Endometriosis: disease pathophysiology and the role of prostaglandins

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1 Endometriosis: disease pathophysiology and the role of prostaglandins Meng-Hsing Wu 1, Yutaka Shoji 2, Pei-Chin Chuang 2 and Shaw-Jenq Tsai 2, * Endometriosis is considered to be a polygenic disease with a complex, multifactorial aetiology that affects about 10% of women in the reproductive age. Women with endometriosis have symptoms that include chronic pelvic pain, dysmenorrhoea and dyspareunia, significantly reducing their quality of life. Endometriosis is also the primary cause of infertility in women, with the prevalence rate ranging from 20% to 50%. The high prevalence and severe outcomes of this disease have made it a major public health concern in modern society. Currently, the mechanism(s) responsible for the initiation and promotion of this disease remains obscure. In this review, we focus on the expression, regulation and action of prostaglandins in the cellular and molecular mechanisms that contribute to the development and/or maintenance of endometriosis. Endometriosis is a common gynaecological disease in women of reproductive age. It is an enigmatic disorder defined as the presence of endometria-like tissues outside of the uterine cavity (ectopic lesions). Endometriosis is diagnosed in 6 10% of women in the general population and in 20% of women who have undergone laparoscopy for pelvic pain or infertility (Ref. 1). The prevalence rate of endometriosis proven by laparoscopy in patients with chronic pelvic pain can be as high as 28% (Ref. 2). The most common symptoms for women who have endometriosis are pelvic pain and infertility; both adversely affect the quality of life. The pregnancy rate in women with endometriosis is about half of women with tubal factor infertility (Ref. 3) and is negatively correlated with the severity of disease. The cause of reproductive failure may be due to poor oocyte development, implantation or embryogenesis. In addition to infertility, a strong cause effect relationship between endometriosis and pelvic pain is commonly observed. 1 Department of Obstetrics and Gynecology, National Cheng Kung University Medical College, Tainan 701, Taiwan, Republic of China. 2 Department of Physiology, National Cheng Kung University Medical College, Tainan 701, Taiwan, Republic of China. *Corresponding author: Shaw-Jenq Tsai, Department of Physiology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan, Republic of China. Tel: (ext. 5426); Fax: ; seantsai@mail.ncku.edu.tw 1

2 Dysmenorrhoea is associated with cyclic recurrent microbleeding within various entities of ectopic endometriotic implants and consequent inflammation (Ref. 4). Endometriosis-related adhesions and compression or infiltration of nerves in the subperitoneal pelvic space by ectopic lesions also cause painful symptoms (Ref. 5). A direct relationship between the severity of dysmenorrhoea and the production of prostaglandins (PGs) in tissue has been observed in endometriosis (Ref. 6). Current medical therapies for endometriosis include the use of combination oral contraceptives, danazol, gonadotropin-releasing hormone (GnRH) analogues, progestins and nonsteroidal antiinflammatory drugs. Most target the suppression of PG levels and thus reduce endometriosis-associated pain (Ref. 7). However, no effective therapeutic regimen has been developed thus far to cure or prevent the recurrence of endometriosis, although there are some novel therapeutic approaches currently under investigation (Ref. 8). Although the aetiology of endometriosis is still poorly defined and several hypotheses have been proposed, Sampson s transplantation and implantation hypothesis published in 1927 is still the most widely accepted (Ref. 9). This hypothesis proposes that endometrial tissue fragments are spread by retrograde menstruation through fallopian tubes into the peritoneal cavity. The pattern of endometriosis supports the theory of retrograde menstruation and is most commonly located in the gravitationally dependent area of the pelvis, including the ovary, anterior and posterior cul-de-sac, uterosacral ligaments, and fallopian tubes (Ref. 10). However, this theory seems over-simplified to describe the disease process; additional factors that increase susceptibility to endometriosis must exist and remain to be identified. For example, the survival and implantation of ectopic endometrial cells must involve escape from apoptosis (Ref. 11), adherence to the peritoneum (Ref. 12), degradation of the underlying extracellular matrix (ECM) (Ref. 13), generation of neovascularity (Ref. 14), acquisition of steroidogenic capacity (Refs 15, 16) and evasion from the immune surveillance system (Ref. 17). Furthermore, biochemical differences in terms of gene expression profile demonstrated between paired eutopic endometrial (i.e. at its usual location) and ectopic endometriotic (i.e. at abnormal locations) tissues (Ref. 18) have raised an important issue regarding epigenetic control of gene expression and the development of endometriosis. Genetic and environmental factors are also important entities to be considered. Endometriosis is a multigenic disease with complex genetic traits (Ref. 10). Although many reports have claimed the identification of endometriosis-susceptible genes, most have failed the test of being confirmed by second or third groups. Recently, a region on chromosome 10q26 was shown to be an important region that highly associated with endometriosis by whole genome screening of affected paired sisters (Ref. 19). Although the candidate gene(s) has not been mapped and the finding needs to be confirmed by other studies, such an approach has shed light on the hunt for a disease-causing gene(s). Environmental factors, such as dioxin, might interact with multiple genetic susceptibility loci to produce the phenotype of endometriosis (Ref. 20). Most of these environmental contaminants exhibiting estrogenic effects will lead to endocrine disruption through various environmental media such as food and water. Dietary intake of dioxin-like compounds with biological activity will increase the body burden (the total amount of these chemicals that are present in the human body at a given point in time), which might contribute to the pathogenesis of endometriosis (Ref. 21). In addition, these chemicals can pass through the placenta to affect fetal environment (Ref. 22). In a prospective cohort study, the rate of laparoscopically confirmed endometriosis was 80% greater among women exposed in utero to diethylstilbestrol (a synthetic estrogen originally prescribed in pregnancy to prevent miscarriage) after 10 years of follow up (Ref. 23). However, the relationship between endocrine-disrupting chemicals and endometriosis remains controversial because of lack of studies with sufficient statistical power (Ref. 24). Notwithstanding the enigmatic aetiology, retrograded endometrial tissues must evolve a system that overcomes two pivotal problems encountered. The first is the escape from elimination by the body s defence system and the second is the development of a 2

3 self-supporting system that enables them to survive and/or proliferate in the hostile microenvironment of the peritoneal cavity. This review focuses on recent progress in dissecting the molecular and cellular mechanisms contributing to the development of endometriosis, with emphasis on how the retrograded endometrial tissue develops both a self-supporting system and an immunesuppressive mechanism. The particular role of PGs will be highlighted. Acquisition of steroidogenic capacity Endometriosis is an estrogen-dependent disorder. Aberrant production of estrogen by endometriotic stromal cells is indispensable for the development and maintenance of endometriosis especially during the period of menstruation when no ovarian estrogen is available (Refs 15, 25, 26, 27). This notion was supported by identification in endometriotic stromal cells of the presence of all proteins/ enzymes required for de novo synthesis of estrogen: steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3b-hydroxysteroid dehydrogenase (3b-HSD), 17a-hydroxylase 17,20 lyase, P450 aromatase and 17b-HSD type 1 (Refs 15, 25). Among these enzymes, StAR and aromatase control the first and last committed steps in the biosynthesis of estrogen. StAR transports cholesterol across the mitochondrial membrane to the inner mitochondrial leaflet, where the first enzymatic reaction occurs. Aromatase catalyses the conversion of androstenedione to estrone. Estrone is further converted to 17b-estradiol (normally referred to as estrogen) by 17b-HSD type 1, whereas 17b-HSD type 2 reverses this process. In disease-free uterine endometrium, no StAR or aromatase are detected but there are increased StAR and aromatase levels in extra-ovarian endometriotic implants and endometriomas (Refs 15, 25, 28, 29). In addition, the absence of 17b-HSD type 2 in pelvic endometriotic implants (Ref. 30) further favours an increase in the local concentration of estrogen. Role of prostaglandin E 2 (PGE 2 ) The mechanism responsible for aberrant expression of StAR and aromatase is not known but accumulated data support the view that PGE 2 plays an indispensable role. PGE 2 is a potent inducer of StAR and aromatase in endometriotic stromal cells (Refs 15, 27), which results in aberrant production of estrogen to support the survival and proliferation of ectopic endometriotic tissues. PGE 2 binds to G-protein-coupled plasma membrane receptors. Four distinct PGE 2 receptors (EP1, 2, 3 and 4), encoded by different genes, have been identified in human tissues (Ref. 31); EP1 and EP3 undergo alternative mrna splicing, generating different isoforms (Ref. 31). Binding of PGE 2 to EP2 or EP4 activates adenylyl cyclase and the protein kinase A (PKA) signalling pathway through G-protein activation. EP1 is a Gi-coupled receptor and its activation leads to an increase in the intracellular free Ca 2þ levels and/or PKA inhibition. Binding of PGE 2 to EP3 causes intracellular Ca 2þ mobilisation, activation of PKA, PKC and mitogen-activated protein kinase (MAPK), or inhibition of the PKA signalling pathway (Ref. 31). Thus, PGE 2 can activate several signalling pathways, depending on the specific EP receptor to which it binds. In human endometrial and endometriotic stroma, EP2, EP3 and EP4 are expressed, whereas the mrna of EP1 is undetectable (Ref. 32). It has been reported that PGE 2 regulates expression of the gene encoding StAR through a mechanism that requires PKA activation, cyclic AMP response element binding protein (CREB) phosphorylation, CREB-binding protein (CBP) recruitment and histone H3 acetylation (Ref. 32) (Fig. 1). This result suggests that PGE 2 -induced StAR expression is mediated by binding to the EP2 or EP4 receptor. Indeed, the specific EP2 agonist butaprost is able to mimic the effects of PGE 2, and pretreatment with EP2 antagonist abrogates PGE 2 -induced StAR expression. By contrast, administration of the EP3 agonist sulprostone or the EP4 agonist PGE1-OH exerts no or minimal effect on StAR expression, suggesting that the regulation of StAR by PGE 2 is mediated mainly by binding to the EP2 receptor (Ref. 32). Induction of StAR expression by PGE 2 is restricted to ectopic endometriotic stromal cells but not epithelial cells (Ref. 15). More importantly, stromal cells isolated from eutopic endometrium do not respond to PGE 2 treatment (Ref. 15). Therefore, ectopic endometriotic stromal cells undergo some sort of transition and become susceptible to PGE 2 treatment. 3

4 E 2 E 2 ER Nucleus Cholesterol STAR and CYP19 genes StAR, CYP19 camp Actions of prostaglandin E 2 (PGE 2 ) in estrogen and fibroblast growth factor (FGF)-9 production The cause of such transition is not known; however, a similar mechanism has also been reported in PGE 2 -induced aromatase expression in ectopic endometriotic stromal cells (Ref. 33). E 2 E 2 a AC CBP HAT EP2 γ β PKA P CREB HO O PGE 2 OH MEK FGF9 gene COOH Raf ERK Expert Reviews in Molecular Medicine C 2007 Cambridge University Press Ac Ac α s α q E 2 ER b EP3 γ β PKCδ PLCβ P ELK-1 Figure 1. Actions of prostaglandin E 2 (PGE 2 ) in estrogen and fibroblast growth factor (FGF)-9 production. PGE 2 can induce the expression of FGF-9 through two parallel pathways by binding to its receptors EP2 and EP3. (a) Binding of PGE 2 to EP2 receptor activates trimeric G-protein (a s, b, g), which leads to activation of adenylyl cyclase (AC) and production of cyclic adenosine monophosphate (camp). Elevation of camp triggers the activation of protein kinase A (PKA), which then translocates to the nucleus and phosphorylates camp response element binding protein (CREB). CREB presumably binds to the CRE site of the target gene, in this case those encoding steroidogenic acute regulatory protein (StAR) and aromatase (CYP19). Phosphorylation (P) of CREB by PKA recruits CREB-binding protein (CBP) to the chromatin to induce acetylation of histone through its histone acetyl transferase (HAT) domain. Acetylation (Ac) of histone results in an increase in STAR and CYP19 gene transcription, which ultimately leads to an increase of estrogen (E 2 ) production from cholesterol. E 2 then binds to its receptor (ER), a nuclear receptor that is also a transcription factor. The E 2 ER complex binds to the estrogen-response element of the FGF9 gene promoter and induces the transcription of FGF-9. (b) Alternatively, PGE 2 binds to a second receptor, EP3, and triggers the activation of Gq-protein (a q, b, g). This leads to activation of phospholipase Cb (PLCb). PLCb cleaves phosphoinositol 3,4-bisphosphate to form inositol trisphosphate and diacylglycerol (DAG). Activation of PKCd by DAG phosphorylates Raf and its downstream signalling cascade proteins, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK). Activation of ERK results in phosphorylation of the transcription factor ELK-1. Phosphorylated ELK-1 binds to the FGF9 gene promoter and induces transcription of FGF-9. DAG In the case of aromatase, aberrant expression of steroidogenic factor (SF)-1 in ectopic endometriotic stromal cells might contribute to the transition of these cells from 4

5 PGE 2 -insensitive to PGE 2 -sensitive status (Ref. 34). However, the mechanism of aberrant expression of SF-1 in ectopic endometriotic tissue remains unknown. Taken together, these data indicate that PGE 2 alone, through activation of the EP2 receptor, is sufficient to cause production of estrogen, a unique feature only observed in ectopic endometriotic stromal cells. By doing so, the ectopic endometriotic tissue can avoid apoptosis and/or maintain proliferation even during menstruation and the early follicular phase, when ovarian estrogen supply is not possible or is limited. Effect of estrogen on endometriosis Although estrogen is one of the most important factors in the development and maintenance of endometriosis (Refs 35, 36), the mechanism(s) of how estrogen exerts its pathological effect is still not completely elucidated. It is known that estrogen per se seldom exerts growthpromoting effects. Instead, the mitogenic effect of estrogen is usually mediated through peptide growth factors in an autocrine/ paracrine manner. Peptide growth factors such as epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and fibroblast growth factors (FGFs) have been shown to exert estrogen-induced growth effects in other cell types (Refs 37, 38, 39, 40). To delineate the effects of peptide growth factors on the development of endometriosis, concentrations of peptide growth factors and their cognate receptors in normal and endometriotic tissues have been examined. However, attempts to characterise the expression patterns of these growth factors in association with the severity of endometriosis have not found strong correlations. For example, the expression of EGF and EGF receptors are not different between ectopic endometriotic implants and eutopic endometrial tissues (Refs 41, 42). The concentration of IGF-1 in the peritoneal fluid of women with endometriosis was higher (Ref. 43), no different (Ref. 44) or even lower (Ref. 45) compared with the concentration in women without endometriosis. Although increased expression of FGF-2 and the FGF receptor FGFR1 in glandular epithelium and stroma was found in the condition adenomyosis (abnormal thickening of the endometrial lining) compared with autologous endometrium (Ref. 46), concentrations of FGF-2 in the peritoneal fluid and immunoreactive FGF-2 in pelvic endometriotic lesions were not different from those of normal or eutopic counterparts (Refs 42, 47, 48). Moreover, transcripts of the gene encoding FGF-2 in ectopic endometriotic stromal cells were lower compared with those in eutopic endometrial stroma (Ref. 49). Nevertheless, the lack of positive correlation does not exclude the importance of peptide growth factors in the development of endometriosis. It is well established that the cyclic demise of uterine endometrium is a result of the lack of support from peptide growth factors regulated by ovarian hormones. The ectopic endometriotic tissue can escape this destiny if it evolves a selfsupporting system that enables autonomous production of growth factors when they are needed the most. Thus, the crucial role of peptide growth factor in contributing to the development of endometriosis may not be determined by the amount but the time of expression. The identification of FGF-9 as an estromedin that regulates endometrial stromal cell proliferation in normal endometrium (Ref. 50) has shed light on the search for functional roles of peptide growth factors in the development of endometriosis and the survival of endometriotic implants. Since expression of FGF-9 is regulated by estrogen and ectopic endometriotic tissue is capable of synthesising estrogen autonomously, it is likely that ectopic endometriotic cells might be able to produce FGF-9 to serve as a survival and/or mitogenic factor. As expected, FGF-9 is consistently expressed by ectopic endometriotic tissue (Fig. 1) with greater amounts in early stages compared with later stages (Ref. 51), which correlates with the concentrations of estrogen in the peritoneal fluid of women with endometriosis (Ref. 15). It is known that FGF-9 binds to the receptors FGFR2IIIc and FGFR3IIIc with high affinity and that binding of FGF-9 to both receptors elicits potent mitogenic effects (Ref. 52). In human endometriotic cells, expression was detected of FGFR2IIIb, FGFR2IIIc, FGFR3IIIb and FGFR3IIIc (Ref. 50). Since FGF-9 is one of the major growth factors for cell proliferation (Refs 50, 53), this suggests that early endometriotic lesions can survive the hostile 5

6 microenvironment of the peritoneal cavity partly owing to the proliferative, and possibly anti-apoptotic, effect exerted by expressing FGF-9 and its high-affinity receptors in the ectopic endometriotic stroma. Another important phenomenon is that expression of FGF-9 in ectopic endometriotic lesions is not changed throughout the menstrual cycle, which is in sharp contrast to expression in eutopic endometrium. Expression of FGF-9 in normal endometrium is the greatest in late follicular phase and is at basal level in late secretory or early proliferative phases (Ref. 50). This difference concurs because ectopic endometriotic tissue is able to synthesise estrogen, thus reducing the dependency on estrogen of ovarian origin. During late follicular phase, ovarian estrogen represents the dominant source for FGF-9 induction in both eutopic and ectopic endometrial stromal cells. When serum estrogen of ovarian origin falls, especially during menstruation or early follicular phase, expression of FGF-9 in eutopic endometrial stroma decreases owing to lack of estrogen stimulation. By contrast, estrogen synthesised by ectopic endometriotic stroma (Ref. 54) serves as a primary source for induction of FGF-9 through an autocrine mechanism when systemic estrogen is low. As a result, the expression of FGF-9 in ectopic endometriotic stroma persists. In accordance with these results, it was reported that FGF-9 and FGFRs are indeed upregulated by treatment with estrogen (Ref. 51). In summary, these results support the notion that expression of the FGF-9 system in ectopic endometriotic tissues is induced by estrogen of endometriotic stromal cell origin in an autocrine fashion, a crucial factor in promoting ectopic retrograded endometrial survival and/or propagation, especially during menstruation or early follicular phase when ovarian estrogen output is at the nadir or is limited. It might also explain, at least in part, the failure of danazol or GnRH agonists, used for the systemic depletion of luteinising hormone-stimulated ovarian estrogen, to eliminate ectopic endometriotic lesions (Ref. 55). By contrast, the use of aromatase inhibitors for the systemic and local inhibition of estrogen biosynthesis has shown promising results in the treatment of endometriosis (Ref. 56). All these reports demonstrate the importance of abnormal local production of estrogen in the aetiology of endometriosis. Molecular and cellular mechanisms of FGF-9-induced endometriotic stromal cell proliferation The biological function of FGF-9 in tissue formation and disease has been demonstrated in many studies (Refs 50, 51, 53, 57, 58, 59); however, the mechanism of how FGF-9 controls these processes remains largely uncharacterised. FGF signalling is mediated through complex interactions between specific members of the FGF family and one or more FGFR isoforms. Receptors for FGF (FGFR1, 2, 3 and 4) are tyrosine kinase receptors comprising two intracellular tyrosine kinase domains, a single transmembrane domain and an extracellular portion that contains three immunoglobulin (Ig)-like domains. The third Ig domain (Ig III), which has the highest impact on FGF binding specificity and tissue-specific expression patterns, is the region in which alternative splicing occurs. Three different splice variants (designated as IIIa, IIIb and IIIc) have been identified for FGFR1 and FGFR2, whereas only the IIIb and IIIc variants have been detected for FGFR3 (Refs 60, 61, 62, 63). No splice variant has yet been identified for FGFR4. The splice variant IIIa is a secreted protein whereas IIIb and IIIc are both membrane-bound receptors containing mutually exclusive Ig III domains. It is generally believed that the IIIb isoform is expressed in epithelial lineages whereas the IIIc variant is restricted to mesenchymal origin (Refs 64, 65, 66). In human endometrial stromal cells, the major FGFR subtype is FGFR2IIIc, which is 100 times more abundant than any other subtypes of FGF-9-specific receptors (Ref. 50). This unique feature of endometrial stromal cells makes it the ideal model for investigating the signalling pathway of FGF-9 without interference from other FGFRs. Study using endometriotic stromal cells shows that FGF-9 stimulates cell proliferation through two parallel but additive pathways (Ref. 67) (Fig. 2). Treatment with FGF-9 induces phosphorylation and nuclear translocation of extracellular signal-regulated kinase (ERK), which is important for transcription of genes encoding cyclin A, cyclin B 1 and cyclin D 1. In a second pathway, FGF-9 stimulates phospholipase Cg (PLCg)-dependent 6

7 Nucleus a Raf MEK ERK Ras SOS mrna Ribosome FGFR2IIIc Grb2 Enhanced protein translation FGF-9 Ca 2þ influx and phosphorylation of mammalian target of rapamycin (mtor). Activation of mtor leads to phosphorylation of p70 ribosomal S6 kinase (S6K1), an important P P P P RNA polymerase p70 PLCγ Enhanced gene transcription IP 3 + mtor PIP 2 DAG CaMK PKC Calmodulin Signalling pathways utilised by fibroblast growth factor (FGF)-9 to induce endometrial stromal cell proliferation Ca 2+ Published in Expert Reviews in Molecular Medicine by Cambridge University Press 2007 Figure 2. Signalling pathways utilised by fibroblast growth factor (FGF)-9 to induce endometrial stromal cell proliferation. Endometrial stromal cells express high levels of FGFR2IIIc, which is the highaffinity receptor for FGF-9. FGF-9 binds to its receptor FGFR2IIIc (P indicates phosphorylation) and activates two parallel but additive pathways that cooperatively induce gene transcription and cell proliferation. (a) The classical pathway is mediated by the Grb2/SOS-dependent Ras signalling cascade. Activation of Ras leads to the sequential activation of its downstream signalling molecules, including Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK). Phosphorylation of ERK results in translocation and activation of its target genes such as those encoding the cyclins that are important for cell-cycle progression. (b) The second pathway is mediated by activation of phospholipase Cg (PLCg), which then converts phosphoinositol 3,4-bisphosphate (PIP 2 ) to form inositol trisphosphate (IP 3 ) and diacylglycerol (DAG). Although DAG was able to induce the activation of PKC, experimental data suggest that PKC is not involved in FGF-9-induced cell proliferation. Instead, IP 3 -induced Ca 2þ influx activates Ca 2þ - calmodulin dependent protein kinase (CaMK). Activation of CaMK phosphorylates the kinase mammalian target of rapamycin (mtor), which then phosphorylates ribosomal p70 S6 kinase (p70). Activation of p70 by mtor enhances protein translation. Collectively, FGF-9 utilises Ras and PLCg/mTOR/p70 to increase transcription and translation efficiency, respectively, which stimulates cell proliferation. Modified figure reproduced with permission from (Ref. 67). mediator that controls protein translation. Intriguingly, FGF-9-induced phosphorylation of mtor is independent of the well-established phosphoinositide 3-kinase/Akt signalling b 7

8 pathway utilised by most peptide growth factors such as IGF-1, EGF and other FGFs (Refs 68, 69, 70, 71). Blockage of either pathway results in only partial inhibition of FGF-9-induced stromal cell proliferation, whereas simultaneous disruption of the Ras/ERK and PLCg/mTOR pathways completely inhibits the action of FGF-9. Taken together, these results demonstrate specific signalling pathways that are utilised by FGF-9 to exert its mitogenic effect in controlling endometrial cell proliferation. The selective ligand-mediated FGFR signalling is important for understanding signalling pathways by different FGF FGFR interactions and the consequence of their physiological and pathological functions. PGE 2 induces FGF-9 expression in ectopic endometriotic stromal cells The fact that expression of FGF-9 is regulated by estrogen (Ref. 51) and that production of estrogen is induced by PGE 2 (Refs 15, 25, 27, 32) implies that the mitogenic effect of PGE 2 on endometriosis might be mediated through upregulation of FGF-9 in endometriotic stromal cells. To test this hypothesis, the effects of PGE 2 on FGF-9 expression in primary cultured human endometriotic stromal cells were recently investigated. It was demonstrated that PGE 2 induces FGF-9 mrna expression in a doseand time-dependent manner (Ref. 72). Administration of cells with 1 mm PGE 2 induced FGF-9 expression at 8 h, reaching a maximum at 12 h and then declining towards the basal level at 24 h after PGE 2 treatment. Interestingly, pretreatment with ICI , an estrogen receptor antagonist, prior to addition of PGE 2 did not inhibit basal or PGE 2 -induced FGF-9 expression. Furthermore, time course experiments also demonstrated that PGE 2 - induced FGF-9 expression (12 h after treatment) preceded that induced by estrogen (24 h after treatment). These data suggest that PGE 2 can induce FGF-9 expression without the involvement of estrogen. Alternatively, it has been reported that some peptide growth factors such as IGF-1 and EGF can transactivate estrogen receptor a (ERa) independently of estrogen. Therefore, it is possible that transactivation of ER by such peptide hormones might contribute to PGE 2 -induced FGF-9 expression. However, experimental data indicate that neither IGF-1 nor EGF affects FGF-9 mrna expression whereas PGE 2 significantly induces FGF-9 expression. As described above, three of four EP receptors (EP2, EP3 and EP4) are expressed by endometrial stromal cells. By using selective EP agonists and antagonists, results demonstrate that induction of FGF-9 by PGE 2 is mediated through the EP3 receptor and its downstream signalling pathways (Ref. 72). Although the downstream signalling pathways of EP3 are the most complicated among all EP receptors (Ref. 31), it is known that the major effector downstream of EP3 is PKCd. This conclusion is drawn from several lines of evidence, including the use of a pharmacological activator (12- myristate 13-acetate) and inhibitors (GF109203, Gö6976 and rottlerin) of PKC, knockdown experiments using small interfering (si)rna, and forced expression of the catalytic domain of PKCd. Activation of PKCd by PGE 2 leads to phosphorylation of ERK, which then phosphorylates Elk-1, a member of the ternary complex factor (TCF) subfamily of ETS-domain transcription factors (Ref. 73). Two putative Elk-1-binding sites have been identified in the FGF-9 promoter region between 2886 and Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation- PCR (ChIP) assay indicate that both Elk-1 sites are important for the activation of the gene encoding FGF-9 by PGE 2. The finding that the EP3 receptor mediates the action of PGE 2 in stimulating FGF-9 expression is intriguing because EP2 has been known to be the major receptor in mediating PGE 2 actions. Previous reports demonstrate that PGE 2 induces estrogen biosynthesis in endometriotic stromal cells through PKA signalling pathways coupled to the EP2 receptor (Refs 27, 32). Since estrogen also induces the expression of FGF-9 (Ref. 50), these data reveal that PGE 2 simultaneously activates two distinct pathways by binding to different receptor isoforms to exert the same function (Fig. 1). Effects mediated by EP3 receptor signalling pathways represent the acute action of PGE 2 whereas upregulation of FGF-9 by EP2 receptor-dependent estrogen action represents a delayed response to PGE 2. Considering that FGF-9 is a survival and mitogenic factor, induction of FGF-9 by PGE 2 at different time points may have different functions. However, why PGE 2 induces FGF-9 8

9 expression through two different signalling pathways remains an open question and further investigation is needed to dissect the significance of actions mediated by different EP receptors in the induction of FGF-9 expression. Role of macrophages in the development of endometriosis As has been described above, although retrograde menstruation is the crucial event in the development of endometriosis, the factors that allow the implantation and propagation of endometriotic lesions are largely unknown. We and others have demonstrated that aberrant production of steroids by ectopic endometriotic lesions is an important factor leading to the survival and proliferation of endometriotic tissues (Refs 15, 54). In addition, the alteration/ dysfunction of the immune system that results in the decreased phagocytotic ability of immune cells may serve as another crucial factor for the development of endometriosis (Ref. 74). The immune system is responsible for the removal of endometrial tissues retrograded to the peritoneal cavity. The central piece of this defensive system is the monocyte/macrophage, which plays dual functional roles in regulating cytokine production and phagocytosis. During endometriosis development, immune cells are recruited to the peritoneal cavity. Among these immune cells, macrophages are the dominant cell type and are involved in phagocytosis and inflammation, especially in clearing the retrograded endometrial debris (Refs 75, 76). Studies of women undergoing laparoscopy have concluded that the incidence of retrograde menstruation can be as high as 90% (Refs 77, 78), whereas only about 10 15% of these women develop endometriosis, indicating that the great majority of peritoneal macrophages successfully accomplish their role. The number of macrophages is increased in the peritoneal fluid of endometriosis patients and the majority are in a hyperactive state and show characteristic features of activated macrophages such as increased CD25 surface marker (Refs 77, 79). In a homogeneous murine model, it has been reported that peritoneal macrophages are increased by 4 h after injection of endometrial epithelial and stromal cells to the peritoneal cavity of the animal. Production of monocyte chemotactic protein 1, interleukin (IL)-1a, tumour necrosis factor (TNF)-1a and IL-6 in the peritoneal milieu is also increased (Refs 80, 81). In a clinical study, IL-1 was elevated in the peritoneal fluid of woman with endometriosis, and this was mainly produced by peritoneal monocytes/macrophages (Ref. 82). IL-1 is a key cytokine in the regulation of inflammation and immune responses. In endometriosis, several molecules are induced by IL-1, such as IL-2, IL-8, RANTES (for regulated upon activation, normal T cell expressed and secreted ), macrophage migration inhibitory factor and PGs (Refs 83, 84, 85). Although peritoneal macrophages recruited to the peritoneal cavity are responsible for the removal of retrograded red blood cells and endometrial debris (Refs 76, 86), hyperactivation of macrophages may have a negative impact on the pathogenesis of endometriosis. For example, it has been reported that peritoneal macrophages isolated from patients with endometriosis tend to have higher ability to produce inflammatory agents and poorer cytotoxic capability (Ref. 74). It is not known whether this functional alteration either results from factors (such as chemokines, cytokines, PGs and growth factors) produced by endometriotic tissue that alter the environment of the peritoneal cavity to provide advantage for attachment and outgrowth of endometrial cells or results from inhibition of phagocytotic ability of macrophage by these factors. PGE 2 suppresses scavenger function of macrophages The scavenger function of macrophages is mediated through at least two mechanisms. The first mechanism is the secretion and activation of matrix metalloproteinases (MMPs) to break down the ECM of foreign entities (Ref. 87). The second mechanism of scavenging activity involves expression of scavenger receptors on the macrophages to enhance the uptake and degradation of cell debris (Refs 88, 89). Macrophages express several scavenger receptors that enable take-up of pathogens and apoptotic cells, phosphatidylserine and oxidised lipoprotein (Refs 88, 89). As a class, these proteins tend to recognise polyanionic macromolecules and may have physiological functions in the recognition and clearance of foreign entities. MMPs are a large family of zinc proteases including 22 human homologues that can be divided into four major subgroups: interstitial 9

10 collagenases, gelatinases, stromelysins and membrane-type MMPs (Ref. 90). Under normal physiological conditions, the activities of MMPs are precisely regulated at the level of transcription, activation of the precursor zymogens, interaction with specific ECM components and inhibition by endogenous inhibitor (Ref. 91). Most MMPs are closely regulated at the level of transcription, with the notable exception of MMP-2, which is often constitutively expressed and controlled through a unique mechanism of enzyme activation and some degree of post-transcriptional mrna stabilisation (Refs 90, 92, 93). The extracellular activation of most MMPs can be initiated by other already activated MMPs or by several serine proteinases that can cleave peptide bonds within MMP pro-domains (Ref. 90). Macrophages can secrete MMP-2, 7, 9 and 12, all of which can degrade elastin and are implicated in the pathogeneses of emphysema and aortic aneurysm (Refs 94, 95). MMP-9 has been suggested to be involved in cell migration occurring in the various physiological and pathological processes such as tumour cell invasion and chronic inflammation by facilitating the destruction of the type IV collagen-containing basement membrane, which separates the epithelial and stromal compartment (Ref. 96). Besides ECM degradation, MMP-9 can activate several cytokines such as latent transforming growth factor (TGF)-b and pro-tnf-a to their active forms (Refs 90, 97). Thus, MMP-9 not only plays important roles in the scavenger activity of macrophages, it also has a significant impact on the defensive mechanism of the immune system. Intriguingly, peritoneal macrophages isolated from patients with endometriosis were found to have phenotypic and functional alterations leading to poor phagocytotic capacity, a mechanism that is highly associated with the severity of endometriosis (Refs 74, 98). The decreased phagocytotic ability of macrophages isolated from the peritoneal fluid of women with endometriosis may be due to decreased MMP-9 activity as MMP-9 / mice have an enhanced bacterial outgrowth in peritoneal lavage fluid, blood and liver (Ref. 99). In a recent study, we found that expression and enzymatic activity of MMP-9 by peritoneal macrophages derived from patients with endometriosis were reduced compared with those from normal women (Ref. 100). Decreased expression and activity of MMP-9 secreted by macrophages might result in reduced phagocytotic ability and attenuated scavenger function in macrophages. It was demonstrated that peritoneal fluid derived from women with endometriosis can inhibit the gelatinase activity of MMP-9 of macrophages whereas peritoneal fluid derived from endometriosis-free women has no such effect (Ref. 100). Decreased gelatinase activity of macrophages by peritoneal fluid of patients with endometriosis is due to a decrease in MMP-9 mrna and protein expression. It is known that cytokines such as IL-1, TNF-a and interferon g can regulate MMP-9 expression and the concentrations of IL-1 and TNF-a are elevated in the peritoneal fluid of women with endometriosis. However, experimental data do not support the notion that these proinflammatory cytokines can inhibit MMP-9 expression and activity in peritoneal macrophages (Ref. 100). Since PGE 2 is also elevated in the peritoneal fluid of women with endometriosis, it is reasonable to test whether PGE 2 can regulate MMP-9 expression by peritoneal macrophages. Interestingly, PGE 2 effectively decreased MMP-9 activity. Inhibition of MMP-9 activity by PGE 2 is not due to an increase in inhibitors of MMP-9 such as tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2 and RECK, but to a downregulation of MMP-9 protein expression. This downregulation effect of PGE 2 is mediated by the EP2/EP4 receptor and through the PKA signalling pathway (Ref. 100). Taken together, these data provide a likely explanation to answer the question as to why peritoneal macrophages in women with endometriosis have lower phagocytotic capability. Further study is needed to identify if any other factors besides PGE 2 are also involved in modulating the function of macrophages. Expression of cyclooxygenase (COX)-2 in peritoneal macrophages A growing body of epidemiological, pharmacological and laboratory studies has provided conclusive evidence to support the important role of PGE 2 in the pathophysiology of endometriosis. As described in previous sections, PGE 2 stimulates StAR, aromatase and FGF-9 expression, which provide proliferative 10

11 and/or anti-apoptotic signals to ectopic endometriotic tissues. By contrast, PGE 2 suppresses immune functions by inhibiting the expression and activity of MMP-9 in peritoneal macrophages and thus prevents ectopic tissues from being removed by macrophages. Since PGs are unstable eicosanoids with very short halflife (Ref. 101), it is generally believed that they must be produced and function locally. Hence, peritoneal macrophages and ectopic endometriotic tissue represent two of the most likely candidates that would contribute to the elevation of peritoneal PGE 2. The rate-limiting step in PGE 2 biosynthesis is regulated by COX, which catalyses the conversion of arachidonic acid to PGH 2. Two isoforms of COX exist, the constitutively expressed COX-1 and the inducible COX-2. COX-1 is expressed ubiquitously and is thought to produce PGs for primary housekeeping functions such as platelet aggregation, vasodilatation in the kidney and cytoprotection of gastric mucosa (Refs 102, 103). By contrast, COX-2 is normally expressed in very low amounts or is even undetectable in most tissues under physiological conditions but is rapidly induced by cytokines, endotoxins, proinflammatory agents, tumour promoters and certain hormones (Refs 102, 103). Therefore, PGs produced by COX-2 usually lead to pathological alteration in various tissues. Recent study demonstrates that both COX-1 and COX-2 are over-expressed in peritoneal macrophages derived from women with severe endometriosis (Ref. 79). The expression of COX (especially COX-2) in peritoneal macrophages is highly associated both with PGE 2 concentration in the peritoneal fluid and with the severity of endometriosis. It is likely that aberrant expression of COX-2 in peritoneal macrophages of patients with endometriosis may result from differences in genetic background. To test this hypothesis, expression patterns of COX-2 in mononuclear cells from the peripheral circulation and from peritoneal fluid have been evaluated. Mononuclear cells isolated from the peripheral blood of women with endometriosis appear to be the same as those obtained from disease-free women in terms of COX-2 expression and the presence of surface marker (Ref. 79). By contrast, significant numbers of mononuclear cells isolated from the peritoneal fluid of women with endometriosis clearly express macrophagespecific marker whereas those isolated from disease-free women predominantly express monocytic cell antigen. Thus, distinct patterns of COX-2 expression in peritoneal macrophages seem unlikely to be due to genetic variation but rather to exposure to microenvironmental stimulants such as IL-1b, TNF-a and even PGE 2 in the peritoneal fluid of women with endometriosis. Clearly, these are not the only factors that regulate the transition of macrophages from naive (in normal peritoneal fluid) to activated (in endometriotic peritoneal fluid) states. More studies are needed before a comprehensive picture regarding factors controlling the recruitment and differentiation of peritoneal macrophages in women with endometriosis can be drawn. Another noteworthy finding is the marked elevation of COX-1 expression in peritoneal macrophages obtained from patients with severe endometriosis (Ref. 79). This finding indicates that prolonged exposure to stimuli in the peritoneal fluid of women with endometriosis results in induction not only of the inducible COX-2 but also of the constitutive COX-1. Although this finding is intriguing, it is not unprecedented as similar findings have been reported in numerous cancer models (Refs 104, 105, 106, 107) and in some cell types during cell differentiation (Refs 108, 109). These data suggest that COX-1 and/or its products might also be involved in pathological and/or physiological processes. However, mechanisms responsible for COX-1 upregulation in peritoneal macrophages remain unknown and further investigation is necessary to identify the cause and effect of COX elevation in peritoneal macrophages during endometriosis formation and progression. Over-expression and distinct regulation of COX-2 in ectopic endometriotic tissue Elevated concentration of PGE 2 in the peritoneal fluid of women with endometriosis is an important factor contributing to the development and symptomatic consequences of endometriosis. Besides the fact that peritoneal macrophages obtained from patients with endometriosis have greater PG synthetic capability compared with those from endometriosis-free women (Ref. 79), an alternative but not necessarily mutually 11

12 exclusive hypothesis is that PGE 2 may be synthesised by pelvic endometriotic implants. Indeed, elevated expression of COX-2 in ectopic endometriotic lesions was detected (Refs 110, 111, 112). Over-expression of COX-2 in ectopic tissues is mirrored by increased production of PGE 2 in primary cultured stromal cells derived from ectopic endometriotic lesions. This phenomenon is indisputably observed in unpaired normal and endometriotic samples as well as in paired eutopic and ectopic samples collected from the same individual (Ref. 112). This result indicates that aberrant expression of COX-2 in ectopic endometriotic tissue is not just a random event due to different genetic backgrounds. Instead, it implies that aberrant COX-2 expression and PGE 2 production in endometriotic tissues may be the consequence of epigenetic alteration. Supported by a growing body of evidence, the biochemical nature of ectopic endometriotic tissues, though emerging from the same origin, is different from its eutopic counterpart (Refs 15, 25, 30, 113, 114). To investigate the potential mechanism underlying distinct biochemical responsiveness in eutopic and ectopic endometria, the expression of COX-2 in response to IL-1b has been evaluated because IL-1b is one of the primary inflammatory cytokines in the peritoneal fluid of women with endometriosis. The data demonstrate that the expression of COX-2 in response to IL-1b challenge in ectopic endometriotic stromal cells is much greater than that in eutopic endometrial stromal cells. Levels of COX-2 protein are increased by treatment with IL-1b in both eutopic and ectopic endometrial stromal cells. However, induction of COX-2 by IL-1b in ectopic endometriotic stromal cells is at least 100 times more sensitive compared with its eutopic counterpart (Ref. 112). The induction of COX-2 expression and PGE 2 secretion by IL-1b is through transcriptional upregulation of the gene encoding COX-2 and by posttranscriptional enhancement of COX-2 mrna stability through ERK/p38 MAPK-dependent CREB phosphorylation in ectopic endometriotic stromal cells. By contrast, in eutopic endometrial stromal cells, IL-1b fails to induce COX-2 promoter activity but is able to increase its mrna stability. Thus, the distinct responsiveness of the gene encoding COX-2 to IL-1b is probably mediated by differential regulation of the COX-2 promoter. Considering the homogeneity of genetic backgrounds of paired eutopic and ectopic endometrial stromal cells, distinct responses of the COX-2 promoter to IL-1b imply that epigenetic regulation of gene expression and/or posttranslational modification of chromatin leading to distinct promoter activity of a gene could be the underlying mechanism. Current working model for the aetiopathogenesis of endometriosis On the basis of most recent studies, a working hypothesis can be proposed (Fig. 3). In this model, retrograded menses causes inflammation in the pelvic cavity and the recruitment of macrophages and other immune cells. It is thought that immune cells remove most endometrial tissues discharged to the pelvic cavity, although this has not been demonstrated in vivo. Under certain circumstances, again through as-yet-unknown mechanisms, the retrograded tissues survive and reside in the peritoneal cavity. The immune cells, lead by macrophages, produce cytokines (IL-1b in this model) that induce over-expression of COX-2 in macrophages and ectopic endometriotic tissues. Expression of COX-2 leads to increased production and accumulation of prostanoids, especially PGE 2, in the peritoneal fluid. Elevated concentration of PGE 2 induces aberrant expression of steroidogenic proteins such as StAR and aromatase in the ectopic endometriotic stromal cells, leading to abnormal biosynthesis of estrogen, a crucial survival factor for endometrium. Autonomous production of estrogen by ectopic tissues induces several known peptide growth factors such as EGF, IGF-1, vascular endothelial growth factor (VEGF) and FGF, which serve as autocrine (for endometriotic cells) and paracrine (for epithelial cells) factors to stimulate cell proliferation and angiogenesis. Simultaneously, PGE 2 exerts direct action on endometriotic and endothelial cell proliferation through induction of FGF and/or VEGF. Implanted endometriotic lesions produce great amounts of PGE 2, and perhaps other factors, which inhibit the secretion and enzymatic activity of MMPs (such as MMP-9) by macrophages. Decreased MMP activity results in attenuation of the scavenger function of macrophages, which in the long-term is in 12

13 Uterus Peritoneal cavity Endometrial tissues Macrophage Peritoneal cavity MMP-9 IL-1β favour of the survival and growth of endometriotic lesions. Furthermore, it is clear that aberrant production of PGE 2 results in autonomous production of estrogen by ectopic endometriotic tissue. Estrogen exerts a stimulatory effect on COX-2 Decreased MMP-9 favours survival and growth of endometriotic tissues Proposed working model of endometriosis PGE 2 Expert Reviews in Molecular Medicine C 2007 Cambridge University Press StAR/CYP19 Ectopic endometriotic stromal cells EGF IGF-1 VEGF FGF Act as autocrine and paracrine factors to stimulate endometriotic cell proliferation and angiogenesis Figure 3. Proposed working model of endometriosis. Retrograded menses enter the peritoneal cavity and, by some unknown mechanism, reside and survive. The retrograded tissues cause inflammation in the pelvic cavity that recruits macrophages and other immune cells. These cells produce cytokines [interleukin (IL)-1b in this model] that induce overexpression of cyclooxygenase (COX)-2 in macrophages and ectopic endometriotic tissues. Expression of COX-2 leads to increased production and accumulation of prostanoids, especially prostaglandin E 2 (PGE 2 ), in the peritoneal fluid. Elevated PGE 2 induces aberrant expression of steroidogenic proteins such as steroidogenic acute regulatory protein (StAR) and P450 cytochrome aromatase (CYP19) in the ectopic endometriotic stromal cells, leading to abnormal biosynthesis of estrogen (E 2 ), a crucial survival factor for endometrium. Autonomous production of E 2 by ectopic tissues induces several known peptide growth factors such as epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGFs), which serve as autocrine (for endometriotic cells) and paracrine (for epithelial cells) factors to stimulate cell proliferation and angiogenesis. Simultaneously, PGE 2 exerts direct action on endometriotic and endothelial cell proliferation through induction of FGF and/or VEGF. Implanted endometriotic lesions produce great amounts of PGE 2, and perhaps other factors, which inhibit the secretion and enzymatic activity of matrix metalloproteinases (MMPs) such as MMP-9 by macrophages. Decreased MMP activity results in attenuation of the scavenger function of macrophages, which favours survival and growth of the endometriotic lesion. COX-2 expression and hence PGE 2 production. Over-expression of COX-2 in endometriotic lesions and peritoneal macrophages isolated from women with endometriosis provide a logic rationale for a positive-feedback loop between PGE 2 and estrogen. Thus, the 13 E 2