Differential Role of PR-A and -B Isoforms in Transcription Regulation of Human GnRH Receptor Gene

Kwai Wa Cheng, Chi-Keung Cheng and Peter C. K. Leung

Department of Obstetrics and Gynaecology, The 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 Gynaecology, The 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
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The presence of progesterone response element (PRE) in the 5'-flanking region of the human GnRH receptor (GnRHR) suggests the possible regulation of this gene by progesterone (P). In the present study, we examined the effects of P in transcriptional regulation of human GnRHR gene expression at the pituitary and placenta levels since the GnRHR has been detected in both tissues. By the use of transient transfection assays, a differential regulation of human GnRHR promoter activity by P was observed. P treatment resulted in a decrease in promoter activity in the pituitary {alpha}T3–1 cells, suggesting a P-mediated inhibitory action. Interestingly, P is found to have a stimulatory role at the placental expression of this gene. Addition of RU486 to, or inhibition of endogenous P production by, the placental JEG-3 cells leads to a decrease in promoter activity, which is reversed by the replacement of P. Further studies have identified a putative PRE, namely human GR-PRE (located between -535 and -521, related to translation start site), that may be responsible for the P action since the mutation of these motifs reversed the P-mediated effects. The binding of PR to this element is confirmed by antibody supershift assays. The physiological effects of P are mediated through two PR isoforms, namely PR-A and PR-B. In the present study, overexpression of human PR-A resulted in a decrease in human promoter activity in both pituitary and placental cells. Interestingly, overexpression of PR-B exhibits a cell-dependent transcriptional activity, whereby it functions as a transcription activator in the placenta but as a transcription repressor in the pituitary. In summary, our results demonstrated a differential usage of PR-A and PR-B in transcriptional regulation of human GnRHR gene expression by P at the pituitary and placenta levels.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IT IS WELL established that hypothalamic GnRH (1) plays a major role in controlling reproductive function. It regulates the biosynthesis and secretion of gonadotropin after binding to its receptor (GnRHR) at the level of the pituitary gonadotrope cells (1). In addition to the hypothalamic-pituitary axis, GnRH and GnRHR have been detected in other reproductive tissue, including the human placenta. Placental GnRH is biochemically and structurally identical to hypothalamic GnRH (2, 3, 4). Functionally, GnRH has been demonstrated to regulate human CG (hCG) secretion (5). This stimulatory effect was inhibited by treatment with a GnRH antagonist (6), suggesting a receptor-mediated process. Recently, we have reported the isolation of full-length GnRHR from human placental cells, including choriocarcinoma JEG-3 cells, immortalized extravillous trophoblast, and primary culture of trophoblast cells (7). The colocalization of GnRH and its receptor and the effect of exogenous GnRH on hCG secretion strongly suggest that the placenta may possess its own GnRH system, analogous to the hypothalamus-pituitary system. We and others (8, 9, 10) have demonstrated that the regulation of GnRHR number is mediated, at least in part, at the transcriptional level of GnRHR gene expression. Therefore, regulation of GnRHR expression has been implicated as one of the important mechanisms in controlling GnRH action.

The change in GnRHR numbers and its mRNA levels on the pituitary gonadotropes throughout the estrous cycle (11, 12, 13) and after gonadectomy (14, 15) suggests a role of gonadal steroids in regulating the expression of the GnRHR gene. It has also been shown that progesterone (P) negatively regulated the hypothalamic- pituitary functions through a negative feedback mechanism in animals (16, 17) and in humans (18, 19, 20). The physiological effects of P are mediated through a specific nuclear receptor protein, PR. Two major isoforms of PR, namely PR-A and PR-B, have been described. The large PR-B form contains an additional 164 amino acids at the N terminus that are missing in the truncated PR-A form (21). Hormonal binding to PRs results in the dissociation of heat shock proteins, thereby allowing receptors to form dimers and bind specific nucleotide sequences known as progesterone response elements (PREs) (22, 23). Receptor interaction with PREs can result in either an increase or decrease in gene transcription. In cell transfection systems, the two PR isoforms have distinct transcriptional properties. In general, PR-B acts as a stronger transcriptional activator, whereas the transcriptional activity of PR-A was cell- and gene-specific dependent. Interestingly, PR-A also functions as a transcriptional inhibitor of PR-B as well as of other steroid receptor families when PR-A itself is transcriptionally inactive (24, 25, 26, 27, 28).

There is evidence that P inhibits expression of ovine GnRHR gene expression. Using primary cultures of ovine pituitary cells, P reduced the binding of GnRH (29) and decreased the amount of GnRHR mRNA (15, 30, 31). Other than these findings, no information is available on the role of P in regulating human GnRHR gene expression. In addition, the exact role of P in regulating GnRHR gene expression at the transcriptional level remains unknown. We have previously isolated the human GnRHR gene (32), and DNA analysis of the human GnRHR 5'-flanking region revealed the presence of a putative PRE (32). In light of these studies, it is reasonable to believe that the P can directly regulate the expression of the human GnRHR. As the GnRHR mRNA was detected in both pituitary and placenta, and the placenta is a major site of P secretion, the goal of the present study was to understand the molecular mechanism underlying the regulatory role of P on GnRHR gene transcription.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
P Regulates Human GnRHR Promoter Activity
To examine the transcriptional regulation of human GnRHR gene expression by P in the pituitary and placenta, full-length human GnRHR promoter-luciferase construct (p2300F-Luc) was transiently transfected into {alpha}T3-1 and JEG-3 cells, respectively, and treated with increasing concentrations of P for 24 h. Although a slight decrease (14%, P > 0.05) in promoter activity in {alpha}T3-1 cells was observed with 10 nM P treatment, a statistically significant decrease of promoter activity was achieved after treatment with 1 µM P (28%, P < 0.01) and 10 µM P (59%, P < 0.001), respectively (Fig. 1AGo). In addition, this inhibitory effect was shown to be time dependent since the degree of inhibition increased with time of treatment (Fig. 1BGo). These results suggested an inhibitory effect of P on human GnRHR gene transcription at the pituitary level. In contrast, no change in GnRHR promoter activity was observed in JEG-3 cells (data not shown). As JEG-3 cells produce a high level of P endogenously, we next used a P antagonist, RU486, to indirectly examine the effects of P on human GnRHR promoter activity in JEG-3 cells. Interestingly, a dose- and time-dependent decrease in GnRHR promoter activity was observed after addition of RU486 (Fig. 2Go, A and B), suggesting that P was important in maintaining the expression of human GnRHR gene expression in the placenta. To further confirm the stimulatory role of P in the placental expression of human GnRHR, the endogenous production of P from JEG-3 was inhibited by the addition of aminoglutethimide (AGT) (38). A dose-dependent inhibition of P production was observed and the maximal inhibition was achieved at 0.1 mM AGT (Fig. 3AGo). Furthermore, inhibition of P production by ATG resulted in a 30% decrease (P < 0.001) in human GnRHR promoter activity, and this decrease in promoter activity was reversed by the replacement of P (Fig. 3BGo). These results strongly implicate a stimulatory role of P in the expression of human GnRHR gene in the placenta.



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Figure 1. Dose- and Time-Dependent Regulation of Human GnRHR-Luciferase Vector (p2300-LucF) Activity in {alpha}T3-1 Cells Treated with P

A, The p2300-LucF transfected cells were treated with varying concentration of P (1 nM to 10 µM) for 24 h. The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. Luciferase units were calculated as luciferase activity/ß-galactosidase activity. B, The p2300-LucF transfected cells were treated with 10 µM P for the indicated time points. Relative promoter activity is shown as percentage of control. Values represent mean ± SE from triplicate assays in three separate experiments. a, P < 0.01 compared with control; b, P < 0.01 vs. the immediately adjacent group on the left.

 


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Figure 2. Dose- and Time-Dependent Regulation of Human GnRHR-Luciferase Vector (p2300-LucF) Activity in JEG-3 Cells Treated with RU486

A, The p2300-LucF transfected cells were treated with varying concentrations of RU486 (1 nM to 10 µM) for 24 h. The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. Luciferase units were calculated as luciferase activity/ß-galactosidase activity. B, The p2300-LucF transfected cells were treated with 10 µM RU486 for the indicated time points. Relative promoter activity is shown as percentage of control. Values represent mean ± SE from triplicate assays in three separate experiments. a, P < 0.01 compared with control; b, P < 0.01 vs. the immediately adjacent group on the left.

 


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Figure 3. Effects of P in Human GnRHR-Luciferase Vector (p2300-LucF) Activity in JEG-3 Cells

A, Human choriocarcinoma JEG-3 cells were treated with varying concentration (1 pM to 0.1 mM) of DL-aminoglutethimide (AGT) for 24 h. The production of P was measured by RIA and shown as percentage of control. Values represent mean ± SE from triplicate assays in two separate experiments. a, P < 0.01 from control; b, P < 0.001 vs. the immediately adjacent group on the left. B, The production of P from JEG-3 cells was inhibited with 0.1 mM AGT treatment during transfection. After transfection, the cells were incubated for an additional 24 h in the presence of 0.1 mM AGT and increasing concentrations of P (0.1 nM to 0.1 µM). Relative promoter activity is shown as percentage of control after being normalized to ß-galactosidase activity. Values represent mean ± SE from triplicate assays in three separate experiments. a, P < 0.01 compared with control.

 
Localization of P Response Region in Human GnRHR 5'-Flanking Region
To localize a specific region that mediates the P effects on the 2.3-kb 5'-flanking region of the human GnRHR gene, a series of 5'-deletion mutants were analyzed in {alpha}T3-1 and JEG-3 cells and treated with 10 µM P and 10 µM RU486, respectively. Transient transfection studies showed similar results for both cell lines (Fig. 4Go). The results revealed that a region between -577 to -227 (relative to translation start site) was involved in P-mediated action. Progressive 5'-deletion up to -577 did not affect the P- and RU486-induced inhibition of the human GnRHR promoter activity in {alpha}T3-1 and JEG-3 cells, respectively (Fig. 4Go, B and C). Further deleting the sequence from -577 to -227 (relative to translation start site) resulted in a loss of the response in P- and RU486-induced inhibition in both cells. Although we have demonstrated that an upstream promoter was predominantly used in JEG-3 cells (39), no RU486-induced decrease in promoter activity was observed in this upstream promoter in JEG-3 cells (data not shown). Taken together, these data suggest that the 350-bp region (located between -577 and -227) containing the putative transcription factor(s) binding site was important in mediating the P-mediated effects on human GnRHR promoter activity in both pituitary and placenta.



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Figure 4. Localization of the P-Responsive Region in the Human GnRHR Gene

A, Diagrammatic representation of progressive 5'-deletion constructs of p2300-LucF. Deletion mutants were transiently transfected into {alpha}T3-1 (panel B) and JEG-3 (panel C) cells by the calcium precipitation method and treated with 10 µM P and 10 µM RU486, respectively, for 24 h. The RSV-lacZ vector was cotransfected to normalize for varying transfection efficiencies. Relative promoter activity of each construct is shown as fold increase over a promoterless luciferase control pGL2-Basic, the activity of which is set to be 1, after being normalized to ß-galactosidase activity. Values represent mean ± SE of triplicate assays in three separate experiments. a, P < 0.01 compared with pGL2-Basic.

 
It has been demonstrated that RU486 binds both GR and PR (40). To eliminate the possible role of GR in mediating the RU486 action in placental cells, JEG-3 cells were transiently transfected with Sty-HLuc (-707 to +1; human GnRHR promoter-luciferase construct) and treated with increasing concentrations of dexamethasone (DEX) or P in the presence of 10 µM RU486 (Fig. 5Go). As expected, addition of 10 µM RU486 resulted in a significant decrease in promoter activity, and P (0.1 µM and 10 µM) replacement reversed this inhibitory effect. In contrast, addition of DEX did not reverse the RU486-induced decrease in promoter activity.



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Figure 5. Blocking of P Action by RU486 in JEG-3 Cells

The JEG-3 cells were transiently transfected with Sty-HLuc. Cells were harvested 24 h post transfection and were treated with vehicle (control), 10 µM RU486, 0.1 nM, 10 nM, or 1 µM DEX in the presence of 10 µM RU486, or 0.1 µM or 10 µM P in the presence of 10 µM RU486. The RSV-lacZvector was cotransfected to normalize for varying transfection efficiencies. Luciferase units were calculated as luciferase activity/ß-galactosidase activity. Values represent mean ± SE from duplicate assays in three separate experiments. a, P < 0.01 compared with control; b, P < 0.01 compared with RU486.

 
Identification of Transcription Binding Sites
DNA analysis of the DNA region between -577 and -227 identified one putative PRE binding site, namely hGR-PRE (5'-TCAACAGTGTGTTTG-3' located at -535 to -521; with 75% homology to the consensus PRE site) and a half-PRE binding site, namely hGR-hPRE (5'-AGAACA-3' located at -402 to -397; with 100% homology to the half-consensus PRE site). To examine the role of these putative PRE motifs in controlling the expression of GnRHR gene, the two putative PRE motifs were mutated in Sty-HLuc, as the highest promoter activity was obtained from this construct (Fig. 4Go). Mutation of the putative hGR-PRE resulted not only in reducing the P-induced inhibition but also increased the basal luciferase activity in {alpha}T3-1 cells (Fig. 6AGo). A 60% increase (P < 0.001) in basal promoter activity was observed after site-directed mutation of the hGR-PRE but not in mutated hGR-hPRE. Although the mutation of hGR-PRE significantly reduced the P-induced decrease in promoter activity from 54% to 19%, it did not completely eliminate the P-induced inhibition of promoter activity. Mutation of hGR-hPRE did not affect the basal promoter activity or eliminate the P-mediated action in transfected {alpha}T3-1 cells.



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Figure 6. Functional Analysis of Putative PRE in Human GnRHR Promoter

Mutations were introduced by three-step PCR mutagenesis as described in Materials and Methods. The wild-type and mutated promoter constructs were cotransfected with RSV-lacZvector, to normalize for varying transfection efficiencies, into {alpha}T3-1 and JEG-3 cells. A, The transfected {alpha}T3-1 cells were treated with 10 µM P for 24 h. B, the PRE mutant-transfected JEG-3 cells were treated with 10 µM RU486 for 24 h. The relative basal activity of each promoter mutant is shown as percentage of vehicle treated (control) Sty-HLuc, the activity of which is taken as 100%, after being normalized to ß-galactosidase activity. The percentage of inhibition is calculated by comparison with individual control. Values represent mean ± SE of triplicate assays in three separate experiments. The names and the relative position of the putative transcription factor binding sites are given, and the mutated element is shown as bars with diagonal lines. The relative change in promoter activity after treatment was indicated. a, P < 0.05 compared with control.

 
In JEG-3 cells, mutation of hGR-PRE led to a 35% decrease (P < 0.001) in basal promoter activity (Fig. 6BGo). Again, no such reduction in promoter activity was observed in mutated hGR-hPRE. In addition, mutation of hGR-PRE completely abolished the RU486-induced decrease in promoter activity in placental cells (Fig. 6BGo). Taken together, these results suggest that the hGR-PRE located at -535 to -521 plays an important role in mediating the effect of P on GnRHR transcription.

Effects of Cotransfection of PR on Human GnRHR Promoter Activity in {alpha}T3-1 and JEG-3 Cells
The expression of PRs in JEG-3 and {alpha}T3-1 cells was examined by Western blot analysis. Aliquots of protein (50 µg), isolated from JEG-3 or {alpha}T3-1 cells, were separated in SDS-PAGE and detected with antibody against PR. As seen in Fig. 7AGo, both PR-A and PR-B were expressed in JEG-3 and {alpha}T3-1 cells. The molecular masses of the detected human PR-A (95 kDa) and human PR-B (114–120 kDa), and mouse PR-A (85 kDa) and mouse PR-B (115 kDa) from JEG-3 and {alpha}T3-1 cells, respectively, were similar to those reported previously (25, 41). Interestingly, the levels of PR-A in JEG-3 cells were very low when compared with PR-B levels. Although both PR-A and PR-B isoforms were detected in {alpha}T3-1 cells, a relative high concentration of P was required to induce the decrease in hGnRHR promoter activity. This may due to the different ability of human and mouse PRs to transactivate a human gene. To further evaluate the role of human PR-A and PR-B in mediating the P action at the pituitary and placenta levels, the human PR-A and PR-B expression constructs were cotransfected into {alpha}T3-1 and JEG-3 cells. Cotransfection of human PR-A and PR-B expression vectors into JEG-3 and {alpha}T3-1 cells resulted in an increase in human PR-A and PR-B levels (Fig. 7Go, B and C).



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Figure 7. Western Blot Analysis of PR

A, Basal expression of PR. Total cellular protein isolated from JEG-3 or {alpha}T3-1 cells was separated and immunoblotted with PR antibody as described in Materials and Methods. Overexpression of human PR in JEG-3 cells (panel B) and {alpha}T3-1 cells (panel C). Total cellular protein was isolated from JEG-3 or {alpha}T3-1 cells 8 h after transfection with 1 µg of human PR-A (lane 2) or PR-B (lane 3) expression vector. The estimated molecular masses of the human PR (hPR-A and hPR-B) and mouse PR (mPR-A and mPR-B) are indicated.

 
As expected, no significant change in promoter activity was observed after treatment with 0.1 µM P for 24 h at the Sty-HLuc-transfected {alpha}T3-1 cells (Fig. 8Go). However, overexpression of human PRs in {alpha}T3-1 cells increased the sensitivity toward P treatment. A significant decrease (52%, P < 0.001) in promoter activity was achieved in 0.1 µM P treatment after cotransfection with 1 µg of PR-B expression vector (Fig. 8AGo). Similarly, cotransfection with 1 µg of PR-A expression vector resulted in a slight decrease (20%, P < 0.05) in the promoter activity after treatment with 0.1 µM P. Nevertheless, these data suggest that PR-B may play a more active role than PR-A in mediating P-induced inhibitory effects on the pituitary, since a higher inhibition in promoter activity was observed in PR-B cotransfected cells after P treatment. The functional role of the PRE in mediating the P-induced decrease in promoter activity at the pituitary was also examined in the hGR-PRE and hGR-hPRE-mutated constructs. Again, an increase in basal promoter activity was observed after the mutation of hGR-PRE, and this mutation eliminated and reduced the degree of inhibition induced by 0.1 µM P after overexpression of human PR-A and PR-B (from 52% to 17%), respectively (Fig. 8AGo).



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Figure 8. Effects of Overexpression of Human PR-A and PR-B in Promoter Activity on PRE Intact or Mutated Constructs

A, The wild-type or PRE mutated construct-transfected {alpha}T3-1 cells were treated with 0.1 µM P for 24 h with or without cotransfection of human PR-A or PR-B expression vector. B, The wild-type or PRE mutated construct-transfected JEG-3 cells were cotransfected with human PR-A and/or PR-B expression vector. The relative basal activity of each promoter mutants is shown as percentage of vehicle-treated (control) Sty-HLuc, the activity of which is taken as 100%, after being normalized to ß-galactosidase activity. The percentage change in promoter activity is calculated by comparison with individual control. Values represent mean ± SE of triplicate assays in three separate experiments. The names and the relative positions of the putative transcription factor binding sites are given, and the mutated element is shown as bars with diagonal lines. The percentage change in promoter activity is indicated. *, P < 0.05 compared with control.

 
In the placenta JEG-3, overexpression of hPR-A resulted in a 51% decrease (P < 0.001) in basal promoter activity (Fig. 8BGo). In contrast, overexpression of hPR-B led to an increase (39%, P < 0.001) in promoter activity (Fig. 8BGo). In addition, coexpression of hPR-A and hPR-B counteract each other’s function in regulating the promoter activity. These results suggested that the balance between PR-A and PR-B expression in the placenta plays an important role in controlling the expression of this gene, whereas PR-B stimulates and PR-A inhibits the promoter activity. Further studies have shown that the hGR-PRE solely mediated the hPR-B-stimulated increase in human GnRHR promoter activity as the mutation of this binding site eliminated the increase in luciferase activity (Fig. 8BGo). Although the mutation of hGR-PRE decreased the inhibitory effect of overexpression of hPR-A (from 51% to 23%), it did not completely eliminate this effect, suggesting that an additional regulatory mechanism is involved in mediating the PR-A action.

The Binding of PR to the Putative hGR-PRE
To confirm the identity of the transcription factor bound to the hGR-PRE, gel mobility shift assay was preformed with a synthetic oligodeoxynucleotide containing the putative hGR-PRE binding site in the presence of consensus and mutated PRE, hGR-PRE, nonrelated oligodeoxynucleotide, or antibody against the PR. Very weak DNA-protein complexes were obtained from nuclear extracts isolated from {alpha}T3-1 cells without transfection with human PR expression vectors (Fig. 9AGo, lane 1). In contrast, strong specific DNA-protein complexes were observed using the nuclear extract isolated from {alpha}T3-1 cells, after cotransfection with both human PR-A and PR-B expression vectors (Fig. 9AGo, lanes 2 and 3: indicated as arrows 1, 2, and 3). These complexes were eliminated with the addition of increasing competitor DNA fragment (200-fold in excess) containing a consensus PRE (lane 5) and hGR-PRE (lane 7) but not with a competitor containing mutated PRE (lane 4), mutated hGR-PRE (lane 6), or nonrelated sequence nuclear factor-{kappa}B (NF-{kappa}B) (lane 8) or transcription factor IID (TFIID) (lane 9). Furthermore, the addition of an antibody against the PR supershifted the DNA-protein complexes, further supporting the binding of the PR to this binding site (Fig. 9BGo, lane 11). Similarly, specific DNA-protein complexes were obtained using nuclear extract isolated from JEG-3 cells (Fig. 10AGo, indicated as arrows 4 and 5). These complexes were also eliminated with the addition of increasing competitor DNA fragment (200-fold in excess) containing a consensus PRE (lane 3) and hGR-PRE (lane 5) but not with a competitor containing mutated PRE (lane 2), mutated hGR-PRE (lane 4), or nonrelated sequence NF-{kappa}B (lane 6) or TFIID (lane 7). As expected, the addition of an antibody against the PR supershifted the DNA-protein complexes (Fig. 10BGo, lane 9).



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Figure 9. Identification of PR Binding to the Putative hGR-PRE Binding Sites in the Human GnRHR Gene by GMSA Using Nuclear Extract Isolated from Human PR-Transfected {alpha}T3-1 Cells

A, Synthetic deoxyribonucleotide containing the putative PRE sequences (hGR-PRE) in the human GnRHR gene was 32P labeled and incubated with nuclear extract isolated from human PR-A and PR-B cotransfected, P-treated {alpha}T3-1 cells in the presence of 200-fold excess specific competitor oligonucleotide. Specific DNA-protein complexes formed to the hGR-PRE (indicated as arrows 1, 2, and 3) were eliminated in the presence of competitor oligonucleotide (lane 3, no competitor; lane 4, mutated consensus PRE; lane 5, consensus PRE; lane 6, mutated hGR-PRE; lane 7, hGR-PRE; lane 8, consensus NF-{kappa}B and lane 9, consensus TFIID). B, GMSA studies were performed in the presence of antibodies specific to PR, Oct-1, and GATA-2. Antibodies were incubated with nuclear extract isolated from human PR-A and PR-B cotransfected, P-treated {alpha}T3-1 cells for 1 h before the addition of the 32P-labeled hGR-PRE probe. DNA-protein complexes were supershifted by the addition of antibody against PR (lane 11, indicated as arrow labeled "Shifted") but not by antibodies against IgG (lane 10), Oct-1 (lane 12), and GATA-2 (lane 13).

 


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Figure 10. Identification of PR Binding to the Putative hGR-PRE Binding Sites in the Human GnRHR Gene by GMSA Using Nuclear Extract Isolated from Human Placental JEG-3 Cells

A, Synthetic deoxyribonucleotide containing the putative PRE sequences (hGR-PRE) in human GnRHR gene was 32P labeled and incubated with nuclear extract isolated from human placental JEG-3 cells in the presence of a 200-fold excess of the indicated specific competitor oligonucleotide. Specific DNA-protein complexes formed to the hGR-PRE (indicated as arrows 4 and 5) were eliminated in the presence of competitor oligonucleotide (lane 1, no competitor; lane 2, mutated consensus PRE; lane 3, consensus PRE; lane 4, mutated hGR-PRE; lane 5, hGR-PRE; lane 6, consensus NF-{kappa}B and lane 7, consensus TFIID). B, Gel mobility assay studies were performed in the presence of antibodies specific to PR, Oct-1, and GATA-2. Antibodies were incubated with nuclear extract isolated from human placental JEG-3 cells for 1 h before the addition of the 32P-labeled hGR-PRE probe. DNA-protein complexes were supershifted by the addition of antibody against PR (lane 9, indicated as arrow labeled "Shifted") but not by antibodies against IgG (lane 8), Oct-1 (lane 10), and GATA-2 (lane 11).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
While P is the dominant steroid present in the circulation during human gestation, pituitary responsiveness to GnRH is reduced during this period (42, 43, 44, 45). Recent studies in the ovine have demonstrated that the numbers of GnRHR were relatively lower during the luteal phase of the estrous cycle (46), and induction of luteolysis by PGF2{alpha} resulted in an increase in both GnRHR numbers and mRNA levels in vivo (47). These data suggested a negative regulatory effect of P in GnRHR expression. In subsequent studies using primary culture of ovine pituitaries, a decrease in GnRHR mRNA levels was observed after P treatment (15, 30, 31), further supporting the negative role of P in regulating the ovine GnRHR gene expression. In spite of these observations, little is known about the role of P in regulating human GnRHR gene expression. Furthermore, the molecular mechanism of P action in mediating the expression of the GnRHR gene remains unknown. In the present study, we have examined the effects of P in controlling the transcription of the human GnRHR gene at the pituitary and placental levels. Because human pituitary gonadotrope cells were unavailable to us, we used the mouse {alpha}T3-1 pituitary tumor cell line as an experimental model to study the transcription regulation of the human GnRHR gene (9, 33, 35). The expression of GnRHR mRNA from the JEG-3 cells indicated the feasibility of using these cells to study the regulation of the human GnRHR gene (7).

We have observed a P-induced decrease in human GnRHR promoter activity in pituitary cells. This observation was in agreement with the results obtained from the studies in the ovine showing that P negatively regulates the expression of the GnRHR gene. Interestingly, this P-induced inhibition of human GnRHR promoter activity could not be observed in the placental JEG-3 cells, which also possess the GnRH-GnRHR system (7, 48). Instead, using RU486 to block endogenous P action and AGT to inhibit P production resulted in a decrease in human GnRHR promoter activity, suggesting the importance of P in maintaining the expression of GnRHR at the placenta. GnRH has been shown to stimulate the secretion of hCG from the placenta (5), which is important in maintaining early pregnancy by stimulating P production from the corpus luteum (49). The lack of the negative regulatory system in the expression of placental GnRHR by P may help sustain the GnRH-stimulated hCG production through pregnancy.

Progressive 5'-deletion of the human GnRHR 5'-flanking region and mutational studies have identified a putative PRE, namely hGR-PRE located at -535 to -521, that participates in P-mediated action. Our data showed that the very same region mediated the P-induced inhibition and stimulation actions on pituitary and placental cells, respectively. Identity of the transcription factor that bound to this region has been demonstrated containing PR by gel mobility shift assay (GMSA) and supershift assay. P mediates its biological activity after its interaction with a specific receptor within the nuclei of the target cells. PRs are ligand-inducible transcriptional regulators that control gene expression upon binding at the PRE in the vicinity of target promoters or influence gene expression by interaction with other transcription factors independently of PRE (50). The expression of PR-A and PR-B isoforms in the JEG-3 and {alpha}T3-1 cells was examined by the use of Western blot analysis. The expression levels of PR-A and PR-B were similar in the {alpha}T3-1 cells, whereas PR-B in the JEG-3 cells is much higher than PR-A. The requirement of a relatively higher concentration (1 µM) of P to achieve the statistically significant decrease in human GnRHR promoter activity in {alpha}T3-1 cells may be due to the use of mouse PRs in studying the regulation of the human GnRHR gene, or the different transcription activity of human and mouse PRs in the human gene. This explanation is supported by the result of GMSA. As seen in Fig. 9AGo, the endogenous mouse PR formed very weak DNA-protein complexes with the putative human hGR-PRE. To examine the role of human PR in controlling the expression of the human GnRHR gene, {alpha}T3-1 cells were cotransfected with human PR-A or PR-B expression vector. Overexpression of human PRs in {alpha}T3-1 cells increased the sensitivity toward P treatment and resulted in a decrease in GnRHR promoter activity after 0.1 µM P treatment. The similar results obtained from untransfected and human PRs cotransfected {alpha}T3-1 cells after P treatment further implicate the potential role of PRs in regulating the GnRHR gene transcription and the value of these cells in studying the P-induced regulation of human GnRHR gene. Although overexpression of both human PR-A and PR-B isoforms resulted in increased sensitivity toward P treatment, it appears that PR-B plays a more important role in mediating the P-induced inhibitory action. This postulation was based on the observations that 1) a higher degree of P-induced decrease in the human GnRHR promoter was obtained from PR-B-cotransfected cells, and 2) mutation of hGR-PRE completely eliminated the PR-A-mediated inhibitory effects but not PR-B-mediated action. Although the presence and distribution of steroid receptors in the normal human pituitary have not been reported, recent studies have demonstrated the expression of PR in human pituitary adenomas and supported the direct action of P in the human pituitary (51).

In agreement with our Western blot results in PR expression in JEG-3 cells, expression of PRs in the human placenta has also been demonstrated (52, 53, 54). Thus, human placenta is very likely a target tissue for the action of P. In fact, recent reports describing the effects of P on expression of CG {alpha}- and ß-subunit genes in the human placenta (55) and CRH gene in human trophoblast cell cultures (56) provide evidence that placenta itself can be a target tissue for the action of P. To further study the role of these receptors in mediating the P-induced effect in placenta, human PR-A and PR-B expression vectors were cotransfected into JEG-3 cells. Interestingly, our results showed that PR-A and PR-B have a differential function in regulating the expression of the human GnRHR gene in placenta cells. Similar to the data obtained from the pituitary cells, overexpression of PR-A resulted in a decrease in human GnRHR promoter activity. However, overexpression of PR-B increased the human GnRHR promoter activity in placental cells, while this overexpression led to a decrease in promoter activity in pituitary cells. These results support the P-stimulatory role of GnRHR gene expression in the placenta, since a much higher level of PR-B was detected in the JEG-3 cells endogenously. Taken together, these results indicate that PR-A is mainly involved in down-regulation, while PR-B up-regulates, the transcription of human GnRHR in the placenta.

Inasmuch as the PR-A and PR-B isoforms arise by transcription of the same gene with the use of different promoter regions (57), it is possible that independent regulation of these promoters may occur (58). It is now clear that their relative levels are under hormonal control (59). Thus, different hormonal input in the pituitary and placenta may affect the expression levels of PRs. As both PR-A and PR-B are capable of binding P, dimerizing and interacting with PRE, the level of PRs as well as the stoichiometric ratio of PR-A and PR-B within a target cell under specific physiological conditions would be expected to alter the relative complement of dimeric complex and exert a significant impact on the overall cellular response to P. To add to the complexity of the PR regulatory system, a third PR isoform has recently been identified in T47D human breast cancer cells and named PR-C (60). This N-terminally truncated PR-C isoform arises from the use of translation start site downstream of the translation site for PR-A and PR-B at the same gene. The PR-C contains the second zinc finger of the DNA-binding domain but is lacking the first one. Alone, PR-C is transcriptionally inactive but able to regulate the PR-A and PR-B transcriptional activity when coexpressed (61). Recent studies have also demonstrated that transactivation by PRs might involve additional cofactors including coactivator or corepressor (62, 63, 64). It is possible that different coactivators or repressors existed in the pituitary and placenta cells, which resulted in a different response in PR-B-mediated action. In addition, there is evidence for the interaction between steroid hormone receptors, including PR, and other transcription factors, such as octamer transcription factor 1 (Oct-1), activating protein-1, and signal transducer and activator of transcription, in controlling gene expression at the transcriptional level (62, 65, 66, 67). Hence, the incomplete elimination of the PR-B- and PR-A-mediated decrease in promoter activity in the mutated hGR-PRE construct in {alpha}T3-1 and JEG-3 cells, respectively, may be due to fact that an additional transcription-regulatory mechanism(s) is used. However, the exact role of these PR-C isoforms, activating protein-1/CREB motifs, and/or other possible mechanism(s) in mediating the PR-A and PR-B action in placental and pituitary cells has yet to be determined.

In summary, we have demonstrated a direct action of P in regulating the human GnRHR gene expression at transcriptional levels through binding to a putative hGR-PRE motif. Overexpression of human PR-A shows a transcriptional inhibition action in GnRHR gene expression. In contrast, PR-B exhibits a cell-dependent differential transcriptional activity in which it acts as transcriptional activator in placenta cells but a transcriptional repressor in pituitary cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
Mouse pituitary gonadotrope-derived {alpha}T3-1 cells and human choriocarcinoma JEG-3 cells were maintained in DMEM, with 4.5 mg/ml glucose (Life Technologies, Inc., Burlington, Ontario, Canada) and RPMI 1640, respectively, supplemented with 10% FBS (Life Technologies, Inc.) 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, 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 (9, 33, 39). Human PR-A and PR-B expression vector was provided by Dr. P. Chambon (INSERM, Universite Louis Pasteur, Paris, France). Plasmid DNA for transfection studies was prepared using QIAGEN 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 DNA was then dissolved in 0.1x TE (1 mM Tris-Cl; pH 7.5, 0.1 mM EDTA) to a final concentration of 1 µg/ml.

Site-Directed Mutagenesis
Human GnRHR 5'-flanking region -707 to +1 (related to translation start site) subcloned into pBSK II (+) vector (Stratagene, La Jolla, CA) was used as a template for the mutagenesis reaction. Mutations were introduced by a three-step PCR mutagenesis method as described previously (9), using universal primers UP-T3F (5'-GTGCCTCTCCTGAACAGGCCTCAAGCAATTAACCCTCACTAAAGG-3'), UP (5'-GTGCCTCTCCTGAACAGGCCTCAA-3'), T7R (5'-CGTAATACGAC- TCACTATAGG-3') and mutagenic primers for mP-PRE (5'-GTTTTCCTTTTCAAAGCGGCCGCTGAGCACTCGAACACT- GGAC-3') and mP-hPRE (5'AAACTATTAGTGTTAGTCGGCCGTCCAACATACAGATGTA3'). Mutation was confirmed by restriction enzyme and sequence analysis.

Transient Transfections and Reporter Assay
Transfections were carried out using the calcium precipitation methodology as previously described (7). To correct for different transfection efficiencies of various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was cotransfected into cells. Briefly, 5 x 105 {alpha}T3-1 cells or 1.5 x 105 JEG-3 cells were seeded into six-well tissue culture plates before the day of transfection in 2 ml culture medium containing 1% charcoal-dextran-treated FBS (HyClone Laboratories, Inc., Logan, UT). Before the transfection, the cells were washed once and cultured in 1 ml of fresh medium (containing 1% charcoal-dextran-treated FBS). Two micrograms of the GnRHR promoter-luciferase construct and 0.5 µg RSV-lacZ were dissolved in 50 µl 0.1x TE containing 0.25 M CaCl2 and mixed with 50 µl 2x 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 with for an additional 24 h with normal culture medium containing 10% charcoal-dextran-treated 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, Ontario, Canada). Luminescence was measured using 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
Progesterone (P), mifepristone (RU486), DEX, and DL-AGT were purchased from Sigma (Sigma-Aldrich Corp. Ltd., Oakville, Ontario, Canada). In experiments in which the effects of P, DEX, and RU486 on luciferase activity were studied, the cells were treated with corresponding drugs for 24 h before luciferase and ß-galactosidase activities were measured. P production in JEG-3 was inhibited by addition of AGT during transfection, and the secreted P levels were measured by RIA as previously described (34).

GMSA
Oligodeoxynucleotides corresponding to the putative hGR-PRE (5'-GAGTGCTCAACAGTGTGTTTGAAAAGG-3') and mutated hGR-PRE (mhG-GR-PRE; 5'GAGTGCTCAGCGGCCGCTTTGAAAAGG-3') elements at the human GnRHR 5'-flanking region and its complements were synthesized by the Oligonucleotide Synthesis Laboratory (University of British Columbia, Vancouver, British Columbia, Canada), and annealed to form a double-stranded DNA. Consensus and mutated PRE oligonucleotides DNA and antibodies for PR (catalog no. sc-538x), Oct-1 (catalog no. sc-232x), and GATA-2 (catalog no. sc-267x) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Oligonucleotides DNA for NF-{kappa}B (catalog no. E3291), and TFIID (catalog no. E3221) were purchased from Promega Corp. (Nepean, Ontario, Canada). Probes for GMSA were end-labeled with [P32]-ATP by T4 polynucleotide kinase (Life Technologies, Inc., Gaithersburg, MD), and separated from unincorporated radionucleotides by passage over Sephadex G-25 column. Nuclear extracts were prepared according to the method described previously (35). Protein concentrations were determined by a modified Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). GMSAs 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 of 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 for 1 h before the addition of labeled probe. The binding mixture was incubated at room temperature for 20 min and separated in 6% polyacrylamide gel containing 1x TBE (Tris-borate-EDTA: 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.

Western Blot Analysis
For Western blot analysis, approximately 1 x 106 cells were incubated in 100 µl of cell lysis RIPA (containing 1 x PBS (pH 7.4), 1% NP 40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 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.). Aliquots of protein from JEG-3 and {alpha}T3-1 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 Pharmacia Biotech, Morgan, Ontario, Canada). The membranes were blocked with 5% (wt/vol) nonfat milk in Tris buffered saline, containing 20 mM Tris-Cl (pH 8.0), 140 mM NaCl, and 0.05% (wt/vol) Tween 20, for at least 1 h before the addition of PR antibody (catalog no. sc-538x, Santa Cruz Biotechnology, Inc.) in 1:2,000 dilution. This antibody reacts with both PR-A and PR-B of mouse, rat, and human origin (product information, Santa Cruz Biotechnology, Inc.. All antibody incubation and washing were performed in Tris buffered saline with 0.05\% Tween 20. The Amersham Pharmacia Biotech enhanced chemiluminescence system (ECL) was used for detection. Membranes were visualized by exposure to Kodak X-Omat film.

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


    ACKNOWLEDGMENTS
 
We thank Dr. P. Chambon for providing human PR-A and PR-B expression vectors.


    FOOTNOTES
 
This work was supported by grants from the Canadian Institutes of Health Research. P.K.C.L. is a career investigator of the British Columbia Research Institute for Children’s and Women’s Health.

Abbreviations: AMG, Aminoglutethimide; DEX, dexamethasone; GMSA, gel mobility shift assay; GnRHR, GnRH receptor; NF-{kappa}B, nuclear factor-{kappa}B; Oct-1, octamer transcription factor-1; P, progesterone; PRE, progesterone response element; RSV, Rous sarcoma virus; Sty-HLuc (-707 to +1), human GnRHR promoter luciferase construct; TFIID, transcription factor IID.

Received for publication August 23, 2000. Accepted for publication August 16, 2001.


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