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Endocrinology Vol. 142, No. 4 1506-1516
Copyright © 2001 by The Endocrine Society

ARTICLES

Functional Mapping of a Placenta-Specific Upstream Promoter for Human Gonadotropin-Releasing Hormone Receptor Gene1

Kwai Wa Cheng, Billy K. C. Chow and Peter C. K. Leung2

Department of Obstetrics and Gynecology, University of British Columbia (K.W.C., P.C.K.L.), Vancouver, Canada V6H 3V5; and Department of Zoology, University of Hong Kong (B.K.C.C.), Hong Kong, China

Address all correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, 2H30-4490 Oak Street, British Columbia Women’s Hospital, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH has been showed to regulate hCG expression and secretion from the placenta through a GnRH receptor (GnRHR)-mediated process. Recently, we have reported the isolation of human GnRHR full-length complementary DNA from the human placental cells including choriocarcinoma JEG-3 cells, immortalized extravillous trophoblasts, and primary cultures of trophoblasts. Despite these observations, the molecular mechanism that controls the transcription regulation of the GnRHR gene expression in the placenta remains unknown. Here we described the identification of an upstream placenta-specific promoter located between nucleotide (nt) -1737 and -1346 (relative to the translation start site) for the human GnRHR gene. Using transient transfection studies, this upstream promoter has been shown to determine the placental cell-specific expression of this gene. Primer extension studies further confirmed the utilization of this promoter in JEG-3 cells in vivo. By mutagenesis coupled to functional studies, we have identified four putative transcription factor-binding sites, namely human glucocorticoid receptor (hGR)-Oct-1 (nt -1718 to -1710), hGR-cAMP response element (CRE; nt -1649 to -1641), hGR-GATA (nt -1602 to -1597), and hGR-activating protein-1 (nt -1518 to -1511), that are essential to the expression of this gene. Mutations of these cis-acting motifs reduced the promoter activity. The CRE and GATA motifs were subsequently shown to be placenta specific, as mutations of these motifs caused a dramatic loss in promoter activities in the placental JEG-3 cells, but not in the ovarian carcinoma OVCAR-3, monkey kidney COS-1, and human embryonic kidney 293 cells. Gel mobility assays confirmed the binding of nuclear proteins Oct-1, CRE-binding protein, GATA-2, GATA-3, c-Fos, and c-Jun from JEG-3 cells to these four elements.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS INCREASING evidence for an extrapituitary function of GnRH in the placenta. Previous studies have reported the presence of GnRH-like material in the placenta that is biochemically and structurally identical to hypothalamic GnRH (1, 2). The complementary DNA encoding the GnRH precursor in placenta has also been isolated (3). Functionally, GnRH has been demonstrated to regulate hCG secretion (4, 5, 6). In addition, the secretion of hCG from placental cells was inhibited in the presence of a GnRH antagonist (7, 8), suggesting a receptor-mediated regulatory role of GnRH in hCG secretion. By the use of in situ hybridization, GnRH receptor (GnRHR) messenger RNAs (mRNAs) were detected in the human placenta and localized to both cytotrophoblast and syncytiotrophoblast cell layers (9). Using primers specific to the human GnRHR, the expression of the GnRHR gene was detected in human placenta cells, choriocarcinoma JEG-3 cells, and immortalized extravillous trophoblast (10, 11, 12). Using a solution hybridization protection assay and an in situ hybridization assay, the level of GnRH mRNA was found to remain constant throughout gestation (13). In contrast, other studies have demonstrated dynamic changes in human GnRH receptor numbers and its mRNA levels in the placental trophoblast cells at various gestation ages, and these changes are functionally correlated to hCG secretion from placental cells (9, 14). Taken together, these findings indicate that the regulation of GnRHR gene expression might play a dynamic role in mediating GnRH action in the placenta.

To understand the molecular mechanism involved in transcriptional regulation of GnRHR gene expression, the 5'-flanking regions of the GnRHR genes have been isolated from the mouse (15, 16), human (17, 18), rat (19, 20), and ovine (21). Among them, the mouse GnRHR promoter has been well characterized using pituitary gonadotrope {alpha}T3–1 cells (15, 16, 22, 23, 24). In humans, we have identified a putative gonadotropin-specific element that controls the basal expression of the human GnRHR gene in the {alpha}T3–1 cell by interacting with steroidogenic factor-1 (SF-1) (25). Despite the presence of significant levels of GnRHR mRNA in the human placenta, the transcriptional regulation of this gene in this tissue remains unknown. In the present study we identified a 351-bp placental cell-specific upstream promoter in the human GnRHR gene by progressive deletion studies. DNA sequence analysis of this region revealed several putative DNA-binding motifs, including Oct-1, GATA, cAMP response element (CRE), and activating protein-1 (AP-1). Functional involvement of these cis-acting motifs in regulating the human GnRHR gene expression was supported by site-directed mutagenesis as well as gel mobility shift studies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture
Mouse pituitary gonadotrope-derived {alpha}T3–1 cells were provided by Dr. P. L. Mellon (Department of Reproductive Medicine, University of California, San Diego, CA). Human embryonic kidney-293 cells (HEK293), African green monkey kidney cells (COS-1), human ovarian carcinoma OVCAR-3 cells, and human choriocarcinoma JEG-3 cells were obtained from American Type Culture Collection (Manassas, VA). Human dermal fibroblasts (HDF) and immortalized human extravillous trophoblast cells (IEVT) were provided by Dr. N Auersperg (Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada) and Dr. P. K. Lala (Department of Anatomy, University of Western Ontario, Ontario, Canada), respectively. The {alpha}T3–1, HEK-293, COS-1, and HDF cells were maintained in DMEM with 4.5 mg/ml glucose (Life Technologies, Inc., Burlington, Canada), supplemented with 10% FBS (Life Technologies, Inc.). The JEG-3 and IEVT cells were maintained in RPMI 1640 containing 10% FBS. Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 in air. Cells were passaged when they reached about 90% confluence using a trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA).

Preparation of human GnRHR promoter-luciferase constructs
Human GnRHR-luciferase construct (p2300-LucF) and progressive 5'- or 3'-deletion constructs were prepared as previously described (26, 27). Plasmid DNA for transfection studies was prepared using QIAGEN Plasmid Maxi Kits (QIAGEN, Chatsworth, CA) following the manufacturer’s suggested procedure. The concentration and quality of DNA were determined by measuring absorbance at 260 nm and by agarose gel electrophoresis, respectively. Purified plasmid DNA was then dissolved in 0.1 x TE (1 mM Tris-Cl, pH 7.5, and 0.1 mM EDTA) to a final concentration of 1 µg/ml.

Primer extension
The transcription initiation site was identified by primer extension studies with oligonucleotides PE-A, PE-B, and PE-C (Table 1Go) as described previously (17). Briefly, each primer was end-radiolabeled with [32P]ATP by T4 polynucleotide kinase (Life Technologies, Inc.) and hybridized with 25 µg polyadenylated RNA for 90 min at 65 C. The reaction mix was then incubated for 1 h at 42 C after the addition of 20 U SuperScript RNase H-reverse transcriptase (Life Technologies, Inc.). The reaction was stopped by the addition of RNase A (20 µg/ml). The extended products were purified and subsequently analyzed on a 6% polyacrylamide/7.0 M urea gel.


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  		Table 1. Oligonucleotides used in the current study

 
Site-directed mutagenesis
Human GnRHR 5'-flanking region -1747 to -1346 subcloned into pBSK II+ vector (Stratagene, La Jolla, CA) was used as a template for mutagenesis reaction. Mutations were introduced by a three-step PCR mutagenesis method as described previously (27) using mutagenic primers mP-Oct-1, mP-CRE, mP-GATA, and mP-AP-1 and universal primers UP-T3F, UP, and T7R (see Table 1Go for complete sequence information). Mutation was confirmed by restriction enzyme digestion and DNA sequence analysis.

Transient transfections and reporter assay
Transfections were carried out using the calcium precipitation methodology as previously described (12). To correct for different transfection efficiencies of various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was cotransfected into cells with the GnRHR promoter-luciferase construct. Briefly, 5 x 105 of {alpha}T3–1 cells; 2.5 x 105 of HEK293, COS-1, OVCAR-3, and HDF cells; or 1.5 x 105 of JEG-3 and IEVT cells were seeded into six-well tissue culture plates before the day of transfection. Two micrograms of the GnRHR promoter-luciferase construct and 0.5 µg RSV-lacZ were dissolved in 50 µl of 0.1 x TE containing 0.25 M CaCl2 and mixed with 50 µl of 2 x BES [50 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulforic acid, 280 mM NaCl, and 1.5 mM Na2HPO4, pH 6.95]. The DNA mixture was incubated for 20 min at room temperature and then applied to the cells. Incubation of the cells with transfection medium was continued for approximately 16 h at 37 C in 3% CO2. After transfection, the cells were washed twice with culture medium and incubated for an addition 24 h with normal culture medium containing 10% FBS. Cellular lysates were collected with 200 µl cell lysis buffer and immediately assayed for luciferase activity with the Enhanced Luiferase Assay Kit (PharMingen, Mississauga, Canada). Luminescence was measured using a Lumat LB 9507 luminometer (E.G.&G, Berthold, Germany). ß-Galactosidase activity was also measured and used to normalize for varying transfection efficiencies. Promoter activity was calculated as luciferase activity/ß-galactosidase activity. A promoterless pGL2-Basic vector was included as a control in the transfection experiments.

Gel mobility shift assay
Overlapping oligodeoxynucleotides corresponding to the putative and mutated human glucocorticoid receptor (hGR)-Oct-1, hGR-CRE, hGR-GATA, and hGR-AP-1 motifs at the human GnRHR 5'-upstream promoter were synthesized by the Oligonucletide Synthesis Laboratory (University of British Columbia, Vancouver, Canada) and annealed to form a double stranded DNA (Table 1Go). Consensus and mutated Oct-1, CRE, GATA, and AP-1 oligonucleotide DNA and antibodies against Oct-1 (catalog no. sc-232X), GATA-2 (catalog no. sc-267X), GATA-3 (catalogue no. sc-268X), CRE-binding protein (CREB; catalogue no. sc-240X), c-Jun/AP-1 (catalogue no. sc-45X), and c-Fos (catalogue no. sc-52X) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Oligonucleotide DNA for nuclear factor-{kappa}B (NF-{kappa}B; catalogue no. E3291) and TFIID (catalogue no. E3221) were purchased from Promega Corp. (Nepean, Canada). Probes for gel mobility shift assay were end-radiolabeled with [32P]ATP by T4 polynucleotide kinase (Life Technologies, Inc.) and separated from unincorporated radionucleotides by a Sephadex G-25 column. Nuclear extracts were prepared from JEG-3 cells according to the method described previously (27). Protein concentrations were determined by a modified Bradford assay (Bio-Rad Laboratories, Inc.). Gel mobility shift assays were carried out in 20 µl containing 20 mM HEPES (pH 7.5), 20 mM KCl, 20 mM NaCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 2 µg poly(dI:dC), 5 µg nuclear proteins, 2 mg/ml BSA, and radiolabeled probe.

For the competition assays, the unlabeled DNA was added simultaneously with the labeled probe. Antibodies used in supershift experiments were added to the nuclear extract at room temperature 1 h before addition of the labeled probe. The binding mixture was incubated at room temperature for 20 min and separated in a 6% polyacrylamide gel containing 1 x TBE (0.09 M Tris-borate and 2 mM EDTA, pH 8.0). Before loading of samples, the gel was prerun for 90 min at 100 V at 4 C. Electrophoresis was carried out at 30 mA at 4 C. The gel was then dried under vacuum and exposed to x-ray film (Kodak X-OMAT AR film, Eastman Kodak Co., Rochester, NY) at -70 C.

Data analysis
For transfection assay, data were shown as the mean ± SD of triplicate assays in at least three independent experiments. All data were analyzed by one-way ANOVA, followed by Dunnett’s test or Tukey’s multiple comparison test using the computer software PRISM (version 2, GraphPad Software, Inc., San Diego, CA). Data were considered significantly different from each other at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of human GnRHR 5'-flanking region in placental cells
Placental JEG-3 and IEVT cells were chosen as the cell models to study the regulation of GnRHR gene promoter in the placenta because these cell lines endogenously express GnRHR mRNA (12). To localize elements within 2.3 kb of the 5'-flanking region of the human GnRHR gene that mediate placental cell-specific expression, various 5'- and 3'-deletion mutants were constructed and analyzed in JEG-3 and IEVT cells. Transient transfection studies showed similar result activity profiles in both cell lines. Our results revealed that a distal and a proximal region are important for promoter activity in the placental cells (Figs. 1Go and 2Go). The proximal region was located between nucleotides (nt) -707 and -167 (relative to the translation start site). Progressive 5'-deletion to the PstI site (nt -1018) did not affect basal promoter activity (Fig. 1Go). Further deleting the sequence from PstI to StyI (nt -707) resulted in an increase in promoter activity (JEG-3 cells: 18-fold vs. pGL2-Basic, P < 0.001; IEVT cells: 13.8 fold vs. pGL2-Basic, P < 0.001), suggesting the presence of a negative regulatory element located within this region. Deletion of the sequences from nt -707 to -407 dramatically reduced the promoter activity. Further removal of DNA sequences from nt -407 to -167 eliminated any residual promoter activity.



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  		Figure 1. Effects of 5'-deletion on the promoter activity of the human GnRHR 5'-flanking region. Progressive 5'-deletion constructs of p2300-LucF were transiently transfected into JEG-3 and IEVT cells by the calcium precipitation method. The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. The relative promoter activity of each construct is shown as the fold increase over a promoterless luciferase control pGL2-Basic, whose activity is set at 1, after being normalized to ß-galactosidase activity. Values represent the mean ± SE of triplicate experiments. a, P < 0.05 vs. pGL2-Basic.

 


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  		Figure 2. Effects of 3'-deletion on the promoter activity of the human GnRHR 5'-flanking region. Progressive 3'-deletion constructs of p2300-LucF were transiently transfected into JEG-3 and IEVT cells by the calcium precipitation method. The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. The relative promoter activity of each construct is shown as the fold increase over a promoterless luciferase control pGL2-Basic, whose activity was set at 1, after being normalized to ß-galactosidase activity. Values represent the mean ± SE of triplicate experiments. a, P < 0.05 vs. pGL2-Basic.

 
The distal promoter region was located between nt -1671 and -1346. Progressive 3'-deletion to nt -421 did not significant affect the basal promoter activity (Fig. 2Go), whereas a dramatic increase in promoter activity was obtained after deletion of the DNA region between nt -421 and -577 in both JEG-3 cells (59-fold vs. pGL2-Basic, P < 0.001) and IEVT cells (38.2-fold vs. pGL2-Basic, P < 0.001). This increase in promoter activity was maintained to the deletion of 951 bp from the 3'-end of the human GnRHR 5'-flanking region to nt -1346. Interestingly, deletion of DNA sequences from nt -577 to -771 resulted in a loss of promoter activity, and further deletion from nt -771 to -1018 resumed the maximal promoter activity (Fig. 2Go). These data suggest that strong positive and negative regulatory regions were located within these two sequences, respectively. The locations of these regulatory regions were the same as those identified by the 5'-deletion.

Transient transfection studies with the identified distal and proximal promoter regions into JEG-3 and {alpha}T3–1 cells indicated a possible differential usage of the promoter in the placental and pituitary cells (Fig. 3Go). Although an average 16-fold increase (P < 0.001) in luciferase activity was observed in the proximal region-transfected JEG-3 cells, the highest promoter activity (55-fold, P < 0.001) was observed from the distal region. In contrast, the proximal region was more active in the pituitary gonadotrope {alpha}T3–1 cells (47.2-fold vs. pGL2-Basic, P < 0.001), whereas only an average 5-fold increase (P < 0.001) in luciferase activity was obtained from the distal region-transfected {alpha}T3–1 cells.



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  		Figure 3. Differential usage of human GnRHR promoters in JEG-3 and {alpha}T3–1 cells. The distal and proximal promoters identified in Figs. 1Go and 2Go were transiently transfection into JEG-3 and {alpha}T3–1 cell by calcium precipitation method. The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. The relative promoter activity of each construct is shown as the fold increase over a promoterless luciferase control pGL2-Basic, whose activity is set at 1, after being normalized to ß-galactosidase activity. Values represent the mean ± SE of triplicate experiments. a, P < 0.001 vs. pGL2-Basic; b, P < 0.001 vs. p2200/-1346Luc.

 
To better define the distal region important for placental cell expression, further deletion mutants were generated between nt -2297 and -1346. These constructs were transiently transfected into JEG-3 cells (Fig. 4Go). Deletion of DNA spanning from nt -2297 to -1737 (related to translation start site) did not affect the basal promoter activity. A dramatic decrease (90%) in luciferase activity was observed after deleting the DNA region between nt -1737 and -1671 (p1671/-1346 Luc), suggesting the importance of this region in mediating the promoter activity. However, removal of DNA sequence from nt -1671 to -1346 (p2300/-1671 Luc) resulted in minimal promoter activity, even though the DNA section from nt -1737 to -1671 was retained. Taken together, these data suggest that the upstream 5'-flanking region between nt -1737 and -1346 was responsible for maintaining maximal basal promoter activity.



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  		Figure 4. Localization of the minimal promoter region for placental expression of GnRHR gene. Several deleted constructs of p2300/-1346 Luc was cotransfected with RSV-lacZ vector into JEG-3 cells. The relative promoter activity of each construct is shown as the fold increase over a promoterless luciferase control pGL2-Basic, whose activity is set at 1, after being normalized to ß-galactosidase activity. Values represent the mean ± SE of triplicate experiments. a, P < 0.05 vs. pGL2-Basic; b, P < 0.001 vs. p1737/-1346Luc.

 
Characterization of the transcription start site in human placental GnRHR
We previously reported the detection of two human GnRHR mRNA transcripts by Northern blotting analysis from JEG-3 cells (12). To examine the activity of the putative distal and proximal promoter regions in vivo, the transcription initiation site for the placental GnRHR gene in JEG-3 cells was examined by primer extension. Using primers PE-A and PE-B (Fig. 5AGo), four (indicated by arrows A–D) and one (indicated by arrow E) extended products were observed using JEG-3 polyadenylated RNA, respectively, but not in HDF cells (Fig. 5BGo). The transcription start sites identified by PE-A were located at nt -1379, -1391, and -1416. The most upstream transcription start site (Fig. 5BGo, arrow D) detected by the PE-A primer was further examined by the use of upstream primer PE-C (Fig. 5CGo). Two transcription start sites were identified at nt -1608 and -1629. More importantly, all transcription start sites detected were located within the distal or proximal promoter regions identified in the present study. These results strongly supported the use of these two promoters in placental JEG-3 cells in vivo.



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  		Figure 5. Identification of human GnRHR transcription start site in JEG-3 cells by primer extension. A, Diagrammatic representation of the human GnRHR 5'-flanking region. The relative positions of extension primers (PE-A, PE-B, and PE-C) and the detected transcription start sites ({blacktriangledown}) are indicated. B, RNA isolated from HDF and JEG-3 cells were extended with primer PE-A and PE-B. Four (A–D) and one (E) extended products were obtained from JEG-3 cells using primers PE-A and PE-B, respectively. No signal was obtained from HDF. GATC, Sequence reaction used to estimate the size of the extended fragments. C, The longest extended product (arrow D in B) identified by PE-A was examined by PE-C. Two signals were detected using RNA isolated from JEG-3 cells, but were not found in that from HDF cells.

 
Cell-specific use of upstream promoter
To further examine the specificity of the upstream promoter region (nt -1737 to -1346, relative to translation start site) in the placental cells, the p1737/-1346 Luc was transiently transfected into seven cell lines from different tissues or species, including {alpha}T3–1, JEG-3, IEVT, HEK293, OVCAR-3, COS-1, and HDF cells (Fig. 6Go). The upstream promoter was not active in HDF, but was highly active in placental JEG-3 and IEVT cells. Lower promoter activities were observed in other cell lines tested. These data suggest that this region is specific to placental cell, but not in other cell lineage.



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  		Figure 6. Cell-specific activity of the upstream human GnRHR promoter. The luciferase construct (p1737/-1346 Luc) was cotransfected into {alpha}T3–1, JEG-3, IEVT, HEK293, OVCAR-3, COS-1, and HDF with the RSV-lacZ vector to normalize for varying transfection efficiencies. The relative promoter activity of each construct is shown as the fold increase over a promoterless luciferase control pGL2-Basic, whose activity is set at 1, after being normalized to ß-galactosidase activity. Values represent the mean ± SE of triplicate experiments. a, P < 0.001 vs. pGL2-Basic.

 
Identification of transcription factor-binding sites
DNA sequence analysis of the upstream promoter between nt -1737 and -1346 revealed the presence of four putative transcription factor-binding sites, namely hGR-Oct-1 (5'-ATACAAAT-3' located at nt -1718 to -1710, in reverse orientation, with 87.5% homology to the consensus Oct-1 site), hGR-CRE (5'-TGACATGA-3' located at nt -1649 to -1641 with 75% homology to the consensus CRE site), hGR-GATA (5'-AGATAA-3' located at nt -1602 to -1597, in reverse orientation, with 100% homology to the GATA site), and hGR-AP-1 (5'-TGAGTCA-3' located at nt -1518 to -1511 with 100% homology to the consensus AP-1 site). The expressions of Oct-1, GATA-2, GATA-3, CREB, c-Jun, and c-Fos in JEG-3 cells, detected by Western blot analysis (data not shown), suggest the possible binding of these factors to their corresponding sites in the human GnRHR upstream promoter. To examine the functional roles of these motifs in regulating the expression of GnRHR gene in the placental cells, site-specific mutants were constructed and transiently transfected into JEG-3, OVCAR-3, HEK-293, and COS-1 cells (Fig. 7Go). Mutation of the hGR-Oct-1 site resulted in a dramatic decrease in luciferase activity in all of the cells tested (77% decrease in JEG-3 cells, P < 0.001; 48% decrease in OVCAR-3 cells, P < 0.001; 35% decrease in COS-1 cells, P < 0.001; and 19% decrease in HEK293 cells, P < 0.05). Similarly, mutation of the hGR-AP-1 site decreased luciferase activity in these cells (72% decrease in JEG-3 cells, P < 0.001; 44% decrease in OVCAR-3 cells, P < 0.001; 30% decrease in COS-1 cells, P < 0.05; and 61% decrease in HEK293 cells, P < 0.001). These results suggest that both Oct-1 and AP-1 functioned constitutively in these cells. On the other hand, mutation of hGR-CRE and hGR-GATA sites caused 36.4% (P < 0.001) and 41.6% (P < 0.001) losses of luciferase activities only in JEG-3 cells, respectively, but not in other cells (Fig. 7Go). These results suggest that these transcription factors play specific roles in the placental cells. The interaction of these transcription factors in regulating the GnRHR promoter activity was further examined by constructs containing multiple mutations. Mutations of all four putative motifs eliminated 96% of the promoter activity (data not shown).



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  		Figure 7. Functional analysis of human GnRHR upstream promoter in different cells. Mutations were introduced by three-step PCR mutagenesis as described in Materials and Methods. The mutated promoter constructs were cotransfected with RSV-lacZ vector, to normalize for varying transfection efficiencies, into JEG-3, OVCAR-3, COS-1, and HEK293 cells. The relative activity of each promoter is shown as percentage of p1737/-1346 Luc whose activity is taken as 100%, after being normalized to ß-gal activity. Values represent the mean ± SE of triplicate experiments. The names and the relative positions of the putative transcription factor-binding sites are given, and the mutated element is shown (). a, P < 0.001 vs. native p1737/-1346Luc; b, P < 0.05 vs. native p1737/-1346Luc.

 
Binding of Oct-1, CREB, GATA-2, GATA-3, c-Jun, and c-Fos to the putative binding sites
To confirm the identities of the transcription factors bound to the upstream GnRHR promoter, gel mobility shift assay was preformed with synthetic oligodeoxynucleotides containing the hGR-Oct-1, hGR-CRE, hGR-GATA, and hGR-AP-1 sites in the presence of consensus, mutated, unrelated oligodeoxynucleotides or antibodies against these transcription factors. Specific DNA-protein complexes were formed with the radiolabeled probes for hGR-Oct-1 (Fig. 8AGo, indicated by arrow A), hGR-CRE (Fig. 8BGo, indicated by arrow B), hGR-GATA (Fig. 8CGo, indicated by arrows C and D), and hGR-AP-1 (Fig. 8DGo, indicated by arrow E) using nuclear extract prepared from JEG-3 cells. These complexes disappeared with competitor DNA fragment (200-fold in excess) containing either the consensus sequences (Oct-1, CRE, GATA, and AP-1) or unlabeled probe (hGR-Oct-1, hGR-CRE, hGR-GATA, and hGR-AP-1), but not with the mutated (m) competitor (mOct-1, mhGR-Oct-1, mCRE, mhGR-CRE, mGATA, mhGR-GATA, mAP-1, mhGR-and AP-1), or unrelated sequences (NF-{kappa}B or TFIID). Furthermore, the addition of antibodies against these transcription factors either supershifted or eliminated the DNA-protein complexes, further supporting the binding of Oct-1 (Fig. 9AGo), CREB (Fig. 9BGo), GATA-2 and GATA-3 (Fig. 9CGo), and AP-1 (Fig. 9DGo) to their corresponding sites in the upstream promoter.



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  		Figure 8. Gel mobility shift assay of JEG-3 nuclear proteins binding to the putative Oct-1-, CRE-, GATA-, and AP-1-binding sites in the human GnRHR upstream promoter. Synthetic deoxyribooligonucleotide containing the putative upstream GnRHR Oct-1-like (hGR-OCT-1), CRE-like (hGR-CRE), GATA-like (hGR-GATA), and AP-1-like (hGR-AP-1) sequences were 32P labeled and incubated with JEG-3 nuclear extracts in the presence of the indicated competitor oligonucleotide at a 200-fold excess. A, Specific binding of Oct-1 complex (indicated as arrow A) to the hGR-Oct-1 in the presence of competitor oligonucleotide (lane 1, no competitor; lane 2, mutated consensus Oct-1; lane 3, consensus Oct-1; lane 4, mutated hGR-Oct-1; lane 5, hGR-Oct-1; lane 6; consensus AP-1). B, Specific binding of the CRE complex (indicated as arrow B) to the hGR-CRE in the presence of competitor oligonucleotide (lane 1, no competitor; lane 2, mutated consensus CRE; lane 3, consensus CRE; lane 4, mutated hGR-CRE; lane 5, hGR-CRE; lane 6; consensus AP-1). C, Specific binding of GATA complexes (indicated as arrows C and D) to the hGR-GATA in the presence of competitor oligonucleotide (lane 1, no competitor; lane 2, mutated consensus GATA; lane 3, consensus GATA; lane 4, mutated hGR-GATA; lane 5, hGR-GATA; lane 6; consensus AP-1). D, Specific binding of the AP-1 complex (indicated as arrow E) to the hGR-AP-1 in the presence of competitor oligonucleotide (lane 1, no competitor; lane 2, mutated consensus AP-1; lane 3, consensus Ap-1; lane 4, mutated hGR-AP-1; lane 5, hGR-AP-1; lane 6; consensus CRE). Unrelated oligonucleotides NF-{kappa}B and TFIID were included in each experiment as shown in lanes 7 and 8, respectively.

 


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  		Figure 9. Identification of transcription factor Oct-1, CREB, GATA-2, GATA-3, c-Fos, and c-Jun binding to the upstream GnRHR promoter. Gel mobility shift assay studies were performed as described in the presence of antibodies specific to Oct-1, CREB, GATA-2, GATA-3, c-Fos, c-Jun, and estrogen receptor (ER). Antibodies were added 1 h before the addition of JEG-3 nuclear extract. A, The 32P-labeled hGR-Oct-1 probe was incubated in the presence of antibody against IgG (lane 1), Oct-1 (lane 2), CREB (lane 3), and ER (lane 4). One DNA complex was supershifted by Oct-1 antibody (indicated as arrow A). B, The 32P-labeled hGR-CRE probe was incubated in the presence of antibody against IgG (lane 1), CREB (lane 2), c-Jun (lane 3), and ER (lane 4). One DNA complex was eliminated by CREB antibody (indicated as arrow B). C, The 32P-labeled hGR-GATA probe was incubated in the presence of antibody against IgG (lane 1), GATA-2 (lane 2), GATA-3 (lane 3), Oct-1 (lane 4), and ER (lane 5). Two DNA complexes were supershifted by the addition of GATA-2 and GATA-3 antibodies (indicated as arrows C and D). D, The 32P-labeled hGR-AP-1 probe was incubated in the presence of antibodies against IgG (lane 1), Oct-1 (lane 2), c-Fos (lane 3), c-Jun (lane 4), and ER (lane 5). One DNA complex was eliminated by c-Fos and c-Jun antibodies (indicated as arrow E).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GnRH has been shown to regulate the secretion and expression of hCG from the placenta through a receptor-mediated process. As the GnRHR number and its mRNA levels change dynamically throughout gestation while the level of GnRH mRNA remains relatively constant, regulation of GnRHR gene expression probably plays an important role in mediating the actions of GnRH in the placenta. As the first step to understand the molecular mechanisms regulating basal GnRHR gene expression in the placenta, the 2.3-kb human GnRHR gene 5'-flanking region was characterized in two placental cell lines, JEG-3 and IEVT. By the use of transient transfection studies, two potential regions, a distal and a proximal, have been identified to control the expression of the human GnRHR gene in the placental cells. Our comparative analysis of the distal and proximal elements in JEG-3 and {alpha}T3–1 cells suggests the differential usage of promoters in placenta and pituitary such that placental expression is predominantly controlled by the distal promoter, and pituitary expression is regulated by the proximal promoter. We and others demonstrated that the SF-1-binding element was important in pituitary-specific expression of GnRHR (22, 25), glycoprotein {alpha}-subunit (28), LH ß-subunit in vitro (29), and in vivo (30). Similar to these studies, our data further support the role of SF-1 in expressing human GnRHR gene at the pituitary level, as the SF-1-containing proximal promoter was more highly active in {alpha}T3–1 cells than the non-SF-1-containing distal promoter. Using RNA isolated from the JEG-3 cells, multiple transcription initiation sites for the GnRHR gene were located in proximity to the distal and proximal promoters identified in the present study. Interestingly, the locations of the transcription start sites identified in placental cells were similar to those identified in brain and pituitary cells and were located correspondingly to the TATA and/or CAAT box cluster as previously reported (17, 18). For instance, the transcription start sites identified at nt -1391 and -1379 were 24 and 36 bp downstream of the TATA box located at nt -1414, respectively. These data not only further confirmed the expression of GnRHR in the placenta, but also suggested the use of these promoters in vivo. Furthermore, the highest promoter activity observed in placental JEG-3 and IEVT cells after they were transfected with the distal promoter (p1737/-1346Luc), compared with other cell lines tested, indicates that this region is predominantly used in the placental cells.

Mapping of this upstream GnRHR promoter identified four major transcription factors-binding sites, hGR-Oct-1, hGR-CRE, hGR-GATA, and hGR-AP-1, which were functionally involved in regulating the expression of this gene. The hGR-Oct-1 was shown to bind transcription factor Oct-1, and deletion or mutation of this motif resulted in a significant decrease in promoter activity. More interestingly, Oct-1 has been demonstrated to position the site of transcription initiation by recruiting TFIIB (31). A number of studies have identified that the promoter- and cell type-specific function of Oct-1 is partly due to its ability to recruit cofactors into the preinitiation complex (32, 33) as well as other transcription factors (34) in an octamer site-dependent manner. In fact, deletion and mutation hGR-Oct-1 motifs resulted in 90% and 77% decreases in promoter activity, respectively, implicating the importance of this motif in the human GnRHR upstream promoter.

The binding of c-Jun and c-Fos to hGR-AP-1 in the human GnRHR upstream promoter was confirmed by gel mobility shift assay. It has recently been shown that AP-1 is involved in controlling the placenta-specific expression of mouse lactogen I gene (35), rat lactogen II (36), ovine P-450 side-chain cleavage (CYP11A1) gene (37), human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (38), and hCS-A and -B genes (39). As a result, we believe that AP-1 plays a role in regulating the expression of the human GnRHR gene in the placenta. In fact, this idea was further confirmed by mutational studies showing that a significant decrease (72%) in luciferase activity was observed after mutation of the hGR-AP-1-binding site. Recently, we also reported that the homologous transcriptional down-regulation of the human GnRHR gene in pituitary gonadotrope {alpha}T3–1 cells was mediated through an AP-1 site located between nt -1000 and -994 (27). Although it abolished the GnRH-induced down-regulation, mutation or deletion of this AP-1 site did not significantly affect the basal GnRHR promoter activity in the pituitary. Hence, AP-1 factor may lead to different responses by interacting with different AP-1 sites in different tissues. As AP-1 protein is a dimeric transcription factor consisting of either Jun/Jun homodimer or Jun/Fos heterodimers (40, 41), and both c-Jun and c-Fos belong to multigene families, the differential expression among these multiple genes leads to various compositions of AP-1 in different cells and may result in different transcriptional activities (41, 42).

Our data strongly suggest that the motifs hGR-CRE and hGR-GATA are important for the placenta-specific expression of the human GnRHR gene, as the mutation of these regions leads to the decrease in promoter activity only in JEG-3 cells, but not in HEK293, OVCAR-3, and COS-1 cells. From gel mobility shift assays and antibody supershift assays, the binding of CREB, GATA-2, and GATA-3 to these elements was confirmed. In human trophoblast cells, cAMP plays a critical role in controlling placenta-specific gene expression. Characterization of the corticotropin-releasing hormone gene promoter indicated that the CRE is essential for gene expression, as mutation or deletion of this cis-acting element resulted in the loss of expression in the placenta (43). Analysis of the human {alpha}-subunit promoter in placental cells revealed a placenta-specific enhancer region that contains two juxtaposed CRE (44, 45, 46). In addition to the CRE, placenta-specific expression of the human {alpha}-subunit gene is also controlled by GATA-binding protein. GATA-2 and GATA-3 have been reported to be present in JEG-3 cells and to regulate human gonadotropin {alpha}-subunit gene (47) and human 17ß-hydroxysteroid dehydrogenase type 1 gene (48) expression in the placenta. Similarly, the trophoblast-specific expression of mouse placental lactogen I was controlled by GATA-2 and GATA-3 (49). The analysis of mouse proliferin gene promoter also revealed functional GATA-2/3-binding sites (49). GATA-2 and GATA-3 knockout mice revealed that placenta lacking GATA-2 and GATA-3 led to a markedly reduction of both placental lactogen I and proliferin mRNA expression (50). These results further support that GATA-2 and GATA-3 are important as in vivo regulators for placenta-specific gene expression. In agreement with these results, our data demonstrated the involvement of GATA elements in controlling the placental expression of the human GnRHR gene. The molecular mechanism(s) leading to the placental cell-specific decrease in promoter activity after mutation of hGR-CRE and hGR-GATA is unclear. It is possible that a placental cell-specific cofactor is needed to mediate CREB and GATA action, as protein-protein interactions play an important role in control of transcription factor activity (40, 51, 52).

In summary, we have identified an upstream promoter in the human GnRHR gene that is primarily used in placental cells. Mutation studies have identified four transcription elements, including Oct-1, CRE, GATA, and AP-1, in which the CRE and GATA elements were shown to be placenta specific. The identification of CRE and AP-1 motifs in the upstream GnRHR promoter provides a means for regulating the expression of this gene by the protein kinase C and protein kinase A pathways at the transcriptional level.


   Acknowledgments
 
We thank S. K. Kang and Elly S. W. Ngan for their technical assistance.


   Footnotes
 
1 This work was supported by the Canadian Institutes of Health Research (to P.C.K.L.) and a Hong Kong government grant (to B.K.C.C.). Back

2 Career investigator with the British Columbia Research Institute for Children’s and Women’s Health. Back

Received September 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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