Báo cáo khoa học: Gonadotropin-releasing hormone: GnRH receptor signaling in extrapituitary tissues docx

MINIREVIEW
Gonadotropin-releasing hormone: GnRH receptor signaling
in extrapituitary tissues
Lydia W. T. Cheung and Alice S. T. Wong
School of Biological Sciences, University of Hong Kong, China
Introduction
The hypothalamic gonadotropin-releasing hormone
(GnRH) is a decapeptide that plays a critical role in
the regulation of reproduction. GnRH-I (pGlu-His-
Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH
2
) is the first
GnRH isoform discovered in mammalian brain. Its
major role is to stimulate pituitary secretion of
gonadotropins, luteinizing hormone and follicle-stimu-
lating hormone, which in turn stimulate the gonads
for steroid production. Subsequently, a second iso-
form of GnRH (His5, Trp7, Tyr8) (GnRH-II) has
been isolated from chicken brain. It is also highly
conserved among vertebrates, including mammals [1].
However, in contrast to GnRH-I, GnRH-II is
expressed at significantly higher levels outside the
Keywords
cross-talk; extrapituitary; GnRH; GnRH
receptor; MAPK; metastasis; pituitary;
receptor tyrosine kinase; signaling; tumor
Correspondence
A. S. T. Wong, School of Biological
Sciences, University of Hong Kong, 4S-14
Kadoorie Biological Sciences Building,
Pokfulam Road, Hong Kong, China

Fax: +852 2559 9114
Tel: +852 2299 0865
E-mail:
(Received 14 April 2008, revised 28 May
2008, accepted 11 June 2008)
doi:10.1111/j.1742-4658.2008.06677.x
Gonadotropin-releasing hormone (GnRH) has historically been known as
a pituitary hormone; however, in the past few years, interest has been
raised in locally produced, extrapituitary GnRH. GnRH receptor
(GnRHR) was found to be expressed in normal human reproductive tissues
(e.g. breast, endometrium, ovary, and prostate) and tumors derived from
these tissues. Numerous studies have provided evidence for a role of GnRH
in cell proliferation. More recently, we and others have reported a novel
role for GnRH in other aspects of tumor progression, such as metastasis
and angiogenesis. The multiple actions of GnRH could be linked to the
divergence of signaling pathways that are activated by GnRHR. Recent
observations also demonstrate cross-talk between GnRHR and growth fac-
tor receptors. Intriguingly, the classical G
aq
–11-phospholipase C signal
transduction pathway, known to function in pituitary gonadotropes, is not
involved in GnRH actions at nonpituitary targets. Herein, we review the
key findings on the role of GnRH in the control of tumor growth, progres-
sion, and dissemination. The emerging role of GnRHR in actin cytoskele-
ton remodeling (small Rho GTPases), expression and⁄ or activity of
adhesion molecules (integrins), proteolytic enzymes (matrix metalloprotein-
ases) and angiogenic factors is explored. The signal transduction mecha-
nisms of GnRHR in mediating these activities is described. Finally, we
discuss how a common GnRHR may mediate different, even opposite,
responses to GnRH in the same tissue ⁄ cell type and whether an additional

receptor(s) for GnRH exists.
Abbreviations
EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-related kinase; FAK, focal adhesion
kinase; FGF, fibroblast growth factor; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor;
JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF-jB, nuclear factor kappa B; PI3K,
phosphatidylinositol 3-kinase; PKC, protein kinase C; Pyk2, proline-rich tyrosine kinase 2; RTK, receptor tyrosine kinase; uPA, urokinase-type
plasminogen activator; VEGF, vascular endothelial growth factor.
FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5479
brain and is particularly abundant in the kidney,
bone marrow, and prostate [2]. This leads to the
speculation that GnRH-II may have distinct physio-
logical functions from those of GnRH-I. In line with
this is the observation that although GnRH-II can
stimulate gonadotropin secretion, its efficiency is
much lower than that of GnRH-I (only about 2% of
that of GnRH-I) [3]. This suggests that the primary
role of GnRH-II is not in the regulation of gonado-
tropin secretion. Instead, this peptide has been shown
to act as a neuromodulator [4]. The exact actions of
GnRH-II in peripheral tissues are not entirely under-
stood, but this is certainly an important topic for
investigation which may offer an opportunity to eluci-
date the undisclosed complexity of GnRH.
In this minireview, we will focus on recent progress
in understanding the roles of GnRH-I and GnRH-II
in extrapituitary tissues, in particular its emerging
role in tumor growth, invasion, and metastasis. We
will also describe the molecular mechanisms underlying
these effects, focusing on the roles of proteolysis,
adhesion, and signaling, as well as our still-emerging

understanding of receptor cross-talk with other
pathways. Finally, we will discuss two important
outstanding questions in the field regarding what might
distinguish the different responses to the same ligand
(GnRH) and whether an additional receptor(s) for
GnRH exists in humans.
Localization of GnRH receptor (GnRHR)
in peripheral reproductive tissues
The initial interest in extrapituitary GnRHR stemmed
primarily from observations in the 1980s that GnRH
analogs can inhibit the growth of nonpituitary tumor
cell lines [5]. Soon after this, a functional type I
GnRHR was demonstrated in a variety of normal
human reproductive tissues (e.g. breast, endometrium,
ovary, and prostate) and tumors derived from these
tissues.
In the ovary, GnRHR mRNAs are expressed in
granulosa-luteal cells, and increased expression of
GnRHR correlates with follicular growth and develop-
ment [6]. GnRHR binding has been demonstrated in
luteinized granulosa cells, late follicles and developing
corpora lutea, but not in primordial, early antral and
preovulatory follicles [7,8]. This stage-specific expres-
sion of GnRHR in the human granulosa and luteal
cells suggests a role for GnRH in the regulation of
ovarian physiology, particularly ovulation, follicular
atresia and luteolysis. The presence of GnRHR protein
and mRNA has also been demonstrated in human
ovarian tumor specimens, ovarian cancer cell lines and
their tissue of origin, ovarian surface epithelium [9,10].

Interestingly, levels of GnRHR seem to be associated
with cancer grading and have been reported to be
elevated in advanced stage (stages III and IV) as
compared to early stage (stages I and II) ovarian
carcinomas [11]. Our recent findings that GnRH can
promote the motility and invasiveness of ovarian can-
cer cells further corroborate the view that GnRH may
play a crucial role in tumor progression ⁄ metastasis
[12,13], and these findings will be discussed in a later
section.
Using [
125
I][d-Trp6]GnRH, specific receptor binding
has been detected in membranes from 24 of 31 (77%)
endometrial carcinomas and from three of 13 (23.1%)
nonmalignant human endometrial specimens [14].
GnRHR mRNA has been clearly detected in surgical
endometrial carcinoma specimens and endometrial
carcinoma cell lines [15,16]. As with normal myome-
trium, most benign neoplasms studied thus far,
including uterine leiomyoma, also possess GnRHR
[17].
Early studies showed that the human placenta con-
tains specific binding sites for GnRH that interact with
GnRH agonists and antagonists [18]. Later on,
GnRHR was localized to the cytotrophoblast and
syncytiotrophoblast cell layers [19,20]. Temporal
expression of GnRHR in the placental cells at different
weeks of gestation has been observed, in parallel with
the time-course of chorionic gonadotropin secretion

during pregnancy [21], suggesting that the expression
of the receptor is a function of pregnancy stage.
The presence of GnRHR has been demonstrated in
numerous human breast cancer cell lines and tumor
biopsy specimens [22–24]. GnRHR was immunolocal-
ized in the cytoplasm in 37 of 58 (64%) invasive ductal
carcinoma cases [23]. The expression of GnRHR in
normal human breast tissue is still controversial, but
the sample size may have been too small to allow any
definite conclusion [22,25].
GnRHR is also present in prostate cancer cells, as
shown by radioligand-binding studies, PCR, and
western blotting analysis [26,27]. GnRHR immunore-
activity is localized to the luminal and basal epithelial
cells in benign and malignant prostate tissues. In this
study, the relative GnRHR mRNA levels showed a wide
range of individual differences that were unrelated to
the histological grades of the 16 cases [27]. There does,
however, appear to be significantly higher expression of
GnRHR in hormone-refractory prostate carcinoma
than in other types of prostate tumor (n = 80) [28].
Although these extrapituitary GnRHRs share the
same cDNA nucleotide sequence and encode tran-
scripts and proteins of the same size as the pituitary
GnRH receptor signaling L. W. T. Cheung and A. S. T. Wong
5480 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS
GnRHR [20,26,29], they also differ in several ways.
First, cell surface receptor expression in extrapituitary
sites is low as compared to that of the pituitary
[15,27]. This may underlie the greater effect of the

GnRHR ligands on the gonadotropes. Second, there
are at least two classes of GnRHR: one has high affin-
ity [with nanomolar dissociation constants (K
d
)] for
GnRH, and one has low affinity (with micromolar K
d
values) for GnRH. The high-affinity GnRH-binding
sites are commonly regarded as being the same as the
GnRHR of the pituitary gland. Whereas in most of
the reported cases, both the low-affinity and high-affin-
ity GnRHR have been found in extrapituitary tissues
[30–33], in some cases, only low-affinity GnRHR could
be detected [10,18,34], and in others, e.g. in endome-
trial cancers and nonmalignant endometrial specimens,
only the high-affinity GnRHR has been demonstrated
[14]. The exact role of each of these receptors and the
implications of differential levels of expression remain
to be elucidated.
Functions of GnRH-I and GnRH-II in
cancers
Tumor growth
Over the last two decades, both GnRH agonists and
antagonists have been widely used as therapeutics in
treating sex steroid-dependent tumors. The majority
of these GnRH analogs, when given continuously,
inhibit gonadotropin synthesis and secretion via
downregulation of the pituitary GnRHRs. This indi-
rect mechanism of action has provided the rationale
for the use of GnRH analogs in the treatment of hor-

mone-dependent tumors for many years. Only since
the detection of GnRHR in extrapituitary tissues has
there been increasing interest in its direct action on
tumor cells.
GnRH-I analogs have direct antiproliferative effects
on ovarian cancer cells, which is linked to the disrup-
tion of the cell cycle at G
0
⁄ G
1
[31,35,36]. On the other
hand, several independent in vitro studies failed to
demonstrate significant growth inhibition by GnRH-I
agonists, even at fairly high concentrations (micromolar
range) [37,38]. In fact, a biphasic impact of GnRH-I
agonists on growth has been reported: whereas GnRH-I
agonists at high dose (1 lm) inhibit cell proliferation
in vitro, cells treated with agonists at low dose (10 nm)
show significant growth stimulation [39]. Further
studies demonstrated that nanomolar concentrations of
GnRH-I agonists also increase cell survival under
multiple stress conditions, including DNA replication-
specific cytotoxic agents and UV radiation [40].
GnRH-II has antiproliferative effects on ovarian cancer
cells [41–43]. Although it has been suggested that this
effect of GnRH-II is mediated through the type I
GnRHR [43], there are other findings implicating a
type I GnRHR-independent action [41,42].
It is interesting to note that although both GnRH-I
agonists and antagonists exert antiproliferative effects,

the effects of GnRH-I antagonists are stronger than
those of the agonists [44]. This difference has also been
seen in an in vivo model, which demonstrates a signifi-
cant inhibition of tumor growth by GnRH-I antago-
nists but not GnRH-I agonists [45]. The advantage of
GnRH antagonists over the agonistic peptides is prob-
ably due to the fact that they inhibit the secretion of
gonadotropins and reduce sex steroid levels immedi-
ately after application, thus achieving rapid therapeutic
effects, whereas repeated exposure to agonistic agents
is required to induce functional desensitization of the
gonadotropes [46].
Treatment of human endometrial cancer cells (cell
line Ishikawa) with the GnRH-I antagonist SB-75
results in growth inhibition, mainly due to the Fas ⁄ Fas
ligand-mediated apoptotic pathway, whereas GnRH-I
agonists have no effect on the same cell line [15,47,48].
Another endometrial carcinoma cell line, HEC-1A, also
exhibits differential responses to different GnRH agon-
ists and antagonists [15,30,36,48]. GnRH-II has been
shown to have antiproliferative effects on endometrial
carcinoma cells [41]. The effects of GnRH-I are
abrogated after type I GnRHR knockout [36], whereas
those of the GnRH-I antagonist cetrorelix and of
GnRH-II persist [41]. These findings suggest that the
antiproliferative effects of cetrorelix and GnRH-II are
not mediated through the type I GnRHR.
GnRH-I has been demonstrated to have antiprolifer-
ative effects on prostate cancer cells [49–51], except in
one in vivo study [52]. This antiproliferative effect

appears to be independent of the androgen receptor
status of the prostate carcinoma cells, as both andro-
gen-sensitive LNCaP cells and androgen-resistant
DU-145 cells remain sensitive to GnRH [49,50]. Acti-
vation of GnRHR may mediate these effects via direct
induction of apoptosis through caspase activation [53].
Compatible with a role for GnRH in survival at low
doses, an enhancing effect of GnRH was observed
when cells were treated with a low concentration
(100 pm) of GnRH-I agonist [54]. GnRH-II was shown
to have an antiproliferative effect on DU-145 cells and
growth-stimulatory effect on TSU-Pr1 cells, but the
type I GnRHR was not involved [55].
The influence of GnRH on the growth of human
breast cancer cells was first studied with MCF-7 cells
[56], and both in vitro and in vivo proliferation of
L. W. T. Cheung and A. S. T. Wong GnRH receptor signaling
FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5481
breast cancer cells could be inhibited by both agonistic
and antagonistic analogs of GnRH [57,58]. However,
higher efficiency of GnRH antagonists in growth inhi-
bition than that of GnRH agonists has been reported
[24,58].
Invasion and metastasis
The observation that GnRH controls tumor growth
suggests a regulatory role for this peptide in the meta-
static behavior of cancer cells. This hypothesis is sup-
ported by studies showing that GnRH-I and GnRH-II
can affect the expression of several extracellular
matrix-degrading enzymes in human extravillous cyto-

trophoblasts and decidual stromal cells to facilitate
implantation [59,60]. However, its potential role in
cancer metastasis has just begun to be revealed.
Metastasis is a complex phenomenon that requires
several specific steps, such as decreased adhesion,
increased motility, and proteolysis. The effects of GnRH
in tumor metastasis are mediated through the regulation
of adhesion molecules, Rho GTPases, and two families
of metastasis-related proteinases, the matrix metallopro-
teinases (MMPs) and the urokinase-type plasminogen
activator (uPA) system, at several levels: mRNA
transcription, secretion, and proenzyme activation.
The ability of GnRH to regulate metastasis was first
reported in melanoma cells [61]. High doses of GnRH-I
analog, at micromolar concentrations, significantly
reduces the ability of melanoma cells to invade
and migrate [61]. Preliminary data (R. M. Moretti,
M. Monagnani Marelli, J. C. van Groeninghen, M.
Motta & P. Limonta, unpublished results, 2003) indicate
that this inhibitory action is due to the effects of
integrins and MMPs [62].
We were the first to report possible metastatic activ-
ity of GnRH-I in tumors of the female reproductive
tract [12]. GnRH-I exerts a biphasic effect on cellular
migration and invasion: whereas lower (nanomolar)
concentrations of the GnRH-I agonist stimulate cellu-
lar migration and invasion in a dose-dependent man-
ner, high (micromolar) concentrations are not as
efficient. This proinvasive effect is mediated through
activation of metastasis-related proteinases, in particu-

lar MMP-2 and MMP-9 [12]. Moreover, GnRH-I is
able to transactivate the MMP-2 and MMP-9 promot-
ers, which means that GnRH can be considered to be
a new member of MMP-2 and MMP-9 transcriptional
modulators. Like GnRH-I, native GnRH-II and its
synthetic analog also induce a similar biphasic regula-
tion of ovarian cancer invasion [13]. The finding that
small interfering RNA-mediated downregulation of
type I GnRHR completely reversed the effects of both
GnRH-I and GnRH-II on cell invasion supports the
view that the same receptor, type I GnRHR, is essen-
tial for the effects of GnRH-I and GnRH-II in ovarian
cancer cells.
The decrease in uPA activity of cytosol from Dun-
ning R3327H rat prostate tumors after treatment with
GnRH-I analogs suggests that GnRH may be an
important factor in reducing the invasiveness of pros-
tate cancer [63]. High doses of GnRH-I agonists and
antagonists have been reported to attenuate the
invading capacity of both androgen-dependent and
androgen-independent prostate cancer cells by modu-
lating E-cadherin-mediated cell–cell contacts and pro-
duction of uPA and its inhibitor (plasminogen
activator inhibitor-1) [64–66]. GnRH has also been
shown to regulate cell motility through its interaction
with the small GTPases Rac1, Cdc42, and RhoA,
which are involved in the regulation of actin polymer-
ization [67].
Up to now, there has been only one study, by Von
Alten et al., investigating the role of GnRH in breast

cancer metastasis, using a coculture system with
human osteosarcoma cells to analyze tumor cell
invasion to bone [68]. The consequences of GnRHR
activation are complex and appear to be cell context
dependent: whereas treatment of cells with the
GnRH-I agonist triptorelin, the GnRH-II agonist
[d-Lys6]GnRH-II and the GnRH-I antagonist cetrorelix
decreases the invasion rate in most breast cancer cell
lines, these agents have no significant effect in the
GnRHR-positive MDA-MB-435 cells [68]. Further
investigations are required to elucidate the reason why
the MDA-MB-435 cell line reacts differently.
Organ-specific homing and colonization of cancer
cells are important and interesting features of metasta-
sis. A role for GnRH has also been suggested in the
regulation of the immune response and metastasis.
GnRH-I and GnRH-II are expressed in human normal
and cancerous T-cells. GnRH triggers laminin receptor
gene expression, adhesion to laminin, in vitro chemo-
taxis, and in vivo homing to specific organs [69].
Angiogenesis
Angiogenesis is crucial to a number of physiological
and pathological processes, such as reproduction,
development, and tissue repair, as well as tumor
growth and metastasis. Vascular endothelial growth
factor (VEGF) is implicated as the most important
angiogenesis inducer, because of its potency in a
variety of normal and tumor cells. Other angiogenic
factors include fibroblast growth factor (FGF), plate-
let-derived growth factor and the angiopoietin family.

GnRH receptor signaling L. W. T. Cheung and A. S. T. Wong
5482 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS
The effect of GnRH on angiogenesis in the ovary, in
which this neovascularization is necessary for follicular
and luteal function, has been demonstrated. A recent
in vivo study using rats revealed that an application of
the GnRH-I agonist leuprolide acetate decreases the
protein expression of VEGF and angiopoietin-1 and
their receptors in ovarian follicles, and that this can be
reversed by coinjection of the GnRH antagonist antide
[70]. A similar inhibitory effect on angiogenesis can be
observed in marmosets injected with the GnRH-I
antagonist antarelix [71]. However, VEGF mRNA
expression is unaffected by the treatment. The clinical
response of uterine shrinkage after GnRH analog
treatment and a pathological role of FGF-2, VEGF
and platelet-derived growth factor in uterine leiomy-
oma growth and vascularization has also been sug-
gested [72]. Considering that angiogenesis is an
important process in many human cancers, it would be
very interesting to determine whether GnRH also plays
a key role in tumor angiogenesis.
Intracellular signal transduction
Upon GnRH binding, GnRHR undergoes a conforma-
tional change and stimulates a unique G-protein. Inter-
estingly, the classical G
aq
–11-phospholipase C signal
transduction pathway, which is known to operate in
the pituitary, is not involved in the antitumor activity

of GnRH analogs. Rather, GnRHRs couple to G
ai
in
these tumors and result in the activation of several
downstream signaling cascades [73,74], such as mito-
gen-activated protein kinase (MAPK), phosphatidyl-
inositol-3-kinase (PI3K), and nuclear factor kappa B
(NF-jB) signaling. The GnRH-induced signaling path-
ways in extrapituitary tissues are shown schematically
in Fig. 1.
Fig. 1. Schematic representation of GnRHR signaling in extrapituitary tissues. Binding of GnRH to GnRHR triggers several intracellular signal-
ing cascades and cross-talk with mitogenic signaling, depending on the cell context. Some of these signaling modules can transduce extra-
cellular signals to the nucleus and thereby regulate genes that are involved in cell growth, metastasis, or survival. Arr, b-arrestin; CREB,
cAMP response element-binding protein; FGFR, fibroblast growth factor receptor; HB-EGF, heparin-binding EGF; IjB, inhibitory factor kap-
pa B; IGFR, IGF receptor; MEK, mitogen-activated protein kinase kinase; MLK3, mixed-lineage kinase 3; PTP, protein tyrosine phosphatase;
Sos, son of sevenless; TNF-a, tumor necrosis factor alpha.
L. W. T. Cheung and A. S. T. Wong GnRH receptor signaling
FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5483
MAPK
The major MAPK cascades include extracellular sig-
nal-regulated kinase (ERK), Jun N-terminal kinase
(JNK), and p38 MAPK. Many studies have shown
that the MAPK pathway is critical for GnRH activi-
ties, which provides an important link for the trans-
mission of signals from the cell surface to the nucleus.
Activation of MAPK by GnRH involves distinct
upstream pathways in generating tissue-specific and
cell-specific signaling (Fig. 1). This can occur at differ-
ent levels via different mechanisms: (a) second messen-
gers [protein kinase C (PKC) and cAMP] [26];

(b) receptor-interacting proteins such as Src and
b-zarrestin [53,75]; and (c) upstream kinases such as
MAPK ⁄ ERK kinase and PI3K [53,76]. For example,
GnRH-I induces apoptosis in DU-145 prostate cancer
cells via JNK, which is activated through two indepen-
dent mechanisms [53]. Activation of the pathway is
dependent on c-Src with concomitant decrease in Akt
activity, and the combination of these two events
relieves the inhibition of the upstream activator of
JNK, MLK3 [53] (Fig. 1). Interestingly, although
ERK1 ⁄ 2 is phosphorylated through epidermal growth
factor receptor (EGFR) under the same conditions,
this pathway is not involved in the apoptotic effects.
These findings demonstrate that activation of the two
MAPKs, which lead to distinct physiological out-
comes, is separated already at the upstream levels. In
the ovarian cancer cell line CaOV-3, prolonged stimu-
lation of ERK1 ⁄ 2 through Shc and son of sevenless is
required for GnRH-I-mediated growth inhibition [76].
Consistent with the fact that sustained activation of
ERK1 ⁄ 2 is often correlated with cell cycle progression,
GnRH-I-induced growth inhibition is attributed to G
1
arrest [76]. Moreover, the signaling cascade was shown
to be initiated by G
bc
, supporting the notion that the
post-GnRHR signaling cascade in extrapituitary cells
is different from that in pituitary cells. GnRH-induced
MAPK activation has also been shown in another

ovarian cancer cell line, OVCAR-3. Both ERK1 ⁄ 2 and
p38 MAPK mediate the antiproliferative effects of
GnRH-I and GnRH-II in a PKC-dependent manner
[43,77]. GnRH-II induces the activation of activator
protein-1 transcription factor via p38 MAPK, suggest-
ing a potential role of activator protein-1 in ovarian
cancer cell growth [77]. The JNK pathway also drives
tumor invasion and migration in ovarian cancer cells
[12], but the activation mechanism(s) remains to be
elucidated.
Temporal and spatial differences in cellular signaling
may have significant phenotypic manifestations [78,79].
For example, sustained activation of ERK1 ⁄ 2 has been
implicated in nerve growth factor-mediated neuronal
differentiation of PC12 cells, whereas a rapid and
transient activation is associated with growth factor-
mediated proliferation of PC12 cells [80]. Thus, the
duration of kinase activation seems to be a major
determinant of signal outcome. We have shown differ-
ential regulation of ERK1 ⁄ 2, p38 MAPK, and JNK
by GnRH-I with sustained signaling through the JNK
pathway in ovarian cancer cells [12]. Consistently,
GnRH-stimulated MMP-2 and MMP-9 expression,
secretion and cell invasion were attenuated by specific
inhibition of JNK but not of ERK1 ⁄ 2 and p38
MAPK, suggesting that prolonged activation of JNK
may contribute to a more invasive phenotype. Strong
and sustained activation of MAPK has been reported
to be necessary for its cytoplasm-to-nucleus transloca-
tion, and thereby contributes to the regulation of gene

expression [79,81]. It will be interesting to see whether
sustained activation of JNK leads to its nuclear trans-
location, which is required for GnRH-stimulated cell
invasion. The JNK pathway targets multiple transcrip-
tion factors, including c-Jun, c-Fos, ATF and PEA,
and putative binding sites for these DNA-binding pro-
teins are present in the MMP promoters [82]. Whether
these putative regulatory elements participate in the
GnRH-dependent activation of the MMP-2 and
MMP-9 genes remains to be determined.
Cross-talk with mitogenic signaling
Cross-talk between cell surface receptors, which has
been recognized as a mechanism capable of generating
signal diversity, is now receiving further interest.
Figure 1 illustrates the cross-talk between GnRHR
and receptor tyrosine kinases (RTKs). For instance,
GnRH causes transactivation of RTKs, such as EGFR
[75,78,83]. MMP-2 and MMP-9 seem to be essential
for GnRH-induced EGFR activation by cleavage of
the heparin-binding epidermal growth factor (EGF)
precursor [84]. Transactivation of EGFR has been
shown to activate ERK1 ⁄ 2, as GnRH-induced
ERK1 ⁄ 2 phosphorylation can be abolished in the pres-
ence of the EGFR inhibitor AG1478 [53,78]. However,
the biological importance of ERK1 ⁄ 2 activation in
response to this cross-talk still remains elusive.
Negative cross-talk between GnRHR and growth
factor receptors has also been described. For instance,
the antiproliferative effects of GnRH-I and GnRH-II
agonists are mediated through attenuation of EGFR

signaling in many reproductive tumor cells [57,66,85–
87]. In prostate cancer cells, cetrorelix is able to coun-
ter EGFR-dependent adhesive signaling through
a PKC-dependent mechanism [66]. Activation of
GnRH receptor signaling L. W. T. Cheung and A. S. T. Wong
5484 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS
GnRHR appears to mediate these effects via activation
of phosphotyrosine phosphatase, thereby reducing
EGF-induced EGFR autophosphorylation, resulting in
downregulation of mitogenic signal transduction and
cell proliferation [85,86,88]. A negative regulatory
interaction between GnRHR and mitogenic signaling
pathways has also been reported in human prostate
cancer cells via insulin-like growth factor. GnRH-I
agonists inhibit expression of the insulin-like growth
factor receptor, receptor tyrosine phosphorylation, and
the subsequent downstream activation of Akt [89–91].
Another example is FGF-2. GnRH analog treatment
has been shown to block cell proliferation and inva-
sion induced by FGF-2 stimulation [65].
PI3K
The PI3K signaling pathway and its downstream
target Akt (also named protein kinase B) has been
implicated in promoting cell survival, proliferation,
and invasion. In uterine leiomyomas, the GnRH-I ago-
nist leuprolide acetate causes a significant reduction in
PI3K ⁄ Akt activity and inhibits the expression of the
antiapoptotic proteins (c-FLIP and PED ⁄ PEA15),
thereby inducing apoptosis [92]. In the SKOV-3 ovar-
ian cancer cell line, GnRH-I and GnRH-II interfere

with activation of the PI3K ⁄ Akt cascade, and this is is
associated with the inhibitory effects of GnRH on cell
invasion [13].
Although PI3K ⁄ Akt and MAPK are two parallel
pathways in some cell types, they are two related path-
ways in the mediation of GnRH actions, as inhibition
of PI3K ⁄ Akt can alter the activation of MAPK. For
instance, in prostate cancer cells, stimulation of
PI3K ⁄ Akt releases mixed-lineage kinase 3, which in
turn activates the JNK pathway, and this positive reg-
ulation is important for the proapoptotic effect of
GnRH-I (Fig. 1) [53]. PI3K ⁄ Akt is also an upstream
kinase of ERK1 ⁄ 2, and EGFR transactivation by
GnRH-I may be required for the activation of this
cascade [75,93].
Other signaling pathways
Activation of NF-jB is important for the protection
against apoptosis in ovarian tumors induced by the
GnRH-I agonist tiptorelin [94]. The effect is probably
mediated by the G
ai
-coupled GnRHR, and receptor
activation causes nuclear translocation of NF-jB [94].
Unlike the other signaling pathways studied, the
GnRH-I-induced NF-jB activation appears to be inde-
pendent of the cross-talk between GnRHR and growth
factor signaling, as treatment with phosphatase inhibi-
tor has no effect on the activation of NF-jB [94]. It
has also been shown that GnRH-I suppresses interleu-
kin-8 expression via attenuation of tumor necrosis fac-

tor alpha-induced NF-jB signaling in endometriotic
stromal cells (Fig. 1) [95]. These data suggest that
modulation of cytokine signal transduction by GnRH
may be one of the mechanisms contributing to its
growth-inhibitory effect.
The non-RTKs focal adhesion kinase (FAK) and
proline-rich tyrosine kinase 2 (Pyk2) are the predomi-
nant mediators of integrin signaling. GnRHR has been
shown to signal through these molecules, suggesting a
role for GnRH in cytoskeletal reorganization. In
human endometrial cancer cells (HEC-1A), b
3
-integrin-
dependent activation of FAK is associated with the
inhibitory effects of GnRH-I and GnRH-II on growth
[96]. Leiomyoma regression induced by GnRH-I agon-
ists has been suggested to be mediated, at least in part,
through a mechanism involving suppression of FAK
[97]. Maudsley et al. demonstrated a novel signaling
cascade of GnRHR that functionally antagonizes the
actions of testosterone and inhibits prostate tumor
growth [98]. GnRH controls the tyrosine phosphoryla-
tion status of the focal adhesion proteins Pyk2 and
Hic-5. This alteration of the focal adhesion dynamics
then results in nuclear translocation of the androgen
receptor, which renders it transcriptionally inactive
[98].
Mechanisms underlying the diverse
responses to GnRH action
As discussed earlier, GnRH and its agonists have a

dual and biphasic action: whereas low concentrations
(0.1–10 nm) of GnRH stimulate cell proliferation,
migration and invasion in a dose-dependent manner,
high concentrations (100 nm to 1 l m) inhibit these
functions [12,13,39]. Moreover, the same dose of
GnRH can elicit completely opposite responses in cells
derived from the same tissue. We demonstrated that in
two human ovarian cancer cell lines, OVCAR-3 and
SKOV-3, GnRH-I and GnRH-II induce invasion of
OVCAR-3 cells, but inhibit the invasiveness of SKOV-
3 cells [13]. A similar difference has been found in the
effects of GnRH on cell proliferation and cell migra-
tion in the prostate carcinoma cell lines TSU-Pr1 and
DU-145 [67]. Whereas GnRH-I and GnRH-II stimu-
lated cell proliferation, induced actin cytoskeleton
remodeling and promoted migration in TSU-Pr1 cells,
they were inhibitory in DU-145 cells [67]. The observa-
tion that GnRH-I and GnRH-II have no significant
effect in cell lines with type I GnRHR depletion indi-
cates that the type I GnRHR is indispensable for the
L. W. T. Cheung and A. S. T. Wong GnRH receptor signaling
FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5485
effects of both GnRH-I and GnRH-II [13,67]. Thus,
one intriguing question is how a common GnRHR
may mediate different, even opposite, responses to
GnRH in the same cell type ⁄ tissue. The reasons are
unknown, but several possibilities (as summarized in
Table 1) can be envisaged. First, the treatment condi-
tion may be one determinant of the outcome. The
pulsatility of GnRH release is necessary for the hor-

mone to stimulate pituitary gonadotropes. On the
other hand, sustained administration of the peptide
brings about a short initial stimulation that is rapidly
followed by a decrease in gonadotropin synthesis and
secretion [99]. In support of this, the signal response is
different at different doses. It has been shown that pul-
satile GnRH stimulates more sustained ERK activity
(more than 8 h), whereas continuous infusion of aT3-1
cells with GnRH stimulates short-term (2 h) ERK
activity [100]. There is also evidence that GnRH treat-
ment stimulates cAMP production at nanomolar con-
centrations, but has an inhibitory effect at micromolar
concentrations [101]. It should be pointed out that the
nanomolar concentration (0.01–1 nm) corresponds to
the physiological circulating level, and the effects
caused by this dose range may represent the physiolog-
ical functions of GnRH [54,102].
Second, GnRH action has been shown to be medi-
ated by coupling to different G
a
-proteins, depending
on the time and dose of exposure [101,103]. In general,
G
aq
and G
as
are associated with a stimulatory effect
[103], whereas G
ai
often mediates the antiproliferative

and proapoptotic effects of GnRH [73,74]. Low GnRH
concentrations promote the coupling of GnRHR to
G
as
[101]. High concentrations of GnRH cause a
switch in receptor coupling from G
as
to G
ai
[101].
Moreover, stimulation of cAMP production by GnRH
is through coupling to G
as
, whereas inhibition of
cAMP production at high concentrations of GnRH is
through coupling to G
ai
[101,104]. These findings sug-
gest that the intracellular milieu in different tissues
results in differential coupling and different phenotypic
effects.
Third, multiple GnRH-dependent signaling path-
ways may occur via different subunits of a single
G-protein [105]. After ligand-induced dissociation,
both the a-subunit and bc-subunits are capable of
activating various effectors, such as adenylyl cyclase,
phospholipase C, and ion channels, thereby conferring
on the receptor the potential for dual signaling
[106,107]. The effector pathway to be activated is
specific to the upstream subunits. For instance,

whereas the a-subunit of G
i
inhibits adenylyl cyclase
activity, the bc-subunits may stimulate the activities of
some adenylyl cyclase subtypes [108,109].
The receptor expression level is also known to be a
determinant for different signal outcomes [6,110,111].
In gonadotropes, different cell surface densities of
GnRHR result in the differential regulation of luteiniz-
ing hormone and follicle-stimulating hormone subunit
gene expression by GnRH-I [112]. We and others have
previously shown that low doses of GnRH upregulate
the expression of its receptor, whereas high doses
decrease it [12,111,113]. This difference in regulation
suggests that high levels of GnRHR expression may
enhance the cellular response to GnRH stimulation,
presumably due to more efficient signal amplification
or altered signaling through coupling to different
G-proteins.
Moreover, ligand selectivity has been proposed to
explain the opposite (stimulatory and inhibitory)
effects of GnRH. For instance, in positively respond-
ing prostate carcinoma cell lines, GnRH-I is more
effective than GnRH-II. On the other hand, in nega-
tively responding cell lines, GnRH-II is much more
effective than GnRH-I. Given the short plasma half-
life of GnRH, efforts have been made to obtain
GnRH analogs, to resist degradation and to increase
potency. However, the different GnRH agonists may
selectively stabilize different receptor-active conforma-

tions and therefore different ligand-induced selective
signaling pathways [114]. In this regard, it has been
shown that the highly variable amino acid at posi-
tion 8 of GnRH plays a discriminating role in selecting
the receptor conformational state [115].
The presence of splice variants of the GnRHR tran-
script may be another possible reason for the different
or opposite responses to GnRH. To date, variant tran-
scripts of GnRHR have been isolated in many species,
e.g. chicken [116], mouse [117] and human [118].
Although these splice variants are totally incapable of
ligand binding or signal transduction, they have been
implicated in the functional regulation of the wild-type
receptor. Previous studies have reported their inhibi-
tory activity on full-length GnRHR function [119].
This inhibition is specific, augmented by increasing
Table 1. Potential mechanisms that underlie the diverse responses
to GnRH.
Determinants References
Treatment conditions, e.g. duration
and dose
[99–101]
Different G
a
subtypes [73,74,101,103,104]
Different G-protein subunits [105–109]
GnRHR expression level [6,110–112]
Ligand selectivity [114,115]
Presence of GnRHR splice variants [118,119]
Intrinsic cellular properties [61,62]

GnRH receptor signaling L. W. T. Cheung and A. S. T. Wong
5486 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS
Table 2. Physiological characteristics and responses to GnRH of common cancer cell lines. fl, decrease; ›, increase; –, no effect; ?, undetermined.
Cell line Origin Isolation
Original
histology
Tumorigenicity
in nude mice
Anchorage-
independent
growth
Invasive
capability
in vitro
Effect of
GnRH on
growth
Effect of GnRH
on motility ⁄
invasion References
CaOV-3 Ovary Primary
carcinoma
Serous Yes High Low fl› [12,76,125,126]
DU-145 Prostate Brain Poorly
differentiated
Yes Low High fl;– fl [50,55,67,120,127,128,132]
HEC-1A Endometrium Primary
carcinoma
Moderately
differentiated

Yes High High fl;– ? [15,30,36,48,129–131]
Ishikawa Endometrium Primary
carcinoma
Well
differentiated
Yes High Low fl;– ? [15,47,48,129–131]
LNCaP Prostate Lymph
node
Well
differentiated
Yes Very low Very low fl;– ? [49,120,127,128]
MCF-7 Breast Pleural
effusion
Ductal Only achievable
with estrogen
added
Low in
absence of
estrogen
Low flfl [56,68,122–124,135]
MDA-MB-435 Breast Pleural
effusion
Ductal Yes High High fl – [68,121,133–135]
OVCAR-3 Ovary Ascites Serous Yes Low Low fl› [12,13,43,136,137]
SKOV-3 Ovary Ascites Serous Yes High High flfl [13,43,125]
TSU-Pr1 Prostate Lymph
node
Poorly
differentiated
Yes High High ›;– › [54,55,67,120,127]

L. W. T. Cheung and A. S. T. Wong GnRH receptor signaling
FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5487
amounts of the cotransfected splice variant cDNA and
possibly by preventing or diverting the normal process-
ing of GnRHR or enhancing GnRHR degradation
[118].
Finally, it is also possible that differences in
response may be ascribed to the intrinsic properties of
the cells. The physiological characteristics of the
human cancer cell lines mentioned in this minireview
are summarized in Table 2. For example, in contrast
to SKOV-3 cells, OVCAR-3 cells have low invasive
potential. Thus, whereas low doses of GnRH-I and
GnRH-II can exert a significant invasive effect in
OVCAR-3 cells, they fail to stimulate SKOV-3 maxi-
mally [12,13]. Both GnRH-I and GnRH-II only exert
inhibitory effects on SKOV-3 cells at high doses [13].
Novel receptor(s) for GnRH in humans?
An important issue that remains unresolved in this
field is whether one or more other GnRHR subtypes
exist in humans. The discovery of GnRH-II has stimu-
lated the search for a cognate type II GnRHR. Molec-
ular cloning of the type II GnRHR in goldfish,
marmoset and monkey has shown that the type II
receptor is structurally and functionally distinct from
the type I receptor [138–140]. In humans, a type II
GnRHR has not been found. However, a search of the
human genome database has revealed a putative
type II GnRHR gene on chromosome 1q21.1
[140,141]. Expression of this type II GnRHR mRNA

has been shown in many human tissues, including
endometrium, ovary, placenta, and prostate cancer
cells [42,55,140–142]. Although these findings suggest
that the human type II receptor gene is transcription-
ally active, the mRNA is disrupted by a frameshift in
coding exon 1 and a premature stop codon in exon 2,
suggesting that a conventional seven-transmembrane
receptor cannot be translated from this gene. The gene
also overlaps two flanking genes and displays alterna-
tive splicing [143]. Thus, the functionality of these
human type II GnRHR splice variants and their
involvement in transmitting signals from GnRH-II are
still in question.
One noteworthy feature of the primate type II
GnRHR is that, unlike the type I receptor, it possesses
a C-terminal tail, which is responsible for the recep-
tor’s susceptibility to rapid desensitization and inter-
nalization [138,144,145]. Finch et al. showed that
GnRH was able to efficiently inhibit the proliferation
of breast cancer cells when engineered with sheep
type I GnRHR, but not with Xenopus type II GnRHR
[145]. This clearly implies that the antiproliferative
effect of GnRH is mediated most efficiently by a recep-
tor that is not rapidly desensitized or internalized.
There is evidence that GnRH-II may act through the
type I GnRHR. In monkey pituitary cultures, in which
the type II GnRHR is functional, GnRH-II has been
found to stimulate gonadotropin secretion exclusively
through the type I GnRHR [146]. In contrast, other
evidence suggests that the neuromodulatory action of

GnRH-II on mammalian behavior is not mediated via
the type I receptor in musk shrews [147]. Thus, it
appears that GnRH-II may selectively interact with
different GnRHRs to mediate its different actions, pre-
sumably due to the structural differences between the
two GnRHR subtypes.
Alternatively, it is possible that the human type II
GnRHR may be encoded by a different gene that has
yet to be identified. Database searches have revealed
the presence of more than two other GnRHR genes in
the human genome apart from the conventional type I
receptor gene [148]. These genes are located on sepa-
rate chromosomes. Whether functional, full-length
transcripts can be produced from these receptor-like
genes remains to be determined. Recently, a novel
GnRH-II-binding protein, in addition to a conven-
tional GnRHR, has been identified by using photo-
affinity labeling with an azidobenzoyl-conjugated
GnRH-II in prostate cancer cells [149]. Taken together,
these observations thus suggest the potential existence
of novel receptors for GnRH-I and GnRH-II.
Concluding remarks
This overview shows that GnRH modulates a variety
of cellular functions in extrapituitary tissues, such as
cell growth, invasion, and angiogenesis. However, the
effects of GnRH are complex and appear to be cell
context dependent. The ability of GnRH to elicit very
different, even opposite (positive and negative),
responses in extrapituitary tissues may arise from dif-
ferential usage of signal transduction pathways and

receptor cross-talk. Clearly, further studies are
required to unravel this complex signaling network
and the coordinated regulatory roles of different
factors in specific cellular events during tumorigenesis.
High-throughput gene profiling and bioinformatics
approaches should be helpful to expand this area of
research. The information may also serve as a basis
for investigators in the field to explore the signaling
mechanisms of other G-protein-coupled receptors.
Most studies thus far have only been conducted in
cellular models, but in vivo approaches will be essential
for a complete understanding of the specific role of
each GnRH isoform, including the putative GnRH-III
isolated from the human brain [150]. Given the clinical
GnRH receptor signaling L. W. T. Cheung and A. S. T. Wong
5488 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS
utility of GnRH analogs in hormone-dependent dis-
eases, better characterization of GnRH actions and its
key molecular effectors is a necessary first step to more
effective and perhaps new therapeutic strategies for
improving clinical outcome.
Acknowledgements
This work was supported by the Hong Kong Research
Grant Council grant 778108 to A. S. T. Wong.
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