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
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 respon-
sible for gonadal steroidogenesis and gametogenesis.
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:
(Received 18 April 2008, revised 4 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 gametogene-
sis. 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 proper-
ties similar to those of in vivo GnRH neurons and also transgenic mice
facilitated GnRH gene regulation research. This minireview provides a sum-
mary 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.
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 gonadotropin-
releasing 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
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-associ-
ated 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 decapep-
tides 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, how-
ever, a second form of GnRH (GnRH-II) was identi-
fied. 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 ubiqui-
tous 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 tis-
sue 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.
Regulation of GnRH gene expression
In view of the fact that GnRH is essential for repro-
ductive processes, understanding the control of its
synthesis and release is therefore of the utmost impor-
tance. However, it is difficult to study the regulation
of GnRH gene transcription in vivo, due to the scar-

city and scattered distribution of the GnRH neurons.
In the past, immortalized GnRH-expressing neuronal
cell lines have probably been the only effective and
manageable resources with which to explore mecha-
nisms regulating the expression, synthesis and release
of GnRH. Studies on transcriptional regulation of the
GnRH gene have been performed largely in GnRH-
secreting 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 neuron-
specific 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 essen-
tial 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, sug-
gesting 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.
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).
V. H. Y. Lee et al. Transcriptional regulation of GnRH gene
FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5459
AT-rich site (–85/ –81)
AT-rich site (–91/ –87)
Msx1 & Dlx2 (–1634/ –1631)
Msx1 & Dlx2 (–1620/ –1617)
RARE(–1494/ –1470)
–1827/ –1819
–

1863
Oct–1 (–1785/ –1771)
Ref.28
Oct–1 (–1702/ –1695)
Ref.28
–1571
–1780/ –1772
Oct–1 (–1569/ –1562)
Ref.49
GATA (–1748/ –1743)
Ref.10
–1746/ –1738
–1736/ –1728
GATA (–1715/ –1710)
Ref.10
–1697/ –1689
C/EBP (–1684/ –1676)
Oct–1
(–110/ –88)
Ref.12
+1
Otx/ bicoid (–153/ –146)
Ref.14
Msx1 & Dlx2 (–54/ –51)
Ref.30
Oct–1 (–47/ –40)
Ref.12
Msx1 & Dlx2 (–41/ –38)
Ref.30
Pulsatile expression

Ref.49
NO responsiveness
Ref.122
RA responsiveness
Ref.127
AP–1 (–99/ –94)
hCG responsiveness
Ref.39
–173
nPRE (–171/ –73)
Ref.31
Melatonin responsiveness
Ref.117
PKC responsiveness (–1800/ –1576)
Ref.43
P
4
res
p
onsiveness
Ref.90
PKC
responsiveness
(–126/ –73)
Ref.42
Pbx/Prep1 (–1753/ –1734)
Ref.36
Pbx/Prep1 (–84/ –61)
Ref.36
Pbx/Prep1 (–1612/ –1593)

Ref.36
Pbx/Prep1 (–109/ –89)
Ref.36
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.
+273
–
5500
–2100
–1700
ER
β
1 (–225/ –201)
ERβ
β
1 (–184/ –150)
Otx2(–257/ –252)
Otx2(–319/ –315)
Insulin
responsiveness
(–1250/ –587)
Ref.116

Enhancer GnRH neuron specificity
Repressor ovary specificity

(–3446/ –2078) Ref.20
E
2
responsiveness
Ref.70
Gn RH neuron
specificity Ref.37
Egr–1 (–75/ –67)
insulin
responsiveness
Ref.112

+1 –1005
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.
–
3832
+8
GnRH neuron specificity
(–992/ –763) Ref.21
Constitutive expression in GT1-7 cell
(–1131/ –350) Ref.24
Brn-2 (–925/ –916)
Brn-2 (–867/ –858)
ERE (–925/ –916) Ref.72
E

2
responsiveness
(–548/ –169) Ref.77
AP-1 (–402/ –396)
IGF-I responsiveness
Ref.133

–551
+1
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.
Transcriptional regulation of GnRH gene V. H. Y. Lee et al.
5460 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS
The transcription start sites
For the determination of the transcription initiation
site in the rat and mouse GnRH genes, primer exten-
sion 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 hypothala-
mus and nonhypothalamic tissues such as ovary, testes,
placenta and mammary gland [24–26]. In the hypothal-

amus, 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 evi-
dence 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 non-
human 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 transcrip-
tion (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 transcrip-
tion start site. It provides 50–100-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 con-
served 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 expres-
sion 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 trans-
genic 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 hypo-
thalamic 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 dif-
ferences 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
V. H. Y. Lee et al. Transcriptional regulation of GnRH gene
FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5461

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].
A5¢-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-spe-
cific 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 signifi-
cant reduction (95% reduction) in transcriptional activ-
ity, showing that OCT1BS-a is the most crucial element
for transcriptional activity of the GnRH-I gene enhan-
cer. 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 path-
way, 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 distal-
less 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 homeo-

domain 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 antennape-
dia class of non-Hox homeodomain transcription
factors, bind to the ATTA consensus sequence [34].
Msx1 is found as a repressor, whereas Dlx2 is an acti-
vator, 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 regula-
tors 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 fam-
ily 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 tran-
scription. 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 overexpres-
sion 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) homeo-
domain transcription factor, Pbx1b, was also found to
Transcriptional regulation of GnRH gene V. H. Y. Lee et al.
5462 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS
be a cofactor of Oct-1 by using a yeast two-hybrid sys-
tem [37]. Moreover, Pbx1b contains the Meis homolo-
gous 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 hetero-
dimers 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 coacti-

vators of Oct-1 in GnRH-I expression, because coex-
pression 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 sig-
nificantly 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-dri-
ven transcription of the GnRH-I gene. Recently, the
critical role of Otx2 in regulating tissue-specific expres-
sion 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]. How-
ever, 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 essen-
tial 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. Over-
expression 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 con-

sensus 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 element-
binding protein (CREB) [40]. Overexpression studies
showed that Fos and CREB, but not Jun, inhibited
rGnRH-I promoter activity in a dose-dependent man-
ner in the )3026 to +116 bp construct, and the inhibi-
tory 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 pro-
teins. 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
V. H. Y. Lee et al. Transcriptional regulation of GnRH gene
FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5463
)1715 bp; GATA-B, )1743 to )1748 bp) [10]. Muta-
tion analysis demonstrated that both GATA sites are
functionally important and that one factor, GATA-4,

present in GT1 cells, can interact with the GATA-
binding 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 fac-
tors 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 acti-
vator 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 TPA-
mediated 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 cis-
elements 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 tran-
scription 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 hypo-
thalamus. 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
–1863
–1571
+1

–173
PKC responding region
(–1800/ –1576)
GnRH-I
PKC responding region
(
–126/–73
)

Dlx2
–
TPA
Ca2+
PKC
Dlx2
P
Dlx2
Oct-1
Oct-1
P
Oct-1
Prep1
Pbx1a
Pbx1a
Pbx1a
Jun
Fos
–
–
–

+
Oct-1 site
Pbx/Prep1 site
Dlx2 site
AP-1 site
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.
Transcriptional regulation of GnRH gene V. H. Y. Lee et al.
5464 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 clo-
sely 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 pulsatili-
ty [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 homeo-
protein. More recently, a new site, OCT1BS-c ()1569
to )1562 bp), was also found to bind Oct-1 [51]. How-
ever, only OCT1BS-a and OCT1BS-c, and not
OCT1BS-b, were shown to be necessary for the pulsa-
tility in mutation analysis.
A recent study also demonstrated the involvement

of Ca
2+
and a novel Ca
2+
-binding protein, down-
stream regulatory element antagonist modulator
(DREAM), in GnRH-I pulsatility [52]. DREAM was
identified as being responsible for the GnRH-I pulsa-
tility, as it was shown to be part of the OCT1BS-b
binding complex, an essential element in the GnRH
enhancer for promoter pulse. Also, immunoneutraliza-
tion 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 sug-
gested that DREAM, via Ca
2+
, may serve as a basis
for the communication between cytoplasm and nucleus
that links the pulsatile secretion and pulsatile expres-
sion 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 medulloblas-
toma 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 tran-
scription. 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 contain-
ing 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 trans-
lated human AP-4 proteins bound to the two E-box-
binding 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
+1
–750
E-box(–790/ –785) Ref.51
Est-like (–779/ –776) Ref.51
E-box (–762/ –757) Ref.51
GII-Sil (–641/ –636)
JEG–3 cells specificity Ref.52
CRE (–67/ –60) Ref.53
–1124
–793
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 ele-
ments and their functional significance are
listed.
V. H. Y. Lee et al. Transcriptional regulation of GnRH gene
FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5465
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 palin-
dromic cAMP response element consensus site
(TGACGTCA, )67 to )60 bp) in the hGnRH-II pro-
moter [55]. In TE-671 cells, treatment with 1 mm
(Bu)
2
cAMP for 12–48 h strongly upregulated GnRH-II
gene expression, and this was verified by RT-PCR and
immunofluorescence staining. An increased concentra-
tion of GnRH-II peptides was also observed in the cell
medium after the treatment. Moreover, strong induc-
tion of promoter activities of the hGnRH-II gene in
response to 1 mm (Bu)
2
cAMP was found in transfec-
tion studies on the hGnRH-II promoter construct cou-
pled 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 treat-
ment. 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 hypo-
thalamic 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 injec-
tion 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, treat-
ment 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 regu-
lated 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. sug-
gested 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, even-
tually 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 pep-
tide 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) stimu-
lated 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 differ-
ences between the actions of GnRH-I and its metabo-
lite 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
Transcriptional regulation of GnRH gene V. H. Y. Lee et al.
5466 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS
during pregnancy. Like GnRHs, gonadotropins have
been shown to differentially regulate the two GnRH
genes via stimulation of cAMP production and activa-
tion of PKA [1]. Through these pathways, gonadotro-
pins 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-bromo-
cAMP, 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 treat-
ment 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 lev-
els 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 differ-
ently in the ovary.
Steroid hormones
The pulsatile secretion of GnRH from the hypotha-
lamic 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 regula-
tors 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 progester-
one (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 neu-
rons 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. There-
fore, steroid receptors were not detected [71]. Also, it
might be due to limitations in the sensitivity of the
detection methods. These steroid receptors, upon form-
ing 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 involve-
ment 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 regu-
lated differently at different stages of the estrous
V. H. Y. Lee et al. Transcriptional regulation of GnRH gene
FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5467
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 acti-
vation 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 ste-
roid hormones has also been reported. In the ovary,
E
2
treatment (1–100 nm) for 24 h resulted in a dose-
dependent decrease in GnRH-I mRNA levels, as deter-
mined 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
-medi-

ated 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. dem-
onstrated 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-depen-
dent manner [81]. Similarly, in placental tumor cells,
E
2
also negatively regulated rGnRH-I promoter
activity [82].
Apart from GnRH-I gene regulation, steroid hor-
mones 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 dem-
onstrated 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 medullo-
blastoma 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 gonado-
tropin 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 differ-
ently 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
produc-
tion 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 sev-
eral 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].
Transcriptional regulation of GnRH gene V. H. Y. Lee et al.
5468 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS
The effects of P
4
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
P
4
(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 P
4
treatment and increased GnRH-I promoter
activity in the presence of P
4
. However, there was no
significant effect on GnRH-II mRNA levels after
either P
4
treatment or overexpression of either PR-A
or PR-B [97].
The role of P
4
in GnRH-I and GnRH-II expression
has also been investigated in hGLCs [76]. Treatment
of the cells with the P
4
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 P

4
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 regula-
tion [71]. GT1 cells were found to have enough
5a-reductase activity to efficiently convert testosterone
to dihydrotestosterone (DHT), a more potent andro-
gen [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 expres-
sion (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.
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. deter-
mined the presence of AR in the plasma membrane by
using western blot analysis and fluorescence staining
[101]. Treatment with DHT, testosterone or a cell-
impermeable BSA-conjugated testosterone (T-3-BSA)
stimulated GnRH-I release in GT1-7 cells. However,
only treatment with DHT or testosterone, but not T-3-

BSA, resulted in downregulation of GnRH-I mRNA
levels. Thus, repression of GnRH-I expression by tes-
tosterone 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 treat-
ment for 6 days with corticosterone in adult male rats
was found to cause significant suppresssion of hypo-
thalamic 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 E
2
-induced gonadotropin surge in immature
female rats, had no effect on GnRH-I mRNA expres-
sion. The authors suggested that although dexametha-
sone 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 treat-
ment in male rhesus monkeys [104]. It also suggests

that another possibility is decreased pituitary respon-
siveness 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 E
2
, testosterone and DHT [106]. The high
V. H. Y. Lee et al. Transcriptional regulation of GnRH gene
FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5469
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 E
2
was unlikely to hap-
pen. 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 conver-

sion to other steroid hormones in GT1-7 cells.
Physiological regulators
Insulin
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 hypotha-
lamic 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 insu-
lin. In GnRH-expressing neuronal cells (GN11), after
treatment with insulin (10 nm), the activity of the
mGnRH-I promoter 1250 bp upstream of the transcrip-
tion start site was found to be increased by up to 4.0-
fold as compared to controls [113]. However, insulin
treatment of GN11 cells transfected with a 587 bp pro-
moter did not cause stimulation of promoter activities,

indicating that the elements mediating insulin stimula-
tion 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
activation of the mouse promoter. This suggested that
insulin regulates mGnRH-I promoter via a MAP
kinase pathway. Recently, insulin-dependent stimula-
tion 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 immunoprecipita-
tion 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 attenu-
ated the insulin-induced stimulation. These data sug-
gested 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].
Melatonin
Melatonin is the principal hormone produced by the
vertebrate pineal gland, which mainly regulates circa-
dian 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 downre-

gulate 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 recep-
tor-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.
Nitric oxide (NO)
Signal transduction pathways utilizing NO as a sec-
ondary messenger in the brain have been well studied.
NO has been shown to be important for GnRH secre-
tion [121–123], and also acts as an intermediate for
induction of N-methyl-d-aspartic acid (NMDA)-medi-
ated induction of GnRH secretion. In GT1-7 cells,
Transcriptional regulation of GnRH gene V. H. Y. Lee et al.
5470 FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 homo-
logy 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 con-
firmed 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 Oct-
1. 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 bind-
ing 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 exp-
ression have been carried out in rat hypothalamic
fragments and GT1-1 cells [13]. Incubation of hypo-
thalamic fragments with atRA (0.01–1.00 lm) for 2 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 sug-
gested 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 identi-
fied 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
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 inhi-
bit GnRH promoter activity and repress GnRH-I
mRNA expression [128]. Deletion analysis showed that
the )230 to )110 bp sequence within the proximal pro-

moter 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 sug-
gested that the transcriptional repression of GnRH by
9-cis-RA may be mediated by other transcription fac-
tors that interact with 9-cis-RA and bind to the proxi-
mal 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 differentia-
tion, 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]. Treat-
ment 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 ele-
ment of IGF-I-mediated induction of hGnRH-I expres-
sion. 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 compo-
nents of the signaling pathways in response to IGF-I

induction in NLT cells.
c-Aminobutyric acid (GABA)
GABA is one of the major neurotransmitters responsi-
ble for modifying GnRH neural activity and GnRH
secretion. Studies have shown that GABA hyperpolar-
izes GnRH neurons [2] and induces rGnRH-I gene
expression [134,135] in mature rat GnRH neurons.
However, in embryonic rat GnRH neurons, GABA
V. H. Y. Lee et al. Transcriptional regulation of GnRH gene
FEBS Journal 275 (2008) 5458–5478 ª 2008 The Authors Journal compilation ª 2008 FEBS 5471
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 3 kb of rGnRH-I
promoter together with the enhanced green fluorescent
protein reporter also demonstrated that GABA acti-
vates 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 transcrip-
tion regulatory factors. Self-regulation by GnRH itself
and feedback regulatory mechanisms by other compo-
nents 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 car-
ried 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 expres-
sion of GnRH genes may have different regulatory
mechanisms in the brain neurons and in the reproduc-
tive organs. However, most of the studies regarding
GnRH gene regulation have been performed in neuro-
nal 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.
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 reg-
ulation of the GnRH-I and GnRH-II genes; for
instance, there are different effects of the steroid hor-
mones E
2
and P
4
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 gonadotro-
pin-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 identifica-
tion 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. Chow.
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