Characterization of a New Upstream GnRH Receptor Promoter in Human Ovarian Granulosa-Luteal Cells

Chi Keung Cheng, Chung Man Yeung, Billy K. C. Chow and Peter C. K. Leung

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

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH has been implicated as an important local autocrine and paracrine factor in regulating ovarian function. However, to date, the transcriptional regulation of GnRH receptor (GnRHR) gene in human ovary remains poorly understood. Here we report the characterization of a new upstream promoter for the GnRHR gene in human granulosa-luteal cells. Using progressive deletion analysis, a region between nucleotide -1300 and -1018 (relative to the translation start site) was shown to exhibit the highest promoter activities in two immortalized human granulosa-luteal cell lines, SVOG-4o and SVOG-4m. Two putative CCAAT/enhancer binding protein (C/EBP) motifs and one GATA motif were identified within this region. Mutational studies showed that these three motifs cooperated synergistically to regulate GnRHR gene transcription in the granulosa cells but not in other cell types including human ovarian carcinoma OVCAR-3, human embryonic kidney-293 (HEK-293) and mouse pituitary gonadotrope-derived {alpha}T3–1 cells. Surprisingly, by competitive EMSAs, we found that an Oct-1 consensus sequence was able to inhibit protein complex formation with the distal C/EBP motif, suggesting a possible cross-talk between the Oct-1 transcription factor and this C/EBP motif. Taken together, our results strongly indicate a role of the C/EBP and GATA motifs in regulating GnRHR gene transcription in human granulosa-luteal cells and further suggest that tissue-specific expression of human GnRHR gene is mediated by differential promoter usage.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HYPOTHALAMIC GnRH plays a pivotal role in mammalian reproduction by stimulating the synthesis and secretion of gonadotropins via binding to the GnRH receptors (GnRHRs) on the pituitary gonadotropes. However, there is increasing evidence for extrapituitary actions of GnRH in the gonads. We and others have previously demonstrated that human ovary and granulosa-luteal cells express GnRH and its receptor mRNA transcripts (1, 2), and functionally, GnRH has been shown to regulate steroidogenesis (3, 4), stimulate MAPK cascade (5) and apoptosis (6), and inhibit FSH-induced cAMP-dependent response (7) in human granulosa-luteal cells. These studies therefore suggest that GnRH may act as an important autocrine and paracrine factor in regulating local ovarian function. However, to date, the molecular mechanisms in regulating GnRHR gene expression in the human ovary remain poorly understood.

To better understand the mechanisms that regulate GnRHR gene transcription, the 5'-flanking region of human GnRHR gene has been isolated (8, 9), and our earlier studies showed that a downstream gonadotrope-specific element located within the first exon was responsible for gonadotrope-specific expression of the GnRHR gene via interaction with steroidogenic factor-1 (10). Moreover, we have demonstrated that mouse gonadotrope {alpha}T3–1 and human ovarian carcinoma OVCAR-3 cells appeared to utilize the same promoters for basal GnRHR gene transcription (11). However, our recent findings suggested that there was a possible differential usage of GnRHR promoters between {alpha}T3–1 and placental JEG-3 cells. A proximal promoter located between nucleotide (nt) -707 and +1 was found to be used by the gonadotropes, whereas a distal promoter located between nt -1737 and -1346 was shown to be specifically employed by the placental cells. A cAMP-responsive element and a GATA motif within this distal promoter region were responsible for the placenta-specific transcription of the GnRHR gene (12). In fact, a similar pattern of differential usage of promoters has also been observed in the GnRH gene for directing tissue-specific expression in hypothalamic GT1–7 and placental JEG-3 cells (13). Taken together, these studies indicate that neuronal and reproductive tissues differentially utilize downstream and upstream promoters for tissue-specific transcription of GnRH and GnRHR genes.

Nevertheless, it remains unclear whether other reproductive tissues such as the ovary would utilize another, but as yet unidentified, upstream promoter to regulate GnRHR gene transcription. To address this issue, two immortalized human granulosa-luteal cell lines, SVOG-4o and SVOG-4m, were used to identify other putative promoter region(s) for the GnRHR gene. Interestingly, a new upstream promoter located between nt -1300 and -1018 (relative to the translation start site) was shown to exhibit the highest activity among four other cell lines in the granulosa cells. DNA sequence analysis revealed two putative CCAAT/enhancer binding protein (C/EBP) motifs and one GATA motif within this upstream promoter, and their functional significance in mediating granulosa cell-specific GnRHR gene transcription was examined by site-directed mutagenesis and EMSAs. Our results clearly demonstrate that these putative C/EBP and GATA motifs are crucial in controlling GnRHR gene transcription, possibly via functional cooperation, in human ovarian granulosa-luteal cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Human GnRHR in the Immortalized Granulosa-Luteal Cell Lines
RT-PCR and Western blot analysis were performed to study the expression of GnRHR at the RNA and protein levels in the immortalized human granulosa-luteal cell lines, SVOG-4o and SVOG-4m, as well as in primary cultured human granulosa-luteal cells. A 373-bp PCR fragment was obtained in both cell lines and the primary culture, with the immortalized granulosa cells showing a relatively higher mRNA expression than the primary cultured cells. The authenticity of the PCR product was confirmed by DNA base sequencing and Southern blot analysis (Fig. 1AGo). A mouse monoclonal antibody specific to the human GnRHR was used in Western blot analysis, and two bands of about 62 kDa were detected in both the immortalized and primary cultured cells (Fig. 1BGo). Consistent with the results of RT-PCR, higher protein expression levels were observed in the SVOG-4o and -4m cells (Fig. 1BGo). Taken together, these results indicate that the human GnRHR mRNA and protein are expressed in the immortalized human granulosa-luteal cells.



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Figure 1. Expression of Human GnRHR mRNA and Protein in Immortalized (SVOG-4m and SVOG-4o) and Primary Cultured (PC) Granulosa-Luteal Cells

A, RT-PCR amplification of GnRHR cDNAs from SVOG-4m, SVOG-4o, and PC using GnRHR-F and GnRHR-R primers (upper panel). The authenticity of the PCR product was confirmed by DNA base sequencing, and the verified product was used as a probe in Southern blot analysis (lower panel). B, Western blot analysis to detect GnRHR protein expression from total cellular extracts (35 µg) of human granulosa-luteal cells using a mouse monoclonal antibody F1G4 against the human GnRHR protein.

 
Mapping of the Human GnRHR Promoter in the Granulosa-Luteal Cells
To localize the active promoter regions, progressive 5'- and 3'-deletion mutants were constructed and analyzed in SVOG-4o and -4m cells. Transient transfection studies revealed similar promoter activity profiles in both cell lines. The promoter activities of all the 5'-deletion mutants were similar and were less than 5-fold when compared with the promoterless pGL2-Basic vector (Fig. 2Go). However, a strong promoter activity (SVOG-4o: 38-fold vs. pGL2-Basic; SVOG-4m: 35-fold vs. pGL2-Basic) was observed when the proximal 1018-bp fragment was deleted [i.e. the p(-2197/-1018)-Luc construct], suggesting the use of an upstream promoter by the immortalized granulosa cells (Fig. 3Go). Further 3'-deletion to nt -1346 reduced the promoter activities by 40% and 45% in the SVOG-4o and -4m cells, respectively. Interestingly, inclusion of the region between nt -1018 and -771 in p(-2197/-771)-Luc completely abolished the promoter activity, indicating the presence of a very strong negative regulatory element within this region (Fig. 3Go).



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Figure 2. Progressive 5'-Deletion Analysis of the Human GnRHR Promoter in Two Immortalized Granulosa-Luteal Cell Lines, SVOG-4o and SVOG-4m

A nested family of 5'-deletion mutants was transiently transfected into the cells by LIPOFECTAMINE PLUS Reagent. The RSV-lacZ vector was cotransfected to normalize the transfection efficiency. The relative promoter activity was represented as the fold increase when compared with the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments, each performed in triplicate. a, P < 0.05 vs. pGL2-Basic.

 


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Figure 3. Progressive 3'-Deletion Analysis of the Human GnRHR Promoter in Two Immortalized Granulosa-Luteal Cell Lines, SVOG-4o and SVOG-4m

A nested family of 3'-deletion mutants was transiently transfected into the cells by LIPOFECTAMINE PLUS Reagent. The RSV-lacZ vector was cotransfected to normalize the transfection efficiency. The relative promoter activity was represented as the fold increase when compared with the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments, each performed in triplicate. a, P < 0.05 vs. pGL2-Basic.

 
To locate the minimal region that is sufficient for producing maximal promoter activity in the granulosa cells, a more detailed 200-bp deletion mapping was performed between nt -2197 and -1018 (Fig. 4Go). 5'-Deletion of a 200-bp fragment from p(-2197/-1018)-Luc reduced the promoter activities by 71% and 68% in the SVOG-4o and -4m cells, respectively, suggesting the presence of a strong positive regulatory element in this distal region. However, further 5'-deletion from p(-1900/-1018)-Luc increased the promoter activities by 2-fold in both cell lines. Additional increases in promoter activities (1.6-fold in SVOG-4o; 1.3-fold in SVOG-4 m) were observed when a 200-bp region was removed from p(-1500/-1018)-Luc. These results indicate that repressor elements are located between nt -1900 and -1300, and the core minimal promoter region, which is sufficient for producing the maximal basal activity in the granulosa cells, is situated between nt -1300 and -1018.



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Figure 4. Fine Mapping of the Minimal Promoter Region for GnRHR Gene Transcription in the SVOG-4o and SVOG-4m Cells

A series of 200-bp deletion constructs was generated between nt -2197 and -1018, and the GnRHR promoter-luciferase constructs were cotransfected with RSV-lacZ vector into the cells. The relative promoter activity was represented as the fold increase when compared with the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments, each performed in triplicate. a, P < 0.01 vs. pGL2-Basic.

 
To examine whether this minimal upstream promoter (nt -1300 to -1018) is granulosa cell specific, the p(-1300/-1018)-Luc was transiently transfected into six different cell lines including SVOG-4o, SVOG-4m, IOSE-29EC, OVCAR-3, HEK-293, and {alpha}T3–1 cells (Fig. 5Go). The highest promoter activity was observed in the immortalized granulosa-luteal cells, whereas other cell lines exhibited various degrees of much lower promoter activities. These results suggest that the upstream promoter segment is predominantly used by the granulosa cells.



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Figure 5. Activities of the Upstream GnRHR Promoter (nt -1300 to -1018) in Different Cell Lines

The GnRHR promoter-luciferase construct p(-1300/-1018)-Luc was cotransfected with RSV-lacZ vector into the SVOG-4o, SVOG-4m, IOSE-29EC, OVCAR-3, HEK-293, and {alpha}T3–1 cells. The relative promoter activity was represented as the fold increase when compared with the promoterless pGL2-Basic vector. Values represent the mean ± SEM of three independent experiments each performed in triplicate. a, P < 0.01 vs. pGL2-Basic.

 
Identification of the Transcription Start Sites for the GnRHR Gene in Human Granulosa-Luteal Cells
To further confirm the usage of the upstream promoter (nt -1300 to -1018) by the granulosa cells, a primer extension analysis was performed using two oligonucleotides located at different positions within the human GnRHR 5'-flanking region (Fig. 6AGo). One extension product was generated by primer PE-1, and two products were obtained from primer PE-2 when total RNA from the SVOG-4o cells was used, and their corresponding positions were located at nt -769, -1375, and -1397, respectively. However, no extension product was obtained from the control human dermal fibroblast (HDF) cells. The major transcription start site was located at nt -769, and when its neighboring sequence was examined carefully, a TATA box and a CAAT box were identified at 79 bp and 104 bp, respectively, upstream of this major transcription start site (Fig. 6BGo).



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Figure 6. Identification of the Transcription Start Sites for GnRHR Gene in Human Granulosa-Luteal Cells by Primer Extension Analysis

A, Total RNA (60 µg) from SVOG-4o cells and HDFs were hybridized and extended with the primers PE-1 and PE-2. One extension product (with a size of 141 bp) was obtained from the SVOG-4o cells using the primer PE-1, whereas two products (with sizes of 90 and 112 bp) were obtained when the primer PE-2 was used. The deduced transcription start sites from these extension products were located at positions -769, -1375, and -1397, respectively. No extension product was detected from the control HDF cells. B, A diagrammatic representation of the human GnRHR 5'-flanking region. The upstream GnRHR promoter primarily used by the granulosa cells was shown as a shaded box, and the locations of the TATA (T) and CAAT (C) boxes were shown. The positions of the primers (PE-1 and PE-2) used for primer extension analysis and the identified transcription start sites were indicated with horizontal arrows and bent arrows, respectively. The major transcription start site was located at -769.

 
Mutational Analysis of Putative C/EBP and GATA Motifs Within the Upstream Promoter
Two putative C/EBP binding sites, namely distal C/EBP (dC/EBP) (5'-TCTGTGGTAACAA-3' located from nt -1244 to -1232, with 91% homology to the consensus C/EBP motif) and proximal C/EBP (pC/EBP) (5'-ATATTTAGTAACCA-3' located from nt -1157 to -1144, with 82% homology to the consensus C/EBP motif) and one GATA binding site (5'-AAGATAATG-3' located from nt -1176 to -1168, with 89% homology to the consensus GATA motif) all in sense orientations were identified within the upstream promoter. To examine the functional significance of these motifs in regulating GnRHR gene transcription in the granulosa cells, site-directed mutants were constructed and transiently transfected into SVOG-4o cells and three other different cell lines including OVCAR-3, HEK-293, and {alpha}T3–1 cells (Fig. 7AGo). Mutation of the dC/EBP motif caused a 35% and a 26% reduction of promoter activities in the SVOG-4o and {alpha}T3–1 cells, respectively. However, no significant change of promoter activities was observed in the OVCAR-3 and HEK-293 cells. On the other hand, mutation of the pC/EBP motif significantly reduced the promoter activities in the SVOG-4o (45% reduction), OVCAR-3 (24% reduction), and HEK-293 cells (27% reduction) but not in the {alpha}T3–1 cells. Interestingly, mutation of the putative GATA motif caused a significant reduction of promoter activity only in the SVOG-4o cells (33% reduction) but not in the three other cell lines, suggesting a specific functional role of this motif in regulating GnRHR gene transcription in the granulosa cells. To examine whether there is any functional cooperation among these motifs, constructs containing double or triple mutations were generated and analyzed in the SVOG-4o cells. Constructs containing two mutations (dC/EBP + pC/EBP or GATA + pC/EBP) had their luciferase activities reduced by 54% (Fig. 7BGo). However, an almost 80% reduction of promoter activity was observed in the granulosa cells (compared with 31% reduction in OVCAR-3 cells; 33% reduction in HEK-293 cells; 17% reduction in the {alpha}T3–1 cells) when all these three motifs were mutated simultaneously (Fig. 7Go, B and C).



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Figure 7. Mutational Studies of the Putative C/EBP and GATA Motifs on the Promoter Activity of p(-1300/-1018)-Luc

Each motif was mutated by a three-step PCR mutagenesis, which artificially introduced a NotI restriction site into the target sequences. A, The GnRHR promoter-luciferase constructs with a single mutation in one of the three motifs (dC/EBP-mut, GATA-mut and pC/EBP-mut) were cotransfected with RSV-lacZ vector into SVOG-4o, OVCAR-3, HEK-293, and {alpha}T3–1 cells. B, Constructs containing single, double, or triple mutations were cotransfected with RSV-lacZ vector into the SVOG-4o cells. C, the construct containing triple mutations was analyzed in the SVOG-4o, OVCAR-3, HEK-293, and {alpha}T3–1 cells. The relative promoter activity was represented as the percentage of the wild-type vector p(-1300/-1018)-Luc whose activity was set as 100% after being normalized by ß-galactosidase activity. Values represent the mean ± SEM of three independent experiments, each performed in triplicate. a, P < 0.05 vs. p(-1300/-1018)-Luc; b, P < 0.05 vs. pC/EBP-mut; c, P < 0.05 vs. (dC/EBP + pC/EBP)-mut; d, P < 0.05 vs. (GATA + pC/EBP)-mut.

 
Analysis of DNA-Protein Interactions of the Putative C/EBP and GATA Motifs by EMSAs
EMSA studies revealed that the same DNA-protein complexes were formed with the putative C/EBP and GATA motifs when nuclear extracts from primary human granulosa-luteal cells or SVOG-4o cells were used. Two DNA-protein complexes, complexes A and B, were formed with the synthetic oligonucleotide containing the dC/EBP motif (Fig. 8AGo). Formation of these complexes was completely abolished in the presence of 200-fold excess of the unlabeled oligonucleotide (Fig. 8BGo, lane 2), whereas addition of other unrelated sequences [nuclear factor-{kappa}ß-c (NF-{kappa}ß-c), cAMP-responsive element binding protein-c (CREB-c), and activating protein-1-c (AP-1-c)] or a mutated distal C/EBP sequence failed to inhibit the complex formation (Fig. 8BGo, lanes 5–7 and 9). Surprisingly, a consensus C/EBP oligonucleotide (C/EBP-c) also failed to compete with this dC/EBP (Fig. 8BGo, lane 4) motif even when a 600-fold excess of the competitor was used (Fig. 8CGo, lanes 6–8). However, a dose-dependent inhibition of complex formation was observed with an increasing amount of a consensus Oct-1 oligonucleotide (Oct-1-c) (Fig. 8CGo, lanes 1–5). On the other hand, one complex (complex C) was formed with the putative GATA motif (Fig. 9AGo) and, similarly, the addition of the unlabeled oligonucleotide (200-fold excess) completely prevented DNA-protein complex formation (Fig. 9BGo, lane 2). Competitive EMSA studies showed that a consensus GATA oligonucleotide (GATA-c) could inhibit DNA-protein complex formation in a dose-dependent manner (Fig. 9BGo, lanes 3–5), whereas a mutated GATA sequence failed to compete with the radiolabeled probe (Fig. 9BGo, lane 7). Meanwhile, one DNA-protein complex (complex D) was formed with the pC/EBP motif (Fig. 9CGo, lanes 1 and 2), and its formation was totally abolished with the addition of 200-fold excess of the unlabeled oligonucleotide (Fig. 9CGo, lane 3). However, the formation of this complex was not affected in the presence of oligonucleotides (200-fold excess) containing consensus sequences of six different transcription factors including C/EBP-c, NF-{kappa}ß-c, Oct-1-c, AP-1-c, CREB-c, and glucocorticoid receptor (GR-c), or a mutated pC/EBP sequence (Fig. 9CGo, lanes 4–9 and 11).



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Figure 8. EMSAs of the dC/EBP Motif Using Nuclear Extracts (NE) from Human Granulosa-Luteal Cells

Synthetic oligonucleotide containing the dC/EBP sequence was end-labeled with 32P and incubated with nuclear extracts from primary cultured (PC) or SVOG-4o cells in the absence or presence of different competitor oligonucleotides. A, Formation of two DNA-protein complexes (complexes A and B) using nuclear extracts from PC (10 µg, lane 1) or SVOG-4o cells (2, 5, and 10 µg, lanes 2–4). B, SVOG-4o nuclear extracts (10 µg) were incubated with 50 fmol of the radiolabeled probe in the presence of 200-fold excess of cold competitor (unlabeled probe), five different competitor oligonucleotides (Oct-1-c, C/EBP-c, NF-{kappa}ß-c, CREB-c, and AP-1-c) or a mutated dC/EBP sequence. C, SVOG-4o nuclear extracts (10 µg) were incubated with 50 fmol of the radiolabeled probe in the presence of an increasing amount of a consensus Oct-1 (Oct-1-c) or C/EBP (C/EBP-c) oligonucleotide.

 


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Figure 9. EMSAs of the Putative GATA and the pC/EBP Motifs Using Nuclear Extracts (NE) from Human Granulosa-Luteal Cells

Synthetic oligonucleotides containing the putative GATA and pC/EBP sequences were end-labeled with 32P and incubated with nuclear extracts from primary cultured (PC) or SVOG-4o cells in the absence or presence of different competitor oligonucleotides. A, Formation of a DNA-protein complex (complex C) using nuclear extracts from PC (10 µg, lane 1) or SVOG-4o cells (2, 5, and 10 µg, lanes 2–4) in the presence of 50 fmol of the radiolabeled probe containing the putative GATA binding motif. B, SVOG-4o nuclear proteins (5 µg) were incubated with 50 fmol of the radiolabeled GATA motif-containing oligonucleotide in the presence of 200-fold excess of cold competitor (unlabeled probe), an increasing amount of a consensus GATA oligonucleotide (GATA-c), or 200-fold excess of a mutated GATA sequence. C, Formation of a DNA-protein complex (complex D) using 10 µg of nuclear extracts from PC (lane 1) or SVOG-4o cells (lane 2) in the presence of 50 fmol of the radiolabeled probe containing the pC/EBP motif. For the competitive assays (lanes 3–11), the radiolabeled probe was incubated with 10 µg of SVOG-4o nuclear extracts in the presence of 200-fold excess of cold competitor, consensus sequences of six different transcription factors including C/EBP-c, NF-{kappa}ß-c, Oct-1-c, AP-1-c, CREB-c, and GR-c, or a mutated pC/EBP sequence.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The presence of GnRH and its receptor in the human ovary and other reproductive tissues (14, 15, 16) strongly suggests that GnRH may act as an important local autocrine and paracrine factor in regulating reproductive functions. Although the transcriptional regulation of the human GnRHR gene has been well studied in gonadotropes (10) and placental cells (12), little is known in the ovary. In this report, two immortalized human granulosa-luteal cell lines expressing GnRHR mRNA transcripts and proteins were used to study the molecular mechanisms in regulating GnRHR gene transcription. Two bands of about 62 kDa were detected by Western blot analysis in the granulosa cells (Fig. 1BGo), and this result is not consistent with a previous study using membrane fractions (17) in which only one band was detected. This discrepancy could be explained by the detection of the unprocessed or unglycosylated receptor proteins in our analysis. The new upstream promoter identified in our present study (i.e. from nt -1300 to -1018) appears to be unnecessary for placenta-specific expression of the human GnRHR gene because deletion of this region does not alter the promoter activities in both JEG-3 and IEVT (immortalized extravillous trophoblast) cells (12). Unfortunately, we could not examine the activity of this upstream promoter in primary granulosa-luteal cells because we repeatedly failed to transfect the upstream promoter construct into these cells using various transfection methods. The identification of this new minimal GnRHR promoter in granulosa-luteal cells and the presence of multiple TATA boxes within the human GnRHR 5'-flanking region (8) further suggest that cell-specific expression of the human GnRHR gene is mediated by differential promoter usage. Nevertheless, this phenomenon is to be confirmed by the analysis of different GnRHR transcripts in various cell types. Moreover, the utilization of this upstream promoter appeared to be restricted to the granulosa cells when compared with other ovarian cell types (Fig. 5Go). These findings indicate that a unique transcriptional machinery may be involved in granulosa cell-specific expression of the GnRHR gene. This differential gene regulation also implies that the local GnRH/GnRHR system may possess distinct biological functions among various ovarian compartments.

Although the usage of the upstream promoter by the granulosa cells was confirmed by primer extension analysis, we cannot rule out the possibility that a further upstream positive regulatory region (nt -2197 to -1900) may also participate in regulating GnRHR gene expression in the granulosa cells because deletion of this region resulted in a dramatic reduction of the promoter activity (Fig. 4Go). Moreover, the identification of two additional transcription initiation sites (nt -1375 and -1397) located downstream of this element may suggest the presence of another upstream GnRHR promoter for the granulosa cells. However, the functional significance of this upstream positive regulatory element remains to be elucidated. Another interesting phenomenon observed from deletion analysis is that addition of the region between nt -1018 and -771 can completely abolish the promoter activity (Fig. 3Go), and the same scenario has also been observed in {alpha}T3–1 (11), OVCAR-3 (11), and JEG-3 cells (12). These findings indicate that the repressor protein(s) interacting with this powerful negative regulatory region may be commonly expressed in gonadotropes and in ovarian and placental cells.

DNA sequence analysis revealed the presence of two putative C/EBP motifs and one GATA motif within the upstream promoter region, and mutational analysis showed that alteration of either one of these motifs did not completely abolish the promoter activity in the granulosa cells. Interestingly, these C/EBP and GATA motifs appear to cooperate synergistically in controlling GnRHR gene transcription in the granulosa cells because only simultaneous mutation of all these motifs can eliminate the activity of the upstream promoter (Fig. 7BGo). The fact that this functional cooperation was not observed in the ovarian carcinoma OVCAR-3 cells (Fig. 7CGo) further suggests that differential regulatory mechanisms regulate GnRHR gene transcription in various ovarian compartments. Although we failed to assess the activity of the upstream promoter in primary granulosa-luteal cells, EMSA studies revealed that the same DNA-protein complexes were formed when nuclear extracts from SVOG-4o or primary cultured cells were used (Figs. 8Go and 9Go). Surprisingly, competitive EMSA studies showed that there was a possible cross-talk between the Oct-1 transcription factor and the distal C/EBP motif. This observation could be explained by the ability of Oct-1 to recognize various degenerate sequences and adopt different conformations (18, 19). Indeed, previous studies have also demonstrated that the POU domain transcription factor Oct-1 could bind to an element overlapping that of C/EBP and act as a transcriptional repressor of the IL-8 gene (20). On the other hand, a GATA transcription factor is believed to be involved in the transcriptional regulation of the GnRHR gene in the granulosa cells, via binding to the putative GATA motif within the upstream promoter. In fact, transcription factors GATA-4 and GATA-6 and a GATA family cofactor FOG-2 have recently been shown to be expressed in human granulosa-luteal cells (21). Among these proteins, GATA-4 has been reported to differentially activate the transcription of several genes expressed in the gonads including steroidogenic acute regulatory protein, aromatase, and inhibin {alpha}-subunit through synergistic interactions with other transcription factors such as steroidogenic factor 1 (22). The possible involvement of GATA-4 in the transcriptional regulation of the GnRHR gene in human granulosa cells is currently under investigation. On the contrary, the identity of the transcription factor binding to the pC/EBP motif remains to be confirmed because the complex formation was not inhibited by consensus oligonucleotides of six different transcription factors including C/EBP (Fig. 9CGo). Indeed, this proximal C/EBP binding site is different from that of the consensus C/EBP oligonucleotide used in the competitive EMSA studies. The failure of the consensus C/EBP sequence to inhibit complex formation may be due to the fact that C/EBP dimerization is a prerequisite to DNA binding (23), and dimerization of different C/EBP proteins would generate different DNA binding specificities that, in turn, precisely modulate the transcriptional activities of various target genes. Therefore, it is possible that the protein factor binding to the pC/EBP motif does not recognize and interact with the consensus C/EBP sequence. But in the meantime, we cannot exclude the possibility that C/EBP proteins are involved in the granulosa cell-specific expression of the GnRHR gene. In fact, C/EBP-{alpha} and -ß proteins have been shown to be present in the nuclear extracts of human granulosa-luteal cells, and C/EBP-ß is involved in the cAMP regulation of steroidogenic acute regulatory gene transcription (24).

In conclusion, we have identified a novel upstream promoter in which two putative C/EBP motifs and one GATA motif function cooperatively to regulate GnRHR gene transcription in human granulosa-luteal cells specifically (Fig. 10Go). Moreover, our results further suggest that tissue-specific expression of the human GnRHR gene is mediated, at least in part, by differential promoter usage in various cell types.



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Figure 10. Diagrammatic Representation of Differential Promoter Usage of the Human GnRHR Gene by Various Cell Types

Promoter regions (upstream or downstream) primarily used by the placental cells, granulosa cells, and gonadotropes were boxed, and their corresponding locations were indicated. The major transcription start site for the human GnRHR gene in the granulosa cells was shown with a bent arrow. The functional role of the putative GATA, proximal C/EBP (pC/EBP), and distal C/EBP (dC/EBP) motifs in controlling GnRHR gene transcription in the granulosa cells was also indicated. The diagram was not drawn to scale.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
The use of human granulosa-luteal cells was approved by the Clinical Screening Committee for Research and Other Studies involving Human Subjects of the University of British Columbia. Follicular aspirates were collected during oocyte retrieval from women undergoing in vitro fertilization. Human granulosa-luteal cells were prepared as previously described (25) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Inc., Logan, UT) for 4 d in humidified atmosphere of 5% CO2 in air at 37 C before RNA and protein extraction.

Immortalized human granulosa-luteal cells (SVOG-4o and SVOG-4 m) were generated with SV40 large T antigen as previously reported (26). Immortalized human ovarian surface epithelial cells (IOSE-29EC) (27) and HDFs were provided by Dr. N. Auersperg (Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada). Human ovarian carcinoma OVCAR-3 cells and human embryonic kidney-293 cells (HEK-293) were obtained from American Type Culture Collection (Manassas, VA). Mouse pituitary gonadotrope-derived {alpha}T3–1 cells were provided by Dr. P. L. Mellon (Department of Reproductive Medicine, University of California, San Diego, CA). The SVOG-4o, SVOG-4m, IOSE-29EC, and OVCAR-3 cells were maintained in M199/MCDB105 (1:1) supplemented with 10% FBS (HyClone Laboratories, Inc.). The HDF, HEK-293, and {alpha}T3–1 cells were maintained in DMEM (Life Technologies, Inc., Burlington, Canada) containing 10% FBS. Cultures were maintained at 37 C in humidified atmosphere of 5% CO2 in air. Cells were passaged when they reached about 80% confluence using trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA).

RNA Extraction, RT-PCR, and Southern Blot Analysis
Total RNA was extracted by TRIZOL Reagent (Life Technologies, Inc.) and reverse transcribed using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Morgan, Canada) following the manufacturer’s suggested protocol. Two primers (Table 1Go) specific for the human GnRHR gene were designed based on the published sequence (GenBank accession no. XM030222.1; forward primer, from nt 342 to 363; reverse primer, from nt 714 to 693) and used for PCR amplification. The PCR was carried out for 35 cycles with denaturation for 30 sec at 94 C, annealing for 1 min at 60 C, extension for 1 min at 72 C, and a final extension for 15 min at 72 C. The authenticity of the PCR product was confirmed by DNA base sequencing, and the verified product was used as a probe in Southern blot analysis. For the Southern blot analysis, the PCR product was separated by agarose gel electrophoresis and transferred onto a nylon membrane (Amersham Pharmacia Biotech). The membrane was hybridized with a digoxigenin-labeled GnRHR cDNA probe (Roche Molecular Biochemicals, Laval, Canada). Detection was carried out following the manufacturer’s recommended procedures (Roche Molecular Biochemicals) and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY).


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Table 1. Nucleotide Sequences of Primers Used in RT-PCR, Plasmid Construction, Primer Extension, Site-Directed Mutagenesis, and EMSA Studies

 
Western Blot Analysis
Cells were lysed in a lysis buffer containing 1 x PBS (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 30 µg/ml aprotinin, and 10 µg/ml leupeptin for 15 min on ice. The cell lysates were collected and debris was cleared by centrifugation. Proteins were resolved using SDS-PAGE and transferred onto a nitrocellulose membrane (Hybond-C, Amersham Pharmacia Biotech) by electroblotting. The membrane was blocked with 5% (wt/vol) nonfat dried milk in Tris-buffered saline, containing 20 mM Tris-Cl (pH 8.0), 140 mM NaCl, and 0.05% (vol/vol) Tween 20 for 2 h at room temperature before incubating with a mouse monoclonal antibody F1G4 specific to human GnRHR (17) overnight at 4 C. Immunostained proteins were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Preparation of Human GnRHR Promoter-Luciferase Constructs
Human GnRHR promoter-luciferase construct p(-2297/+1)-Luc (the numbering is relative to the ATG translation start site) and progressive 5'- and 3'-deletion constructs were prepared as previously described (11, 28). Deletion constructs for fine mapping were generated by PCR amplification of the corresponding regions of the GnRHR 5'-flanking region and subsequent cloning of the fragments into the promoterless pGL2-Basic vector. The PCR was carried out for 30 cycles with denaturation for 30 sec at 94 C, annealing for 1 min at 60 C, extension for 1 min at 72 C, and a final extension for 15 min at 72 C. Restriction site (XhoI or HindIII) was artificially introduced into the primers (Table 1Go). Plasmid DNA for transient transfection was prepared using Plasmid Midi Kits (QIAGEN, Chatsworth, CA) following the manufacturer’s suggested procedures. The concentration and quality of DNA were determined by measuring absorbance at 260 nm and by agarose gel electrophoresis, respectively.

Transient Transfection and Reporter Gene Assay
Transient transfections were carried out using LIPOFECTAMINE PLUS Reagent (Life Technologies, Inc.) following the manufacturer’s suggested procedures. To correct for different transfection efficiencies of various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was cotransfected into cells with the GnRHR promoter-luciferase construct. Briefly, 4 x 105 of cells were seeded into six-well tissue culture plates before the day of transfection. One microgram of the GnRHR promoter-luciferase construct and 1 µg of RSV-lacZ vector were cotransfected into the cells under serum-free conditions. After 3 h of transfection, 1 ml of medium containing 20% FBS was added, and the cells were further incubated for 24 h. After incubation, the old medium was removed and the cells were cultured for another 24 h with normal fresh medium containing 10% FBS. Cellular lysates were collected with 150 µl reporter lysis buffer (Promega Corp., Nepean, Canada) and immediately assayed for luciferase activity with the Luciferase Assay System (Promega Corp.). Luminescence was measured using a Lumat LB 9507 luminometer (EG&G, Berthold, Germany). ß-Galactosidase activity was measured using the ß-Galactosidase Enzyme Assay System (Promega Corp.) 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.

Primer Extension Analysis
The transcription start site was identified by primer extension analysis as previously described (29) using oligonucleotides PE-1 and PE-2 (Table 1Go). Briefly, each primer was end-radiolabeled with [32P]ATP by T4 polynucleotide kinase (Life Technologies, Inc.) and hybridized with 60 µg total RNA for 42 C overnight. The RNA was then reverse transcribed at 42 C for 2 h with 20 U SuperScript RNaseH-reverse transcriptase (Life Technologies, Inc.), and the reaction was stopped by the addition of ribonuclease A (20 µg/ml). The extended products were purified by phenol-chloroform extraction and analyzed on a 6% polyacrylamide/7.0 M urea gel. A sequencing ladder (A, C, G, T) was generated from M13 mp18 DNA using the universal primer provided by the T7 Sequencing Kit (Amersham Pharmacia Biotech) and used as a size standard.

Site-Directed Mutagenesis
Human GnRHR 5'-flanking region from nt -1300 to -1018 subcloned into the pGL2-Basic vector (Promega Corp.) was used as a template for the mutagenesis reaction. Mutations were introduced by a three-step PCR mutagenesis method as described previously (28) using mutagenic primers MP-dC/EBP, MP-GATA, and MP-pC/EBP and universal primers MP-B, MP-D, and pGL2-BasicR (Table 1Go). Mutation was confirmed by restriction enzyme digestion and DNA sequence analysis.

EMSA
Overlapping oligonucleotides corresponding to the putative C/EBP and GATA motifs and mutated sequences used in competitive EMSA (Table 1Go) were synthesized by the Oligonucleotide Synthesis Laboratory (University of British Columbia, Vancouver, Canada) and annealed to form double-stranded DNA. Consensus C/EBP (C/EBP-c), Oct-1 (Oct-1-c), GATA (GATA-c), AP-1-c, CREB-c, NF-{kappa}ß-c, and GR-c oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Probes for EMSA were end-radiolabeled with [32P]ATP by T4 polynucleotide kinase (Life Technologies, Inc.) and separated from unincorporated radionucleotides by Microspin G-25 columns (Amersham Pharmacia Biotech). Nuclear extracts were prepared from primary granulosa-luteal cells and SVOG-4o cells according to the method described previously (28). Protein concentrations were determined by a modified Bradford assay (Bio-Rad Laboratories, Inc., Richmond, CA). Gel mobility shift assays were carried out in 20 µl containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 1 µg poly (dI:dC), 2–10 µg nuclear proteins, and 50 fmol radiolabeled probe (30,000 cpm). For the competitive assays, the competitor oligonucleotides were added simultaneously with the labeled probe. The binding reaction was incubated at room temperature for 30 min and separated on a 6% polyacrylamide gel containing 1 x 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 and exposed to the Kodak x-ray film (Eastman Kodak Co.) at -70 C.

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


    ACKNOWLEDGMENTS
 
We thank Professor Karande for providing the mouse monoclonal antibody F1G4 specific to human GnRHR and Dr. K. W. Cheng for his technical assistance.


    FOOTNOTES
 
This work was supported by grants from the Canadian Institutes of Health Research. P.C.K.L. is a Distinguished Scholar of the Michael Smith Foundation for Health Research.

Abbreviations: AP-1, Activating protein-1; C/EBP, CCAAT/enhancer binding protein; CREB, cAMP-responsive element binding protein; dC/EBP, distal C/EBP; FBS, fetal bovine serum; GnRHR, GnRH receptor; GR, glucocorticoid receptor; HDF, human dermal fibroblast; NF-{kappa}ß, nuclear factor-{kappa}ß; nt, nucleotide; pC/EBP, proximal C/EBP; RSV, Rous sarcoma virus.

Received for publication January 31, 2002. Accepted for publication March 18, 2002.


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