Gonadotropin-releasing hormone - Reproduction

Page 1 of 34 Reproduction Advance Publication first posted on 18 February 2009 as Manuscript REP-08-0397

Gonadotropin-releasing hormone (GnRH) signaling in intrauterine tissues

Hsien-Ming Wu1,2,3, Hsin-Shih Wang2,3, Hong-Yuan Huang2 ,Yung-Kuei Soong2,Colin D. MacCalman1 and Peter CK Leung1

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Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H3V5 2

Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital Linkou Medical Center, Taoyuan, Taiwan R.O.C. 3

Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan R.O.C.

Correspondence should be addressed to Peter CK Leung at Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H3V5 Fax no. : 1-604-8752725 E-mail address : [email protected]

Running head : GnRH signaling in intrauterine tissues.

Copyright © 2009 by the Society for Reproduction and Fertility.

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Abstract Type I GnRH (GnRH-I) and type II GnRH (GnRH-II), each encoded by separate genes, have been identified in humans. The tissue distribution and functional regulation of GnRH-I and GnRH-II clearly differ despite their comparable cDNA and genomic structures. These hormones exert their effects by binding to cell surface transmembrane G protein coupled receptors (GPCR) and stimulating the Gq/11 subfamily of G proteins. The hypothalamus and pituitary are the main origin and target sites of GnRH, but numerous studies have demonstrated that extra-hypothalamic GnRH and extra-pituitary GnRH receptors exist in different reproductive tissues such as the ovary, endometrium, placenta, and endometrial cancer cells. In addition to endocrine regulation, GnRH is also known to act in an autocrine and paracrine manner to suppress cell proliferation and activate apoptosis in the endometrium and endometrial cancer cells through several mechanisms. Both GnRH-I and GnRH-II exhibit regulatory roles in tissue remodelling during embryo implantation and placentation, which suggests that these hormones may have important roles in embryo implantation and early pregnancy. The presence of varied GnRH and GnRH receptor systems demonstrates their different roles in distinct tissues using dissimilar mechanisms. These may result in the generation of new GnRH analogues used for several hormone-related diseases.

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Introduction

Gonadotropin-releasing hormone (GnRH) , a hypothalamic neuronal secretory decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), is essential for human reproduction. The GnRH is processed in specialized hypothalamic neurons and is released in a pulsatile manner into the hypothalamo-hypophyseal portal circulation before being transported to the gonadotrope cells in the anterior pituitary. The GnRH stimulates the biosynthesis and secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from pituitary gonadotropes after binding to its cognate receptor (GnRH receptor). Both LH and FSH are released in asynchronous pattern of responses to changing GnRH pulse frequency, and in turn regulates gonadal steroidgenesis and gametogenesis(Fink 1988). Since its discovery, approximately one thousand GnRH analogues have been identified and widely studied (Cheung, et al. 2006; Conn and Crowley 1994). Besides its well-known endocrine function, GnRH may directly regulate some extrapituitary reproductive tissues such as endometrium, ovary, and placenta(Chou, et al. 2003; Grundker, et al. 2004; Islami, et al. 2001; Kim, et al. 2006). Recent studies revealed that both GnRH and GnRH receptors are expressed in the human endometrium and endometrial cancer. Functional studies have also demonstrated that GnRH regulates cell proliferation, apoptosis and tissue

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remodeling. Autocrine or paracrine regulation results in varying responses depending on physiological conditions and the endometrial tissue. Consequently, the extra-pituitary roles of GnRH have attracted interest in the fields of reproductive biology, clinical reproductive medicine and tumor biology. Here, we will summarize the scientific literature regarding the extra-pituitary GnRH and GnRH receptor system in endometrium and endometrial cancer.

Applications of GnRH analogues to uterine related diseases

. . GnRH analogues are used in the treatment of many hormone-related diseases, uterine related diseases especially(Table 1). Prolonged use of GnRH analogue and tibolone as add-back therapy acted efficiently for the treatment of endometrial hyperplasia(Agorastos, et al. 2004). GnRH analogues seem to be suitable drugs for an efficacious and less toxic endocrine therapy for endometrial cancer(Fister, et al. 2007). The effect of GnRH analogues on cervical cancer risk is difficult to predict, but the reduced steroid concentrations are likely to result in less proliferation of the cervix than normal ovulatory cycles and hence reduces the risk of cervical cancer(Pike and Spicer 2000). Desensitization and thereby reduction of steroid hormones are widely used in clinical medicine to treat many hormone-related diseases (Emons and Schally

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1994; Millar, et al. 1987; Moghissi 1992). Non-peptide orally-active GnRH antagonists are possible substitutes for agonists as they avoid the undesirable stimulation that leads to desensitization (Millar, et al. 2000). The GnRH peptide antagonists compete with endogenous GnRH, and then inhibit the reproductive system (Griesinger, et al. 2006). The numerous clinical applications of GnRH analogues in uterine related diseases have prompted many studies of cellular and molecular hormone function which have increased the understanding of the hormone regulation system and optimal applications.

GnRH isoforms in humans

Because it was first isolated and sequenced in mammals, hypothalamic GnRH is often referred to as type I mammalian GnRH (mGnRH) or GnRH-I (Millar, et al. 2004). GnRH-I is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) that has a key role in the process of reproduction. It is produced by hypothalamic neurosecretory cells and released in a pulsatile manner into the hypothalamo-hypophyseal portal circulation, through which the hormone is transported to the anterior pituitary gland(Cheng and Leung 2005). The human GnRH-I gene is composed of four exons separated by three introns and is present as a

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single gene copy on chromosome 8p11.2-p21(Radovick, et al. 1990; Yang-Feng, et al. 1986). The function for GnRH has been highly conserved in vertebrate evolution regardless of the variation of its amino acids sequence(Kasten, et al. 1996). The second type, GnRH-II or midbrain GnRH, is expressed in the midbrain, hippocampus and discrete nuclei of the hypothalamus. GnRH-II differs from GnRH-I by three amino acid residues at positions 5, 7, and 8 (His5Trp7Tyr8GnRH-I) and is conserved from primitive fish to humans(Millar 2003; White, et al. 1998). The human GnRH-II gene has been cloned and mapped to chromosome 20p13 by fluorescence in situ hybridization. GnRH-II also comprises four exons interrupted by three introns, and the predicted GnRH-II preprohormone is organized identically to the GnRH-I precursor. The GnRH-II hormone is expressed at significantly higher levels in extra-brain tissues such as the reproductive tissues (White et al. 1998). Researchers have made substantial effort to elucidate the roles of varying GnRH-II expression in the human reproductive system.

GnRH receptors

The GnRH receptor is a member of the rhodopsin-like G protein-coupled receptor (GPCR) superfamily, and contains a characteristic seven-transmembrane (TM)-domain

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structure(Cheng and Leung 2005; Cui, et al. 2000). However, a unique feature of the GnRH receptor is its lack of a carboxy-terminal cytoplasmic tail, which is known to contribute to various aspects of GPCR regulation by desensitizing and internalizing a network of receptor-associated proteins (Bockaert, et al. 2003). The classical GPCR internalization pathway involves GPCR kinases, β-arrestin, clathrin-coated pits, and the GTPase dynamin(Millar et al. 2004). The finding of GnRH-II in humans and cloning of its receptor from fishes, amphibians and primates have increased the likelihood of this receptor in humans (Neill, et al. 2001). Primate studies reveal that GnRH-II receptor complementary DNA encodes a 379-amino acid G protein-coupled/seven-transmembrane (TM) receptor having a C-terminal cytoplasmic tail not found in the GnRH-I receptor. The existence of a C-terminal tail is characteristic of GPCR, which modulates receptor desensitization following serial ligand stimulation. This phenomenon implies that these receptors are coupled to different signal transduction pathways and may play distinct roles in reproductive functions. The GnRH-II receptor is shown to be highly selective for GnRH-II and is widely expressed in reproductive tissues and in the central nervous system(Millar 2003). In addition, GnRH-II receptor is expressed in the majority of gonadotropes suggesting its role in the regulation of gonadotropin secretion.

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Binding of GnRH to GnRH receptor

The binding interaction between the ligand and the receptor is a basic mechanism of receptor function. The GnRH and GnRH receptor interaction initiates intracellular signaling through the receptor. Ligand binding is the first step in receptor activation or receptor-mediated transduction of hormone signals across the cell membrane. The GnRH and GnRH receptor interaction was elucidated by analyzing the molecular mechanisms of receptor conformation and receptor activation. The active conformation is associated with a triple network of ligand, receptor and G-protein. This system has one initial binding step common to both agonist and antagonist and one transition step leading to formation of the ternary network. This system also exhibits formation of receptor-G-protein complex with a high affinity for agonistic ligands. Ligand-stabilised and ligand-induced conformation changes are required for receptor activation. Studies of the GnRH receptor have improved understanding of the molecular mechanism of receptor activation. Several GnRH receptor active conformations are specific for GnRH analogues and intracellular signaling pathways(Millar et al. 2004).

Interaction of GnRH and GnRH-R in reproductive tissues

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Expression of GnRH mRNA has been demonstrated in human reproductive tissues, including human endometrium. The GnRH receptor mRNA has also been recognized in human ovary (Kang, et al. 2000; Raga, et al. 1999). Two homologous genes for GnRH-II receptor are present in the human genome, one on chromosome 14 and the second on chromosome 1. The chromosome 14 gene lacks exon 1, required to encode a full-length receptor. In comparison, the chromosome 1 gene contains all three exons. The exon 1-containing transcripts of GnRH-II receptor have been identified in mature human sperm. This suggests a relationship between GnRH and male reproduction such as spermatogenesis, sperm maturation and fertilization (van Biljon, et al. 2002). The growing evidence of endogenous GnRH and GnRH receptors in reproductive tissues has prompted researchers to examine autocrine and paracrine mechanisms in reproductive tissues(Hsueh and Jones 1981). The GnRH receptor mRNA is expressed by both normal and neoplastic endometrial cells, including those derived from stromal and ectopic endometrial tissues. Two classes of GnRH-I binding sites are found in endometrial carcinoma cell lines, but only one class of high-affinity binding sites can be detected in endometrial cancer cells and normal endometrial tissue. Sequence analysis reveals no mutations or alternative splicing patterns throughout the entire coding region of endometrial GnRH receptor

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RNA transcripts. (Borroni, et al. 2000; Grundker, et al. 2001b).

Ligand receptor-mediated intracellular signal transduction in pituitary and extra-pituitary tissues

Guanyl nucleotide binding protein (G-protein) has an important role in GnRH-mediated action. Increased GTP induces inositol phosphate production with a resultant decrease in GnRH binding affinity to GnRH receptor(Perrin, et al. 1989). The Gq/11α is the major G-protein coupled to GnRH receptor. In human female reproductive tissues and cells, GnRH receptor is coupled to a 41 kDa Giα protein. The G-proteins have a general sequence for palmitoylation, and receptor-mediated palmitoylation of G-proteins is a common specific phenomenon acting in a time- and dose-dependent manner (Stanislaus, et al. 1998). The second and third intracellular loops are important for receptor-G protein interaction and signaling functions in GPCRs. Differences in levels of GnRH receptor affect the GnRH-stimulated production of inositol phosphate within cells and contribute to the cell type-specific effects of GnRH(Morgan, et al. 2008). A specific alanine residue is involved in certain GPCRs, the equivalent of which is Ala-261 in the GnRH receptor. Ala on the human GnRH receptor cannot be involved in ligand binding but is critical for coupling of the

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receptor to its cognate G protein. Mutation of Ala to Pro or Val partially uncouples G protein whereas Lys, Leu, Glu or Ile substitutions completely block GnRH-mediated IP production. This suggests that GnRH receptor can couple to multiple G proteins at the second and third intracellular loops. For instance, GnRH agonists have poor activation of Gq, and some antagonists can activate Gi. Therefore, the activation of G-proteins in different cells induces several intracellular signaling pathway through GnRH and GnRH receptor. This pathway is also affected by the ligands, indicating ligand receptor-dependent signaling(Chabre, et al. 1994; Myburgh, et al. 1998). Intracellular transmission of extracellular signals is partially activated by some groups of sequentially activated protein kinase such as MAPK cascade (Fig. 1). The MAPKs play an important role in GPCR-mediated intracellular signaling (Luttrell 2002). The GnRH receptor activates MAPK cascades, including ERK1/2, c-Jun amino-terminal kinase (JNK), p38 MAPK and big MAPK (BMK1/ERK5) to different levels via a tyrosine kinase-, Ca2+- and PKC-dependent mechanism (Johnson and Lapadat 2002). The ERK is the key protein kinase in the signaling of growth factors. The ERK1/2 controls the intracellular signaling pathway which regulates the cellular growth and differentiation. The activation of ERK1/2 is mainly PKC-dependent and involves two different pathways that meet at Raf-1. Constitutive localization of the GnRH receptor to low-density membrane microdomains is necessary for GnRH

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signaling to ERK(Navratil, et al. 2003). The GnRH activates ERK1/2 by phosphorylating Sos and Shc through a Gi-protein coupled pathway. The GnRH receptor can couple with either Gq/11- or Gi/o-mediated activation of the MAPK cascade and activate the MAPK cascade by mechanisms similar to that of other GPCRs (Kimura, et al. 1999).The actual mechanisms of MAPK cascade activation by GnRH receptor and the regulation of intracellular loops in coupling to MAPK cascades, however, is still undetermined. Activation of JNK is highly dependent on cytosolic Ca2+ and is regulated through the pathway by serial stimulation of PKC, c-Src, CDC42/Rac1 and MAPK kinase (MEK)K1 (Mulvaney and Roberson 2000; Weston and Davis 2007). Although JNK may affect the survival, proliferation, apoptosis and invasion of cancer cells, its physiological and genetic mechanisms are not well understood. Activated p38 MAPK is involved in the PKC-dependent cascade. The p38 MAPK kinase participates in the activation of biological effects in cell cultures in response to varying stimuli. These effects depend on the particular stimuli and cell types. The p38 MAPK kinase is known to induce apoptosis in some cells but prevent apoptosis in others. Similarly opposing effects of the kinase have been observed in cell cycle regulation (Bradham and McClay 2006). The GnRH-I receptor in extra-pituitary tissues resembles that in the pituitary gonadotrophs, but GnRH signaling in extra-pituitary tissues and tumors may differ from those in the pituitary gonadotrophs.

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In extra-pituitary cells, activation of MAPK cascades by the GnRH receptor has been investigated. The intracellular mechanisms seemed to involve transactivation of the epidermal growth factor (EGF) receptor (Grundker, et al. 2002b; Shah, et al. 2003). The GnRH analogues reduce the expression of growth factor receptors and growth factor-induced tyrosine kinase activity. Growth factor-induced tyrosine phosphorylation is likely neutralized by GnRH analogues via activation of the phosphotyrosine phosphatase (PTP), which is likely coupled to the GnRH receptor through a G-proteinαi in human gynecological tumors (Imai, et al. 1996). In endometrial and ovarian cancer cells, the GnRH receptor stimulates the PTP, which neutralizes EGF-induced tyrosine phosphorylation of the EGF receptor with a resultant downregulation of mitogenic signal transduction and cell growth. However, this phenomenon can be negated by pertussis toxin, which suggest the the involvement of pertussis toxin-sensitive G-proteinαi in GnRH-induced PTP activity (Grundker et al. 2001b). However, whether GnRH signaling in extra-pituitary tissues differs from that in pituitary gonadotroph is still unresolved. Differing GnRH signaling between pituitary gonadotrophs and extra-pituitary tissues may result from varied coupling to G-proteins. In human endometrial cancer cells, GnRH agonist activates the activator protein-1 (AP-1) mediated through the pertussis toxin-sensitive G-proteinαi. Further, GnRH agonist activates AP-1 by simulating JNK (Grundker, et al. 2001a) (Fig. 1). The

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GnRH agonist suppresses growth factor-induced MAPK activity and does not activate PLC and PKC in endometrial cancer cells (Grundker et al. 2002b). Therefore, PKC and MAPK may not be involved in the GnRH agonist-induced activation of the JNK/AP-1 pathway. Further studies are needed to elucidate GnRH agonist-induced AP-1 activity related to the antiproliferative action of GnRH analogues through the control of cell cycle. The c-fos proto-oncogene is a key regulator of normal cell growth and differentiation. Extracellular stimuli, including several mitogens and steroid hormones, rapidly induce a c-fos response (Kovacs 1998). Transcriptional regulation of c-fos partly depends on the interactions of nuclear proteins with multiple cis-elements in the c-fos gene promoter. Serum response element (SRE) is one of the cis-elements. The SRE is essential for c-fos induction, which activates MAPK pathways by extracellular stimuli (Karin 1994). Several studies have shown that estrogen receptorα (ERα) mediates 17β -estradiol (E2)-activated expression of c-fos, which is induced as an immediate early-response gene in ERα-positive cancer cell line such as endometrial cancer (Bonapace, et al. 1996; Duan, et al. 1998). The GnRH agonists counteract EGF-induced proliferation and c-fos gene expression via Ras/MAPK signaling. The transcriptional activation of SRE by E2 is due to ERα activation of the MAPK pathways. The pathway is blocked by GnRH with a resulting reduction of E2-induced

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SRE activation and consequent reduction of E2-induced c-fos expression. This activity then supresses E2-induced cell proliferation in endometrial cancer (Grundker et al. 2004).

GnRH and apoptosis

Studies of rodent ovarian granulosa cells reveal that GnRH-I analogues induce apoptosis (Yano, et al. 1997). Continuous treatment of corpus luteum with a GnRH-I analogue induces apoptosis in vivo, however, the underlying mechanism of the proapoptotic effect of the GnRH remains unresolved. In cancer cells, the role of GnRH-stimulated apoptosis is still uncertain. GnRH treatment in vitro induces a dose-dependent stimulation of Fas ligand expression in several reproductive cancer cell lines and cells. Fas is usually expressed in GnRH receptor-positive tumors, which suggests that GnRH is an autocrine proapoptotic factor in Fas-positive tumors and that this proapoptotic property may partially reflect its antitumor effect (Imai, et al. 1998). The GnRH-I analogues have two possible counteracting effects, antiapoptotic and antiproliferative, mediated by two different signalling cascades but triggered by the same Gα protein in some ovarian cancer

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cells(Cheung et al. 2006). In the uterus, GnRH-I analogues in vivo or in vitro can suppress myometrium cell growth by activating apoptosis (Wang, et al. 2002). Conversely, GnRH-I analogue can suppress the expression of some proapoptotic factors while upregulating the in vivo antiapoptotic effects of Bcl-2 protein (Huang, et al. 2002). Numerous studies consistently show that GnRH-I analogues can induce apoptotic cell death in endometriotic cells in vitro (Bilotas, et al. 2007; Meresman, et al. 2003)(Table 2). Different responses to GnRH analogues occur in some cell lines. The possibility is that differences in the levels of GnRH receptor expression exist during cell passage in vitro or as a result of varied culture conditions. Consequently, differences in levels of GnRH receptor and signaling pathway individually contribute to the induction of apoptosis and play an important role in the regulation of GnRH on cell type-specific growth (Morgan et al. 2008)

Endometrial cancer

Endometrial cancer is one of the most common gynecological cancer in the world and each year accounts for approximately 50,000 deaths worldwide. The disease occurs primarily in postmenopausal women although 25% of patients are premenopausal, and 5% are younger than 40 years old (Parkin, et al. 2005). Prolonged

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exposure to endogenous or exogenous estrogens is one of the risk factors for endometrial cancer. Adjuvant treatment with tamoxifen for breast cancer is associated with increased incidence of endometrial cancer. Medical treatment for endometrial cancer includes aromatase inhibitors, progestational agents, and tamoxifen. The effectiveness of aromatase inhibitors for treating endometrial cancer remains unclear, but some reports have documented a lower response rate than that for progestagens (Rose, et al. 2000). Tamoxifen, which clearly enhances survival and reduces recurrence in hormone-sensitive breast cancer, is a widely used therapeutic antiestrogenic agent in endometrial cancer. However, many reports reveal that tamoxifen may worsen the endometrial cancer related to ERβ expression and hormone-resistant phenotype (Wilder, et al. 2004). GnRH is an important molecule of the hypothalamus-pituitary-gonadal axis related to estrogen steroidogenesis in vertebrates. The GnRH agonists and antagonists targeting the hypothalamic-pituitary-ovarian axis may have an antiproliferative role in endometrial cancer(Jeyarajah, et al. 1996).

Endocrine effects of GnRH analogues on human endometrial cancer Most endometrial cancers depend on estrogen for development and growth. The GnRH analogues offer an alternative treatment for endometrial cancer through estrogen deprivation by inhibiting the hypothalamic-pituitary-ovarian axis and thereby

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inhibiting estrogen production(Kullander 1992). The GnRH analogues may be used for conservative treatment of endometrial cancer. Therefore, the cancer stage and histopathology result should be the main consideration for GnRH analogue treatment. More studies are necessary to elucidate the endocrine role of GnRH analogues in endometrial cancer.

Autocrine and paracrine effects of GnRH analogues on human endometrial cancer Specific low affinity and high capacity binding sites for GnRH analogues were described in studies of endometrial cancer almost 20 years ago, which demonstrates the specific binding of GnRH in membrane preparations from endometrial cancer (Srkalovic, et al. 1990). The GnRH agonist and antagonist exert time- and dose-dependent inhibitory effects on the proliferation of endometrial cancer cell lines. The GnRH may play an autocrine or paracrine regulatory role in the growth of reproductive tissue tumors. Many studies have clearly demonstrated the participation of GnRH in endometrial cancer. Researchers suggest that the autocrine regulatory system stimulates cancer development and growth through direct effects on the endometrium based on GnRH receptor expression and GnRH peptide generation in

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endometrial cancers (Eicke, et al. 2006; Furui, et al. 2002). A second GnRH system has been described in primates. The antiproliferative effects of GnRH-I or GnRH-II agonists have been demonstrated in endometrial cancer cell lines positively expressing GnRH-II receptor mRNA. The antiproliferation of GnRH-II receptor positive endometrial cancer cell lines is induced by GnRH-II in a time- and dose-dependent manner, and the antiproliferative effects are significantly greater than those of GnRH-I agonist (Grundker, et al. 2002a; Volker, et al. 2002). However, the underlying signal transduction mechanisms of GnRH-II system are still unknown. Recent studies reveal that the mitogenic effects of growth factors in endometrial cancer cell lines are counteracted by GnRH-II agonist , implying an interaction with the mitogenic signal transduction. The GnRH-II hormone may reduce EGF-induced tyrosine auto-phosphorylation of EGF-receptors by activating PTP, and EGF-induced activation of mitogen-activated protein kinase is obstructed in cells treated with GnRH-II. The EGF-induced expression of the immediate early gene c-fos is suppressed by GnRH-II treatment. The GnRH-II agonist still stimulates PTP and suppresses the EGF-induced mitogenic signal transduction following the knock-out of GnRH-I receptor expression, which suggests that the effects of GnRH-II may be independent of GnRH-I receptor (Eicke et al. 2006).

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GnRH and endometrium

Expression of GnRH mRNA has been demonstrated in human endometrium. The expression of GnRH mRNA isolated from endometrial cells reveals a dynamic pattern such as significantly elevated GnRH mRNA expression detected in the secretory phase of the menstrual cycle. Further, GnRH-I immunoreactivity has been demonstrated in various endometrial cells with the strongest staining observable in the luteal phase (Dong, et al. 1998; Raga, et al. 1998). The expression of GnRH-II has been explored in human endometrium throughout the entire reproductive cycle, and two splice variants of GnRH-II mRNA are expressed with the shorter transcript carrying a 21-bp deletion. Dynamic expression of GnRH-II has been demonstrated in all endometrial cell types with stronger immunoreactivity in the early and midsecretory phases, which is similar to GnRH-I and suggests that GnRH-II may have an important role in human embryo implantation (Cheon, et al. 2001). The GnRH-I and GnRH-II hormones are known to activate urokinase-type plasminogen activator in human extravillous cytotrophoblasts and decidual stroma cells in vitro, indicating that the hormones may have a possible role in regulating the proteolytic degradation of the extracellular matrix of the endometrial stroma, an essential process for decidualization and trophoblast invasion (Chou et al. 2003; Paria, et al. 2002). Taken together, clinical studies of GnRH-I and

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GnRH-II indicate that these hormones may have a critical role in autocrine and paracrine regulation in human embryo implantation and early pregnancy.

GnRH and endometriosis

Endometriosis is one of the most common diseases in women of reproductive age. The disease is characterized by the presence of endometrial tissue outside the uterine cavity. The prevalence of endometriosis is 6-10% in women in the general population and 20% in women who have undergone laparoscopy for infertility or pelvic pain (Eskenazi and Warner 1997). The pathogenetic mechanisms of endometriosis remain unclear. Endocrine, genetic, immune, and environmental factors are believed to participate in the pathogenesis of endometriosis. The hypothesis that peritoneal endometriosis is due to menstrual dissemination of endometrial tissue into the peritoneal cavity is currently the most widely accepted. Endometriosis is highly related to infertility, probably resulting from poor folliculogenesis, oogenesis, fertilization, embryo development and implantation. Endometriosis is known to be an estrogen-dependent disease. The development and maintenance of endometriosis mainly depends on deranged generation of estrogen from endometriotic stromal cells.

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Several proteins such as 17ß-hydroxysteroid dehydrogenase (17ß –HSD) type I, 3ß-HSD, P450 aromatase, steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage enzyme ( P450scc) required for synthesizing estrogen occur in endometriotic stromal cells (Tsai, et al. 2001). Long term administration of GnRH analogues is applied to obtain a condition equivalent to hypogonadotropic hypogonadism by downregulating pituitary GnRH receptors with a resulting suppression of gonadotropin production. Because of its unique functions, GnRH anlogues have been applied for medical treatment of many hormone-related diseases such as endometriosis. Further, some studies report that GnRH system may play a role of autocrine and paracrine regulation in endometriosis since GnRH and GnRH receptor mRNA have been detected in human eutopic and ectopic endometrium (Borroni et al. 2000; Imai, et al. 1994). Concerning the apoptosis in endometriosis induced by GnRH analogues, it was demonstrated that GnRH analogues may have a direct effect by enhancing the apoptotic index, decreasing the secretion of pro-mitogenic cytokines such as IL-1ß and VEGF, increasing the expression of the pro-apoptotic proteins Bax and FasL, decreasing the expression of the anti-apoptotic protein Bcl-2 and suppressing cell proliferation in endometriosis (Bilotas et al. 2007; Meresman et al. 2003). The GnRH-II peptide also has shown antiproliferative and anti-inflammatory effects in endometrial stromal cells. The decreased expression of GnRH II on eutopic

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and ectopic endometrium of women with endometriosis implies that endogenous cytostatic regulation of GnRH-II may be weakened in the development of endometriosis(Morimoto, et al. 2005).

Conclusion Estrogen deprivation induced by GnRH analogues is now considered a conservative treatment for some hormone-related diseases such as endometrial cancer and endometriosis through the effects of endocrine regulation. Additionally, direct inhibition of growth induced by GnRH analogues has also been demonstrated in vitro in endometrial cancer and endometriosis through different mechanisms. Different GnRH isoforms have been demonstrated in mammals. These may play important roles in reproductive and non-reproductive tissues since GnRH exists in varied tissues. The emergence of different GnRH systems in widely distinct tissues reveals the varied mechanisms of action and the likely resultant evolution of a new generation GnRH analogues. Specific GnRH analogues binding sites occur in hormone-related diseases such as endometrial cancer. The evolution and clinical evaluation of GnRH analogues may play a role in targeted chemotherapy with better efficacy and lower systemic toxicity than traditional chemotherapy. Future studies will elucidate the connection of GnRH-GnRH receptor system, histopathology- and other markers, as well as the roles

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of the various GnRH-GnRH receptor systems in endometrium, endometrial cancer.

Acknowledgment This work was supported by the Canadian Institutes for Health Research. PCKL is a Distinguished Scientist of the Child and Family Research Institute.

References Agorastos T, Vaitsi V, Paschopoulos M, Vakiani A, Zournatzi-Koiou V, Saravelos H, Kostopoulou E, Constantinidis T, Dinas K, Vavilis D, et al. 2004 Prolonged use of gonadotropin-releasing hormone agonist and tibolone as add-back therapy for the treatment of endometrial hyperplasia. Maturitas 48 125-132. Bilotas M, Baranao RI, Buquet R, Sueldo C, Tesone M & Meresman G 2007 Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls. Hum Reprod 22 644-653. Bockaert J, Marin P, Dumuis A & Fagni L 2003 The 'magic tail' of G protein-coupled receptors: an anchorage for functional protein networks. FEBS Lett 546 65-72. Bonapace IM, Addeo R, Altucci L, Cicatiello L, Bifulco M, Laezza C, Salzano S, Sica V, Bresciani F & Weisz A 1996 17 beta-Estradiol overcomes a G1 block induced by HMG-CoA reductase inhibitors and fosters cell cycle progression without inducing ERK-1 and -2 MAP kinases activation. Oncogene 12 753-763. Borroni R, Di Blasio AM, Gaffuri B, Santorsola R, Busacca M, Vigano P & Vignali M 2000 Expression of GnRH receptor gene in human ectopic endometrial cells and inhibition of their proliferation by leuprolide acetate. Mol Cell Endocrinol 159 37-43. Bradham C & McClay DR 2006 p38 MAPK in development and cancer. Cell Cycle 5 824-828. Chabre O, Conklin BR, Brandon S, Bourne HR & Limbird LE 1994 Coupling of the alpha 2A-adrenergic receptor to multiple G-proteins. A simple approach for estimating receptor-G-protein coupling efficiency in a transient expression system. J Biol Chem 269 5730-5734. Cheng CK & Leung PC 2005 Molecular biology of gonadotropin-releasing hormone 22

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(GnRH)-I, GnRH-II, and their receptors in humans. Endocr Rev 26 283-306. Cheon KW, Lee HS, Parhar IS & Kang IS 2001 Expression of the second isoform of gonadotrophin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol Hum Reprod 7 447-452. Cheung LW, Leung PC & Wong AS 2006 Gonadotropin-releasing hormone promotes ovarian cancer cell invasiveness through c-Jun NH2-terminal kinase-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9. Cancer Res 66 10902-10910. Chou CS, MacCalman CD & Leung PC 2003 Differential effects of gonadotropin-releasing hormone I and II on the urokinase-type plasminogen activator/plasminogen activator inhibitor system in human decidual stromal cells in vitro. J Clin Endocrinol Metab 88 3806-3815. Conn PM & Crowley WF, Jr. 1994 Gonadotropin-releasing hormone and its analogs. Annu Rev Med 45 391-405. Cui J, Smith RG, Mount GR, Lo JL, Yu J, Walsh TF, Singh SB, DeVita RJ, Goulet MT, Schaeffer JM, et al. 2000 Identification of Phe313 of the gonadotropin-releasing hormone (GnRH) receptor as a site critical for the binding of nonpeptide GnRH antagonists. Mol Endocrinol 14 671-681. Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G & Roberts JL 1998 Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue. Mol Hum Reprod 4 893-898. Duan R, Porter W & Safe S 1998 Estrogen-induced c-fos protooncogene expression in MCF-7 human breast cancer cells: role of estrogen receptor Sp1 complex formation. Endocrinology 139 1981-1990. Eicke N, Gunthert AR, Emons G & Grundker C 2006 GnRH-II agonist [D-Lys6]GnRH-II inhibits the EGF-induced mitogenic signal transduction in human endometrial and ovarian cancer cells. Int J Oncol 29 1223-1229. Emons G & Schally AV 1994 The use of luteinizing hormone releasing hormone agonists and antagonists in gynaecological cancers. Hum Reprod 9 1364-1379. Eskenazi B & Warner ML 1997 Epidemiology of endometriosis. Obstet Gynecol Clin North Am 24 235-258. Fink G 1988 Gonadotropin secretion and its control. New York: Raven Press. Fister S, Gunthert AR, Emons G & Grundker C 2007 Gonadotropin-releasing hormone type II antagonists induce apoptotic cell death in human endometrial and ovarian cancer cells in vitro and in vivo. Cancer Res 67 1750-1756. Furui T, Imai A & Tamaya T 2002 Intratumoral level of gonadotropin-releasing hormone in ovarian and endometrial cancers. Oncol Rep 9 349-352. Griesinger G, Diedrich K, Devroey P & Kolibianakis EM 2006 GnRH agonist for 23

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triggering final oocyte maturation in the GnRH antagonist ovarian hyperstimulation protocol: a systematic review and meta-analysis. Hum Reprod Update 12 159-168. Grundker C, Gunthert AR, Hellriegel M & Emons G 2004 Gonadotropin-releasing hormone (GnRH) agonist triptorelin inhibits estradiol-induced serum response element (SRE) activation and c-fos expression in human endometrial, ovarian and breast cancer cells. Eur J Endocrinol 151 619-628. Grundker C, Gunthert AR, Millar RP & Emons G 2002a Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. J Clin Endocrinol Metab 87 1427-1430. Grundker C, Gunthert AR, Westphalen S & Emons G 2002b Biology of the gonadotropin-releasing hormone system in gynecological cancers. Eur J Endocrinol 146 1-14. Grundker C, Schlotawa L, Viereck V & Emons G 2001a Protein kinase C-independent stimulation of activator protein-1 and c-Jun N-terminal kinase activity in human endometrial cancer cells by the LHRH agonist triptorelin. Eur J Endocrinol 145 651-658. Grundker C, Volker P & Emons G 2001b Antiproliferative signaling of luteinizing hormone-releasing hormone in human endometrial and ovarian cancer cells through G protein alpha(I)-mediated activation of phosphotyrosine phosphatase. Endocrinology 142 2369-2380. Hsueh AJ & Jones PB 1981 Extrapituitary actions of gonadotropin-releasing hormone. Endocr Rev 2 437-461. Huang SC, Tang MJ, Hsu KF, Cheng YM & Chou CY 2002 Fas and its ligand, caspases, and bcl-2 expression in gonadotropin-releasing hormone agonist-treated uterine leiomyoma. J Clin Endocrinol Metab 87 4580-4586. Imai A, Ohno T, Iida K, Fuseya T, Furui T & Tamaya T 1994 Presence of gonadotropin-releasing hormone receptor and its messenger ribonucleic acid in endometrial carcinoma and endometrium. Gynecol Oncol 55 144-148. Imai A, Takagi A, Horibe S, Takagi H & Tamaya T 1998 Evidence for tight coupling of gonadotropin-releasing hormone receptor to stimulated Fas ligand expression in reproductive tract tumors: possible mechanism for hormonal control of apoptotic cell death. J Clin Endocrinol Metab 83 427-431. Imai A, Takagi H, Horibe S, Fuseya T & Tamaya T 1996 Coupling of gonadotropin-releasing hormone receptor to Gi protein in human reproductive tract tumors. J Clin Endocrinol Metab 81 3249-3253. Islami D, Chardonnens D, Campana A & Bischof P 2001 Comparison of the effects of GnRH-I and GnRH-II on HCG synthesis and secretion by first trimester trophoblast. 24

Page 27 of 34

Mol Hum Reprod 7 3-9. Jeyarajah AR, Gallagher CJ, Blake PR, Oram DH, Dowsett M, Fisher C & Oliver RT 1996 Long-term follow-up of gonadotrophin-releasing hormone analog treatment for recurrent endometrial cancer. Gynecol Oncol 63 47-52. Johnson GL & Lapadat R 2002 Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298 1911-1912. Kang SK, Cheng KW, Nathwani PS, Choi KC & Leung PC 2000 Autocrine role of gonadotropin-releasing hormone and its receptor in ovarian cancer cell growth. Endocrine 13 297-304. Karin M 1994 Signal transduction from the cell surface to the nucleus through the phosphorylation of transcription factors. Curr Opin Cell Biol 6 415-424. Kasten TL, White SA, Norton TT, Bond CT, Adelman JP & Fernald RD 1996 Characterization of two new preproGnRH mRNAs in the tree shrew: first direct evidence for mesencephalic GnRH gene expression in a placental mammal. Gen Comp Endocrinol 104 7-19. Kim KY, Choi KC, Auersperg N & Leung PC 2006 Mechanism of gonadotropin-releasing hormone (GnRH)-I and -II-induced cell growth inhibition in ovarian cancer cells: role of the GnRH-I receptor and protein kinase C pathway. Endocr Relat Cancer 13 211-220. Kimura A, Ohmichi M, Kurachi H, Ikegami H, Hayakawa J, Tasaka K, Kanda Y, Nishio Y, Jikihara H, Matsuura N, et al. 1999 Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line. Cancer Res 59 5133-5142. Kovacs KJ 1998 c-Fos as a transcription factor: a stressful (re)view from a functional map. Neurochem Int 33 287-297. Kullander S 1992 Treatment of endometrial cancer with GnRH analogs. Recent Results Cancer Res 124 69-73. Luttrell LM 2002 Activation and targeting of mitogen-activated protein kinases by G-protein-coupled receptors. Can J Physiol Pharmacol 80 375-382. Meresman GF, Bilotas MA, Lombardi E, Tesone M, Sueldo C & Baranao RI 2003 Effect of GnRH analogues on apoptosis and release of interleukin-1beta and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod 18 1767-1771. Millar RP 2003 GnRH II and type II GnRH receptors. Trends Endocrinol Metab 14 35-43. Millar RP, King JA, Davidson JS & Milton RC 1987 Gonadotrophin-releasing hormone--diversity of functions and clinical applications. S Afr Med J 72 748-755. 25

Page 28 of 34

Millar RP, Lu ZL, Pawson AJ, Flanagan CA, Morgan K & Maudsley SR 2004 Gonadotropin-releasing hormone receptors. Endocr Rev 25 235-275. Millar RP, Zhu YF, Chen C & Struthers RS 2000 Progress towards the development of non-peptide orally-active gonadotropin-releasing hormone (GnRH) antagonists: therapeutic implications. Br Med Bull 56 761-772. Moghissi KS 1992 Clinical applications of gonadotropin-releasing hormones in reproductive disorders. Endocrinol Metab Clin North Am 21 125-140. Morgan K, Stewart AJ, Miller N, Mullen P, Muir M, Dodds M, Medda F, Harrison D, Langdon S & Millar RP 2008 Gonadotropin-releasing hormone receptor levels and cell context affect tumor cell responses to agonist in vitro and in vivo. Cancer Res 68 6331-6340. Morimoto C, Osuga Y, Yano T, Takemura Y, Harada M, Hirata T, Hirota Y, Yoshino O, Koga K, Kugu K, et al. 2005 GnRH II as a possible cytostatic regulator in the development of endometriosis. Hum Reprod 20 3212-3218. Mulvaney JM & Roberson MS 2000 Divergent signaling pathways requiring discrete calcium signals mediate concurrent activation of two mitogen-activated protein kinases by gonadotropin-releasing hormone. J Biol Chem 275 14182-14189. Myburgh DB, Millar RP & Hapgood JP 1998 Alanine-261 in intracellular loop III of the human gonadotropin-releasing hormone receptor is crucial for G-protein coupling and receptor internalization. Biochem J 331 ( Pt 3) 893-896. Navratil AM, Bliss SP, Berghorn KA, Haughian JM, Farmerie TA, Graham JK, Clay CM & Roberson MS 2003 Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK. J Biol Chem 278 31593-31602. Neill JD, Duck LW, Sellers JC & Musgrove LC 2001 A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun 282 1012-1018. Paria BC, Reese J, Das SK & Dey SK 2002 Deciphering the cross-talk of implantation: advances and challenges. Science 296 2185-2188. Parkin DM, Bray F, Ferlay J & Pisani P 2005 Global cancer statistics, 2002. CA Cancer J Clin 55 74-108. Perrin MH, Haas Y, Porter J, Rivier J & Vale W 1989 The gonadotropin-releasing hormone pituitary receptor interacts with a guanosine triphosphate-binding protein: differential effects of guanyl nucleotides on agonist and antagonist binding. Endocrinology 124 798-804. Pike MC & Spicer DV 2000 Hormonal contraception and chemoprevention of female cancers. Endocr Relat Cancer 7 73-83. Radovick S, Wondisford FE, Nakayama Y, Yamada M, Cutler GB, Jr. & Weintraub BD 26

Page 29 of 34

1990 Isolation and characterization of the human gonadotropin-releasing hormone gene in the hypothalamus and placenta. Mol Endocrinol 4 476-480. Raga F, Casan EM, Kruessel JS, Wen Y, Huang HY, Nezhat C & Polan ML 1998 Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod 59 661-669. Raga F, Casan EM, Wen Y, Huang HY, Bonilla-Musoles F & Polan ML 1999 Independent regulation of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-3 in human endometrial stromal cells by gonadotropin-releasing hormone: implications in early human implantation. J Clin Endocrinol Metab 84 636-642. Rose PG, Brunetto VL, VanLe L, Bell J, Walker JL & Lee RB 2000 A phase II trial of anastrozole in advanced recurrent or persistent endometrial carcinoma: a Gynecologic Oncology Group study. Gynecol Oncol 78 212-216. Shah BH, Soh JW & Catt KJ 2003 Dependence of gonadotropin-releasing hormone-induced neuronal MAPK signaling on epidermal growth factor receptor transactivation. J Biol Chem 278 2866-2875. Srkalovic G, Wittliff JL & Schally AV 1990 Detection and partial characterization of receptors for [D-Trp6]-luteinizing hormone-releasing hormone and epidermal growth factor in human endometrial carcinoma. Cancer Res 50 1841-1846. Stanislaus D, Ponder S, Ji TH & Conn PM 1998 Gonadotropin-releasing hormone receptor couples to multiple G proteins in rat gonadotrophs and in GGH3 cells: evidence from palmitoylation and overexpression of G proteins. Biol Reprod 59 579-586. Tsai SJ, Wu MH, Lin CC, Sun HS & Chen HM 2001 Regulation of steroidogenic acute regulatory protein expression and progesterone production in endometriotic stromal cells. J Clin Endocrinol Metab 86 5765-5773. van Biljon W, Wykes S, Scherer S, Krawetz SA & Hapgood J 2002 Type II gonadotropin-releasing hormone receptor transcripts in human sperm. Biol Reprod 67 1741-1749. Volker P, Grundker C, Schmidt O, Schulz KD & Emons G 2002 Expression of receptors for luteinizing hormone-releasing hormone in human ovarian and endometrial cancers: frequency, autoregulation, and correlation with direct antiproliferative activity of luteinizing hormone-releasing hormone analogues. Am J Obstet Gynecol 186 171-179. Wang Y, Matsuo H, Kurachi O & Maruo T 2002 Down-regulation of proliferation and up-regulation of apoptosis by gonadotropin-releasing hormone agonist in cultured uterine leiomyoma cells. Eur J Endocrinol 146 447-456. 27

Page 30 of 34

Weston CR & Davis RJ 2007 The JNK signal transduction pathway. Curr Opin Cell Biol 19 142-149. White RB, Eisen JA, Kasten TL & Fernald RD 1998 Second gene for gonadotropin-releasing hormone in humans. Proc Natl Acad Sci U S A 95 305-309. Wilder JL, Shajahan S, Khattar NH, Wilder DM, Yin J, Rushing RS, Beaven R, Kaetzel C, Ueland FR, van Nagell JR, et al. 2004 Tamoxifen-associated malignant endometrial tumors: pathologic features and expression of hormone receptors estrogen-alpha, estrogen-beta and progesterone; a case controlled study. Gynecol Oncol 92 553-558. Yang-Feng TL, Seeburg PH & Francke U 1986 Human luteinizing hormone-releasing hormone gene (LHRH) is located on short arm of chromosome 8 (region 8p11.2----p21). Somat Cell Mol Genet 12 95-100. Yano T, Yano N, Matsumi H, Morita Y, Tsutsumi O, Schally AV & Taketani Y 1997 Effect of luteinizing hormone-releasing hormone analogs on the rat ovarian follicle development. Horm Res 48 Suppl 3 35-41.

Legends Table 1. Application of GnRH analogues in uterine related diseases.

Table 2. Summary of extrapituitary effects of GnRH-I and GnRH-II in human endometrium and endometrial cancer

Fig. 1. GnRH-I and GnRH-II signaling in endometrial cancer cell: A) GnRH induces apoptosis through Gαi and activation of Fas ligand. B) GnRH-II induces apoptosis through Gαi and activation of p38 and AP-1. C) GnRH arrests cell

28

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cycle by activating JNK, increasing AP-1 activity and the binding of JunD-DNA. D) Gβγ subunit activates ERK and mediates GnRH-induced growth inhibition. E) Through Gαi, GnRH analogs activate PTP to dephosphorylate EGFR and abolish EGF-induced ERK activation, c-fos expression and proliferation. Dashed arrows represent EGF-stimulated mitogenic signaling pathway.

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Table 1. Application of GnRH analogues in uterine related diseases Benign tumors Endometrial hyperplasia Uterine leiomyoma Adenomyosis Endometriosis Malignant tumors Endometrial cancer Cervical cancer

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Table 2. Summary of extrapituitary effects of GnRH-I and GnRH-II in human endometrium and endometrial cancer Target site

Extrapituitary effect

Nature of GnRH-I

Reference

and/or GnRH-II

Native GnRH-I

Meresman et al. (2003) Meresman et al. (2003) Chou et al. (2003)

Native GnRH-II

Chou et al. (2003)

Leuprolide

Bilotas et al. (2007)

Native GnRH-II

Raga et al. (1998)

Inhibition of

Triptorelin/Native

Grundker et al. (2004) and

cancer

proliferation

GnRH-II

Grundker et al. (2002a)

Endometrial

Inhibition of

GnRH-II agonist

Eicke et al. (2006)

cancer

proliferation

[D-Lys6]GnRH-II

Endometriotic cell

Inhibition of

Leuprolide

proliferation Endometriotic cell

Activation of

Buserelin

apoptosis Decidual stromal

Activation of PAIa-1

cell

expression

Decidual stromal

Suppression of PAIa-1

cell

expression

Endometrial

Activation of

epithelial cell

apoptosis

Endometriotic

Inhibition of

stromal cell

proliferation

Endometrial

a

PAI, plasminogen activator inhibitor.

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Figure 1

GnRH-I / GnRH-II

EGF

GnRH-I receptor

GnRH-II receptor ?

Gαi β γ

Gαi

A

D

B Fas

p38

C

E

PTP

c-jun JunD

P

P P Sos Shc

JNK

AP-1

EGFR

MEK

ERK1/2

apoptosis

EGFR expression

AP-1 c-fos expression cell cycle

proliferation