Expression and transcriptional regulation of the GnRH receptor gene ...

Molecular Human Reproduction Vol.11, No.11 pp. 837–842, 2005 Advance Access publication December 19, 2005

doi:10.1093/molehr/gah241

Expression and transcriptional regulation of the GnRH receptor gene in human neuronal cells Chung-Man Yeung1,4, Beum-Soo An1, Chi Keung Cheng1, Billy K.C.Chow2 and Peter C.K.Leung1,3 1

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada and 2Department of Zoology, University of Hong Kong, Hong Kong, China

3

To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of British Columbia, 2H30-4490 Oak Street, British Columbia Women’s Hospital, Vancouver, Canada. E-mail: [email protected]

4

Present address: Institute of Molecular Biology, University of Hong Kong, Hong Kong, China

GnRH, acts via the GnRH receptor (GnRHR), plays a pivotal role in human reproduction by stimulating the synthesis and secretion of gonadotropins from pituitary gonadotropes. Studies have also suggested that it has other extra-pituitary functions. To date, the transcriptional regulation of human GnRHR gene in the brain remains largely unknown. Recently, the human cerebellar medulloblastoma cell line TE-671 is found to express GnRH. We report here for the first time that GnRHR is also expressed in this neuronal cell line. Treatment with GnRHR agonist stimulated the phosphorylation of both ERK1/2 and JNK in the cells. Moreover, transient transfection of various human GnRHR promoter-luciferase constructs into the cells identified an upstream promoter region located between –2197 and –1018. Important cis-acting regulatory elements were found at –1300/–1018 and –2197/– 1900, as deletion of either region caused a dramatic decrease in the promoter activity. An upstream GnRHR promoter element was identified to be important for basal transcription in the human neuronal TE-671 cells, in contrast to the previous finding that a downstream promoter is responsible for the gonadotrope-specific expression. Furthermore, we showed that antide (GnRHR antagonist) significantly stimulated the GnRHR promoter activity and inhibition of protein kinase C (PKC) pathway by staurosporine could also up-regulate the promoter activity in dose- and time-dependent manners. Taken together, these data suggest that activation of the GnRHR by interacting with GnRH may transcriptionally down-regulate itself via the PKC pathway in human neuronal cells. Key words: GnRH receptor/PKC/TE-671/staurosporine/transcriptional regulation

Introduction The hypothalamic decapeptide, GnRH, plays a pivotal role in mammalian reproduction by stimulating the synthesis and secretion of gonadotropins via binding to the GnRH receptor (GnRHR) on the pituitary gonadotropes (Gharib et al., 1990). Besides, other extrapituitary functions of GnRH have also been reported. GnRH and its receptor transcripts have been detected in the ovary and placenta (Peng et al., 1994; Lin et al., 1995; Minaretzis et al., 1995), and these findings strongly suggest that GnRH has important autocrine and/or paracrine roles to play in those organs (Bussenot et al., 1993; Furger et al., 1996; Kang et al., 2001). Due to the lack of human pituitary cells, we have previously used the mouse αT3-1 cell line as an experimental model system to study the transcriptional regulation of the human GnRHR gene at the pituitary level (Ngan et al., 1999; Cheng et al., 2000a, 2001a; Kang et al., 2000). We have also studied the regulation of GnRHR expression in placental cells (Cheng et al., 2000b) and identified an upstream placenta-specific promoter (Cheng et al., 2001b). More recently, we have characterized another novel upstream promoter for the human GnRHR gene in ovarian granulosa-lutein cells (Cheng et al., 2002a). Taken together, these studies indicate that tissue-specific expression of the human GnRHR gene is mediated, at least partly, by differential

usage of various promoter regions. Apart from its expression in the pituitary and other peripheral tissues, GnRHR transcripts were also detected in the whole brain (Fan et al., 1995). Notably, it was found that the multiple transcriptional start sites identified by primer extension using human brain total RNA (Fan et al., 1995) were different from those identified in the pituitary (Kakar, 1997). This observation suggests that the regulation of GnRHR expression in other brain regions may be different from the pituitary. To date, the transcriptional regulation of human GnRHR gene in the brain remains largely unknown. Recently, the human cerebellar medulloblastoma cell line TE-671 was found to express GnRH (Chen et al., 2001a). In this study, we showed that GnRHR is also expressed in the TE-671 cells. Furthermore, the transcriptional activity of the GnRHR promoter in these neuronal cells was found to be up-regulated by a protein kinase C (PKC) inhibitor (staurosporine), and hence suggesting the involvement of PKC signalling pathway in the regulation of human GnRHR gene expression in the brain.

Materials and methods Cells and cell culture The human cerebellar medulloblastoma cell line TE-671 was obtained from American Type Culture Collection (Manassas, VA, USA). The sources of

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C.-M.Yeung et al. other cell lines used in this study were described earlier (Cheng et al., 2002a, 2001b). The neuronal TE-671 cells and placental JEG-3 cells were maintained in DMEM (Invitrogen, Burlington, Canada) supplemented with 10% Fetal Bovine Serum (FBS) (Hyclone, Logan, UT, USA). The ovarian OVCAR-3 cells were maintained in M199/MCDB105 (1:1) supplemented with 10% FBS (Hyclone). Cultures were maintained at 37°C in humidified atmosphere of 5% CO2 in air.

RNA extraction, RT–PCR and Southern blot analysis Total RNA was isolated from cell cultures by RNesay Mini Kit (Qiagen, Chatsworth, CA, USA). Five μg total RNA extracted from each cell line were reverse transcribed using the Superscript™ II Reverse Transcriptase (Invitrogen) according to the manufacturer’s suggested protocol. Two gene specific primers (forward: 5′-GGGATGTGGAACATTACAGTCC-3′ and backward: 5′-GGATGATGAAGAGGCAGCTGAA-3′) as described in our earlier study (Cheng et al., 2002a) were used in the RT–PCR (with the expected product size of 373 bp) for detecting human GnRHR expression. The PCR was carried out for 36 cycles with denaturation for 1 min 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 PCR products were separated by agarose gel electrophoresis and transferred onto nylon membranes (Amersham Pharmacia Biotech, Morgan, Canada) for Southern blot analyses according to the manufacturer’s suggested protocols. 32P-labelled probes were synthesized using full-length human GnRHR cDNA as the template by ready-to-go DNA labelling beads (Amersham Pharmacia Biotech, Morgan, Canada) and purified using ProbeQuant G-50 micro columns (Amersham Pharmacia Biotech, Morgan, Canada) for subsequent hybridization. The membranes were finally washed and exposed to Kodak X-OMAT films (Eastman Kodak Co., Rochester, NY, USA). In addition, two glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers (forward: 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and backward: 5′-CATGTGGGCCATGACGTCCACCAC-3′) were used in the RT–PCR (with the expected product size of 983 bp) to demonstrate for the quality of cDNA template from each sample. The PCR was performed for 20 cycles with denaturation for 30 s at 94°C, annealing for 1 min at 65°C and extension for 1 min at 72°C.

harvested for β-galactosidase and luciferase assays. The cellular lysates were collected with 150 μl reporter lysis buffer (Promega Corp., Nepean, Canada) and immediately assayed for luciferase and β-galactosidase activities with the Luciferase Assay System and β-Galactosidase Enzyme Assay System (Promega Corp.), respectively. Luminescence was measured using a Lumat LB 9507 luminometer (E.G&G, Berthold, Germany). The promoter activity was calculated as luciferase activity/β-galactosidase activity and expressed as fold of increase relative to the activity of the promoterless pGL2-basic control vector. In experiments where the effects of GnRHa [the GnRHR agonist, D-(Ala6)GnRH], antide (the GnRHR antagonist) and staurosporine (the PKC inhibitor) on luciferase activities were studied, both the control vector-transfected and promoter construct-transfected cells were treated with the corresponding drugs in culture medium containing 2% FBS for the designated periods of time before doing the β-galactosidase and luciferase assays. The pharmacological reagents used in this study were purchased from Sigma-Aldrich Corp. (Oakville, Ontario, Canada).

Data analysis Data were shown as the mean ± SEM of at least three independent experiments and were analysed by one-way analysis of variance (ANOVA) followed by either Dunnett’s or Tukey’s test using the computer software PRISM (GraphPad Software Inc., San Diego, CA, USA). Data were considered significantly different from each other at P < 0.05.

Results Expression of GnRHR in TE-671 cells The expression of human GnRHR in the TE-671 cells was analysed by RT–PCR, followed by Southern blot analysis of the PCR products. GnRHR mRNA is found in this neuronal TE-671 cell line (Figure 1) as well as in two positive control cell lines, the placental JEG-3 and ovarian OVCAR-3, which have been shown earlier that they express the GnRHR (Cheng et al., 2000b; Kang et al., 2000).

Immunoblot analysis The activation of ERK1/2 and JNK by GnRH in the TE-671 cells was analysed by immunoblot assay as previously described (Kang et al., 2001). Briefly, protein samples were subject to 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred to a nitrocellulose membrane. The membrane was immunoblotted using a rabbit polyclonal antibody for phosphorylated ERK1/2 (1:500) and JNK/SAPK (1:500) (Biosource International Inc., Camarillo, CA, USA). Alternatively, the membrane was probed with pan ERK1/2 (1:500) and pan JNK/SAPK1 (1:500) antibody (Biosource International Inc.) for detecting the total ERK1/2 and JNK/SAPK1 levels, respectively.

Preparation of human GnRHR promoter-luciferase constructs and plasmid DNA The human GnRHR promoter-luciferase construct p(–2297/+1)-Luc (numbering is relative to the ATG) and the progressive 5′- or 3′-deletion promoter constructs were prepared as previously described (Cheng et al., 2000a, 2002a). All plasmid DNA used in this study were prepared using Qiagen Plasmid Midi Kits (Qiagen) according to the manufacturer’s suggested procedures.

Activation of ERK1/2 and JNK by GnRH in TE-671 cells It is known that activation of GnRHR is capable of triggering multiple signal transduction pathways including the mitogen-activated protein kinase (MAPK) cascades (Cheng and Leung, 2000). In this study, we showed that treatment with GnRHR agonist stimulated the phosphorylation of both ERK1/2 and JNK in the TE-671 cells (Figure 2).

Identification of an upstream GnRHR promoter (–2197 to –1018) in TE-671 cells The finding of GnRHR expression in the TE-671 cells suggests that this neuronal cell line is a useful model system for studying the transcriptional regulation of human GnRHR gene. Various human

Transient transfection, reporter gene assay and pharmacological treatments Transient transfections were carried out using the Lipofectamine Reagent (Invitrogen) according to the manufacturer’s suggested protocol. To correct for the different transfection efficiencies of various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was co-transfected with the human GnRHR promoter-luciferase construct into the cells. In summary, 4 × 105 cells (per well) were seeded into six-well tissue culture plates before the day of transfection. For each well, 1 μg GnRHR promoter-luciferase construct and 0.5 μg RSV-lacZ DNA were co-transfected into the cells in 1 ml serum-free medium. After 5 h of transfection, 1 ml medium containing 20% FBS was added, and the cells were further incubated for a total transfection time of 24 h. After incubation, the old medium was removed, and the cells were cultured for another 24 h with fresh medium containing 10% FBS before the cellular lysates were

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Figure 1. Detection of human GnRHR expression by RT–PCR coupled to Southern blot analysis—GnRHR transcripts were expressed in the TE-671, JEG-3 and OVCAR-3 cells. Control PCR, using a pair of glyceraldehyde-3phosphate dehydrogenase (GAPDH)-specific primers, was performed in each sample to demonstrate for the quality of the cDNA template. No PCR product was obtained in the negative control that used water instead of cDNA as the template.

GnRH receptor in human neuronal cells might also be present within that region. Analysis of other 5′ deletion constructs demonstrated that the promoter activity of p(–2197/– 1018)-Luc was significantly reduced when the most distal region (from –2197 to –1901) was removed (Figure 4). Further 5′ deletions had no significant effects and could not restore its promoter function (Figure 4). Taken together, these results indicate that the upstream GnRHR 5′ flanking region located from –2197 to –1018 is transcriptionally active and able to direct a basal promoter activity in the TE-671 cells.

Stimulatory effect of antide and staurosporine on the GnRHR promoter activity in TE-671 cells

Figure 2. Activation of ERK1/2 and JNK by GnRHa in the TE-671 cells—the phosphorylation status of ERK1/2 and JNK in the cells treated with 100 nM GnRHa for 5, 10, 15, 30 and 60 min was analysed by immunoblot assay.

GnRHR promoter-luciferase constructs were transfected into the TE-671 cells to determine their promoter activities. The construct p(–2197/– 1018)-Luc was found to have the maximal promoter activity in the cells and further 3′ deletion of this construct [to generate the p(–2197/ –1346)-Luc construct] could completely abolish the promoter activity (Figure 3), suggesting that the region within –1346/–1018 contains important element(s) for the promoter function. The relative promoter activity of p(–2197/–1346)-Luc was found to be no different from that of the pGL2-basic. The promoter function was also completely lost when using the construct p(–2197/–771)-Luc which contains the nucleotides from –1017 to –771. In fact, this result was consistent with our previous finding that construct containing this region could invariably suppress the GnRHR promoter activity in different cell types due to the presence of a strong repressor element located between the nucleotide –1017 and –771 (Cheng et al., 2002b). Inclusion of the region –771/–167 in p(–2197/–167)-Luc could restore the promoter activity of p(–2197/–771)-Luc with a two-fold increase in activity (Figure 3), indicating that important regulatory element(s)

We next determined the effects of treating TE-671 cells with specific GnRHR agonist and antagonist (antide) on the GnRHR promoter activity. There were no significant changes observed in the promoter activities when the cells were treated with GnRHa (Figure 5). However, the promoter activity was stimulated by antide treatment (Figure 6), suggesting that blockage of the GnRHR signalling pathway in the TE671 cells could enhance its own promoter activity. Our present data showed that GnRH was able to activate the MAPK cascade in the TE671 cells (Figure 2). It has also been demonstrated that GnRH can activate the MAPK cascade by a PKC-dependent mechanism (Benard et al., 2001; Farshori et al., 2003; Shah et al., 2003). The cells were therefore treated with staurosporine to determine the effect of blocking the PKC pathway on the promoter activity. The results indicated that staurosporine dose- and time-dependently stimulated the GnRHR promoter activity in the TE-671 cells (Figure 7).

Discussion In this study, we demonstrated for the first time that GnRHR mRNA is expressed in human neuronal TE-671 cells. We further showed that treatment with GnRHR agonist stimulated the phosphorylation of ERK1/2 and JNK, indicating that functional GnRHR is expressed in the TE-671 cells. The transcriptional regulation of human GnRH genes has been previously characterized in the TE-671 cells (Chen et al., 2001b, 2002; Cheng et al., 2003). Using the same cell line, we herein identified an upstream GnRHR promoter located between –2197 and –1018. Within this 5′-flanking region, important basal regulatory elements are located at –1300/–1018 and –2197/–1900, as deletion of either region caused a dramatic decrease in the promoter activity. These results therefore

Figure 3. Functional characterization of various human GnRHR promoter-luciferase constructs in the TE-671 cells—the promoter activity was expressed as fold of increase relative to the activity of the promoterless pGL2-basic control vector. a, P < 0.01 versus pGL2-basic; b, P < 0.001 versus other constructs.

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Figure 4. Fine mapping of the human GnRHR promoter region between –2197 and –1018 by progressive 5′-deletion analysis in the TE-671 cells. The promoter activity was expressed as fold of increase relative to the activity of the promoterless pGL2-basic control vector. a, P < 0.01 versus pGL2-basic; b, P < 0.001 versus p(–2197/–1018)-Luc.

Figure 5. Promoter activities of various human GnRHR promoter-luciferase constructs in the TE-671 cells treated with 100 nM GnRHa for 18 h. Similar results (data not shown) were obtained when the cells were treated for a shorter period of time (4 h). The promoter activity was expressed as fold of increase relative to the activity of the promoterless pGL2-basic control vector.

Figure 6. Stimulation of the human GnRHR promoter construct p(–2197/ –1018)-Luc in the TE-671 cells treated with 1 μM antide (GnRHR antagonist). a, P < 0.05 versus control; b, P < 0.01 versus control.

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indicate that in contrast to the downstream promoter that is responsible for gonadotrope-specific expression of the GnRHR gene (Ngan et al., 1999), an upstream GnRHR promoter element is important for basal transcription in the human neuronal TE-671 cells. The results further suggest that tissue-specific expression of the human GnRHR gene is mediated by differential usage of various promoter regions. It is not uncommon that tissue-specific gene expressions are regulated using multiple promoters. For example, the expression of human CYP19 gene is similarly controlled by differential usage of tissue-specific promoters (Kamat et al., 2002). Previously, upstream promoters located at –1737/ –1346 and –1300/–1018 were also identified to be responsible for GnRHR expression in the placental cells (Cheng et al., 2001b) and ovarian granulosa cells (Cheng et al., 2002a), respectively. Our present study found that an upstream promoter comprising both distal (–2197/ –1900) and proximal (–1346/–1018) regions seems to be important for its function in the neuronal TE671 cells. Many putative transcription factor binding sites, including Oct–1, AP-1, C/EBP and GATA, are present within these regions. The functional significance of these elements in regulating the GnRHR gene expression in the TE671 cells awaits further investigation.

GnRH receptor in human neuronal cells pathway involving PKC. This study also supports our previous finding that activation of the GnRHR via the PKC pathway is important for transcriptional down-regulation of the human GnRHR gene (Cheng et al., 2000a). In the previous study, a putative AP-1 binding site located at –1000/–994 has been found to be involved in the molecular mechanism of this down-regulation in the pituitary cells. Many putative transcription factor binding sites, including an AP-1 site, are present within the upstream GnRHR promoter region (–2197/–1018) identified in our present study. Further investigation will be required to determine whether the AP-1 binding site also plays a role in mediating the downregulation of human GnRHR gene in the neuronal TE-671 cells. The TE-671 cell line represents one of few continuous cell lines that exhibit a neuronal phenotype (Hemmick et al., 1995). Moreover, the cells possess high levels of functional nicotinic (Syapin et al., 1982; Lukas, 1991) and muscarinic (Bencherif and Lukas, 1991) cholinergic receptors. It has been demonstrated that nicotine was able to induce GnRH release in the bullfrog sympathetic ganglia (Cao and Peng, 1998), and acetylcholine could modulate GnRH release from the mouse GT1-7 hypothalamic neurons through muscarinic receptors (Krsmanovic et al., 1998). Hence, TE-671 cells may represent a useful cell line for studying the functional interaction between cholinergic receptors and GnRHR in human neuronal cells.

Acknowledgements This work was supported by grants from the Canadian Institutes of Health Research. PCKL is the recipient of a Distinguished Scholar award from the Michael Smith Foundation for Health Research. Figure 7. Effect of the protein kinase C (PKC) inhibitor, staurosporine, on the activity of the human GnRHR promoter construct p(–2197/–1018)-Luc in the TE-671 cells—(A) dose-dependent stimulation of the promoter activity by treating the cells with different concentrations of staurosporine for 18 h. (B) Time-dependent stimulation of the promoter activity in the cells exposed to 100 nM staurosporine. Similar stimulatory effects were observed when using the full-length promoter construct p(–2297/+1)-Luc in the time-dependent experiments (data not shown), indicating that DNA regulatory element(s) responsible for mediating this stimulation should be present in the p(–2197/ –1018)-Luc construct. a, P < 0.05 versus control; b, P < 0.01 versus control.

We showed here that treatment with GnRHa had no effect on the GnRHR promoter activity in the TE-671 cells. This observation suggests that the promoter might already be auto-regulated by GnRH, which is endogenously expressed in the TE-671 cells (Chen et al., 2001a). We therefore tested the effect of blocking the endogenous GnRH action by treating the cells with GnRHR antagonist. The results demonstrated that antagonist treatment could significantly increase the promoter activity, suggesting a down-regulation of the GnRHR promoter by the endogenous GnRH. It is known that multiple signal transduction pathways including the MAPK cascades are involved in the regulation of GnRHR gene expression (Cheng and Leung, 2000). This study has demonstrated that GnRH is able to activate the phosphorylation of both ERK1/2 and JNK in the human neuronal TE-671 cells. Similarly, it has also been shown that GnRH can stimulate the phosphorylation of ERK1/2 in a mouse hypothalamic neuronal cell line (Shah et al., 2003). Moreover, previous studies have indicated that GnRH-induced phosphorylation of ERK1/2 is dependent on the activation of PKC (Benard et al., 2001; Farshori et al., 2003; Shah et al., 2003). We therefore treated the TE-671 cells with staurosporine (PKC inhibitor) to determine the effect of PKC inhibition on the GnRHR promoter activity. Treatment with staurosporine was found to be capable of stimulating the promoter activity in the cells. Taken together, these data thus suggest for the transcriptional down-regulation of the GnRHR gene in the neuronal TE-671 cells by its own signalling

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