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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0318
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1106-1113
Copyright © 2005 by The Endocrine Society

Differential Role of Progesterone Receptor Isoforms in the Transcriptional Regulation of Human Gonadotropin-Releasing Hormone I (GnRH I) Receptor, GnRH I, and GnRH II

Beum-Soo An, Jung-Hye Choi, Kyung-Chul Choi and Peter C. K. Leung

Department of Obstetrics and Gynecology, British Columbia Research Institute for Children’s and Women’s Health, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

Address all correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypothalamic GnRH is a decapeptide that plays a pivotal role in mammalian reproduction by stimulating the synthesis and secretion of gonadotropins via binding to the GnRH receptor on the pituitary gonadotropins. It is hypothesized that sex steroids may regulate GnRH I (a classical form of GnRH), GnRH II (a second form of GnRH), and GnRH I receptor (GnRHRI) at the transcriptional level in target tissues. Thus, in the present study a role for progesterone (P4) in the regulation of GnRH I, GnRH II, and GnRHRI was investigated using a human neuronal medulloblastoma cell line (TE671) as an in vitro model. The cells were transfected with human GnRHRI promoter-luciferase constructs, and promoter activities were analyzed after P4 treatment by luciferase and ß-galactosidase assay. The mRNA levels of GnRH I and GnRH II were analyzed by RT-PCR. Treatment of TE671 cells with P4 resulted in a decrease in GnRHRI promoter activity compared with the control level in a dose- and time-dependent manner. Cotreatment of these cells with RU486, an antagonist of P4, reversed P4-induced inhibition of GnRHRI promoter activity, suggesting that the P4 effect is mediated by P4 receptor (PR). In the cells transfected with a full-length of PR A- or PR B-expressing vector, overexpression of PR A increased the sensitivity toward P4 in an inhibition of GnRHRI promoter, whereas PR B increased transcriptional activity of GnRHRI promoter in the presence of P4. However, PR B itself did not act as a transcriptional activator of GnRHRI promoter. Because TE671 cells have been recently demonstrated to express and synthesize two forms of GnRHs, we also investigated the regulation of GnRH mRNAs by P4. In the present study, P4 increased GnRH I mRNA levels in a time- and dose-dependent manner. This stimulatory effect of P4 in the regulation of GnRH I mRNAs was significantly attenuated by RU486, whereas no significant difference in the expression level of GnRH II was observed with P4 or RU496. Interestingly, although the expression level of PR B was low compared with that of PR A, P4 action on the GnRH I gene was mediated by PR B. In conclusion, these results indicate that P4 is a potent regulator of GnRHRI at the transcriptional level as well as GnRH I mRNA. This distinct effect of P4 on the GnRH system may be derived from different pathways through PR A or PR B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS A decapeptide that plays a pivotal role in mammalian reproduction by stimulating the synthesis and secretion of gonadotropins via binding to the GnRH I receptors (GnRHRI) on the pituitary gonadotropes. However, GnRH I and its receptor mRNA transcripts have also been detected in some extrapituitary tissues, including ovary and placenta (1, 2, 3), and there is increasing evidence that GnRH I may act as an autocrine or paracrine factor in regulating local cellular functions in these organs (3, 4, 5). Besides a classical form (GnRH I or GnRH), a second form of GnRH (chicken GnRH, or GnRH II) has been detected in human tissues (6). GnRH II is encoded by a different gene and expressed at significantly higher levels outside the brain, especially in kidney, bone marrow, and prostate (6). We and others (7, 8, 9) have demonstrated that the expressions of GnRH I, GnRH II, and GnRHRI mRNAs are regulated at least in part at the transcriptional levels. The change in GnRHRI numbers and its mRNA levels on the pituitary gonadotropes throughout the estrous cycle (10, 11) and after gonadectomy (11, 12) suggests a role for gonadal steroids in regulation of the GnRHRI gene. It is well established that gonadal steroids can influence GnRH I secretion. Estrogen acts in a classic feedback loop between the gonads and the brain (13, 14). It has both a positive and a negative effect on GnRH I secretion. For the greater part of the ovarian cycle, estrogen restrains GnRH I and LH secretion by a negative feedback action (15). Progesterone (P4) is also a dominant ovarian steroid of the mammalian reproductive cycle and serves a number of important regulatory roles. P4 regulates hypothalamic-pituitary functions through a feedback mechanism in animals (16, 17) and humans (18, 19). An elevation of P4 in luteal phase inhibits pulsatile secretion of GnRH I and LH (20, 21, 22) and prevents the occurrence of GnRH I (23) and LH (24) surges in response to fluctuations in peripheral estradiol (E2) levels that accompany the waves of follicular growth occurring in the ovary (25).

These effects of P4 are basically mediated through its binding to a specific intranuclear receptor, the P4 receptor (PR) (26). PR belongs to the nuclear receptor superfamily, and to date, two isoforms of the receptor have been identified, PR A and PR B (94 and 114 kDa, respectively) (27). The PR B isoform is a full-length receptor, whereas the PR A isoform lacks 164 amino acids in the N terminus of the PR B isoform. Both isoforms have been demonstrated to function as ligand-activated transcription factors, but they are not always equal in their functional properties and P4 actions (28). In general, PR B is transcriptionally more active than PR A, but compared with PR A, the activity of PR B is also cell specific (29). In addition, the PR A isoform has been demonstrated to repress the transcriptional activities of other steroid hormone receptors, including the estrogen receptor and PR B (28, 30, 31). Therefore, the relative levels of PR A and PR B within target cells may contribute to the nature and magnitude of functional responses to P4. After binding to the P4 response element (PRE), the receptors can modulate target gene transcription by directly contacting components of the transcriptional machinery (32) or indirectly by means of coactivators, such as steroid receptor coactivator-1 (33) and p300/cAMP response element-binding protein-binding protein) (34).

Even though several studies demonstrated that P4 regulates GnRH I secretion through a negative feedback mechanism via PRs, this scenario of P4-induced gene regulation for GnRH I and GnRHRI is still controversial, and the regulation of GnRH II by P4 is virtually unknown. Moreover, the transcriptional regulation of human GnRHs and GnRHRI is poorly understood due to the lack of appropriate cell models. Recently, a human cerebellar medulloblastoma cell line, TE671, was found to express and secrete both GnRH I and GnRH II, thus allowing us to investigate transcriptional regulation of the human GnRH system. In the present study we examined the transcriptional regulation of GnRH I, GnRH II, and GnRHRI by P4 and demonstrated the distinct functions of P4 and PR A/PR B in the regulation of the human GnRH-GnRHRI system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture

Human cerebellar medulloblastoma (TE671) cells were obtained from American Type Culture Collection (Manassas, VA). Neuronal TE671 cells were maintained in DMEM (Invitrogen Life Technologies, Inc., Burlington, Canada) supplemented with 10% fetal bovine serum (HyClone, Logan, UT). Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 in air. The cells were passaged when they reached about 90% confluence using a trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA).

Preparation of plasmid constructs

Human GnRHRI-luciferase construct (p2300-LucF) was prepared as previously described (8, 35). PR B expression vector was provided by Dr. P. Chambon (Institut National de la Santé et de la Recherche Médicale, University Louis Pasteur, Paris, France), and pOP13-hPR A plasmid was a gift from Dr. Graham (University of Sydney Westmead Hospital, Sydney, Australia). The 2xPRE-tk-Luc vector contains two copies of a consensus PRE upstream of the thymidine kinase promoter and was provided by Dr. McDonnell (Duke University Medical Center, Durham, NC). Plasmid DNAs for transfection studies were prepared using Plasmid Maxi Kits (Qiagen, Chatsworth, CA) following the manufacturer’s suggested procedure. The concentration and integrity of DNA were determined by measuring absorbance at 260 nm and agarose gel electrophoresis, respectively. Purified plasmid DNAs were then dissolved in 0.1x TE (1 mM Tris-Cl, pH 7.5, and 0.1 mM EDTA) to a final concentration of 1 µg/ml.

Transient transfection and reporter assay

Transfection was carried out using FuGene 6.0 (Roche Diagnostics, Indianapolis, IN) following the manufacturer’s procedure. To correct different transfection efficiencies of various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was cotransfected into the cells. Briefly, 4 x 105 TE671 cells were seeded into six-well tissue culture plates before the day of transfection in 2 ml phenol red-free DMEM (Invitrogen Life Technologies, Inc.) containing 10% charcoal-dextran-treated fetal bovine serum (HyClone, Logan, UT). One microgram of the GnRHRI promoter-luciferase construct, 0.5 µg RSV-lacZ, and the indicated amount of expression plasmids were dissolved in 100 µl phenol red-free DMEM containing 3 µl FuGene 6.0. The DNA mixture was incubated for 45 min at room temperature, then applied to the cells. Incubation of the cells with transfection medium continued for approximately 24 h at 37 C in 5% CO2. After 24-h transfection, the cells were treated with various concentration of P4 or RU486 for different time periods before harvest. Ethanol was added to the control medium in the same final solvent concentration (typically 0.1%). The cellular lysates were collected with 150 µl reporter lysis buffer (Promega Corp., Nepean, Canada) and cell lysis buffer, and assayed for luciferase activity and ß-galactosidase activity immediately with the Luciferase Assay System and ß-Galactosidase Enzyme Assay System (Promega Corp.). Luminescence was measured using a Lumat LB 9507 luminometer (EG&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.

Pharmacological treatments

P4 and mifepristone (RU486) were purchased from Sigma-Aldrich Corp. (Oakville, Canada). In the experiments investigating the effects of P4 and RU486 on luciferase activity, cells were treated with corresponding drugs for the indicated periods of time before experiments. The concentrations of P4 (36, 37, 38, 39) and RU486 (9, 36) to treat the cells were decided based on other studies in the regulation of GnRH system.

Western blot analysis

For Western blot analysis, approximately 1 x 106 cells were incubated in 100 µl cell lysis RIPA [containing 1x PBS (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 mg/ml phenylmethylsulfonylfluoride, 30 mg/ml aprotinin, and 10 mg/ml leupeptin] for 15 min on ice. The cellular debris was removed by centrifugation, and the protein concentration in the cell lysates was determined using a modified Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). Aliquots of protein from TE671 cells were taken from the total cell lysates and subjected to SDS-PAGE under reducing conditions. The separated proteins were then electrophoretically transferred onto nitrocellulose paper (Hybond-C, Amersham Biosciences, Morgan, Canada). The membranes were blocked with 5% (wt/vol) nonfat milk in Tris-buffered saline containing 20 mM Tris-Cl (pH 7.40), 500 mM NaCl, and 0.1% (wt/vol) Tween 20 for at least 1 h before the addition of PR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in 1:1500 dilution. This antibody reacts with both PR A and PR B of human origin. Incubation with an antibody incubation and washing were performed in Tris-buffered saline with 0.1% Tween 20. The enhanced chemiluminescence system (ECL, Amersham Biosciences) was used for detection. Membranes were visualized by exposure to Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY).

RNA extraction and RT-PCR

Total RNA was isolated from cell cultures using the RNeasy Mini Kit (Qiagen). One microgram of total RNA extracted from each cell line was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen Life Technologies, Inc., San Diego, CA) according to the manufacturer’s suggested protocol. PCR amplifications were carried out in 20-µl reactions containing 1 µl cDNA, 2.5 U Taq polymerase (Invitrogen Life Technologies, Inc.) and its buffer, 1.5 mm MgCl2, 2 mM deoxynucleotide triphosphate, and 50 pmol of forward and reverse primers. Primers for GnRH I and GnRH II were designed based on the published sequence (9, 40). The forward and reverse primers for GnRH I (accession no. M12578) were 5-ATTCT ACTGA CTTGG TGCGTG-3 and 5-GGAAT ATGTG CAACT TGGTG T-3, respectively. Forward and reverse primers for GnRH II (accession no. AF036329) were 5-GCCCA CCTTG GACCC TCAGA G-3 and 5-CCAAT AAAGT GTGAG GTTCT CCG-3, respectively. PCR amplification for GnRH I was carried out for 27 cycles with denaturing at 94 C for 60 sec, annealing at 60 C for 60 sec, and extension at 72 C for 90 sec, followed by a final extension at 72 C for 15 min. The PCR for GnRH II was performed with denaturing for 1 min at 94 C, annealing for 60 sec at 60 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C for 29 cycles (40). Ten microliters of PCR products were fractionated on a 1.5% agarose gel with ethidium bromide. The expected PCR products of GnRH I and GnRH II were isolated from the gel and sequenced by the dideoxynucleotide chain termination method. Sequence analysis revealed that GnRH I and GnRH II cDNAs have sequences identical to those of the published human GnRH I and GnRH II. The sizes for GnRH II and GnRH I cDNAs were 327 and 380 bp, respectively.

Data analysis

Data are presented as the mean ± SD. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P4 regulates human GnRHRI promoter activity

The presence of endogenous GnRHRI was observed in TE-671 cells using RT-PCR/Southern blot analysis (data not shown). To examine the transcriptional regulation of the human GnRHRI gene by P4, a full-length human GnRHRI promoter-luciferase construct (p2300-LucF) was transiently transfected into TE671 cells and treated with increasing concentrations of P4 for 24 h. A significant decrease in promoter activity was observed after treatment with P4 at 10–6 and 10–5 M (87% and 60%, respectively; Fig. 1AGo). This inhibitory effect on the transcriptional level of GnRHRI was shown after 12 and 24 h of treatment with P4 by 71% and 55%, respectively (Fig. 1BGo). To confirm the specificity of P4 in the expression of human GnRHRI, the human neuronal cells were cotreated with RU486 (10–5 M), an antagonist of PR. Although RU486 itself had no effect on expression of GnRHRI transcript, cotreatment with P4 plus RU486 reversed the P4-induced decrease in GnRHRI promoter activity up to 49% in these cells (Fig. 2Go).



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  		FIG. 1. Dose- and time-dependent effects of P4 on GnRHRI promoter activity. The human GnRHRI promoter-luciferase construct p2300-LucF was transiently transfected into TE671 cells by FuGene 6.0 reagent. The RSV-lacZ vector was also cotransfected to normalize the transfection efficiency. After 24-h transfection, the cells were treated with different concentrations of P4 for 24 h (A) or with P4 (10–5 M) for various time periods (B). The relative promoter activity is represented as luciferase activity/ß-galactosidase activity. Values represent the mean ± SD of three independent experiments performed in duplicate. a, P < 0.05 vs. control.

 


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  		FIG. 2. Effect of RU486 on P4-induced GnRHRI promoter activity. TE-671 cells were transiently transfected with p2300-LucF and were treated with vehicle (control), P4 (10–5 M), RU486 (10–5 M), or P4 plus RU486 (10–5 M). The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. Values represent the mean ± SD from two independent experiments performed in triplicate. a, P < 0.05 compared with control; b, P < 0.05 compared with P4.

 
PR A, but not PR B, mediates P4-induced repression of GnRHRI promoter

The presence of endogenous PR A and PR B in TE671 cells was observed by immunoblot analysis. As shown in Fig. 3AGo, the protein of PR A was highly expressed in these cells, whereas PR B protein was weakly detected. The ratio of PR B and PR A was 1:8 relatively. The molecular masses of the detected human PR A (95 kDa) and PR B (114–120 kDa) were similar to those reported previously (41). To further evaluate the mechanism of P4 action in the expression level of GnRHRI, the full-length of the PR A or PR B construct was cotransfected into TE671 cells. Transient transfection with PR A- or PR B-expressing vectors resulted in an increase in human PR A and PR B levels, respectively, by immunoblot analysis (Fig. 3AGo).



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  		FIG. 3. Effect of PR A or PR B overexpressing constructs on the GnRHRI or PRE promoter activities. A, Basal expression levels of PRs (lane 1) and overexpression of PR A (lane 2) and PR B (lane 3) were investigated by Western blot analysis. B, TE671 cells were cotransfected with p2300-LucF construct and increasing amounts of PR A or PR B plasmid DNA (0.1, 0.5, and 1 µg) in the regulation of GnRHRI promoter activity. The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. After 24 h of transfection, the cells were treated with P4 (10–5 M) for 24 h. C, The reporter plasmid 2xPRE-tk-Luc containing two copies of PRE was cotransfected with 0.5 µg of either PR A or PR B into TE671 cells. After transfection, the cells were treated with P4 (10–5 M) for 24 h. The relative promoter activity of PRE is represented as the percentage of the respective control group (vehicle-treated), for which the activity is set at 100% after being normalized by ß-galactosidase activity. Values in all panels represent the mean ± SD of three independent experiments performed in triplicate. a, P < 0.05 vs. control; b, P < 0.05 vs. P4-treated control.

 
The overexpression of PR A increased by 2-fold the sensitivity toward P4 treatment in the promoter activity of GnRHRI (Fig 3BGo). The decreased effect of P4 on the GnRHRI promoter activity was amplified (P < 0.05) after cotransfection with 0.1 and 0.5 µg PR A. Interestingly, P4 action in the GnRHRI promoter activity was reversed by PR B in a dose-dependent fashion after the cells were cotransfected with PR B. This result suggests distinct functions of PR A and PR B on GnRHRI promoter activity, and diverse regulation of PR A and PR B in the regulation of GnRHRI at the transcriptional level.

Distinct effect of PR A or PR B overexpression on the PRE promoter activity

To rule out the basal effect of P4, we cotransfected the reporter plasmid 2x PRE-tk-Luc with either PR A or PR B into TE 671 cells and then treated the cells in the presence or absence of P4 (Fig. 3CGo). The reporter plasmid 2xPRE-tk-Luc contains two copies of a consensus PRE upstream of the thymidine kinase. The activity of the heterologous PRE promoter was repressed by 3-fold and increased by 4-fold in the response to P4 (10–5 M) when PR A or PR B was overexpressed in these cells, respectively (Fig. 3CGo). However, without overexpression of PR A or PR B, P4 had no significant effect on PRE promoter activity. These results indicate that P4 basically represses PRE promoter activity via PR A and increases it via a PR B-dependent mechanism.

Effect of P4 on human GnRH I and GnRH II mRNAs

Semiquantitative RT-PCR was performed to examine the mRNA levels of GnRH I and GnRH II by P4 in TE671 cells. The 380-bp product corresponding to GnRH I, a 327-bp product corresponding to GnRH II, and a 372-bp product for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control were detected. A linear relationship was found between the cycle numbers and OD for GAPDH, GnRH I, and GnRH II, respectively (data not shown). As a result, 29 cycles for GnRH I, 27 cycles for GnRH II, and 20 cycles for GAPDH were employed for semiquantification, and the PCR products were sequenced to assure authenticity. Treatment with P4 (10–6 and 10–5 M) resulted in increase in the mRNA level of GnRH I (40% and 100%, respectively) compared with the control (Fig. 4AGo). In a time-dependent study, treatment with P4 (10–6 M) increased the expression of GnRH I mRNA significantly at 12 and 24 h, as shown in Fig. 4BGo. In contrast, there was no significant difference in the expression level of GnRH II mRNA by P4 (see Fig. 6Go). The P4-induced increase in GnRH I expression was completely reversed by RU486 (10–5 M), whereas RU486 itself had no significant effect on the expression of GnRH I mRNA (Fig. 5Go).



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  		FIG. 4. Time and dose-dependent effects of P4 on the expression level of GnRH I mRNA. TE671 cells were treated with P4 for 24 h in a dose-dependent manner (A) or were treated for various times with P4 (10–6 M; B). Total RNA was extracted from TE671 cells, and 1 µg total RNA was reverse transcribed. The expression level of GnRH I mRNA was estimated by semiquantitative RT-PCR and normalized by GAPDH. Values in all panels represent the mean ± SD of three independent experiments performed in triplicate. a, P < 0.05 vs. control.

 


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  		FIG. 6. Effect of P4 on the expression of GnRH I and GnRH II mRNA in PRs-overexpressing TE-671 cells. The overexpressing vectors of PR A and PR B (0.5 µg) were transiently transfected into TE671 cells, and after 24 h of transfection, the cells were treated with P4 (10–6 M). The expression levels of GnRH I (A) and GnRH II (B) mRNAs were estimated by semiquantitative RT-PCR and normalized by GAPDH. Values in all panels represent the mean ± SD of three independent experiments performed in triplicate. a, P < 0.05 vs. control; b, P < 0.05 vs. P4-treated.

 


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  		FIG. 5. Effect of RU486 on P4-induced GnRH I mRNA. TE671 cells were treated with P4 (10–6 M), or P4 plus RU486 (10–6 M). The expression level of GnRH I mRNA was estimated by semiquantitative RT-PCR and normalized by GAPDH. Values in all panels represent the mean ± SD of three independent experiments performed in triplicate. a, P < 0.05 vs. control; b, P < 0.05 vs. P4-treated.

 
Effects of PR A and PR B on human GnRH I and GnRH II mRNAs

PR A or PR B was overexpressed to investigate an involved signaling pathway of the P4-induced increase in the expression level of GnRH I mRNA. Treatment with P4 (10–6 M) induced an increase in the expression level of the GnRH I gene, and overexpression of PR B resulted in an increased sensitivity to P4 treatment (1.5-fold vs. P4-only group), as shown in Fig. 6Go. However, the overexpression of PR A or PR B failed to affect the expression level of GnRH II mRNA in the absence or presence of P4.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it is now well established that feedback actions of gonadal steroid hormones play an important role in regulating the function of the GnRH neurons, the precise nature of this influence is not well understood (42, 43). P4, one of the principal ovarian steroid hormones, is known to exert inhibitory and stimulatory effects on gonadotropin secretion in several species, including humans, and these actions involve the modulation of pulsatile GnRH I secretion (44, 45). The precise mechanism by which P4 influences the activity of the GnRH neurons is presently unknown. To date, few studies have been performed examining the regulation of GnRH in humans, because of the lack of appropriate cell models. Previously, we have shown that P4 treatment resulted in a decrease in GnRHRI promoter activity in mouse pituitary cells, whereas P4 increased its activity in human placenta cells (41). We also identified a putative PRE located between –535 and –521 at P2300-LucF, related to the translation start site, which is responsible for the P4 action. Due to the limit of human cell lines as a model to study GnRH system in the brain, the TE671 cell line was chosen as originally derived from a human cerebellar biopsy specimen designated a medulloblastoma (46), representing one of few continuous cell lines to exhibit a neuronal phenotype. This cell line was positively stained for neuron-specific enolase and contained acetylcholinesterase and glutamic acid decarboxylase activity (46). In addition, we selected the TE671 cell line to study the effect of P4 on the GnRH system because a recent study demonstrated that TE671 cells express and secrete GnRH I and GnRH II peptides (47). To further elucidate the molecular mechanism of P4 in the regulation of different components of the human GnRH-GnRHRI, we investigated the transcriptional regulation of GnRH I, GnRH II, and GnRHRI by P4 in TE671 cells. The present results indicate that P4 had a negative role in human GnRHRI promoter activity, and this effect was reversed by RU486, which has been known as a PR and glucocorticoid receptor antagonist, suggesting that the P4 effect on GnRHRI promoter activity is mainly mediated by PRs.

To further investigate the mechanism of P4 function on the expression of GnRHRI gene, we cotransfected the cells with the P2300-LucF construct and PR A or PR B. Overexpression of PR A in TE671 cells increased the sensitivity to P4 and resulted in an additional repression of GnRHRI promoter activity after P4 treatment. Interestingly, overexpression of PR B reversed the PR A-induced repression of P4 in the GnRHRI promoter in a dose-dependent manner after cotransfection with PR B, indicating that the P4-induced negative effect on the GnRHRI expression at the transcriptional level is mediated by PR A, not PR B. PRs are ligand-inducible transcriptional regulators that control gene expression upon binding to the PRE in the vicinity of target promoters or influence gene expression by interaction with other transcriptional factors, i.e. nuclear factor-{kappa}B and signal transducer and activator of transcription, and these interactions result in the repression of transcriptional activities (48). Two PR isoforms have distinct transcriptional properties in a cell-specific manner. In general, PR B acts as a potent transcriptional activator, whereas the transcriptional activity of PR A proceeds in a cell- or gene-specific manner (28, 29, 31, 49). Interestingly, where PR A is inactive, it can act as a potent trans-dominant repressor of PR B-mediated gene transcription (28). To investigate the transcriptional activity of PR A and PR B, the cells were cotransfected with 2xPRE-tk-Luc vector and PR A or PR B. In TE-671 cells, PR A was shown to be a transcriptional repressor, whereas PR B acted as a transcriptional activator in the presence of P4. These results suggest that PR A and PR B act in a gene-specific manner on the GnRHRI promoter in the presence of P4. Although PR B acts as a strong transcriptional activator of the PRE promoter, P4 did not accelerate the PR B-induced transcriptional activity of GnRHRI promoter even at a high dose. It can be assumed that the overexpression of PR B may induce the overexpression of PR A, and then increased PR A probably reverses the function of PR B on the GnRHRI promoter. Taken together, PR A is more functional on the GnRHRI promoter in a gene-specific manner, and PR B could play a role as a transdominant regulator of PR A action.

Because of their differential transcriptional properties, the relative levels of PR A and PR B within target cells may direct the overall functional responses to P4. The expression of PR A and PR B isoforms was examined by immunoblot blot analysis. Both PR isoforms were expressed in TE671 cells; however, the expression level of PR A is high, whereas PR B expression is relatively very low in these cells. This finding supports the idea that inhibitory role of P4 on GnRHRI gene expression is mediated by PR A, because a much higher endogenous level of PR A was detected in this cell line. In addition, these results indicate that PR A is more dominant than PR B and is involved in the down-regulation of human GnRHRI at the transcriptional level. In the present study only a high dose of P4 had a significant effect on the GnRH system. To explain this, we need to consider the local synthesis of P4 and its metabolites in the brain.

Steroid hormones, including P4, are synthesized within the central and peripheral nervous system, mostly by glial cells known as neurosteroids (50). They are essential for important nervous functions, and their local synthesis may protect the brain from drops in circulating steroid levels, because they are synthesized, for example, during the estrous cycle and during ageing (50). Because of the local synthesis, the levels of P4, its metabolites, and dehydroepiandrosterone in the nervous system are much higher than plasma levels (up to 10 times higher) (50, 51). Due to this local synthesis of P4 in the brain, the GnRH system in these neuronal cells requires a higher concentration of exogenous P4 to obtain a significant effect. In contrast, in the central nervous system, P4 may exert membrane effects through the opioidergic, cholinergic, and {gamma}-aminobutyric acid systems (52). The role of opioids in the mediation of P4 action on GnRH neurons has been suggested in monkeys (52). Thus, a high dose of P4 may also induce these alternative signaling pathways in this cell line.

In the present study the expression levels of GnRH I and GnRH II were differentially regulated by P4. Differential regulation of two forms of GnRHs has been recently observed. In goldfish, the ratio between salmon GnRH I and GnRH II changed with sexual maturation (53). A smaller increase in the level of GnRH II than in salmon GnRH I was observed in the pituitary (53). In the chicken, castration led to a change in the level of only chicken GnRH I, but not GnRH II (54). In addition, the differential regulation of GnRH I and GnRH II by E2 has been recently demonstrated in TE671 cells, indicating that E2 increased endogenous GnRH II mRNA levels and decreased endogenous GnRH I mRNA levels (47). Taken together, the differential regulation of two forms of GnRHs by sex steroid hormones suggests that GnRH I and GnRH II may be temporally regulated by steroids during different phases of menstrual cycle. In the present study treatment with P4 in TE671 cells resulted in an increase in GnRH I mRNA levels, whereas P4 had no significant effect on the GnRH II transcriptional level. Because the transcriptional activities of PR A and PR B on the GnRHRI promoter were distinct in this cell line, we also examined the biological pathway of P4 action in the regulation of GnRH I gene. Surprisingly, the transfection of these cells with PR B resulted in an increase in the P4-induced effect on the GnRH I gene. This suggests that even though the expression level of PR B is low, the effect of P4 on the expression of GnRH I mRNA is mediated by PR B, not PR A. A possible mechanism of distinct action of P4 is derived from the recruitment of different coregulators depending on promoter context.

To date, the effect of P4 on the expression of GnRH I is still controversial; it has both stimulatory and inhibitory actions. Even though P4 has a negative or no effect on the expression of GnRH, cotreatment of P4 with E2 can induce the secretion of GnRH I, and the reason for this difference may be the induction of PRs by E2 (55, 56). The differential expression and gene-specific regulation of PR A or PR B could be one of the clues for explaining various P4 actions on regulation of the GnRH system. In conclusion, these results indicate that P4 is a potent regulator of GnRHRI at the transcriptional level as well as GnRH I mRNA. This distinct effect of P4 on the GnRH system may be derived from different pathways, through PR A or PR B.


   Acknowledgments
 
We thank Drs. Graham and P. Chambon for generously providing the PR A and PR B expression vectors. In addition, the 2XPRE-tk-Luc vector was a gift from Dr. McDonnell. We express special thanks to Dr. C. M. Yeung for his assistance and discussion during the course of this study.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research. P.C.K.L. is a distinguished scholar of the Michael Smith Foundation for Health Research. B.-S.A. is a recipient of a bursary award from the Strategic Training Initiative in Research in the Reproductive Health Sciences.

First Published Online November 23, 2004

Abbreviations: E2, Estradiol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GnRHRI, GnRH I receptor; P4, progesterone; PR, P4 receptor; PRE, P4 response element; RSV, Rous sarcoma virus.

Received February 18, 2004.

Accepted October 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Peng C, Fan NC, Ligier M, Vaananen J, Leung PC 1994 Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells. Endocrinology 135:1740–1746[Abstract]
  2. Minaretzis D, Jakubowski M, Mortola J, Pavlou S 1995 Gonadotropin-releasing hormone receptor gene expression in human ovary and granulosa-lutein cells. J Clin Endocrinol Metab 80:430–434[Abstract]
  3. Lin L, Roberts V, Yen S 1995 Expression of human gonadotropin-releasing hormone receptor gene in the placenta and its functional relationship to human chorionic gonadotropin secretion. J Clin Endocrinol Metab 80:580–585[Abstract]
  4. Kang SK, Tai C-J, Nathwani PS, Choi K-C, Leung PCK 2001 Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone in human granulosa-luteal cells. Endocrinology 142:671–679[Abstract/Free Full Text]
  5. Bussenot I, Azoulay-Barjonet C, Parinaud J 1993 Modulation of the steroidogenesis of cultured human granulosa-lutein cells by gonadotropin-releasing hormone analogs. J Clin Endocrinol Metab 76:1376–1379[Abstract]
  6. White RB, Eisen JA, Kasten TL, Fernald RD 1998 Second gene for gonadotropin-releasing hormone in humans. Proc Natl Acad Sci USA 95:305–309[Abstract/Free Full Text]
  7. Duval DL, Nelson SE, Clay CM 1997 The Tripartite basal enhancer of the gonadotropin-releasing hormone (GnRH) receptor gene promoter regulates cell-specific expression through a novel GnRH receptor activating sequence. Mol Endocrinol 11:1814–1821[Abstract/Free Full Text]
  8. Ngan ESW, Cheng PKW, Leung PCK, Chow BKC 1999 Steroidogenic factor-1 interacts with a gonadotrope-specific element within the first exon of the human gonadotropin-releasing hormone receptor gene to mediate gonadotrope-specific expression. Endocrinology 140:2452–2462[Abstract/Free Full Text]
  9. Khosravi S, Leung PC 2003 Differential regulation of gonadotropin-releasing hormone (GnRH)I and GnRHII messenger ribonucleic acid by gonadal steroids in human granulosa luteal cells. J Clin Endocrinol Metab 88:663–672[Abstract/Free Full Text]
  10. Bauer-Dantoin AC, Hollenberg AN, Jameson JL 1993 Dynamic regulation of gonadotropin-releasing hormone receptor mRNA levels in the anterior pituitary gland during the rat estrous cycle. Endocrinology 133:1911–1914[Abstract]
  11. Funabashi T, Brooks PJ, Weesner GD, Pfaff DW 1994 Luteinizing hormone-releasing hormone receptor messenger ribonucleic acid expression in the rat pituitary during lactation and the estrous cycle. J Neuroendocrinol 6:261–266[Medline]
  12. Sakurai H, Adams BM, Adams TE 1997 Concentration of GnRH receptor and GnRH receptor mRNA in pituitary tissue of orchidectomized sheep: effect of oestradiol, progesterone, and progesterone withdrawal. J Endocrinol 152:91–98[Abstract]
  13. Gore AC, Roberts JL 1997 Regulation of gonadotropin-releasing hormone gene expression in vivo and in vitro. Front Neuroendocrinol 18:209–245[CrossRef][Medline]
  14. Herbison AE 1998 Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocr Rev 19:302–330[Abstract/Free Full Text]
  15. Chongthammakun S, Terasawa E 1993 Negative feedback effects of estrogen on luteinizing hormone-releasing hormone release occur in pubertal, but not prepubertal, ovariectomized female rhesus monkeys. Endocrinology 132:735–743[Abstract]
  16. Sagrillo CA, Grattan DR, McCarthy MM, Selmanoff M 1996 Hormonal and neurotransmitter regulation of GnRH gene expression and related reproductive behaviors. Behav Genet 26:241–277[CrossRef][Medline]
  17. Schumacher M, Coirini H, Robert F, Guennoun R, El-Etr M 1999 Genomic and membrane actions of progesterone: implications for reproductive physiology and behavior. Behav Brain Res 105:37–52[CrossRef][Medline]
  18. Poindexter III AN, Dildy GA, Brody SA, Snabes MC, Brodyand SA 1993 The effects of a long-acting progestin on the hypothalamic-pituitary-ovarian axis in women with normal menstrual cycles. Contraception 48:37–45[CrossRef][Medline]
  19. Alexandris E, Milingos S, Kollios G, Seferiadis K, Lolis D, Messinis IE 1997 Changes in gonadotrophin response to gonadotrophin releasing hormone in normal women following bilateral ovariectomy. Clin Endocrinol (Oxf) 47:721–726[CrossRef][Medline]
  20. Goodman R, Karsch F 1980 Pulsatile secretion of luteinizing hormone: differential suppression by ovarian steroids. Endocrinology 107:1286–1290[Abstract]
  21. O’Byrne K, Thalabard J, Grosser P, Wilson RC, Williams CL, Chen MD, Ladendorf D, Hotchkiss J, Knobil E 1991 Radiotelemetric monitoring of hypothalamic gonadotropin-releasing hormone pulse generator activity throughout the menstrual cycle of the rhesus monkey. Endocrinology 129:1207–1214[Abstract]
  22. Karsch F, Cummins J, Thomas G, Clarke I 1987 Steroid feedback inhibition of pulsatile secretion of gonadotropin-releasing hormone in the ewe. Biol Reprod 36:1207–1218[Abstract]
  23. Kasa-Vubu J, Dahl G, Evans N, Thrun LA, Moenter SM, Padmanabhan V, Karsch FJ 1992 Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone. Endocrinology 131:208–212[Abstract]
  24. Scaramuzzi RJ, Tillson SA, Thorneycroft IH, Caldwell BV 1971 Action of exogenous progesterone and estrogen on behavioral estrus and luteinizing hormone levels in the ovariectomized ewe. Endocrinology 88:1184–1189[Medline]
  25. Souza C, Campbell B, Baird D 1997 Follicular dynamics and ovarian steroid secretion in sheep during the follicular and early luteal phases of the estrous cycle. Biol Reprod 56:483–488[Abstract]
  26. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  27. Horwitz K, Alexander P 1983 In situ photolinked nuclear progesterone receptors of human breast cancer cells: subunit molecular weights after transformation and translocation. Endocrinology 113:2195–2201[Abstract]
  28. Vegeto E, Shahbaz M, Wen D, Goldman M, O’Malley B, McDonnell D 1993 Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol 7:1244–1255[Abstract]
  29. Sartorius C, Melville M, Hovland A, Tung L, Takimoto G, Horwitz K 1994 A third transactivation function (AF3) of human progesterone receptors located in the unique N-terminal segment of the B-isoform. Mol Endocrinol 8:1347–1360[Abstract]
  30. Kraus W, Weis K, Katzenellenbogen B 1995 Inhibitory cross-talk between steroid hormone receptors: differential targeting of estrogen receptor in the repression of its transcriptional activity by agonist- and antagonist-occupied progestin receptors. Mol Cell Biol 15:1847–1857[Abstract]
  31. McDonnell D, Goldman M 1994 RU486 exerts antiestrogenic activities through a novel progesterone receptor A form-mediated mechanism. J Biol Chem 269:11945–11949[Abstract/Free Full Text]
  32. Ing NH, Beekman JM, Tsai SY, Tsai MJ, O’Malley BW 1992 Members of the steroid hormone receptor superfamily interact with TFIIB (S300-II). J Biol Chem 267:17617–17623[Abstract/Free Full Text]
  33. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  34. Smith CL, Onate SA, Tsai MJ, O’Malley BW 1996 CREB binding protein acts synergistically with steroid receptor coactivator-1 to enhance steroid receptor-dependent transcription. Proc Natl Acad Sci USA 93:8884–8888[Abstract/Free Full Text]
  35. Cheng KW, Chow BKC, Leung PCK 2001 Functional mapping of a placenta-specific upstream promoter for human gonadotropin-releasing hormone receptor gene. Endocrinology 142:1506–1516[Abstract/Free Full Text]
  36. Han S, Sidell N 2003 RU486-induced growth inhibition of human endometrial cells involves the nuclear factor-{kappa}B signaling pathway. J Clin Endocrinol Metab 88:713–719[Abstract/Free Full Text]
  37. Christoffers AB, Kreisler M, Willershausen B 2003 Effects of estradiol and progesterone on the proliferation of human gingival fibroblasts. Eur J Med Res 8:535–542[Medline]
  38. Schumacher M, Guennoun R, Mercier G, Desarnaud F, Lacor P, Benavides J, Ferzaz B, Robert F, Baulieu EE 2001 Progesterone synthesis and myelin formation in peripheral nerves. Brain Res Brain Res Rev 37:343–359[CrossRef][Medline]
  39. Seeger H, Wallwiener D, Mueck AO 2003 The effect of progesterone and synthetic progestins on serum- and estradiol-stimulated proliferation of human breast cancer cells. Horm Metab Res 35:76–80[CrossRef][Medline]
  40. Nathwani PS, Kang SK, Cheng KW, Choi KC, Leung PC 2000 Regulation of gonadotropin-releasing hormone and its receptor gene expression by 17ß-estradiol in cultured human granulosa-luteal cells. Endocrinology 141:1754–1763[Abstract/Free Full Text]
  41. Cheng KW, Cheng C-K, Leung PCK 2001 Differential role of PR-A and -B isoforms in transcription regulation of human GnRH receptor gene. Mol Endocrinol 15:2078–2092[Abstract/Free Full Text]
  42. Levine J 1997 New concepts of the neuroendocrine regulation of gonadotropin surges in rats. Biol Reprod 56:293–302[Abstract]
  43. Herbison AE 1998 Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocr Rev 19:302–330
  44. Leipheimer R, Bona-Gallo A, Gallo R 1984 The influence of progesterone and estradiol on the acute changes in pulsatile luteinizing hormone release induced by ovariectomy on diestrous day 1 in the rat. Endocrinology 114:1605–1612[Abstract]
  45. Skinner DC, Evans NP, Delaleu B, Goodman RL, Bouchard P, Caraty A 1998 The negative feedback actions of progesterone on gonadotropin-releasing hormone secretion are transduced by the classical progesterone receptor. Proc Natl Acad Sci USA 95:10978–10983[Abstract/Free Full Text]
  46. Hemmick LM, Ross ME, Evinger MJ 1995 Regulation of PNMT gene promoter constructs transfected into the TE 671 human medulloblastoma cell line. Neurosci Lett 201:77–80[CrossRef][Medline]
  47. Chen A, Zi K, Laskar-Levy O, Koch Y 2002 The transcription of the hGnRH-I and hGnRH-II genes in human neuronal cells is differentially regulated by estrogen. J Mol Neurosci 18:67–76[Medline]
  48. Truss M, Beato M 1993 Steroid hormone receptors: interaction with deoxyribonucleic acid and transcription factors. Endocr Rev 14:459–479[Abstract]
  49. Wen D, Xu Y, Mais D, Goldman M, McDonnell D 1994 The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells. Mol Cell Biol 14:8356–8364[Abstract/Free Full Text]
  50. Baulieu E, Schumacher M 2000 Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination. Steroids 65:605–612[CrossRef][Medline]
  51. Warner M, Gustafsson JA 1995 Cytochrome P450 in the brain: neuroendocrine functions. Front Neuroendocrinol 16:224–236[CrossRef][Medline]
  52. Chabbert-Buffeta N, Skinner DC, Caraty A, Bouchard P 2000 Neuroendocrine effects of progesterone. Steroids 65:613–620[CrossRef][Medline]
  53. Rosenblum PM, Goos HJ, Peter RE 1994 Regional distribution and in vitro secretion of salmon and chicken-II gonadotropin-releasing hormones from the brain and pituitary of juvenile and adult goldfish, Carassius auratus. Gen Comp Endocrinol 93:369–379[CrossRef][Medline]
  54. Sharp PJ, Talbot RT, Main GM, Dunn IC, Fraser HM, Huskisson NS 1990 Physiological roles of chicken LHRH-I and -II in the control of gonadotrophin release in the domestic chicken. J Endocrinol 124:291–299[Abstract]
  55. Robinson JE, Healey AE, Harris TG, Messent EA, Skinner DC, Taylor JA, Evans NP 2000 The negative feedback action of progesterone on luteinizing hormone release is not associated with changes in GnRH mRNA expression in the Ewe. J Neuroendocrinol 12:121–129[CrossRef][Medline]
  56. Barni T, Maggi M, Fantoni G, Granchi S, Mancina R, Gulisano M, Marra F, Macorsini E, Luconi M, Rotella C, Serio M, Balboni GC, Vannelli GB 1999 Sex steroids and odorants modulate gonadotropin-releasing hormone secretion in primary cultures of human olfactory cells. J Clin Endocrinol Metab 84:4266–4273[Abstract/Free Full Text]



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