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 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: awong1@hku.hk (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 factor receptors Intriguingly, the classical Gaq–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, progression, and dissemination The emerging role of GnRHR in actin cytoskeleton remodeling (small Rho GTPases), expression and ⁄ or activity of adhesion molecules (integrins), proteolytic enzymes (matrix metalloproteinases) and angiogenic factors is explored The signal transduction mechanisms 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 Introduction The hypothalamic gonadotropin-releasing hormone (GnRH) is a decapeptide that plays a critical role in the regulation of reproduction GnRH-I (pGlu-HisTrp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) is the first GnRH isoform discovered in mammalian brain Its major role is to stimulate pituitary secretion of gonadotropins, luteinizing hormone and follicle-stimulating hormone, which in turn stimulate the gonads for steroid production Subsequently, a second isoform 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 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 GnRH receptor signaling L W T Cheung and A S T Wong brain and is particularly abundant in the kidney, bone marrow, and prostate [2] This leads to the speculation that GnRH-II may have distinct physiological 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 gonadotropin 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 understood, but this is certainly an important topic for investigation which may offer an opportunity to elucidate 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 development [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 expression 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 5480 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 cancer 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 [125I][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 myometrium, most benign neoplasms studied thus far, including uterine leiomyoma, also possess GnRHR [17] Early studies showed that the human placenta contains 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 immunolocalized 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 immunoreactivity 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 transcripts and proteins of the same size as the pituitary FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong 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 affinity [with nanomolar dissociation constants (Kd)] for GnRH, and one has low affinity (with micromolar Kd 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-affinity 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 endometrial 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 indirect mechanism of action has provided the rationale for the use of GnRH analogs in the treatment of hormone-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 disruption of the cell cycle at G0 ⁄ G1 [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 replicationspecific cytotoxic agents and UV radiation [40] GnRH receptor signaling 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 significant inhibition of tumor growth by GnRH-I antagonists but not GnRH-I agonists [45] The advantage of GnRH antagonists over the agonistic peptides is probably due to the fact that they inhibit the secretion of gonadotropins and reduce sex steroid levels immediately 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 agonists 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 antiproliferative 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 androgen-sensitive LNCaP cells and androgen-resistant DU-145 cells remain sensitive to GnRH [49,50] Activation 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 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5481 GnRH receptor signaling L W T Cheung and A S T Wong breast cancer cells could be inhibited by both agonistic and antagonistic analogs of GnRH [57,58] However, higher efficiency of GnRH antagonists in growth inhibition 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 metastatic behavior of cancer cells This hypothesis is supported by studies showing that GnRH-I and GnRH-II can affect the expression of several extracellular matrix-degrading enzymes in human extravillous cytotrophoblasts 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 metalloproteinases (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 activity 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 cellular migration and invasion in a dose-dependent manner, high (micromolar) concentrations are not as efficient This proinvasive effect is mediated through activation of metastasis-related proteinases, in particular MMP-2 and MMP-9 [12] Moreover, GnRH-I is able to transactivate the MMP-2 and MMP-9 promoters, 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 regulation of ovarian cancer invasion [13] The finding that small interfering RNA-mediated downregulation of type I GnRHR completely reversed the effects of both 5482 GnRH-I and GnRH-II on cell invasion supports the view that the same receptor, type I GnRHR, is essential for the effects of GnRH-I and GnRH-II in ovarian cancer cells The decrease in uPA activity of cytosol from Dunning R3327H rat prostate tumors after treatment with GnRH-I analogs suggests that GnRH may be an important factor in reducing the invasiveness of prostate 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 modulating E-cadherin-mediated cell–cell contacts and production 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 polymerization [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 metastasis 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 chemotaxis, 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), platelet-derived growth factor and the angiopoietin family FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong 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 leiomyoma growth and vascularization has also been suggested [72] Considering that angiogenesis is an important process in many human cancers, it would be GnRH receptor signaling very interesting to determine whether GnRH also plays a key role in tumor angiogenesis Intracellular signal transduction Upon GnRH binding, GnRHR undergoes a conformational change and stimulates a unique G-protein Interestingly, the classical Gaq–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 Gai in these tumors and result in the activation of several downstream signaling cascades [73,74], such as mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), and nuclear factor kappa B (NF-jB) signaling The GnRH-induced signaling pathways in extrapituitary tissues are shown schematically in Fig Fig Schematic representation of GnRHR signaling in extrapituitary tissues Binding of GnRH to GnRHR triggers several intracellular signaling cascades and cross-talk with mitogenic signaling, depending on the cell context Some of these signaling modules can transduce extracellular 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 kappa 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 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5483 GnRH receptor signaling L W T Cheung and A S T Wong MAPK The major MAPK cascades include extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 MAPK Many studies have shown that the MAPK pathway is critical for GnRH activities, which provides an important link for the transmission 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 different levels via different mechanisms: (a) second messengers [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 independent 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 ⁄ 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 outcomes, is separated already at the upstream levels In the ovarian cancer cell line CaOV-3, prolonged stimulation of ERK1 ⁄ through Shc and son of sevenless is required for GnRH-I-mediated growth inhibition [76] Consistent with the fact that sustained activation of ERK1 ⁄ is often correlated with cell cycle progression, GnRH-I-induced growth inhibition is attributed to G1 arrest [76] Moreover, the signaling cascade was shown to be initiated by Gbc, 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 ⁄ 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, suggesting 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 ⁄ has been 5484 implicated in nerve growth factor-mediated neuronal differentiation of PC12 cells, whereas a rapid and transient activation is associated with growth factormediated proliferation of PC12 cells [80] Thus, the duration of kinase activation seems to be a major determinant of signal outcome We have shown differential 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 ⁄ 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 translocation, 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 translocation, which is required for GnRH-stimulated cell invasion The JNK pathway targets multiple transcription factors, including c-Jun, c-Fos, ATF and PEA, and putative binding sites for these DNA-binding proteins 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 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 ⁄ phosphorylation can be abolished in the presence of the EGFR inhibitor AG1478 [53,78] However, the biological importance of ERK1 ⁄ 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 counter EGFR-dependent adhesive signaling through a PKC-dependent mechanism [66] Activation of FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong 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 invasion 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 agonist 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 ovarian 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 pathways 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 regulation 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 Gai-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 independent of the cross-talk between GnRHR and growth factor signaling, as treatment with phosphatase inhibi- GnRH receptor signaling tor has no effect on the activation of NF-jB [94] It has also been shown that GnRH-I suppresses interleukin-8 expression via attenuation of tumor necrosis factor 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 (Pyk2) are the predominant 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), b3-integrindependent activation of FAK is associated with the inhibitory effects of GnRH-I and GnRH-II on growth [96] Leiomyoma regression induced by GnRH-I agonists 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 phosphorylation 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 lm) 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 SKOV3 cells [13] A similar difference has been found in the effects of GnRH on cell proliferation and cell migration in the prostate carcinoma cell lines TSU-Pr1 and DU-145 [67] Whereas GnRH-I and GnRH-II stimulated cell proliferation, induced actin cytoskeleton remodeling and promoted migration in TSU-Pr1 cells, they were inhibitory in DU-145 cells [67] The observation that GnRH-I and GnRH-II have no significant effect in cell lines with type I GnRHR depletion indicates that the type I GnRHR is indispensable for the FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5485 GnRH receptor signaling L W T Cheung and A S T Wong Table Potential mechanisms that underlie the diverse responses to GnRH Determinants References Treatment conditions, e.g duration and dose Different Ga subtypes Different G-protein subunits GnRHR expression level Ligand selectivity Presence of GnRHR splice variants Intrinsic cellular properties [99–101] [73,74,101,103,104] [105–109] [6,110–112] [114,115] [118,119] [61,62] 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 condition may be one determinant of the outcome The pulsatility of GnRH release is necessary for the hormone 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 pulsatile GnRH stimulates more sustained ERK activity (more than h), whereas continuous infusion of aT3-1 cells with GnRH stimulates short-term (2 h) ERK activity [100] There is also evidence that GnRH treatment stimulates cAMP production at nanomolar concentrations, 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 physiological functions of GnRH [54,102] Second, GnRH action has been shown to be mediated by coupling to different Ga-proteins, depending on the time and dose of exposure [101,103] In general, Gaq and Gas are associated with a stimulatory effect [103], whereas Gai often mediates the antiproliferative and proapoptotic effects of GnRH [73,74] Low GnRH concentrations promote the coupling of GnRHR to Gas [101] High concentrations of GnRH cause a switch in receptor coupling from Gas to Gai [101] Moreover, stimulation of cAMP production by GnRH is through coupling to Gas, whereas inhibition of cAMP production at high concentrations of GnRH is through coupling to Gai [101,104] These findings suggest that the intracellular milieu in different tissues results in differential coupling and different phenotypic effects 5486 Third, multiple GnRH-dependent signaling pathways 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 Gi 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 luteinizing 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 responding prostate carcinoma cell lines, GnRH-I is more effective than GnRH-II On the other hand, in negatively responding cell lines, GnRH-II is much more effective than GnRH-I Given the short plasma halflife 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 conformations and therefore different ligand-induced selective signaling pathways [114] In this regard, it has been shown that the highly variable amino acid at position of GnRH plays a discriminating role in selecting the receptor conformational state [115] The presence of splice variants of the GnRHR transcript may be another possible reason for the different or opposite responses to GnRH To date, variant transcripts 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 inhibitory activity on full-length GnRHR function [119] This inhibition is specific, augmented by increasing FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS Origin Ovary Prostate Endometrium Endometrium Prostate Breast Breast Ovary Ovary Prostate Cell line CaOV-3 DU-145 HEC-1A Ishikawa LNCaP MCF-7 MDA-MB-435 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS OVCAR-3 SKOV-3 TSU-Pr1 Pleural effusion Ascites Ascites Lymph node Primary carcinoma Primary carcinoma Lymph node Pleural effusion Primary carcinoma Brain Isolation Serous Serous Poorly differentiated Ductal Poorly differentiated Moderately differentiated Well differentiated Well differentiated Ductal Serous Original histology Yes Yes Yes Only achievable with estrogen added Yes Yes Yes Yes Yes Yes Tumorigenicity in nude mice Low High High Low in absence of estrogen High Very low High High Low High Anchorageindependent growth Low High High High Low Very low Low High High Low Invasive capability in vitro Effect of GnRH on motility ⁄ invasion › fl ? ? ? fl – › fl › Effect of GnRH on growth fl fl;– fl;– fl;– fl;– fl fl fl fl ›; – Table Physiological characteristics and responses to GnRH of common cancer cell lines fl, decrease; ›, increase; –, no effect; ?, undetermined [12,13,43,136,137] [13,43,125] [54,55,67,120,127] [68,121,133–135] [56,68,122–124,135] [49,120,127,128] [15,47,48,129–131] [15,30,36,48,129–131] [50,55,67,120,127,128,132] [12,76,125,126] References L W T Cheung and A S T Wong GnRH receptor signaling 5487 GnRH receptor signaling L W T Cheung and A S T Wong amounts of the cotransfected splice variant cDNA and possibly by preventing or diverting the normal processing 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 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 maximally [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 stimulated the search for a cognate type II GnRHR Molecular 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 transcriptionally active, the mRNA is disrupted by a frameshift in coding exon 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 alternative 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 receptor’s susceptibility to rapid desensitization and internalization [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 recep5488 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, presumably 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 separate 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 conventional GnRHR, has been identified by using photoaffinity 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 differential 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 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signaling in extrapituitary tissues Binding of GnRH to GnRHR triggers several intracellular signaling cascades and cross-talk with mitogenic signaling, depending on the cell... protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), and nuclear factor kappa B (NF-jB) signaling The GnRH- induced signaling pathways in extrapituitary tissues are shown schematically in. .. 84, 285–300 GnRH receptor signaling 99 Kaiser UB, Conn PM & Chin WW (1997) Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor- expressing pituitary cell lines Endocr Rev