Gonadotropin-releasing hormone: regulation of the GnRH gene

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1 MINIREVIEW Gonadotropin-releasing hormone: regulation of the GnRH gene Vien H. Y. Lee, Leo T. O. Lee and Billy K. C. Chow School of Biological Sciences, The University of Hong Kong, China Keywords estrogen; follicle-stimulating hormone; GnRH; gonadotropin; luteinizing hormone; PKC signalling; progesterone; promoter; steroid hormone; transcriptional regulation Correspondence B. K. C. Chow, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China Fax: Tel: (Received 18 April 2008, revised 4 August 2008, accepted 29 August 2008) doi: /j x As the key regulator of reproduction, gonadotropin-releasing hormone (GnRH) is released by neurons in the hypothalamus, and transported via the hypothalamo-hypophyseal portal circulation to the anterior pituitary to trigger gonadotropin release for gonadal steroidogenesis and gametogenesis. To achieve appropriate reproductive function, mammals have precise regulatory mechanisms; one of these is the control of GnRH synthesis and release. In the past, the scarcity of GnRH neurons and their widespread distribution in the brain hindered the study of GnRH gene expression. Until recently, the development of GnRH-expressing cell lines with properties similar to those of in vivo GnRH neurons and also transgenic mice facilitated GnRH gene regulation research. This minireview provides a summary of the molecular mechanisms for the control of GnRH-I and GnRH-II gene expression. These include basal transcription regulation, which involves essential cis-acting elements in the GnRH-I and GnRH-II promoters and interacting transcription factors, and also feedback control by gonadotropins and gonadal sex steroids. Other physiological stimuli, e.g. insulin and melatonin, will also be discussed. Introduction Gonadotropin-releasing hormone (GnRH) is a central regulator in the hypothalamic pituitary gonadal axis of the reproductive hormonal cascade. It is expressed in a discrete population of neurosecretory cells located throughout the basal hypothalamus of the brain, and is released into the hypothalamo-hypophyseal portal circulation in a pulsatile manner and in surges during the female preovulatory period [1]. The released GnRH is transported to the anterior pituitary gland, where the hormone binds to its receptor on the gonadotropes. This triggers the synthesis and release of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are responsible for gonadal steroidogenesis and gametogenesis. Abbreviations AP-1, activator protein-1; AR, androgen receptor; atra, all-trans-retinoic acid; C EBP, CCAAT enhancer-binding protein; CREB, camp response element-binding protein; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; Dlx2, distal-less homeobox 2; DREAM, downstream regulatory element antagonist modulator; E 2,17b-estradiol; EMSA, electrophoretic mobility shift assay; ER, estrogen receptor; FSH, follicle-stimulating hormone; GABA, c-aminobutyric acid; GnRH, gonadotropin-releasing hormone; GRG, Groucho-related gene; hcg, human chorionic gonadotropin; hglc, human granulosa-luteal cell; hgnrh-i, human gonadotropin-releasing hormone type I; IGF-I, insulin-like growth factor-i; LH, luteinizing hormone; mgnrh-i, mouse gonadotropin-releasing hormone type I; Msx, muscle segment homeobox; NIRKO, neuron-specific insulin receptor knockout; NMDA, N-methyl-D-aspartic acid; NO, nitric oxide; npre, negative progesterone response element; Oct-1, octamer-binding transcription factor-1; Otx, orthodenticle homeobox; P 4, progesterone; POU, homeodomain protein family of which the founder members are Pit-1, Oct-1/2 and Unc-86 ; PKA, protein kinase A; PKC, protein kinase C; POA, preoptic area; PR, progesterone receptor; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; rgnrh-i, rat gonadotropinreleasing hormone type I; RXR, retinoid X receptor; TPA, 12-O-tetradecanoyl phorbol-13-acetate; b-gal, b-galactosidase FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS

2 V. H. Y. Lee et al. Transcriptional regulation of GnRH gene During embryogenesis, GnRH-expressing neurons arise in the olfactory placode, migrate into the preoptic area (POA), and then extend axons to the median eminence [2]. The hypothalamic expression of GnRH increases gradually during postnatal development and puberty, and is believed to be crucial for the onset of puberty [3]. GnRH is a peptide hormone composed of 10 amino acids (pglu-his-trp-ser-tyr-gly-leu-arg-pro-gly- NH 2 ). Gene expression first gives rise to the prepro- GnRH polypeptide, which consists of a signal peptide, a functional decapeptide, an amidation proteolytic processing signal (Gly-Lys-Arg), and a GnRH-associated peptide [4]. According to the differences in amino acid sequences, localizations and embryonic origins, 24 GnRHs have been identified in the nervous tissues, from vertebrates to protochordates [5,6]. Despite the above divergences, all of these variants are decapeptides that share highly similar structures. Generally, two or three forms of these GnRHs can be found in most vertebrate species. It had been thought that mammals have only one, classical, form of GnRH (GnRH-I). GnRH-I molecules in different mammals have identical amino acid sequences, except in guinea pig, in which the second and seventh amino acids are substituted [7]. In humans, this gene is located on chromosome 8p11.2-p21, with four exons that contain a 276 bp ORF coding for a precursor protein of 92 amino acids. Recently, however, a second form of GnRH (GnRH-II) was identified. As GnRH-II was originally isolated from chicken brain, it was termed chicken GnRH-II [8]. GnRH-II is encoded by a different gene and differs from GnRH-I by amino acids [5,7 9]. GnRH-II is the most ubiquitous peptide of the GnRH neuropeptide family, being present in animals from jaw fish to humans. The highly conserved amino acid sequence of GnRH-II in a wide range of species and over millions of years of evolution suggests the importance of this neuropeptide. The tissue distribution pattern of GnRH-II is dissimilar to that of GnRH-I. Whereas GnRH-I is expressed mostly in the brain, the expression level of GnRH-II is much higher in other organs. cell lines have probably been the only effective and manageable resources with which to explore mechanisms regulating the expression, synthesis and release of GnRH. Studies on transcriptional regulation of the GnRH gene have been performed largely in GnRHsecreting cell lines, such as GT1, GT1-7, and human granulosa-luteal cells (hglcs) [1,10 14]. The GT1 cell is recognized as a good model for studying neuronspecific expression of the GnRH gene, as GT1 cells retain many characteristics of in vivo GnRH neurons. These include distinct neuronal morphology [15], expression of differentiated neuronal markers [16], the pulsatile release of GnRH in cell culture [17,18], and secretion of GnRH in response to particular signals. It should be noted that although in vitro studies in cell lines have been widely employed, they are unlikely to resemble the actual complexity of gene regulation in the brain or other organs. Only recently has the development of various transgenic mice enabled the investigation of GnRH gene expression and regulation in vivo [19 21]. In this minireview, we summarize studies regarding GnRH-I and GnRH-II gene regulation, including essential cis-acting elements in the promoter, and also the interaction of transcription factors in achieving the basal expression levels. In addition, other components of the hypothalamic pituitary gonadal axis with roles in the control of GnRH-I gene expression will also be discussed. These include gonadotropin, gonadal sex steroids and other physiological regulators. GnRH-I Analysis of the promoters of the GnRH-I and GnRH-II genes showed that they contain essentially different putative transcription factor-binding sites that are important for their basal transcription activities, suggesting that the two genes are probably differentially regulated. Because of the recent discovery of GnRH-II as a new isoform of GnRH, the majority of the studies have been done on the GnRH-I promoter, and there are only a few regarding regulation of the GnRH-II gene. Regulation of GnRH gene expression In view of the fact that GnRH is essential for reproductive processes, understanding the control of its synthesis and release is therefore of the utmost importance. However, it is difficult to study the regulation of GnRH gene transcription in vivo, due to the scarcity and scattered distribution of the GnRH neurons. In the past, immortalized GnRH-expressing neuronal The GnRH-I promoter Among the studies on the transcriptional regulation of GnRH genes, most have been performed on GnRH-I. The 5 -flanking region of the GnRH-I gene is highly homologous between species [22], especially human, rat and mouse. A summary of the essential elements in the promoter regions of the rat, mouse and human GnRH-I genes is given in this minireview (Figs 1 3). FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 5459

3 Transcriptional regulation of GnRH gene V. H. Y. Lee et al. Pulsatile expression Ref.49 hcg responsiveness Ref.39 Oct 1 ( 1785/ 1771) Ref.28 Pbx/Prep1 ( 1753/ 1734) Ref.36 GATA ( 1748/ 1743) Ref.10 GATA ( 1715/ 1710) Ref.10 Oct 1 ( 1702/ 1695) Ref.28 C/EBP ( 1684/ 1676) NO responsiveness Ref.122 Pbx/Prep1 ( 1612/ 1593) Ref.36 Oct 1 ( 1569/ 1562) Ref Otx/ bicoid ( 153/ 146) Ref.14 Pbx/Prep1 ( 109/ 89) Ref.36 AP 1 ( 99/ 94) AT-rich site ( 91/ 87) AT-rich site ( 85/ 81) Pbx/Prep1 ( 84/ 61) Ref.36 Msx1 & Dlx2 ( 54/ 51) Ref.30 Msx1 & Dlx2 ( 41/ 38) Ref / / / / / 1689 Melatonin responsiveness Ref.117 Msx1 & Dlx2 ( 1634/ 1631) Msx1 & Dlx2 ( 1620/ 1617) Ref.31 PKC responsiveness ( 1800/ 1576) Ref RARE( 1494/ 1470) RA responsiveness Ref.127 Oct 1 ( 110/ 88) Ref.12 PKC responsiveness ( 126/ 73) Ref.42 npre ( 171/ 73) Oct 1 ( 47/ 40) Ref.12 P4 responsiveness Ref.90 Fig. 1. Diagrammatic representation of the rgnrh-i gene 5 -region. The promoter region is indicated by the green box [22] and the enhancer by the yellow box [23]. Also, the locations of key regulatory elements and their functional significance are listed Insulin responsiveness ( 1250/ 587) Ref Gn RH neuron specificity Ref.37 Otx2( 319/ 315) Otx2( 257/ 252) E 2 responsiveness Ref.70 ERβ 1 ( 225/ 201) ERβ 1 ( 184/ 150) Enhancer--GnRH neuron specificity Repressor--ovary specificity ( 3446/ 2078) Ref.20 Egr 1 ( 75/ 67) insulin responsiveness Ref.112 Fig. 2. Diagrammatic representation of the mgnrh-i gene 5 -region. The promoter region is indicated by the green box [20] and the enhancer by the blue box [19]. Also, the locations of key regulatory elements and their functional significance are listed. GnRH neuron specificity ( 992/ 763) Ref.21 E 2 responsiveness ( 548/ 169) Ref.77 Brn-2 ( 925/ 916) ERE ( 925/ 916) Ref.72 Brn-2 ( 867/ 858) AP-1 ( 402/ 396) IGF-I responsiveness Ref Constitutive expression in GT1-7 cell ( 1131/ 350) Ref.24 Fig. 3. Diagrammatic representation of the hgnrh-i gene 5 -region. The promoter region is indicated by the green box [25], and the locations of key regulatory elements and their functional significance are listed FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS

4 V. H. Y. Lee et al. Transcriptional regulation of GnRH gene The transcription start sites For the determination of the transcription initiation site in the rat and mouse GnRH genes, primer extension analysis was employed [23]. In the study, the first exons in the rat and mouse GnRH genes were found to be 145 and 58 bp respectively, using polya + RNA from rat hypothalamus and total RNA from mouse hypothalamic GT1-7 cells. In humans, studies showed that transcription of the GnRH gene can be initiated at two distinct transcription start sites in the hypothalamus and nonhypothalamic tissues such as ovary, testes, placenta and mammary gland [24 26]. In the hypothalamus, the transcription start site was characterized at 61 bp upstream of the first exon intron junction, whereas a discrete upstream transcription start site, which is 579 bp upstream from the hypothalamic start site, was identified later in a human placental tumor cell line (JEG) and a human breast tumor cell line (MDA), using primer extension and RT-PCR assays. The placental GnRH cdnas were reported to have a longer 5 -UTR than that found in the hypothalamus. Radovick et al. found that the alternative mrna was produced by differential splicing of the GnRH gene. The first intron is removed in the hypothalamus, whereas it is retained in the placenta. Also, the human upstream promoter has been found to have a higher level of transcriptional activity than the downstream one. However, it is not homologous to the upstream region of rat and mouse genes, and there is no evidence showing the use of the upstream promoter in any of the rat and mouse tissues [24]. A later study was carried out to compare the GnRH gene of nonhuman primates with that of humans [25]. The study showed the presence of an upstream transcription start site in the cynomolgus monkey, 504 bp upstream of the hypothalamic promoter, and 75 bp downstream of the human upstream start site. Sequence analysis showed that the cynomolgus monkey and the human upstream promoter share high similarity (94%). Rat GnRH-I promoter Two key regions, including a proximal promoter and a distal enhancer, have been identified in the rat GnRH-I (rgnrh-i) gene that are important for gene transcription (Fig. 1). The promoter is located 173 bp upstream of the transcription start site [22]. It is evolutionarily conserved, with about 80% nucleotide homology among human, rat and mouse. The 300 bp enhancer is located at )1863 to )1571 bp relative to the transcription start site. It provides fold activation of GnRH gene transcription as compared to the activity of the promoter alone [22,27]. The rat promoter has been widely studied for many years; specific binding sites for a number of different transcription factors have been found within the promoter and enhancer. Mouse GnRH-I promoter The two promoter regions of GnRH-I are highly conserved in rat and mouse (Fig. 2). The development of transgenic mice provided in vivo models for GnRH gene regulation studies. Transgenic mice carrying various deletion fragments of the mouse GnRH-I (mgnrh-i) gene fused to a reporter gene have been used for identifying essential sequences for GnRH-I gene expression in GnRH neurons and in the ovary in vivo [19,20]. Pape et al. demonstrated that a 5.5-kb fragment of the 5 -region of the mgnrh-i gene was sufficient to target b-galactosidase (b-gal) and thus GnRH-I expression in about 85% of GnRH neurons [19]. Deletion of the 5 -flanking sequence to 2.1 kb resulted in a 40% reduction in the number of b-gal-expressing GnRH neurons. This suggests that enhancer element(s) are present in the region between )5.5 and )2.1 kb of the mgnrh-i gene. More importantly, further 5 -deletion to )1.7 kb resulted in total loss of b-gal detection. This indicates that the 400 bp region ()2.1 to )1.7 kb) is a critical enhancer region for the mgnrh-i gene in mouse brain in vivo. Later, Kim et al. also worked on transgenic mice, and demonstrated that specific expression of the mgnrh-i gene in the hypothalamus and ovary depends on a proximal region ()1005 bp) of the mgnrh-i promoter in the hypothalamus and ovary [20]. This indicates the presence of elements specific to the hypothalamus and ovary within the )1005 bp region of the mouse promoter. Moreover, through generation of transgenic mice with deletion fragments, the region between )3446 and )2078 bp, which was found to have about 90% homology with the rgnrh enhancer, was shown to be an enhancer for in vivo expression of hypothalamic mgnrh, as well as a repressor that represses mgnrh gene expression in the mouse ovary. Human GnRH-I promoter Sequence alignment has revealed similarities and 8 differences between the human GnRH-I (hgnrh-i) gene and the rgnrh-i gene [28] (Fig. 3). Within the distal promoter of the hgnrh gene, three regions ()3036 to )2923 bp, )2766 to )2539 bp, and )1775 to )1552 bp) were found to be similar to sequences in the rgnrh promoter. In the proximal promoter region, the )343 to +8 bp region of the hgnrh-i gene was found to have marked homology with the )332 to +96 bp FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 5461

5 Transcriptional regulation of GnRH gene V. H. Y. Lee et al. region of the rgnrh-i gene. However, the sequence between )1552 and )579 bp in the hgnrh-i gene has little similarity with the rat promoter. In Gn10 cells, Dong et al. identified the hgnrh promoter in the )551 to +1 bp region [24]. In agreement with this, Kepa et al. found that the )3832 to +8 bp fragment of the hgnrh-i gene gave high levels of expression of reporter and GnRH-I genes in GT1-7 cells [28]. A 5 -deletion to )1131 bp had no effect, whereas a 3 -deletion to )350 bp led to a significant reduction (70%) of promoter activity, showing the importance of the )1131 to )350 bp region for regulation of the hgnrh-i gene. In in vivo studies using transgenic mice with 5 -deletion fragments of the hgnrh-i gene, the )1131 to )484 bp region was found to include cell-specific elements for hgnrh gene expression [29]. Later studies further determined that the )992 to )763 bp region is essential and sufficient for specific hgnrh gene expression in GnRH neurons [21]. Regulation of GnRH-I by essential transcription factors Oct-1, Msx1, Dlx2 and cofactors Octamer-binding transcription factor-1 (Oct-1) plays a critical role in the regulation of rgnrh-i transcription, binding functional elements in the proximal promoter region [12]. DNase I protection experiments revealed that a 51-bp sequence ()76 to )26 bp) conferred a 20- fold induction of the rgnrh-i gene in GT1-7 cells. This region contains an octamer-like motif ()47 to )40 bp) to which Oct-1, a member of the homeodomain protein family of which the founder members are Pit-1, Oct-1/2 and Unc-86 (POU), was found to bind. Oct-1 was also found to bind the octamer motifs in another promoter region ()110 to )88 bp). Within the enhancer of the rgnrh-i gene, two POU homeoprotein Oct-1-binding sites, OCT1BS-a ()1785 to )1771 bp) and OCTBS-1b ()1702 to )1695 bp), which share a 6-bp sequence with the octamer consensus sequence (ATGCAAAT), were identified [30]. Electrophoretic mobility shift assays (EMSAs) showed the binding of Oct-1 proteins to both sites. Block mutation of OCT1BS-a resulted in a significant reduction (95% reduction) in transcriptional activity, showing that OCT1BS-a is the most crucial element for transcriptional activity of the GnRH-I gene enhancer. However, mutation of OCT1BS-b had no effect on the enhancer activity. Consistently, OCT1BS-b was also reported to be not involved in basal or unstimulated enhancer activity of the rgnrh-i gene in GT1-7 cells [31]. Mutation of OCT1BS-b resulted in elimination of repression by the glutamine NO cgmp signaling pathway, but did not influence the nonrepressed GnRH gene expression. This suggested that OCT1BS-b may play a role in modulated but not basal transcriptional activity of the rgnrh-i gene. Two other homeodomain proteins, Mex1 and distalless homeobox 2 (Dlx2), have also been identified as being responsible for GnRH-I gene regulation. Within the proximal promoter and the enhancer of the rgnrh-i gene, four conserved consensus homeodomain sites (ATTA) ()41 to )38 bp, )54 to )51 bp, )1620 to )1617 bp, and )1634 to )1631 bp) have been identified as being essential for basal and cell-specific expression of rgnrh-i in GT1-7 cells [32,33]. Also, Givens et al. found that muscle segment homeobox (Msx) and Dlx, which are members of the antennapedia class of non-hox homeodomain transcription factors, bind to the ATTA consensus sequence [34]. Msx1 is found as a repressor, whereas Dlx2 is an activator, and they functionally antagonize each other by competing for the ATTA elements in the rgnrh-i gene regulatory regions. The majority of the identified transcriptional regulators of the GnRH genes are homeodomain proteins with promiscuous DNA-binding properties, and most are not solely expressed in GnRH neurons. To achieve specific activity of the promoter and target expression of GnRH in GnRH neurons, specific interactions of the transcription regulators with specific cofactors are required [35]. These cofactors can enhance or inhibit the interactions between the homeodomain proteins and the transcriptional regulatory regions of the GnRH promoter and or enhancer to achieve specific GnRH expression in the hypothalamic GnRH neurons and in nonhypothalamic tissues, such as the ovary. Ravel-Harel et al. reported that the Groucho-related gene (GRG) proteins, which belong to the GRG family of coregulators, associate with the GnRH promoter in vivo and interact with Oct-1 and Msx1 in GT1-7 cells [36]. GRG proteins mediate the dynamic switch between activation and repression of GnRH-I transcription. Using glutathione S-transferase pull-down assays, the long-form GRG proteins (GRG1 and GRG4) and the short truncated form (GRG5) were found to interact with Oct-1 and Msx1, probably through the POU domain of Oct-1 and the engrailed homology domain of Msx1. As shown in overexpression studies, the long GRG forms are repressors of GnRH-I gene transcription. They repress the Oct- 1-mediated activation and act as corepressors of Msx1, mediating downregulation of GnRH-I expression. In contrast, the short form GRG5 is an enhancer that reverses the repressive activity of GRG4. A three amino acid loop extension (TALE) homeodomain transcription factor, Pbx1b, was also found to 5462 FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS

6 V. H. Y. Lee et al. Transcriptional regulation of GnRH gene be a cofactor of Oct-1 by using a yeast two-hybrid system [37]. Moreover, Pbx1b contains the Meis homologous regions in the N-terminus and the Prep1 homeodomain in the C-terminus for interaction with its cofactors Prep1 and Meis [38]. A GST pull-down assay showed the in vitro interaction of Oct-1 with Pbx1b and its cofactor Prep1. EMSA showed heterodimers containing TALE proteins, Pbx Prep1 and Pbx Meis1, in GT1-7 nuclear extract bound to four binding sites within both the promoter (at )100 and )75 bp) and enhancer (at )1749 and )1603 bp). These binding sites are in close proximity to or even overlap with the Oct-1 sites. Both Pbx1 and Prep1 are coactivators of Oct-1 in GnRH-I expression, because coexpression of Oct-1 with either Pbx1b or Prep1 resulted in significant activation of GnRH-I gene transcription, whereas no significant change was observed when these constructs were overexpressed individually without Oct-1. Within those Pbx1 Prep1-binding motifs, only mutation in the )1749 bp binding site can eliminate the activation, indicating that the transactivation is specifically dependent on this motif. Otx2 In the proximal promoter of the rgnrh-i gene, an orthodenticle homeobox (Otx) bicoid site ()153 to )146 bp) is conserved across several vertebrate species [14]. This element in the rgnrh-i promoter was found to bind Otx2 proteins. The promoter activity was significantly reduced in both the promoter alone and the promoter with enhancer constructs after the Otx bicoid site was mutated. Moreover, overexpression of Otx2 in GT1-7 cells resulted in induction of rgnrh-i promoter activity. This showed that the Otx bicoid element was important for basal and also enhancer-driven transcription of the GnRH-I gene. Recently, the critical role of Otx2 in regulating tissue-specific expression of the mgnrh-i gene has been discovered. In a transgenic mice study, high luciferase activities were only detected in the hypothalamus and gonads when a DNA construct containing the )356 to +28 bp region of the mgnrh-i gene fused to a luciferase reporter gene was used to generate transgenic mice [39]. However, transgenic mice with the 5 -deletion construct )249 to +28 bp showed high luciferase expression only in gonads. This difference indicated that the DNA sequence between )356 and )249 bp was essential for neuron-specific expression of the GnRH-I gene. Within this region, Kim et al. identified two consensus Otx2-binding sites, a low-affinity binding site (TTATC, )319 to )315 bp) and a high-affinity binding site (TA ATCC, )257 to )252 bp). EMSA demonstrated that Otx2 binds both consensus sites specifically. Overexpression of Otx2 in GN11 cells increased mgnrh gene transcriptional activity by more than fivefold. Moreover, in vivo studies using Otx2-binding sites in mutated transgenic mice showed that elimination of these Otx2 sites resulted in reduced GnRH promoter activities in the mouse brain. This further confirmed the importance of Otx2 binding for appropriate neuronal expression of GnRH. Brn2 Another POU homeodomain protein, Brn2, expressed in the hypothalamus and olfactory tissues, has also been found to regulate hgnrh-i gene expression [21]. In vivo studies of transgenic mice showed the region between )992 and )795 bp to be important for GnRH neuron-specific expression of the hgnrh-i gene. Within this region, two POU protein-binding sites ()925 to )916 bp and )867 to )858 bp) have been identified. These sites have high homology with the Brn2 consensus binding site. EMSA showed that Brn2 proteins in NLT nuclear extract bound to the Brn2 consensus binding site, but not to a mutated Brn2 consensus site. Also, overexpression of Brn2 increased mgnrh mrna expression in cultured GnRH neurons and GN11 cells, and also enhanced hgnrh promoter activities in GN11 cells. Fos and CREB A previous study demonstrated that treatment of GT1-7 neurons with human chorionic gonadotropin (hcg), a GnRH inhibitor, resulted in an increase in phosphorylated Fos, Jun and camp response elementbinding protein (CREB) [40]. Overexpression studies showed that Fos and CREB, but not Jun, inhibited rgnrh-i promoter activity in a dose-dependent manner in the )3026 to +116 bp construct, and the inhibitory action of CREB was more effective than that of Fos [41]. However, these proteins were found not to bind to the hcg responsive region ()126 to )73 bp) of the rgnrh-i proximal promoter, as shown by supershift assays using antibodies against these proteins. This suggests that Fos and CREB might bind to other motifs or might interact with other proteins that bind the rgnrh-i promoter to achieve its regulation. GATA-4, GBF-A1/A2 and GBF-B1 In GT1 cells, two GATA factor-binding motifs that occur in tandem repeats have been found within the rgnrh-i enhancer region (GATA-A, )1710 to FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 5463

7 Transcriptional regulation of GnRH gene V. H. Y. Lee et al. )1715 bp; GATA-B, )1743 to )1748 bp) [10]. Mutation analysis demonstrated that both GATA sites are functionally important and that one factor, GATA-4, present in GT1 cells, can interact with the GATAbinding motifs. Later, Lawson et al. also reported the binding of two GATA factors, GBF-A1 A2 and GBF-B1, to the GATA factor-binding sites in the GnRH-I enhancer [42]. GATA-4 and GBF-B1 were found to be necessary for full enhancer activity; both factors are able to activate the GnRH-I promoter. However, GBF-B1 was also shown to modulate the GATA-4-mediated activation of the GnRH-I enhancer by competing with GATA-4 for binding to the GATA-binding motifs. Protein kinase C (PKC) signaling pathway The above studies revealed multiple transcription factors binding to a number of regulatory elements in the proximal promoter and the enhancer that are crucial for the regulation of GnRH-I gene transcription. Other studies have shown that GnRH-I gene regulation is also mediated through the PKC signaling pathway (Fig. 4). Treatment of GT1-7 cells with the protein kinase A (PKA) pathway activator forskolin (10 lm) did not produce any effect on the rgnrh-i mrna levels, whereas treatment with the PKC pathway activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) (100 nm) caused a significant reduction (70%) in rgnrh-i mrna expression [43]. Bruder et al. showed that TPA caused dose- and time-dependent repression of rgnrh-i promoter activity and a decrease in rgnrh-i transcript levels, which was mediated by increased c-fos and c-jun mrna levels [44]. This TPAmediated repression was found to be dependent on the proximal promoter region at )126 to )73 bp, within which an activator protein-1 (AP-1) site is present (at )99 bp). However, Fos and Jun have been shown not to bind the AP-1 site directly, but to interact with other protein(s) that bind to this site in the proximal promoter. Recently, not only the proximal promoter, but also the enhancer, of the rgnrh-i gene was found to participate in PKC repression [45]. Various ciselements within the enhancer region, as described above, are required for the repression of rgnrh-i expression by PKC. These include Oct-1, Prep Pbx1a, and Dlx2. TPA causes activation of PKC, which in turn leads to increased phosphorylation of these transcription factors, and therefore reduces binding to their interacting sites within the enhancer region of the rgnrh-i gene. The study also revealed a novel site ()1793 to )1785 bp), to which an unknown protein from GT1-7 nuclear extract bound, was involved in this PKC repression of the rgnrh-i gene expression. Pulsatile GnRH-I gene expression GnRH-I is released in a pulsatile manner in the hypothalamus. This intrinsic property of GnRH neurons was first observed in isolated hypothalamic fragments [46,47] and dispersed hypothalamic neuron cultures [48]. Pulsatility is not restricted only to GnRH-I release, but is also associated with GnRH-I gene expression. Recent studies revealed that GnRH-I Pbx1a Pbx1a TPA PKC Ca2+ Dlx2 Oct-1 site Pbx/Prep1 site Dlx2 site AP-1 site Oct-1 Oct-1 P + Dlx2 P Prep1 Pbx1a Oct-1 Dlx PKC responding region ( 1800/ 1576) Jun Fos PKC responding region ( 126/ 73) GnRH-I Fig. 4. PKC repression of rgnrh-i gene expression. The PKC responsive sites within the proximal promoter and the enhancer of the rgnrh-i gene are indicated by colored boxes FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS

8 V. H. Y. Lee et al. Transcriptional regulation of GnRH gene promoter activity operates in a pulsatile manner [49,50]. In fact, the secretory pulse of GnRH-I and the episodic GnRH-I gene expression were found to be closely associated. Analysis of the rgnrh-i promoter revealed that a certain region between )2012 and )1597 bp, which includes the enhancer, was responsible for the pulsatility [28]. Within this region, three Oct-1-binding sites were identified [30,51]. OCT1BS-a ()1785 to )1771 bp) and OCTBS-1b ()1702 to )1695 bp) were shown by Clark and Mellon to bind the Oct-1 POU homeoprotein. More recently, a new site, OCT1BS-c ()1569 to )1562 bp), was also found to bind Oct-1 [51]. However, only OCT1BS-a and OCT1BS-c, and not OCT1BS-b, were shown to be necessary for the pulsatility in mutation analysis. A recent study also demonstrated the involvement of Ca 2+ and a novel Ca 2+ -binding protein, downstream regulatory element antagonist modulator (DREAM), in GnRH-I pulsatility [52]. DREAM was identified as being responsible for the GnRH-I pulsatility, as it was shown to be part of the OCT1BS-b binding complex, an essential element in the GnRH enhancer for promoter pulse. Also, immunoneutralization of DREAM in single GT1-7 cells resulted in a loss of episodic GnRH-I gene expression. An L-type Ca 2+ blocker, nimodipine, which markedly reduced the GnRH-I secretory pulse, was also shown to abolish GnRH-I gene expression pulses. These findings suggested that DREAM, via Ca 2+, may serve as a basis for the communication between cytoplasm and nucleus that links the pulsatile secretion and pulsatile expression of GnRH-I. GnRH-II The GnRH-II promoter Cheng et al. found the core promoter region of the human GnRH-II gene to be located between )1124 and )750 bp relative to the translation start codon by transient transfection studies in neuronal medulloblastoma TE-671 cells, placental choriocarcinoma JEG-3 cells and ovarian carcinoma OVCAR-3 cells [53] (Fig. 5). Moreover, the untranslated exon 1 ()793 to )750 bp) was found to be an enhancer element for stimulation of GnRH-II gene expression [53]. Regulation of GnRH-II by essential transcription factors AP-1 and AP-4 Within the untranslated exon 1 of the hgnrh-ii gene, two E-box-binding sites ()790 to )785 bp and )762 to )757 bp) and one Ets-like element ()779 to )776 bp) were found [53]. These three regulatory elements work in a cooperative manner for basal hgnrh-ii gene transcription. Studies showed in vitro specific binding of the basic helix loop helix transcription factor AP-1 to the two E-box-binding sites, whereas an unknown protein bound to the Est-like element. EMSA using TE-671 nuclear extracts with oligonucleotides containing the two E-box motifs showed the formation of DNA protein complexes, which was abolished by a consensus AP-4-binding sequence. Also, in vitro translated human AP-4 proteins bound to the two E-boxbinding sites formed a complex with similar electrophoretic mobility to that formed with TE-671 extracts. Overexpression studies revealed that AP-4 is an enhancer that upregulates hgnrh-ii promoter activity. p65, retinoic acid receptor-a (RARa) and retinoid X receptor-a (RXRa) Recently, our group has identified a repressor element GII-Sil within the first introns ()641 to )636 bp) of the hgnrh-ii promoter [54]. EMSA showed that proteins in TE-671 nuclear extracts formed two specific DNA protein complexes with GII-Sil. In vitro supershift assays and an in vivo chromatin immunoprecipitation Fig. 5. Diagrammatic representation of the hgnrh-ii gene 5 -region. The promoter region is indicated by the green box [51] and the enhancer by the yellow box [51]. Also, the locations of key regulatory elements and their functional significance are listed E-box( 790/ 785) Ref.51 Est-like ( 779/ 776) Ref.51 E-box ( 762/ 757) Ref GII-Sil ( 641/ 636) JEG 3 cells specificity Ref.52 CRE ( 67/ 60) Ref FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 5465

9 Transcriptional regulation of GnRH gene V. H. Y. Lee et al. assay also showed that nuclear factor kappa B (NF-jB) p65 subunit, and retinoic acid receptors (RARa and RXRa), bind to the GII-Sil element. Also, functional analysis revealed that p65 is a downregulator of the hgnrh-ii promoter. Overexpression of p65 in both TE-671 and JEG-3 cells led to a dramatic decrease in hgnrh-ii promoter activities and endogenous gene expression. Moreover, differential regulation of the GnRH-II gene in two different GnRH-II-expressing human cell lines was observed in assays involving overexpression of RARa and cotransfection of RARa and RXRa. In the studies, an increase in promoter activity was found in placental JEG-3 cells, but no effect could be observed in neuronal TE-671 cells. (Bu) 2 camp Chen et al. revealed the presence of an 8 bp palindromic camp response element consensus site (TGACGTCA, )67 to )60 bp) in the hgnrh-ii promoter [55]. In TE-671 cells, treatment with 1 mm (Bu) 2 camp for h strongly upregulated GnRH-II gene expression, and this was verified by RT-PCR and immunofluorescence staining. An increased concentration of GnRH-II peptides was also observed in the cell medium after the treatment. Moreover, strong induction of promoter activities of the hgnrh-ii gene in response to 1 mm (Bu) 2 camp was found in transfection studies on the hgnrh-ii promoter construct coupled to luciferase. Self-regulation of GnRH gene The role of GnRH in the regulation of synthesis and secretion of gonadotropins in the pituitary is well known. Recent studies have revealed GnRH as an autocrine and paracrine regulator of gonadotropins in the hypothalamus and ovary. It has been demonstrated that GnRH-I gene expression is regulated by itself through an ultrashort loop feedback mechanism in rat hypothalamus [56] and ovarian cells [57]. Also, it has been shown that the GnRH-I and GnRH-II genes are differentially regulated by themselves. In vitro studies using GT1-7 cells and hypothalamic tissue cultures, and in vivo studies in a rat ovary model, showed that GnRH-I treatment inhibited the expression and secretion of GnRH-I [56,58 61]. In hglcs, GnRH-I has been shown to be regulated by its own ligand [62]. Treatment with a GnRH-I analog (leuprolide) produced a biphasic effect on GnRH-I mrna levels, depending on the concentration of treatment. Low concentrations of leuprolide (10 )11 and 10 )10 m) resulted in upregulation of GnRH-I gene expression, whereas high concentrations (10 )8 and 10 )7 m) led to gene repression. This type of biphasic regulation has also been shown in immortalized hypothalamic GT1-7 cells [63] and human OSE cells [64]. Treatment with an antagonist (antide) prevented this biphasic effect in OSE cells, proving the specificity of the response. Moreover, intracerebroventricular injection of a GnRH-I analog into the lateral ventricle of rat brain resulted in a considerable decrease in GnRH-I mrna levels, in a dose- and time-related manner, as detected in the POA [56]. For GnRH-II, treatment with different concentrations of the homologous ligand and GnRH-II analog (10 )11 and 10 )7 m) in hglcs resulted in a large decrease in GnRH-II mrna levels. In a human endometrial cell line, Ishikawa, treatment with GnRH-I increased GnRH-I expression in a time-dependent manner, but did not cause any change in GnRH-II mrna levels [65]. These data showed that the GnRH-I and GnRH-II genes are differentially regulated by their own ligands, suggesting the differential regulation of the two forms of GnRH in different stages of the estrous cycle. The exact mechanism for this differential regulation is unclear. Kang et al. suggested the possibility of different characteristics of binding of the two forms of GnRH to their receptor, which might lead to different conformations of the receptor [62]. The ligand-specific conformation might therefore lead to differential coupling to G-proteins and or different intracellular cellular pathways, eventually leading to differential regulation of GnRH-I and GnRH-II gene expression. GnRH-I(1 5) is a pentapeptide that comprises the first five amino acids of GnRH-I. It is a processed peptide formed by cleavage of the Try5-Gly6 bond by a zinc metalloendopeptidase, EC (EP24.15) [65]. Wu et al. demonstrated that GnRH-I(1 5) stimulated GnRH-I mrna expression in neuronal GT1-7 cells through a different pathway from that used by the parent peptide GnRH-I [66]. In Ishikawa cells, GnRH-I(1 5) was found to have no effect on GnRH-I mrna expression, but induced GnRH-II mrna expression. Baldwin et al. suggested that the differences between the actions of GnRH-I and its metabolite GnRH-I(1 5) on the regulation of the GnRH-I and GnRH-II genes could be caused by the two peptides acting through different GnRH receptors [65]. Gonadotropins Gonadotropins, including FSH and LH, are secreted by gonadotropes of the pituitary under the control of GnRH. A third gonadotropin that is also present in humans is hcg, which is produced in the placenta 5466 FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS

10 V. H. Y. Lee et al. Transcriptional regulation of GnRH gene during pregnancy. Like GnRHs, gonadotropins have been shown to differentially regulate the two GnRH genes via stimulation of camp production and activation of PKA [1]. Through these pathways, gonadotropins may regulate the ratio between GnRH-I and GnRH-II, leading to distinct spatial expressions of the two hormones. In GT1-7 cells, gonadotropins are downregulators of the GnRH-I gene. Lei and Rao showed that GnRH-I is coexpressed with LH hcg receptor in rat POA and GT1-7 cells [67]. Treatment of GT1-7 neurons with LH or hcg resulted in a decrease in steady-state GnRH-I mrna levels. This decrease was found to be dose- and time-dependent, and required the presence of cellular LH hcg receptors. The same group then investigated the signaling pathway and factors involved in the action of hcg [40]. A camp analog, 8-bromocAMP, was reported to mimic the downregulation action of hcg, and application of a PKA inhibitor H89, but not a PKC inhibitor, blocked the action of hcg and that of the camp analog. These findings suggested that PKA signaling and transcription factors such as CREB, Fos and Jun are probably involved in transcriptional inhibition of GnRH gene expression by hcg in GT1-7 cells. Later, the group further extended the study to investigate the cis-acting elements and trans-acting proteins involved in the inhibition by hcg [41]. Deletion analysis revealed that the region between )126 and )73 bp is important for the hcg inhibition. Within this region, the )99 to )94 bp region contained an imperfect AP-1 site, and the )91 to )81 bp region contained two AT-rich sites ()91 to )87 bp and )85 to )81 bp). Also, southwestern blots showed that a 110-kDa protein and a 95-kDa protein bound to the )126 to )73 bp region. Mutagenesis of the AT-rich site, but not the AP-1 site, resulted in complete loss of the inhibitory effect of hcg and also DNA binding of the 95 kda protein. However, supershift assays have not yet been able to determine the identity of the 95 kda protein. The actions of gonadotropins on GnRH-I and GnRH-II gene expression have been shown to be diverse. In the past, treatment with hcg (1 IUÆmL )1 ) in hglcs did not affect the expression level of the rgnrh-i gene, but decreased GnRH receptor mrna levels [68]. However, later studies showed that treatment with FSH or hcg in hglcs resulted in a decrease in GnRH-I mrna levels, but a significant dose-dependent increase in GnRH-II mrna levels [62]. Recently, it was found that GnRH-II mrna levels were significantly reduced following FSH or LH treatment (100 ngæml )1 and 1000 ngæml )1 ) for 24 h in the two IOSE cell lines (IOSE-80 and IOSE-80PC) and three ovarian cancer cell lines (A2780, BG-1 and OVC- AR-3) [69]. In contrast, treatment with either FSH or LH had no effect on GnRH-I mrna levels in the cell lines employed. These findings suggested that gonadotropins regulate the two forms of GnRH differently in the ovary. Steroid hormones The pulsatile secretion of GnRH from the hypothalamic neurons regulates the synthesis and release of gonadotropins in the pituitary. The gonadotropins then regulate both steroidogenesis and gametogenesis. The gonadal steroid hormones, which are key regulators of reproduction, in turn act tightly to regulate GnRH-I and GnRH-II synthesis and release through a negative feedback system between the gonads and the brain. The effects of 17b-estradiol (E 2 ) and progesterone (P 4 ) and their receptors on GnRH gene expression have been well studied. Previously, a number of studies found an absence of steroid receptors in GnRH neurons [70]. It was believed that GnRH neurons synapse with other neurons that act as potential gonadal steroid-sensitive interneurons to modulate GnRH neurons through a number of neurotransmitters and neuropeptides [71]. However, recent studies revealed the expression of different steroid receptors in various hypothalamic and ovarian cell lines [72,73]. Belsham et al. suggested that the apparent absence of steroid receptors was probably due to the scarcity and scattered distribution of GnRH neurons, or to the fact that only specific subgroups of GnRH neurons may contain steroid receptors. Therefore, steroid receptors were not detected [71]. Also, it might be due to limitations in the sensitivity of the detection methods. These steroid receptors, upon forming complexes with their specific steroid hormone ligands, act as intracellular transcription factors, and exert their effects on the expression of GnRH genes. Estrogen The discovery of the estrogen response element ()441 to )428 bp) in the hgnrh-i gene [74] and the presence of both forms of nuclear estrogen receptors (ERa and ERb) in hypothalamic and ovarian cell lines, GT1-7 and hglcs [11,75,76], suggested the possible involvement of estrogen (E 2 ) in regulation of the GnRH-I gene. Inconsistent results have been reported regarding the regulation of GnRH-I gene expression by E 2 in the hypothalamus. This is because GnRH-I is regulated differently at different stages of the estrous FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 5467

11 Transcriptional regulation of GnRH gene V. H. Y. Lee et al. cycle. The results obtained have therefore varied according to the time at which the animals were killed, the dose and duration of estrogen treatment, and the region of the brain analyzed [75]. During the estrous cycle, the levels of GnRH-I in the anterior hypothalamus were found to be inversely related to plasma estrogen profiles, suggesting that E 2 may reduce hypothalamic GnRH-I mrna levels [77]. However, E 2 was found to induce GnRH-I gene expression, which contributes to the gonadotropin surge before ovulation in rats [78]. In animal models, administration of E 2 for 7 days in ovariectomized rats increased GnRH-I mrna levels, but administration for 2 days resulted in a decrease in GnRH-I mrna levels [77]. Moreover, exogenous administration of estrogen in women with a normal menstrual cycle resulted in a simultaneous increase in GnRH-I levels in blood [78]. In the hypothalamic cell lines, E 2 was shown to repress expression of the GnRH-I gene. In GT1-7 cells, E 2 treatment for 48 h was shown to downregulate GnRH-I mrna expression to about 55%. This effect was found to take place via the ER, as a complete ER antagonist blocked the repression by E 2 [75]. Recently, three different splice variants of ERb, including ER-b1, ER-b1d3, and ER-b2, were found to cause significant activation of the mgnrh-i promoter in the absence of the ligand hormone E 2 in hypothalamic GT1-7 cells [72]. Treatment with E 2 abolished the activation by ER-b1 and ER-b2, but not by ER-b1d3. EMSA showed that ER-b1 binds to the )225 to )201 bp and )184 to )150 bp regions, and deletion studies demonstrated that these regions are critical for ER-b1-induced promoter activity. GnRH-I regulation in extrapituitary tissues by steroid hormones has also been reported. In the ovary, E 2 treatment (1 100 nm) for 24 h resulted in a dosedependent decrease in GnRH-I mrna levels, as determined in hglcs [11]. It is interesting that short-term treatment (6 h) had no significant effect on GnRH-I expression, whereas long-term treatment (48 h) resulted in a 40% reduction in GnRH-I mrna levels. The E 2 -induced regulation is mediated through the E 2 receptor, as tamoxifen (a selective ER modulator) blocked the effect of E 2. In CHO-K1 cells, E 2 -mediated repression of the GnRH-I promoter was found to be related to the )169 to )548 bp region of the hgnrh-i promoter [79]. Moreover, Kang et al. demonstrated that E 2 caused a significant reduction in GnRH mrna levels in an ovarian cancer cell line but not in normal human ovarian surface epithelial cells [80], and this effect was mediated via ERs. In a human placenta cell line, E 2 was also shown to decrease GnRH-I promoter activity in a dose-dependent manner [81]. Similarly, in placental tumor cells, E 2 also negatively regulated rgnrh-i promoter activity [82]. Apart from GnRH-I gene regulation, steroid hormones have also been shown to regulate the GnRH-II gene, but with different overall effects. In contrast to repression of the GnRH-I gene, treatment of hglcs with E 2 significantly increased GnRH-II mrna levels in a dose- and time-dependent manner [76]. This demonstrated the differential regulation of the GnRH-I and GnRH-II genes by steroid hormones in the ovary, suggesting that the GnRH-I and GnRH-II genes may be temporally regulated during the different phases of the menstrual cycle. Similarly, the hgnrh-i and hgnrh-ii genes have been found to be differentially regulated by E 2 in TE671 human neuronal medulloblastoma cells [83]. E 2 decreased hgnrh-i mrna levels, but increased hgnrh-ii mrna levels. These effects were found to be promoter-mediated, and a partial putative ERE site in the human GnRH-II promoter is involved. Progesterone P 4 is another dominant ovarian steroid hormone that is known to be involved in the regulation of gonadotropin secretion in several species, including human, rat and mouse. P 4 regulates the GnRH-I gene through a feedback mechanism in humans and other animals [84 86]. Like E 2,P 4 regulates the GnRH-I gene differently under different physiological conditions. Just before ovulation, P 4 activates GnRH neurons and stimulates GnRH release in adult rats after E 2 priming, and thus enhances LH release to trigger ovulation [87,88]. After ovulation, in the luteal phase of the estrous cycle, the corpus luteum increases P 4 production to prepare for possible implantation. This P 4 surge inhibits the pulsatile secretion of GnRH-I and LH [89,90]. In the past, Toranzo et al. showed that P 4 decreased hypothalamic GnRH-I mrna expression in rats [91]. Similarly, P 4 was also found to repress rgnrh gene expression via the progesterone receptor (PR) in GT1-7 cells [92]. Deletion analysis mapped the effects of P 4 to the region between )171 and )73 bp of the rgnrh-i proximal promoter, which included several negative progesterone response elements (npres). EMSA further confirmed the binding of PR to the npre at regions )171 to )126 bp, )126 to )73 bp, and )111 to )73 bp. In contrast, P 4 was found to increase GnRH-I mrna expression levels following E 2 priming in the hypothalamus of ovariectomized immature rats [93] FEBS Journal 275 (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS