Báo cáo khoa học: Gonadotropin-releasing hormone: regulation of the GnRH gene pot

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: +852 2559 9114 Tel: +852 2299 0850 E-mail: bkcc@hkusua.hku.hk (Received 18 April 2008, revised August 2008, accepted 29 August 2008) doi:10.1111/j.1742-4658.2008.06676.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; E2, 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; P4, 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 5458 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 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-GlyNH2) Gene expression first gives rise to the preproGnRH 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 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 The GnRH-I promoter 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 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) 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 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5459 Transcriptional regulation of GnRH gene V H Y Lee et al Pulsatile expression Ref.49 Msx1 & Dlx2 (–54/ –51) Ref.30 Msx1 & Dlx2 (–41/ –38) Ref.30 Pbx/Prep1 (–84/ –61) Ref.36 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) Oct–1 (–1569/ –1562) Ref.49 Pbx/Prep1 (–1612/ –1593) Ref.36 C/EBP (–1684/ –1676) NO responsiveness Ref.122 GATA (–1715/ –1710) Ref.10 Oct–1 (–1702/ –1695) Ref.28 Oct–1 (–1785/ –1771) Ref.28 Pbx/Prep1 (–1753/ –1734) Ref.36 GATA (–1748/ –1743) Ref.10 hCG responsiveness Ref.39 +1 –173 Ref.12 PKC responsiveness (–126/ –73) –2100 –1700 ERβ (–225/ –201) β ERβ (–184/ –150) Otx2(–319/ –315) Otx2(–257/ –252) Insulin responsiveness (–1250/ –587) Ref.116 E2 responsiveness Ref.70 nPRE (–171/ –73) P4 responsiveness Ref.90 Ref.43 +1 Fig 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 AP-1 (–402/ –396) IGF-I responsiveness Ref.133 E2 responsiveness (–548/ –169) Ref.77 Brn-2 (–867/ –858) Brn-2 (–925/ –916) ERE (–925/ –916) Ref.72 GnRH neuron specificity (–992/ –763) Ref.21 +1 +8 –551 Constitutive expression in GT1-7 cell (–1131/ –350) Ref.24 5460 +273 Egr–1 (–75/ –67) insulin responsiveness Ref.112 –1005 Enhancer GnRH neuron specificity Repressor ovary specificity (–3446/ –2078) Ref.20 –3832 Fig 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 Ref.42 PKC responsiveness (–1800/ –1576) –5500 Oct–1 (–47/ –40) Ref.12 Oct–1 (–110/ –88) Gn RH neuron specificity Ref.37 Ref.117 Msx1 & Dlx2 (–1634/ –1631) Msx1 & Dlx2 (–1620/ –1617) Ref.31 –1697/ –1689 –1736/ –1728 –1746/ –1738 –1827/ –1819 –1780/ –1772 Melatonin responsiveness RARE(–1494/ –1470) RA responsiveness Ref.127 –1571 –1863 Fig 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) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al 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%) Transcriptional regulation of GnRH gene 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 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 50–100-fold activation of GnRH gene transcription as compared to the activity of Human GnRH-I promoter Sequence alignment has revealed similarities and 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) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5461 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 20fold 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 5462 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 (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 Oct1-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 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al 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 Transcriptional regulation of GnRH gene 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) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5463 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 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 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 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 TPA Pbx1a Oct-1 site PKC Ca2+ Pbx/Prep1 site Dlx2 site Pbx1a AP-1 site Dlx2 Oct-1 + – Oct-1 P Dlx2 – PKC responding region (–1800/ –1576) 5464 GnRH-I –1571 –1863 – Jun Fos Dlx2 Oct-1 Prep1 Pbx1a – P –173 +1 PKC responding region (–126/–73) Fig 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) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 Ca2+ and a novel Ca2+-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 Ca2+ 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 Ca2+, may serve as a basis for the communication between cytoplasm and nucleus that links the pulsatile secretion and pulsatile expression of GnRH-I 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 ()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 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) –793 –750 E-box(–790/ –785) Ref.51 Est-like (–779/ –776) Ref.51 E-box (–762/ –757) Ref.51 –1124 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS CRE (–67/ –60) Ref.53 Fig 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 The GnRH-II promoter GII-Sil (–641/ –636) JEG–3 cells specificity Ref.52 Cheng et al found the core promoter region of the human GnRH-II gene to be located between )1124 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 GnRH-II +1 5465 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)2cAMP Chen et al revealed the presence of an bp palindromic cAMP response element consensus site (TGACGTCA, )67 to )60 bp) in the hGnRH-II promoter [55] In TE-671 cells, treatment with mm (Bu)2cAMP for 12–48 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 mm (Bu)2cAMP 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 5466 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 3.4.24.15 (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 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al 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 ImL)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 ngỈmL)1 and 1000 ngỈmL)1) for 24 h in the two IOSE cell lines (IOSE-80 and IOSE-80PC) and Transcriptional regulation of GnRH gene three ovarian cancer cell lines (A2780, BG-1 and OVCAR-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 (E2) and progesterone (P4) 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 (E2) in regulation of the GnRH-I gene Inconsistent results have been reported regarding the regulation of GnRH-I gene expression by E2 in the hypothalamus This is because GnRH-I is regulated differently at different stages of the estrous FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5467 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 E2 may reduce hypothalamic GnRH-I mRNA levels [77] However, E2 was found to induce GnRH-I gene expression, which contributes to the gonadotropin surge before ovulation in rats [78] In animal models, administration of E2 for days in ovariectomized rats increased GnRH-I mRNA levels, but administration for 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, E2 was shown to repress expression of the GnRH-I gene In GT1-7 cells, E2 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 E2 [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 E2 in hypothalamic GT1-7 cells [72] Treatment with E2 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, E2 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 E2-induced regulation is mediated through the E2 receptor, as tamoxifen (a selective ER modulator) blocked the effect of E2 In CHO-K1 cells, E2-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 E2 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, E2 was also shown to 5468 decrease GnRH-I promoter activity in a dose-dependent manner [81] Similarly, in placental tumor cells, E2 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 E2 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 E2 in TE671 human neuronal medulloblastoma cells [83] E2 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 P4 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 P4 regulates the GnRH-I gene through a feedback mechanism in humans and other animals [84–86] Like E2, P4 regulates the GnRH-I gene differently under different physiological conditions Just before ovulation, P4 activates GnRH neurons and stimulates GnRH release in adult rats after E2 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 P4 production to prepare for possible implantation This P4 surge inhibits the pulsatile secretion of GnRH-I and LH [89,90] In the past, Toranzo et al showed that P4 decreased hypothalamic GnRH-I mRNA expression in rats [91] Similarly, P4 was also found to repress rGnRH gene expression via the progesterone receptor (PR) in GT1-7 cells [92] Deletion analysis mapped the effects of P4 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, P4 was found to increase GnRH-I mRNA expression levels following E2 priming in the hypothalamus of ovariectomized immature rats [93] FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al The effects of P4 are basically mediated through its binding to the intranuclear receptor, PR Up to now, two isoforms of PR (PR-A and PR-B) have been identified [94] PR-B is a full-length receptor, whereas PR-A lacks 164 amino acids in the N-terminus as compared to PR-B PR-A and PR-B have distinct transactivation properties when expressed individually, and it was found that PR-A can modulate PR-B activity [95,96] The differential effects of PR-A and PR-B on GnRH-I and GnRH-II gene expression have been shown by An Beum-Soo et al [97] The human neuronal medulloblastoma cell line TE-671 was shown to express both GnRH-I and GnRH-II, and therefore to be suitable for studies of regulation of the two forms of GnRH [83] Treatment of TE-671 cells with P4 (10)6 and 10)5 m) resulted in increases in GnRH-I mRNA levels of 40 and 100%, respectively, and the increase in GnRH-I expression was greatest with 12 and 24 h treatments Although the level of PR-B was found to be lower in TE-671 cells than that of PR-A, the stimulatory effect on GnRH-I expression was found to be mediated by PR-B but not PR-A, as overexpression of PR-B increased the sensitivity towards P4 treatment and increased GnRH-I promoter activity in the presence of P4 However, there was no significant effect on GnRH-II mRNA levels after either P4 treatment or overexpression of either PR-A or PR-B [97] The role of P4 in GnRH-I and GnRH-II expression has also been investigated in hGLCs [76] Treatment of the cells with the P4 antagonist RU486 did not affect the level of GnRH-I mRNA, whereas the level of GnRH-II mRNA was found to be increased in a dose- and time-dependent manner This suggests an inhibitory role of endogenous P4 on GnRH-II in the ovary Androgens The discovery of the expression of androgen receptor (AR), as well as ARA70, an AR-specific coactivator that can enhance AR expression, in GT1-7 cells, led to the study of the effects of androgen on GnRH regulation [71] GT1 cells were found to have enough 5a-reductase activity to efficiently convert testosterone to dihydrotestosterone (DHT), a more potent androgen [98] In GT1-7 cells, treatment of GT1-7 cells with DHT at physiologically relevant doses (1 and 10 nm) resulted in downregulation of GnRH-I mRNA expression (55% reduction) This showed that DHT directly mediated GnRH-I expression via AR [71] However, androgen response elements were not present within the rGnRH-I promoter region Transcriptional regulation of GnRH gene In addition to the classical steroid hormone action, which involves binding of steroids to intracellular receptors, in modulating target gene transcription, increasing evidence for rapid and nongenomic steroid effects mediated via membrane receptors has been obtained [99,100] In GT1-7 cells, Shakil et al determined the presence of AR in the plasma membrane by using western blot analysis and fluorescence staining [101] Treatment with DHT, testosterone or a cellimpermeable BSA-conjugated testosterone (T-3-BSA) stimulated GnRH-I release in GT1-7 cells However, only treatment with DHT or testosterone, but not T-3BSA, resulted in downregulation of GnRH-I mRNA levels Thus, repression of GnRH-I expression by testosterone must be mediated via membrane receptors, whereas testosterone-stimulated secretion can act through both membrane and nuclear receptors This study indicated the differential action of androgen on GnRH-I gene expression and secretion via specific nuclear and membrane-mediated mechanisms Glucocorticoids Studies have also been carried out on the effects of glucocorticoids on GnRH-I expression Glucocorticoids were found to repress GnRH-I gene expression and release through functional glucocorticoid receptors expressed in GT1 cells [102] Recently, chronic treatment for days with corticosterone in adult male rats was found to cause significant suppresssion of hypothalamic GnRH-I mRNA levels (35–40% reduction), as well as serum LH but not FSH levels, as compared to controls [103] On the other hand, acute treatment with a synthetic glucocorticoid (dexamethasone), which blocks the E2-induced gonadotropin surge in immature female rats, had no effect on GnRH-I mRNA expression The authors suggested that although dexamethasone does not affect GnRH expression, it might decrease the release of GnRH to the pituitary This is consistent with the finding that there was a reduction in hypothalamic release of GnRH after cortisol treatment in male rhesus monkeys [104] It also suggests that another possibility is decreased pituitary responsiveness to GnRH, like the effect of progesterone Dehydroepiandrosterone Dehydroepiandrosterone (DHEA) and its sulfated metabolite, are secreted from the adrenal gland and are the most abundant circulating steroids in humans [105] It has been believed that DHEA is capable of being converted into other active sex steroids, including E2, testosterone and DHT [106] The high FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5469 Transcriptional regulation of GnRH gene V H Y Lee et al concentration of DHEA in rodent brain suggests the possibility that steroidogenesis may also take place in the nervous system [107] DHEA has different effects on rat GnRH-I gene expression in different sexes; it inhibited rGnRH-I gene expression in male rats, but stimulated it in female rats [108] In GT1-7 cells, DHEA significantly downregulated GnRH-I mRNA expression, and the GnRH-I mRNA level steadily declined over a 48 h period [109] However, the absence of amromatase in GT1-7 cells suggested that the conversion of DHEA to E2 was unlikely to happen Also, testosterone, DHT and androgen were not detected as the metabolites of DHEA These findings suggested that DHEA itself could directly mediate the effect on GnRH downregulation without any conversion to other steroid hormones in GT1-7 cells Physiological regulators activation of the mouse promoter This suggested that insulin regulates mGnRH-I promoter via a MAP kinase pathway Recently, insulin-dependent stimulation of the mGnRH-I promoter in GN11 cells has been found to be mediated by an immediate early gene, early growth response (Egr-1) [114] The importance of Egr-1 to reproduction and GnRH expression was shown by the use of Egr-1 knockout mice as well as Egr-1 small interfering RNA knock-down experiments [115,116] Moreover, by chromatin immunoprecipitation assays, in vivo binding of Egr-1 to a GC-rich region between )75 and )67 bp of the proximal mGnRH-I promoter was detected only in the presence of insulin Mutation of the putative Egr-1 site attenuated the insulin-induced stimulation These data suggested that this site is the sole specific Egr-1-binding site that is crucial to the insulin-induced increase in mGnRH-I promoter activity [82] Insulin Melatonin Nutritional status is an important determinant of reproductive capacity in mammals Impediments to reproduction during chronic and short-term food restriction are thought to be integrated at the hypothalamic level [110] The development of neuron-specific insulin receptor knockout (NIRKO) mice revealed the involvement of insulin signaling in the regulation of the reproductive axis in rodents NIRKO mice are obese, with elevated insulin levels, reduced fertility and lower LH levels as compared to the wild-type mice [111] Insulin can serve as a peripheral signal to inform the brain reproductive centers about the nutritional status of the body The starvation-induced inhibition of the reproductive axis can be reversed rapidly by refeeding, due to the rise in insulin level after feeding [112] Owing to the strong relationship between insulin and the reproductive axis, it is reasonable that the expression of GnRH, as an important part of the reproductive hormonal cascade, is influenced by insulin In GnRH-expressing neuronal cells (GN11), after treatment with insulin (10 nm), the activity of the mGnRH-I promoter 1250 bp upstream of the transcription start site was found to be increased by up to 4.0fold as compared to controls [113] However, insulin treatment of GN11 cells transfected with a 587 bp promoter did not cause stimulation of promoter activities, indicating that the elements mediating insulin stimulation of the mGnRH-I promoter could be located within the region between )1250 and )587 bp Moreover, treatment with the mitogen-activated protein kinase kinase inhibitor PD98059 blocked the insulin-induced Melatonin is the principal hormone produced by the vertebrate pineal gland, which mainly regulates circadian rhythms and reproductive physiology Melatonin mediates its reproductive effects through specific G-protein-coupled receptors, mt1 and MT2 [117,118] Melatonin (1 nm) was shown to significantly downregulate rGnRH-I mRNA expression in a 24 h cyclical manner in GT1-7 cells [119] The potential regulator elements of melatonin were localized to five regions, including )1827 to )1819 bp, )1780 to )1772 bp, )1746 to )1738 bp, )1736 to )1728 bp, and )1697 to )1689 bp, within the rGnRH-I enhancer These regions have been found to bind a number of transcription factors, such as Oct-1, GATA-4 and Otx2 In addition, two direct repeats of consensus binding sites for orphan nuclear receptors, including retinoic acid receptor-related orphan receptor ⁄ retinoid Z receptor and COUP-TFI, and also other consensus binding sites for AP-1 and CCAAT ⁄ enhancer-binding protein (C ⁄ EBP), were discovered within the )1736 to )1728 bp region [120] Supershift assays demonstrated that only COUP-TFI and C ⁄ EBPb bind to this enhancer region of the rGnRH-I gene 5470 Nitric oxide (NO) Signal transduction pathways utilizing NO as a secondary messenger in the brain have been well studied NO has been shown to be important for GnRH secretion [121–123], and also acts as an intermediate for induction of N-methyl-d-aspartic acid (NMDA)-mediated induction of GnRH secretion In GT1-7 cells, FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al Belsham et al have shown that NMDA and NO are responsible for the downregulation of rGnRH-I gene expression [124] They also found that the enhancer of the rGnRH-I gene is necessary for this repression Within the enhancer region, OCTBS-1b ()1702 to )1695 bp) and an adjacent element with high homology with C ⁄ EBP ()1684 to )1676 bp) were found to be critical for the NO-mediated repression of GnRH-I In vitro binding of Oct-1 proteins to OCTBS-1b and of C ⁄ EBPb to the adjacent C ⁄ EBP element were confirmed by supershift assays Moreover, treatment with the components of the glutamate–NO–cGMP pathway, NMDA, single nucleotide polymorphisms and 8-bromo-cGMP, enhanced the binding affinity of Oct1 Therefore, it is possible that cGMP activates cGMP-dependent protein kinase, which causes increased phosphorylation of Oct-1 Phosphorylation of Oct-1 inhibits the Oct-1 binding activity [125], thus the repression of GnRH-I gene expression [31] Retinoic acid (RA) The role of vitamin A and its derivative RA in the maintenance of normal reproductive functions was shown more than 70 years ago [126] There are two active metabolites of vitamin A, all-trans-RA (atRA) and 9-cis-RA, and they mediate their effects via binding to specific receptors, RAR and retinoid X receptor There is evidence that RA is involved in the regulation of the hypothalamic–pituitary–gonadal axis Studies on the effects of atRA on GnRH-I expression have been carried out in rat hypothalamic fragments and GT1-1 cells [13] Incubation of hypothalamic fragments with atRA (0.01–1.00 lm) for h resulted in a significant reduction in GnRH-I mRNA levels (40–50% reduction) However, in GT1-1 cells, different results have been obtained There was no effect on GnRH-I expression upon short-term atRA treatment, whereas with long-term treatment for up to 48 h, a dose- and time-dependent increase in GnRH-I mRNA levels was demonstrated The authors suggested that these differences might be due to the fact that hypothalamus fragments are heterogeneous and contain various cell types, while GT1-1 is only one of those cell types in the hypothalamus Also, they identified the region between )1640 and )1438 bp within the rGnRH promoter as being responsible for the atRA induction [13] Within this distal promoter region, three putative repeats of AGGTCA-related sequences ()1637 to )1617 bp, )1579 to )1562 bp, and )1494 to )1470 bp) were found [127] Among these, only the )1494 to )1470 bp sequence was shown to have similar binding characteristics as the consensus Transcriptional regulation of GnRH gene retinoic acid response element (RARE), as determined by EMSA Moreover, treatment of GT1-1 cells with atRA or 9-cis-RA was able to increase the specific binding of proteins in GT1-1 cell nuclear extracts to the )1494 to )1470 bp sequence and consensus RAREs In contrast to the stimulatory effect of atRA on GnRH-I gene expression, 9-cis-RA was found to inhibit GnRH promoter activity and repress GnRH-I mRNA expression [128] Deletion analysis showed that the )230 to )110 bp sequence within the proximal promoter could be responsible for the 9-cis-RA-mediated GnRH inhibition As no retinoid X response element or related sequence is present in this region, it is suggested that the transcriptional repression of GnRH by 9-cis-RA may be mediated by other transcription factors that interact with 9-cis-RA and bind to the proximal elements The above studies revealed that GnRH-I expression is differentially regulated by atRA and 9-cis-RA Therefore, by changing the relative amounts of these two retinoid metabolites, fine-tuning of GnRH transcription activity can be achieved Insulin-like growth factor In addition to its roles in proliferation and differentiation, insulin-like growth factor-I (IGF-I) has also been shown to be involved in reproductive regulation For example, in female rats, infusion of IGF-I was shown to stimulate GnRH-I release and accelerate the onset of puberty [129,130] In addition, IGF-I increased LH release following GnRH stimulation [131,132] Treatment of a GnRH-expressing neuronal cell line (NLT) with IGF-I resulted in a growth-independent increase in mGnRH-I mRNA levels and a significant increase in hGnRH-I promoter activity [133] An AP-1-binding site ()402 to )396 bp) was found to be the responsive element of IGF-I-mediated induction of hGnRH-I expression Moreover, the study showed that IGF-I could activate the p42 ⁄ p44 MAP kinase pathway and induce c-fos expression in NLT cells These findings showed that Ras ⁄ Raf-1 ⁄ MAP kinases and c-fos are the components of the signaling pathways in response to IGF-I induction in NLT cells c-Aminobutyric acid (GABA) GABA is one of the major neurotransmitters responsible for modifying GnRH neural activity and GnRH secretion Studies have shown that GABA hyperpolarizes GnRH neurons [2] and induces rGnRH-I gene expression [134,135] in mature rat GnRH neurons However, in embryonic rat GnRH neurons, GABA FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5471 Transcriptional regulation of GnRH gene V H Y Lee et al was shown to depolarize the neurons and reduce rGnRH-I gene expression [136,137] Also, in another study, activation of the GABA-A receptor resulted in reduced GnRH mRNA levels in nasal explants [137] However, studies on transgenic rats with the transgene construct containing approximately kb of rGnRH-I promoter together with the enhanced green fluorescent protein reporter also demonstrated that GABA activates GnRH gene expression via GABA-A receptor in embryonic GnRH neurons, as GABA and the GABA-A receptor agonist muscimol, but not the GABA-B receptor agonist baclofen, enhanced enhanced green fluorescent protein expression in transgenic rat embryo (E18.5) primary cultures Future prospects The development of cell lines and transgenic mice has enabled a considerable amount of information to be obtained on the regulation of GnRH genes, especially the GnRH-I gene, of human, rat and mouse Various cis-acting motifs have been discovered in the GnRH promoter and enhancer regions with crucial transcription regulatory factors Self-regulation by GnRH itself and feedback regulatory mechanisms by other components of the hypothalamic–pituitary–gonadal axis, including the gonadotropins and sex steroids, have also been widely studied Despite our extensive knowledge concerning GnRH gene regulation, further investigations need to be carried out on certain aspects The differential activities of GnRH promoter constructs in different organs (hypothalamus and gonads) in transgenic mice and in different cell lines (neuronal and placental cells) have revealed the specific expression of the GnRH-I and GnRH-II genes in the brain and gonads For example, there is neuron-specific expression of )356 to )249 bp of the mGnRH-I promoter [39] and the )992 to )795 bp construct of the hGnRH-I promoter [21] in transgenic mice This raises the possibility that expression of GnRH genes may have different regulatory mechanisms in the brain neurons and in the reproductive organs However, most of the studies regarding GnRH gene regulation have been performed in neuronal cell lines The current promoter models for the GnRH genes established using studies in neuronal cells might not be applicable to the gonads Future studies are needed to investigate any possible differences, including the gonad-specific elements of the GnRH promoters and corresponding transcription factors involved Studies have found that GnRH-I and GnRH-II have different tissue distribution patterns in the body 5472 GnRH-I is expressed mostly in the brain, whereas GnRH-II has the highest expression level outside the brain Also, several studies have shown differential regulation of the GnRH-I and GnRH-II genes; for instance, there are different effects of the steroid hormones E2 and P4 on the two GnRH genes Therefore, it is possible that the two forms of GnRH might have distinct physiological functions in the body, although most studies have shown that GnRH-II has similar functions as GnRH-I in stimulating FSH and LH expression and release, and in repressing gonadotropin-regulated steroidogenesis in the ovary Specific GnRH-II responses have also been identified in certain cell types, and there is increasing evidence for GnRH-II actions on extrapituitary organs Moreover, previous studies have been performed largely on GnRH-I only There are many regulators identified in the GnRH-I gene that have not yet been tested on the GnRH-II gene, such as the actions of the steroid hormones androgen and glucocorticoid, and DHEA, insulin, and melatonin Future efforts are therefore needed to explore specific stimulation and regulation of the GnRH-II gene, with the aim of facilitating the identification of specific and even novel functions of GnRH-II in the body Acknowledgements This work was supported by a research grant from Research Grant Council HKU7639/07M and HKU7566/06M to Billy K C 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(2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS ... regulation of the GnRH- I and GnRH- II genes; for instance, there are different effects of the steroid hormones E2 and P4 on the two GnRH genes Therefore, it is possible that the two forms of GnRH. .. the GnRH- I promoter, and there are only a few regarding regulation of the GnRH- II gene Regulation of GnRH gene expression The GnRH- I promoter In view of the fact that GnRH is essential for reproductive... 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,