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Endocrinology Vol. 141, No. 7 2340-2349
Copyright © 2000 by The Endocrine Society

ARTICLES

Regulation of Human Gonadotropin-Releasing Hormone Receptor Gene Expression in Placental Cells1

Kwai Wa Cheng, Parimal S. Nathwani and Peter C. K. Leung2

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada V6H 3V5

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H30–4490 Oak Street, British Columbia Women’s Hospital, Vancouver, Canada V6H 3V5.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH has been suggested to regulate hCG secretion in the placenta. In the present study, we report isolation of full-length GnRH receptor (GnRHR) complementary DNA from human placental cells, including a choriocarcinoma cell line (JEG-3), immortalized extravillous trophoblasts (IEVT), and first trimester cytotrophoblast cells in primary culture. Sequence analysis of the placental GnRHR complementary DNA revealed a 100% similarity to its pituitary counterpart. Northern blot analysis using polyadenylated RNA isolated from JEG-3 and IEVT cells revealed a 2.5- and 1.2-kb GnRHR transcripts. Using semiquantitative RT-PCR, regulation of placental GnRHR gene expression was examined. In contrast to pituitary gonadotrope {alpha}T3–1 cells, down-regulation of GnRHR messenger RNA (mRNA) levels was not observed in placental cells after 24 h of 0.1-µM GnRH agonist (GnRHa) treatment. Instead, a 43% (P < 0.01) and 30% (P < 0.05) increase in GnRHR mRNA levels was observed in JEG-3 and IEVT cells, respectively. In addition, 10 µM phorbol ester or forskolin treatments resulted in a significant increase in GnRHR expression in both JEG-3 and IEVT cells. The GnRHa-induced increase in GnRHR expression was shown to be a receptor- mediated process, as cotreatment of GnRH antagonist abolished the effect. It has also been demonstrated that these stimulatory effects on GnRHR gene expression were regulated at least in part at the transcriptional level. Pretreatment of JEG-3 cells with a specific protein kinase C inhibitor (GF109203X), adenylate cyclase inhibitor (SQ22536), or protein kinase A inhibitor [PKI-(14–22) amide, myristylated] reversed GnRHa-induced GnRHR gene expression, suggesting that the placental GnRHR couples to the protein kinase C (PKC) and cAMP/protein kinase A (PKA) pathways. By Northern blot analysis, we observed a 100% (P < 0.001) increase in hCGß mRNA levels after 0.1 µM GnRHa treatment in JEG-3 cells. Again, this effect was prevented in the presence of either protein kinase C inhibitor or adenylate cyclase inhibitor, further supporting the role of the PKC and PKA pathways in GnRHR-coupled signaling in placental cells. In summary, these data strongly support the idea that 1) GnRH plays an autocrine/paracrine role in regulating placental function through a receptor-mediated mechanism; and 2) the placental GnRHR couples to both the PKC and PKA pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS increasing evidence for an extrapituitary function of GnRH in the gonads and placenta. Previous studies have reported the presence of GnRH-like material in the placenta that is biochemically and structurally identical to the hypothalamic GnRH (1, 2). The complementary DNA (cDNA) for the GnRH precursor in placenta was isolated (3), and the major site of placental GnRH production was identified to be the cytotrophoblasts (4). The human placenta contains specific binding sites for GnRH that interact with GnRH agonists and antagonists (5, 6, 7, 8). By in situ hybridization, GnRH receptor (GnRHR) messenger RNAs (mRNAs) were detected in the human placenta and localized to the cytotrophoblast and syncytiotrophoblast cell layers. (9). Using primers specific to the human GnRHR, the predicted PCR product was obtained from human placenta cells (10, 11) and JEG-3 cells (12). These preliminary results suggest that the GnRHR is expressed in human placental cells.

It is hypothesized that placental GnRH might be involved in the autocrine/paracrine regulation of the biosynthesis of hCG (13, 14, 15, 16). This hypothesis is supported by the observation that the highest GnRH concentrations in the placenta are present in the first trimester of pregnancy, coinciding with the temporal distribution of hCG synthesis (17). GnRH stimulated hCG biosynthesis and secretion from human placental explants (13, 15, 16) through mobilization of extracellular Ca2+ (18, 19, 20). The secretion of hCG from placental cells was inhibited by treatment with a GnRH antagonist (21, 22). Using immunohistochemistry, the most intense staining for GnRH in the placenta was found during the eighth week of gestation, with low staining during the remainder of the gestation period (4). Using solution hybridization protection assay and in situ hybridization assay, the level of GnRH mRNA remained constant throughout gestation (23). In contrast, other studies have demonstrated dynamic changes in human GnRHR number and mRNA levels in the placental trophoblast cells at various gestation ages that is functionally correlated to hCG secretion from placental cells (9, 24). Taken together, these findings implicate an important role for the GnRHR in regulating hCG secretion during pregnancy.

In the present study, we isolated the full-length cDNA encoding the GnRHR from primary trophoblasts and two placental cell lines, including JEG-3 cells and immortalized extravillous trophoblasts (IEVT). In addition, regulation of GnRHR mRNA levels in the placental cells was investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture
The immortalized human extravillous trophoblasts cells were provided by Dr. P. K. Lala (Department of Anatomy, University of Western Ontario, Ontario, Canada). The human choriocarcinoma cell line JEG-3 was obtained from American Type Culture Collection (Manassas, VA). Both placental cell lines were routinely maintained in RPMI 1640 containing 10% FBS. Cells were passaged with trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA). Primary culture of extravillous cytotrophoblasts, isolated from first trimester placental tissues from women undergoing elective termination of pregnancies (25), was provided by Dr. C. D. MacCalman (Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada). The use of these tissues was approved by the committee for ethical review of research involving human subjects at University of British Columbia.

RNA isolation, RT-PCR amplification, and Southern blot analysis
Total RNA isolation, first strand cDNA synthesis, and PCR amplification were performed as described previously (26). Two sets of primers specific for GnRHR were designed based on the published sequence of human pituitary GnRHR cDNA (27). Primer pairs (sense, 5'-AATATGGCAAACAGTGCCTCTCC-3'; antisense, 5'-CAATCACAGAGAAAAATATCCA-3'; ATG start codon and TGA stop codon are underlined), were used for amplifying the full-length GnRHR cDNA. PCR primers F1 and R1 (see Ref. 26 for sequence information) were used for quantifying the GnRHR mRNA levels. Several controls were included to determine the accuracy of the PCR. First, PCR amplification was performed in the absence of both cDNA and RT reaction to examine the cross-contamination of samples. Second, the integrity of RNA samples was confirmed by gel electrophoresis, and PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was run in parallel to rule out the possibility of RNA degradation. Specific primers for GAPDH (sense, 5'-ATGTTCGTCATGGGTGTGAACCA-3'; antisense, 5'-TGGCAGGTTTTTCTAGACGGCAG-3') were used to justify the quality of the isolated RNA. Finally, as all primer pairs spanned at least one intron, the size of the predicted PCR products ruled out the presence of contaminating genomic DNA in the RNA sample.

For semiquantitative PCR of GnRHR mRNA levels, amplification of placental GnRHR and GAPDH cDNA were carried out for 30 and 18 cycles, respectively. The PCR products were separated by agarose gel electrophoresis and transferred onto a nylon membrane (Amersham Pharmacia Biotech, Morgan, Canada). The membrane was hybridized with a digoxigenin-labeled GnRHR cDNA probe (Roche Molecular Biochemicals, Laval, Canada). After washing in high stringency conditions, detection was carried out following the manufacturer’s recommended procedures (Roche Molecular Biochemicals) and exposed to Kodak Omat x-ray film (Eastman Kodak Co., Rochester, NY). The radioautograms were scanned and quantified with Scion Image-Released ß 3b (Scion Corp., Bethesda, MD). The relative GnRHR mRNA level was calculated by normalizing the PCR products for GnRHR against the GAPDH expression.

Cloning and sequencing of PCR products
The putative GnRHR PCR products were cloned into PCR II vector using the TA Cloning Kit (Invitrogen, San Diego, CA) and sequenced by the dideoxy nucleotide chain termination method using a T7 DNA Polymerase Sequencing Kit (Pharmacia Biotech).

Northern blot analysis
Polyadenylated RNA [poly(A) mRNA] was separated from total RNA by two bindings to oligo(deoxythymidine) cellulose as previously described (28). Forty micrograms of total RNA and 20–50 µg poly(A) mRNA were resolved by formaldehyde denaturing agarose gel electrophoresis and prepared for Northern blotting analysis for hCGß and GnRHR mRNA, respectively. Radioactive labeled hCGß and GnRHR probes were prepared with the Random Labeling Kit (Life Technologies, Inc., Burlington, Canada) according to the manufacturer’s suggested procedure. The membranes were prehybridized and hybridized in standard hybridization solution [50% formamide, 5 x SSPE (containing 0.75 M NaCl, 0.05 M NaH2PO4, and 5 mM EDTA at pH 7.4), 5 x Denhardt’s, 0.5% SDS, and 100 µg/ml denatured herring sperm DNA] at 42 C, followed by washing in high stringency condition (0.1 x SSPE and 0.1% SDS at 65 C for 10 min). Subsequently, the hybridized blots were exposed to Kodak Omat x-ray film.

Transient transfections and reporter assay
Transfections were carried out using the calcium precipitation methodology as previously described (29). To correct for varying transfection efficiencies, the Rous sarcoma virus (RSV)-lacZ vector was cotransfected. Briefly, 1.5 x 105 JEG-3 and IEVT cells were seeded into six-well tissue culture plates before the day of transfection. Five micrograms of the 2.3-kb GnRHR promoter-luciferase construct (p2300-LucF) (30) and 2.5 µg RSV-lacZ were dissolved in a 50 µl 0.1 x TE containing 0.25 M CaCl2 and mixed with 50 µl 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 1 ml serum-free medium and incubated for an additional 24 h with normal culture medium containing 10% FBS. Cellular lysates were collected with 200 µl cell lysis buffer and assayed for luciferase activity immediately with the Enhanced Luciferase Assay Kit (PharMingen, Mississauga, Canada). Luminescence was measured using Lumat LB 9507 luminometer (E.G.&G. Berthold, Bad Wildbad, Germany). ß-Galactosidase activity was also measured and used to normalize for varying transfection efficiencies. Luciferase units were calculated as luciferase activity/ß-galactosidase.

Pharmacological treatments
Pharmacological reagents, including the GnRH agonist, D-(Ala6)-GnRH (GnRHa); the GnRH antagonist (antide), forskolin; and the phorbol ester, phorbol 12-myristate 13-acetate (TPA), were purchased from Sigma (Sigma-Aldrich Corp., Oakville, Ontario, Canada). The protein kinase C (PKC) inhibitor, bisindolymaleimide I (GF109203), the adenylate cyclase inhibitor (SQ22536), and the protein kinase A (PKA) inhibitor (PKI 14–22, cell-permeable, myristylated) were obtained from Calbiochem (La Jolla, CA). In the experiments reported herein, the effects of the GnRH analogs, forskolin and TPA, on GnRHR expression were studied. The cells were treated with the corresponding drugs for 24 h before RNA isolation.

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of human GnRHR full-length cDNA from placental cells
Using GnRHR PCR primers F1 and R1, the expression of GnRHR from JEG-3, IEVT, and primary trophoblast cells was detected in the present study. In addition, the possibility of cross-contamination can be ruled out because no PCR product was observed and detected in all control experiments by both ethidium bromide staining and Southern blotting analysis, respectively (results not shown). Using full-length GnRHR-specific primers, an expected 1003-bp PCR product was obtained from JEG-3, IEVT, and primary trophoblast cells (Fig. 1Go). Human breast carcinoma MCF-7, previously shown to express GnRHR mRNA (27), was used as a positive control. The amplified fragment was identified as GnRHR cDNA by Southern blot hybridization (Fig. 1Go). PCR products were cloned, and sequence analysis revealed that they were identical to the published pituitary GnRHR cDNA sequence. The expression of GnRHR mRNA was also detected by Northern blotting analysis. As shown in Fig. 2Go, 2Go.5- and 1.2-kb signals were observed in JEG-3, IEVT, and breast cancer (MCF-7) cells. Using the same probe, no hybridized signal was observed in human dermal fibroblasts, whereas two bands (3.5 and 1.6 kb) were observed in mouse pituitary gonadotrope ({alpha}T3–1) cells, as previously reported (31) (Fig. 2Go).



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  		Figure 1. RT-PCR amplification of GnRHR full-length cDNA from human choriocarcinoma JEG-3, IEVT, primary cytotrophoblast cells (TB), human breast carcinoma MCF-7 cells, and negative control cells (top panel). Southern hybridization of transferred PCR products with digoxigenin-labeled GnRHR cDNA probe (lower panel).

 


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  		Figure 2. Northern blot analysis for the placental GnRHR mRNA. Poly(A) mRNA isolated from IEVT (44 µg), human choriocarcinoma JEG-3 (58 µg), and human breast carcinoma MCF-7 (37 µg) were detected under high stringency conditions. The relative mol wt (0.24–9.5 kb RNA ladder; Life Technologies, Inc., Burlington, Canada) and the positions of 28S and 18S ribosomal RNA are shown on the left and right, respectively (left panel). Total RNA and poly(A) RNA isolated from mouse pituitary gonadotrope ({alpha}T3–1; 50 µg )and human dermal fibroblasts (HDF; 40 µg), respectively, were hybridized with the same probe as the control. The positions of 28S and 18S ribosomal RNA are shown on the right side [right panel].

 
Regulation of GnRHR mRNA levels in placental cells
To study the regulation of GnRHR in placental cells, a semiquantitative RT-PCR method was used. PCR optimization showed a linear amplification of placental GnRHR and GAPDH cDNA up to 35 and 25 cycles of amplification, respectively, in both JEG-3 and IEVT cells (Fig. 3Go, A and B). Similarly, linear amplification of mouse GnRHR and GAPDH cDNA was obtained up to 30 and 25 cycles using RNA isolated from {alpha}T3–1 cells (Fig. 3CGo). As a result, 30 and 18 cycles of PCR amplification of human placental GnRHR and GAPDH cDNA, and 25 and 18 cycles of PCR amplification of mouse GnRHR and GAPDH cDNA were carried out for subsequent studies.



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  		Figure 3. Validation of quantitative RT-PCR for GnRHR. Total RNA isolated from human choriocarcinoma JEG-3 (A), IEVT (B), and mouse pituitary gonadotrope ({alpha}T3–1; C) was reversed transcribed. An aliquot of first strand cDNA was amplified for GnRHR (left panel) or GAPDH (right panel) using different numbers of PCR cycles. A linear relationship was observed between PCR products and amplification cycle when plotted.

 
It has been reported that GnRH (32, 33) and forskolin (34) decrease GnRHR mRNA levels in mouse pituitary gonadotrope ({alpha}T3–1) cells. In the present study, 23% (P < 0.05 vs. control) and 49% (P < 0.001 vs. control) decreases in GnRHR mRNA levels were observed after 24-h treatment of {alpha}T3–1 cells with 0.1 µM GnRHa and 10 µM forskolin, respectively (Fig. 4Go, A and B). In contrast, no down-regulation of GnRHR mRNA levels was observed in the two placental cell lines after GnRH agonist treatment. Instead, 43% (P < 0.01 vs. control) and 50% (P < 0.01 vs. control) increases in GnRHR mRNA levels were obtained in JEG-3 cells after 24 h of 0.1 µM and 1 nM GnRHa treatment, respectively (Fig. 5AGo). In IEVT cells, no significant change in GnRHR mRNA levels was observed after 1 nM GnRHa treatment, whereas a stimulation of GnRHR mRNA levels (30%; P < 0.05) was observed after 0.1 µM GnRHa treatment. Treatment with 10 µM phorbol ester, 10 µM forskolin, or 2IU/ml hCG resulted in a significant increase in GnRHR expression from both JEG-3 and IEVT cells (Fig. 5Go, A and B).



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  		Figure 4. Effects of GnRHa and forskolin on GnRHR expression in {alpha}T3–1 cells. Cells were treated with vehicle (control) or corresponding drugs for 24 h before total RNA isolation and were analyzed for GnRHR mRNA by semiquantitative RT-PCR assays. GAPDH mRNA was used as an internal control. Autoradiograms of Southern blot analysis were scanned and quantified. The GnRHR mRNA signal was normalized to the GAPDH internal control for each sample. Mouse pituitary gonadotrope ({alpha}T3–1) cells were treated with 0.1 µM GnRHa (A) or 10 µM forskolin (B). Results are the mean ± SD from four individual experiments and are represented as a percentage of the control value. *, P < 0.05 vs. control.

 


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  		Figure 5. Effects of GnRHa, forskolin, TPA, and hCG on GnRHR expression in placental cells. Cells were treated with vehicle (control) or corresponding drugs for 24 h before total RNA isolation and analyzed for GnRHR mRNA by semiquantitative RT-PCR assays. GAPDH mRNA was used as an internal control. Autoradiograms of Southern blot analyses were scanned and quantified. The GnRHR mRNA signal was normalized to the GAPDH internal control for each sample. Human choriocarcinoma JEG-3 and IEVT were treated with GnRHa (0.1 µM and 1 nM) or hCG (2 IU/ml; A) and TPA (10 µM) or forskolin (10 µM; B). Results are the mean ± SD from four individual experiments and are represented as a percentage of the control value. *, P < 0.05 vs. control.

 
To confirm that the GnRHa-induced effect on GnRHR is a receptor-mediated event, a GnRH antagonist (antide) was used to treat the placental cells for 24 h, alone or in combination with GnRHa. No effect on human GnRHR expression was observed after antide treatment alone. However, the GnRHa-induced increase in GnRHR expression in JEG-3 and IEVT cells was reversed in the presence of the GnRH antagonist (Fig. 6Go). A similar result was observed using primary trophoblasts culture isolated from term placenta. A 60% increase (P < 0.01 vs. control) in GnRHR mRNA levels was obtained after GnRH agonist treatment, and this effect was blocked in the presence of the GnRH antagonist.



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  		Figure 6. The GnRH antagonist, antide, blocks GnRHa-induced GnRHR mRNA expression. Human choriocarcinoma JEG-3, IEVT, and primary trophoblast culture (Trophoblasts) were treated with vehicle (control), antide (0.1 µM), or GnRHa (0.1 µM) in the presence or absence of antide (0.1 µM) for 24 h. Total RNA was isolated and analyzed for GnRHR mRNA by semiquantitative RT-PCR. Autoradiograms of Southern blot analysis were scanned and quantified. The GnRHR mRNA signal was normalized to the GAPDH internal control for each sample. Results are the mean ± SD from four individual experiments and are represented as a percentage of the control value. *, P < 0.05 vs. control.

 
Transcriptional regulation of GnRHR gene expression
The expression of GnRHR mRNA from both JEG-3 and IEVT cells suggests the feasibility of using these placental cells, in addition to pituitary cells, to study the transcriptional regulation of GnRHR gene expression. To study the homologous regulation of the human GnRHR at the transcriptional level, both JEG-3 and IEVT cells as well as {alpha}T3–1 cells were transiently transfected with p2300-LucF and treated with GnRHa for 24 h before measurement of luciferase activity. A significant decrease in luciferase activity was observed in the transfected {alpha}T3–1 cells after 24 h of GnRHa treatment (data not shown). No such inhibition was observed in placental cells. In contrast, a 31% (P < 0.01) and 25% (P < 0.05) increase in luciferase activity were observed in p2300-LucF transiently transfected JEG-3 and IEVT cells after GnRHa treatment (Fig. 7Go). Interestingly, a 40% (P < 0.05 vs. control) and 60% (P < 0.01 vs. control) increase in luciferase activity were measured in the transfected JEG-3 and IEVT cells, respectively, after 10 µM TPA treatment (Fig 7Go). Furthermore, 134% (P < 0.001 vs. control) and 92% (P < 0.001 vs. control) increases in GnRHR promoter activity were detected after 10 µM forskolin treatment in the transfected JEG-3 and IEVT cells, respectively (Fig. 7Go).



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  		Figure 7. Effects of GnRH agonist, TPA, and forskolin on human GnRHR-luciferase vector (p2300-LucF) activity. The p2300-LucF transfected JEG-3 and IEVT cells were treated with vehicle (control), 0.1 µM GnRHa, 10 µM TPA, or 10 µM forskolin for 24 h before luciferase activity measurement. The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. Luciferase units were calculated as luciferase activity/ß-galactosidase activity. Results are the mean ± SD from triplicate assays in four separate experiments. *, P < 0.05.

 
Placental GnRH signaling pathway
As an initial attempt to examine the GnRH-mediated signaling pathway used in placental cells to regulate GnRHR gene expression, JEG-3 cells were pretreated with a PKC inhibitor (2 µM), an adenylate cyclase inhibitor (0.5 mM), or a PKA inhibitor (5 µM) for 30 min before GnRH agonist treatment. Our results indicated that both the PKC and PKA pathways were involved in regulating GnRHR gene expression, as the GnRH-induced increase in GnRHR expression was attenuated by pretreatment with these inhibitors (Fig. 8Go). In addition, cotreatment of PKC inhibitor and adenylate cyclase inhibitor abolished this stimulatory effect.



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  		Figure 8. Effects of a specific PKC inhibitor (GF109203X), adenylate cyclase inhibitor (SQ22536), and PKA inhibitor [PKI-(14–22) amide, myristylated] on GnRHa-induced GnRHR mRNA expression. Human choriocarcinoma JEG-3 cells were treated with vehicle (control), GnRHa (0.1 µM), or GnRHa (0.1 µM) in the presence of 2 µM PKC inhibitor (PKCI), 0.5 mM adenylate cyclase inhibitor (ACI), 5 µM PKA inhibitor (PKAI), or 2 µM PKCI plus 0.5 mM ACI. Total RNA was isolated and analyzed for GnRHR mRNA by semiquantitative RT-PCR. Autoradiograms of Southern blot analysis for GnRHR and GAPDH (upper part of the graph) were scanned and quantified. The GnRHR mRNA signal was normalized to the GAPDH internal control for each sample. Results are the mean ± SD from three individual experiments and are represented as a percentage of the control value. a, P < 0.05 vs. control; b, P < 0.05 vs. 0.1 µM GnRHa treatment.

 
GnRH effect on hCGß mRNA levels
Functionally, we observed an increase in hCGß gene expression after GnRHa treatment in JEG-3 cells (Fig. 9AGo). Increases of 100% and 65% in hCGß mRNA levels were observed after 0.1 µM and 1 nM GnRH agonist treatments, respectively. Similarly, a 240% and 740% increase in hCGß mRNA levels was measured after 10 µM phorbol ester and forskolin treatments, respectively (Fig. 9BGo). However, no such stimulatory effect was observed in IEVT cells (data not shown). Furthermore, the TPA- and forskolin-induced increases in hCGß expression were inhibited in the presence of a PKC inhibitor and an adenylate cyclase inhibitor, respectively (Fig. 10Go, A and B). The signal transduction inhibitors used in the present studies was rather specific, as no significant nonspecific effect was observed. Similarly, the GnRHa-induced hCGß expression was reversed in the presence of either the PKC inhibitor or the adenylate cyclase inhibitor, which was further reduced by cotreatment of PKC inhibitor and adenylate cyclase inhibitor (Fig. 10C).



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  		Figure 9. Regulation of hCGß mRNA expression. Total RNA was isolated from human choriocarcinoma JEG-3 after 24-h treatment of treatment with vehicle (control) or corresponding drugs. The hCGß and GAPDH mRNA was detected by Northern blot analysis (upper part of each graph). The radioautograms were scanned and normalized to the corresponding GAPDH signal for each treatment. A, JEG-3 cells were treated with 0.1 µM or 1 nM GnRHa. B, Cells were treated with 10 µM forskolin or 10 µM phorbol ester (TPA). Results are the mean ± SD from three experiments. *, P < 0.05 vs. control.

 


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  		Figure 10. Effects of a specific PKC inhibitor (GF109203X) and adenylate cyclase inhibitor (SQ22536) on GnRHa-induced hCGß mRNA expression from human choriocarcinoma JEG-3. Total RNA were isolated from JEG-3 cells after 24-h treatment of treatment with vehicle (control) or corresponding drugs. The hCGß and GAPDH mRNA was detected by Northern blot analysis (upper part of each graph). The radioautograms were scanned and normalized to the corresponding GAPDH signal for each treatment. JEG-3 cells were treated with 10 µM phorbol ester (TPA; A), 10 µM forskolin (B), 0.1 µM GnRHa (C) in the presence or absence of 2 µM PKC inhibitor (PKCI) and/or 0.5 mM adenylate cyclase inhibitor (ACI). Results are the mean ± SD from three experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. TPA, forskolin, or GnRHa treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The identification and characterization of GnRH in the placenta (1, 2, 3, 4) and its effects in stimulating hCG biosynthesis and secretion (5, 6, 7, 8, 13, 14, 15, 16) suggest that the placenta possesses an intrinsic GnRH-mediated regulatory system that influences the dynamics of hCG secretion during pregnancy. Regulation of GnRHR gene expression appears to be an important determinant of the concentration of GnRHR in the placenta (9). Recently, it has been demonstrated that placental cells express GnRHR (9, 10, 11, 12). In the present study we isolated the cDNA encoding the full-length human GnRHR cDNA from two placental cell lines, including JEG-3 and IEVT, and from primary trophoblast cells by RT-PCR amplification. Sequence analysis of the cDNA revealed that the placental GnRHR was the same as its counterpart in the pituitary, at least at the mRNA level. The difference in GnRH binding observed in placental cells vs. pituitary cells may be explained by different posttranslational modifications. Moreover, GnRHR expression in the placenta was further substantiated by Northern analysis using poly(A) RNA isolated from IEVT and JEG-3 cells. In the human pituitary, Northern blot analysis detected a predominant mRNA transcript of 4.7–5.0 kb and two minor bands of 2.5 and 1.5 kb (35). The 2.5- and 1.2-kb transcripts were observed in both placental cell lines and MCF-7 cells by Northern blot analysis. Interestingly, the major 4.7- to 5.0-kb transcript was not observed in the extrapituitary cells used in our study. We suspect that only the shorter GnRHR transcripts are synthesized in extrapituitary tissues.

We have successfully used semiquantitative RT-PCR to quantitate GnRH and its receptor expression in ovarian cells (36, 37). Although we were able to detect the human GnRHR mRNA by Northern blot analysis, the amount of total RNA required for isolating enough poly(A) RNA for regulation studies was enormous. As a result, semiquantitative RT-PCR was employed to examine GnRHR gene expression in the placental cells. Previous reports have demonstrated that GnRHa (0.1 nM to 1 µM) and 10 µM forskolin treatments resulted in a decrease in GnRHR mRNA in {alpha}T3–1 cells (32, 33, 34). In agreement with these earlier reports, a significant decrease in GnRHR mRNA levels was detected after GnRHa treatment of {alpha}T3–1 cells in our study. A similar GnRH-induced decrease in GnRHR mRNA levels was observed in human ovarian cells (36, 37). Interestingly, homologous down-regulation of GnRHR mRNA was not observed in placental cells. Instead, an increase in the GnRHR mRNA level was observed after 0.1 µM GnRHa treatment in the two placental cell lines. These results suggest that a different regulatory mechanism may be used in the placenta to control GnRHR mRNA expression.

Regulation of gene expression can be controlled at several steps. Recent studies have reported the transcriptional regulation of GnRHR gene expression in the mouse (38, 39) and human (30, 40). The detection of GnRHR mRNA from JEG-3 and IEVT cells suggests the feasibility of using these cells to study the possible transcriptional regulation of the GnRHR gene in the placenta. We have demonstrated that the human 5'-flanking region is functionally active in mouse gonadotrope {alpha}T3–1 cells (30). A statistically significant decrease in luciferase activity was observed in human GnRHR promoter-luciferase construct-transfected {alpha}T3–1 cells after GnRHa and TPA treatments (data not shown), suggesting homologous down-regulation of human GnRHR gene expression at the pituitary level. However, no inhibition of luciferase activity was observed in human GnRHR promoter-transfected placental cells after GnRHa treatment. Furthermore, an increase in GnRHR promoter activity was obtained after GnRH (0.1 µM), TPA (10 µM), and forskolin (10 µM) treatments. These data indicate that activation of the PKC and PKA pathways in the placenta may regulate GnRHR expression at the transcriptional level. These results suggest that the homologous down-regulation of the GnRHR gene seen in the pituitary is absent in the placenta, which may help maintain GnRH-stimulated hCG secretion throughout pregnancy.

Functionally, we demonstrated an increase in hCGß mRNA levels after GnRH agonist treatment in JEG-3 cells, which was mimicked by forskolin and phorbol ester treatments. However, no such effect was observed in IEVT cells. It has been reported that hCG was only detected in the immortalized IEVT cells, not in the parental cell (41). Hence, a different regulatory mechanism may exist in IEVT cells compared with JEG-3 cells. The roles of PKC and PKA pathways in mediating GnRH action in placental cells were further examined pharmacologically. The increases in placental GnRHR and hCGß mRNA levels after GnRHa treatment were reversed by pretreating JEG-3 cells with inhibitors of PKC, PKA, or adenylate cyclase alone and were completely abolished by cotreatment with PKC inhibitor and adenylate cyclase inhibitor. These findings further suggest that both PKA and PKC pathways participate in mediating GnRH action in the placental cells. It is well documented that Gq/11{alpha} couples to GnRHR (42, 43). We and others have shown the stimulation of Ca2+ mobilization in human syncytiotrophoblast cells by GnRH through a receptor-mediated event, implicating coupling of the GnRHR to Gq/11{alpha} in the placenta (18, 19). However, others have reported the coupling of Gs{alpha} to rat GnRHR (44) and of Gi{alpha} to GnRHR identified from human prostate cancer cells (45). A recent study demonstrated the direct coupling of Gs{alpha} and adenylyl cyclase to mouse GnRHR at the first intracellular loop (46). In addition, the lack of complete abolition of GnRHa-induced LH release in Gq{alpha} and G11{alpha} knockout mice further supports the coupling of GnRHR to multiple G proteins in vivo (47). Our present finding of a relatively stronger effect of forskolin vs. GnRHa on the hCGß mRNA level as well as an attenuation of the GnRHa-induced increase in GnRHR and hCGß expression by an adenylate cyclase inhibitor further implicate the coupling of Gs{alpha} to the placental GnRHR.

In summary, the present results strongly substantiate the expression of GnRHR in the human placenta and further strengthen the role of GnRH in the autocrine/paracrine regulation of hCG biosynthesis. The demonstration of GnRHa-mediated up-regulation of the GnRHR mRNA level in placental cells suggests that the regulation of this gene is different from that in the pituitary and ovary. In addition, our results further implicate a role for both the PKC and PKA pathways in placental GnRHR signaling.


   Acknowledgments
 
We thank Dr. Chi-Hsin Chiang and Spiro Getsios for their technical assistance.


   Footnotes
 
1 Presented in part at the 32nd Annual Meeting of the Society for the Study of Reproduction, Pullman, Washington, 1999. This work was supported by the Medical Research Council of Canada. Back

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

Received December 9, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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