmid alone showed no increase in activity in response to GnRH agonist and/or activin A stimulation. These results were similar to those seen in the -765/+62 and -387/+62 constructs of the mGnRHR gene promoter (Fig. 1
), suggesting that the region -387/-264 is sufficient to mediate response to both GnRH agonist and activin A stimulation. 5'-Deletion to -308/-264 completely abrogated the augmented response to GnRH in the presence of activin A (Fig. 3). The GH50/-387/-308 construct did not respond to either GnRH agonist or activin A stimulation (Fig. 3). These data suggest that an element(s) within the region -387/-308 of the mGnRHR gene promoter is necessary to mediate the augmented response to GnRH in the presence of activin A. This region contains a putative SBE at position -331/-324 (5'-GTCTAG[T]C-3'; Ref. 20 and Fig. 4).
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) and transfection experiments carried out in T3-1 cells as described. Results show that mutation of the SBE in GH50/-387/-264 completely abrogated the augmented response to GnRH agonist in the presence of activin A, without affecting the response to GnRH agonist alone (Fig. 3). No difference was noted between the SBE-XhoI and SBE-NotI mutant constructs in GH50/-387/-264 (Fig. 3).
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.
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T3-1 cells, either singly or in combination, along with the GH50/-387/-264 construct of the mGnRHR gene promoter, and response to GnRH agonist stimulation was measured. All SMAD proteins were shown to be functionally active by demonstrating significantly increased luciferase expression in T3-1 cells transfected with SMAD expression vectors and the A3-1-pCS2-Luc reporter plasmid (data not shown), which is known to be responsive to SMAD2, SMAD3, and SMAD4 in vitro (28).
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.
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T3-1 cells were treated with activin A and transfected with GH50/-387/-264 with or without expression vectors for SMAD3 and SMAD4 or pCS2 control. Thereafter, the response to GnRH agonist stimulation was measured. Consistent with the data presented in Fig. 6, overexpression of SMAD3+SMAD4 significantly increased mGnRHR gene promoter activity by 3.5 ± 0.2-fold, compared with pCS2 control [P < 0.05; ANOVA (Fig. 7)]. Treatment with activin A increased expression in both the pCS2 and SMAD3+SMAD4 groups by 2.1 ± 0.1- and 2.4 ± 0.5-fold, respectively [P < 0.05, compared with control; ANOVA (Fig. 7)]. Similarly, stimulation with GnRH agonist increased mGnRHR gene expression in the pCS2 and SMAD3+SMAD4 groups by 4.6 ± 0.4- and 4.9 ± 1.3-fold, respectively (P < 0.05, compared with control), which was further increased following activin A treatment by 4.2- and 5.1-fold, respectively (Fig. 7). In all instances, the magnitude of the response was significantly greater in the SMAD3+SMAD4 group, compared with the pCS2 group (Fig. 7). These data demonstrate that the response of the mGnRHR gene promoter to SMAD overexpression can be further augmented by treatment with activin A.
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T3-1 cells and [32P]-end-labeled -337/-259 of the mGnRHR gene promoter as probe, three distinct protein-DNA complexes could be identified on EMSA that were not present with probe alone (Fig. 8). GnRH agonist stimulation (100 nM for 1 h) of T3-1 cells before preparation of nuclear extract increased the intensity of the middle band, suggesting the presence of a specific, GnRH-responsive protein(s) within the complex. The intensity of the upper and lower bands did not change significantly with GnRH agonist stimulation.
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), but not with unrelated sequences (data not shown), suggesting that the binding is specific. Competition EMSA studies using unlabeled -281/-261 (that contains the AP-1 binding site; Fig. 8) as well as EMSA supershift experiments (data not shown) suggest that the middle band represents an AP-1 (Fos/Jun heterodimer) complex, consistent with prior EMSA studies (11, 31). Further competition EMSA studies show that region -337/-317 of the mGnRHR gene promoter (containing the putative SBE) consistently reduces or eliminates the upper and middle DNA-protein bands, with little if any effect on the lower band.
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).
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T3-1 cells was 1036 ± 165 cpm/µg protein and increased to 1174 ± 217 cpm/µg protein following activin A treatment. This 13.3% increase in specific binding was not statistically significant (P = 0.44; t test). |
Discussion |
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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.
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Acknowledgments |
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T3-1 cells, and Dr. P. Michael Conn for providing iodinated buserelin. |
Footnotes |
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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.
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and ß subunits in male rats. Proc Natl Acad Sci USA 83:4026–4029, ßA, and ßB subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247–251-subunit gene. J Biol Chem 268:3903–3910-subunit gene expression and secretion by activin in T3-1 cells. Mol Endocrinol 9:1737–1749This article has been cited by other articles:
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 |
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 L. M Bilezikjian, A. L Blount, C. J Donaldson, and W. W Vale Pituitary actions of ligands of the TGF-{beta} family: activins and inhibins. Reproduction, August 1, 2006; 132(2): 207 - 215. [Abstract] [Full Text] [PDF]
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N. Safwat, J. Ninomiya-Tsuji, A. J. Gore, and W. L. Miller Transforming Growth Factor {beta}-Activated Kinase 1 Is a Key Mediator of Ovine Follicle-Stimulating Hormone {beta}-Subunit Expression Endocrinology, November 1, 2005; 146(11): 4814 - 4824. [Abstract] [Full Text] [PDF]
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|
|
|
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 |
|
 C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
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S. J. Gregory, C. T. Lacza, A. A. Detz, S. Xu, L. A. Petrillo, and U. B. Kaiser Synergy between Activin A and Gonadotropin-Releasing Hormone in Transcriptional Activation of the Rat Follicle-Stimulating Hormone-{beta} Gene Mol. Endocrinol., January 1, 2005; 19(1): 237 - 254. [Abstract] [Full Text] [PDF]
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 G S Harrison, M E Wierman, T M Nett, and L M Glode Gonadotropin-releasing hormone and its receptor in normal and malignant cells Endocr. Relat. Cancer, December 1, 2004; 11(4): 725 - 748. [Abstract] [Full Text] [PDF]
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 L. Ding, J. Xu, X. Luo, and N. Chegini Gonadotropin Releasing Hormone and Transforming Growth Factor {beta} Activate Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase and Differentially Regulate Fibronectin, Type I Collagen, and Plasminogen Activator Inhibitor-1 Expression in Leiomyoma and Myometrial Smooth Muscle Cells J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5549 - 5557. [Abstract] [Full Text] [PDF]
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 |
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 J. P. Moore Jr., L. Wilson, A. C. Dalkin, and S. J. Winters Differential Expression of the Pituitary Gonadotropin Subunit Genes During Male Rat Sexual Maturation: Reciprocal Relationship Between Hypothalamic Pituitary Adenylate Cyclase-Activating Polypeptide and Follicle-Stimulating Hormone {beta} Expression Biol Reprod, July 1, 2003; 69(1): 234 - 241. [Abstract] [Full Text] [PDF]
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 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]
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 E. R. Norwitz, S. Xu, J. Xu, L. B. Spiryda, J. S. Park, K.-H. Jeong, E. A. McGee, and U. B. Kaiser Direct Binding of AP-1 (Fos/Jun) Proteins to a SMAD Binding Element Facilitates Both Gonadotropin-releasing Hormone (GnRH)- and Activin-mediated Transcriptional Activation of the Mouse GnRH Receptor Gene J. Biol. Chem., September 27, 2002; 277(40): 37469 - 37478. [Abstract] [Full Text] [PDF]
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