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Aromatase: a key molecule in the pathophysiology of endometriosis and a therapeutic target
FERTILITY AND STERILITY威 VOL. 72, NO. 6, DECEMBER 1999
MODERN TRENDS
Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Edward E. Wallach, M.D. Associate Editor
Aromatase: a key molecule in the pathophysiology of endometriosis and a therapeutic target Khaled M. Zeitoun, M.D.,* and Serdar E. Bulun, M.D. Department of Obstetrics and Gynecology, The University of Texas Southwestern Medical Center, Dallas, Texas
Received April 23, 1999; revised and accepted May 11, 1999. Supported in part by an unrestricted research grant from the American Society for Reproductive Medicine– Organon, National Cancer Institute Grant CA-67167 and USAMRMC Grant DAMD17-97-1-7025 (to S.E.B.), and by an American Association of Obstetricians and Gynecologists Foundation Fellowship Award (to K.M.Z.). Reprint requests and present address: Serdar E. Bulun, M.D., Department of Obstetrics-Gynecology, University of Illinois at Chicago, 820 South Wood Street M/C 808, Chicago, Illinois 60610 (FAX: 312996-4238; E-mail: sbulun@uic .edu). * Present address: Department of Obstetrics and Gynecology, Columbia-Presbyterian Medical Center, 622 W. 168th Street, New York, New York 10032-3702. 0015-0282/99/$20.00 PII S0015-0282(99)00393-3
Objective: To provide a clinically useful model illustrating the molecular aberrations affecting estrogen biosynthesis and metabolism in endometriosis and to discuss the therapeutic role of aromatase inhibitors. Design: Literature review. Result(s): Several molecular aberrations were found in endometriotic lesions (in contrast to eutopic endometrium) that favor increased local concentrations of E2. Endometriotic stromal cells aberrantly express aromatase, which converts C19 steroids to estrogens. Aromatase activity in these cells is stimulated by prostaglandin (PG)E2. Estrogen stimulates cyclooxygenase-2, giving rise to increased PGE2 formation. Thus, this positive feedback loop produces increasing quantities of E2 and PGE2 in endometriosis. The lack of aromatase expression in eutopic endometrium is maintained by binding of an inhibitory transcription factor, COUP-TF, to the aromatase promoter. In endometriosis, however, an aberrantly expressed factor, SF-1, displaces COUP-TF to bind to this same promoter and activates aromatase expression and thus local estrogen biosynthesis. Additionally, endometriotic glandular cells are deficient in 17-hydroxysteroid dehydrogenase type 2, which converts E2 to estrone in the eutopic endometrium in response to P. Deficiency of this enzyme in endometriosis impairs the inactivation of E2 and may be a consequence of insensitivity to P. Conclusion(s): Molecular aberrations that increase local E2 concentrations may be important in the etiology of endometriosis. These molecules may be targeted to develop novel therapeutic strategies. The clinical relevance of aromatase expression in endometriosis was shown recently by the successful treatment of an unusually aggressive case of postmenopausal endometriosis with use of an aromatase inhibitor. (Fertil Steril威 1999;72:961–9. ©1999 by American Society for Reproductive Medicine.) Key Words: Aromatase, endometriosis, endometrium, estrogen, progesterone, 17-hydroxysteroid dehydrogenase type 2, aromatase inhibitor
Endometriosis is characterized by the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites and is linked to pelvic pain and infertility. It is estimated to affect 2%–10% of women in the reproductive age group (1–5). Although endometriosis is traditionally considered a disease of women in the reproductive age period, the natural history of this disorder, including onset and progression, has not been well established. For example, the incidences of endometriosis in teenagers and postmenopausal women are probably underestimated (6 –10). Endometriosis is considered a polygenically inherited disease with a complex, multifactorial etiology (11). Sampson’s theory (12, 13) of transplantation of endometrial tissue on the pelvic peritoneum through retrograde menstru-
ation is the most widely accepted explanation for the development of pelvic endometriosis because of convincing circumstantial and experimental evidence. Because retrograde menstruation is observed in almost all cycling women, endometriosis is postulated to develop as a result of a coexisting defect in clearance of the menstrual efflux from the pelvic peritoneal surfaces, possibly involving the immune system (14 –16). Alternatively, intrinsic molecular aberrations in pelvic endometriotic implants were proposed to contribute significantly to the development of endometriosis. Some of these molecular defects include aberrant expression of aromatase (17, 18), certain cytokines (19), and tissue metalloproteinases (20 –26); deficiency of 17-hydroxysteroid dehydrogenase 961
(17HSD) type 2 (27); and resistance to the protective action of P (21, 23, 25, 26). Because endometriosis is an estrogen-dependent disorder, aromatase expression and deficiency of 17-HSD type 2 are of paramount importance in the pathophysiology. In this article, we review the aberrant mechanisms of estrogen biosynthesis and metabolism in women with endometriosis, with an emphasis on identifying targets for new treatment strategies.
ESTROGEN BIOSYNTHESIS AND METABOLISM IN HUMANS
FIGURE 1 Extraovarian estrogen synthesis in women. Estradiol in women is either directly secreted by the ovary or produced in the extraglandular sites (adipose tissue and skin). The principal substrate for extraglandular aromatase activity in ovulatory women is androstenedione of adrenal and ovarian origin. In women receiving GnRH agonists or postmenopausal women, the adrenal gland remains the primary source of androstenedione. Androstenedione is converted by aromatase to estrone in adipose and skin fibroblasts. Estrone is further converted to estradiol by 17-HSD type 1 activity in peripheral tissues. Thus, peripheral aromatization is the major source of circulating estradiol in the postmenopausal period or during ovarian suppression.
The conversion of androstenedione (A) and T to estrone (E1) and E2 is catalyzed by aromatase P450 (P450arom), which is expressed in a number of human tissues and cells, such as ovarian granulosa cells, placental syncytiotrophoblast, adipose and skin fibroblasts, and the brain. In the reproductive-age woman, the ovary is the most important site of estrogen biosynthesis, and this takes place in a cyclic fashion. There is no marked E2 secretion from the ovary in the beginning of the menstrual cycle (during menstruation), but as the dominant follicle matures, granulosa cells replicate and express increasing levels of aromatase under the influence of FSH (28 –32). Aromatase catalyzes the conversion of A to E1, which is further converted to the potent estrogen E2 by the enzyme 17-HSD type 1 in the granulosa cell. At midcycle, peak serum E2 levels are attained just before ovulation, and ovarian E2 secretion from the corpus luteum then continues at lower levels. Upon binding of FSH to its G-protein– coupled receptor in the granulosa cell membrane, levels of intracellular cyclic adenosine monophosphate (cAMP) rise, which gives rise to the binding of two critical transcription factors, i.e., steroidogenic factor-1 (SF-1) and cAMP response element binding protein (CREB), to the classically located proximal promoter II of the aromatase gene (33, 34). This, in turn, activates aromatase mRNA and protein formation and is responsible for the striking stimulation of aromatase activity and, consequently, estrogen secretion from the preovulatory follicle (34, 35). In postmenopausal women, on the other hand, estrogen formation takes place in extraglandular tissues such as adipose and skin (36 –39) (Fig. 1). In contrast to cAMP regulation of aromatase expression in the ovary, this is controlled primarily by cytokines (interleukin [IL]-6, IL-11, tumor necrosis factor [TNF]-␣) and glucocorticoids through the alternative use of promoter I.4 in adipose and skin fibroblasts (35). It should be pointed out here that the major substrate for adipose/skin aromatase is A of adrenal origin. In postmenopausal women, approximately 2% of circulating A is converted to E1, which is further converted to E2 in peripheral tissues. This gives rise to pronounced serum levels of E2 962
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capable of causing endometrial hyperplasia or even carcinoma (36, 39 – 45).
AROMATASE EXPRESSION IN ¨ LLERIAN-DERIVED TISSUES MU Until recently, estrogen action was classically viewed to occur only through an “endocrine” mechanism. In other words, it was thought that only circulating E2, whether secreted by the ovary or formed in the adipose tissue, could exert an estrogenic effect after delivery to target tissues through the bloodstream. Studies on aromatase expression in breast cancer have demonstrated that paracrine mechanisms play an important role in estrogen action in this tissue (46 – 49). Estrogen produced by aromatase activity in breast adipose fibroblasts was demonstrated to promote the growth of adjacent malignant breast epithelial cells (50). Finally, we demonstrated a possible “intracrine” effect of estrogen in uterine leiomyomas and endometriosis (17, 51, 52). Estrogen produced by aromatase activity in the cytoplasm of leiomyoma smooth muscle cells or endometriotic stromal cells can exert its effects by readily binding to its nuclear receptor within the same cell. Vol. 72, No. 6, December 1999
Mu¨llerian tissues are known targets of estrogen action. Thus, the presence or absence of aromatase expression (i.e., estrogen biosynthesis) in the endometrium and myometrium has important physiologic and clinical consequences. Disease-free endometrium and myometrium lack aromatase expression (51, 53). On the other hand, neoplastic counterparts of these tissues display high levels of aromatase expression and activity (51, 54 –56). The expression of aromatase mRNA and protein was demonstrated in stromal cells of endometrial cancer and in smooth muscle cells of uterine leiomyomas (51, 55, 57, 58). Aromatase expression in both of these tumors is regulated by cAMP through the ovariantype promoter II.
FIGURE 2 Aromatase activity in endometriosis-derived stromal cells. Confluent stromal cells in primary culture were maintained for 24 hours in serum-free medium. Treatments consisted of the following: [1] Bt2cAMP (0.5 mmol/L); [2] sulprostone (10⫺6 mol/L), an Ep3 (Ep1, Ep2, Ep3 are PGE2 receptor subtypes) receptor agonist; [3] 11-deoxy-16,16-dimethyl prostaglandin E2 (10⫺6 mol/L), an Ep2 receptor agonist; and [4] 17-phenyl trinor prostaglandin E2 (10⫺6 mol/L), an Ep1 receptor agonist. The stimulation of Ep2 receptors has been demonstrated to increase intracytoplasmic cAMP levels.
In the normal ovary, aromatase expression is limited to granulosa cells and is absent in theca and other stromal cells (30, 59). It should also be pointed out that aromatase expression was demonstrated in the stromal cells of epithelial ovarian neoplasms (55, 59, 60). At present, the importance of aromatase expression (i.e., estrogen biosynthesis) in epithelial ovarian tumors is not known, but it is of interest that most of these aromatase-positive ovarian tumors were of the endometrioid type. Because endometrioid ovarian tumors are proposed to arise from foci of endometriosis, aromatase expression in these epithelial ovarian cancers and endometriosis may share common origins (55, 60, 61). In fact, the growth of endometrioid carcinomas arising from foci of endometriosis in the ovary may be stimulated by estrogen produced by local aromatase activity. Zeitoun. Pathophysiology of endometriosis. Fertil Steril 1999.
THE ROLE OF AROMATASE EXPRESSION IN ENDOMETRIOSIS Despite its role in other estrogen-responsive pelvic disorders, aromatase expression has been studied in greatest detail in endometriosis (17, 18, 52, 56). First, extremely high levels of aromatase mRNA were found in extraovarian endometriotic implants and endometriomas. Second, endometriosis-derived stromal cells in culture incubated with a cAMP analogue displayed extraordinarily high levels of aromatase activity comparable to that in placental syncytiotrophoblast (52). These exciting findings led us to test a battery of growth factors, cytokines, and other substances that might induce aromatase activity through a cAMP-dependent pathway in endometriosis.
Additionally, aromatase mRNA was detected in the eutopic endometrial samples of women with moderate to severe endometriosis but not in those of disease-free women, albeit in much smaller quantities compared with endometriotic implants (17). This might suggest a genetic defect in women with endometriosis, which is manifest by this subtle finding in the eutopic endometrium. We propose that when defective endometrium with low levels of aberrant aromatase expression reaches the pelvic peritoneum by retrograde menstruation, it causes an inflammatory reaction that exponentially increases local aromatase activity, i.e., estrogen formation, induced directly or indirectly by PGs and cytokines (52, 62). This may be a major contributing factor in the formation of endometriotic implants.
PGE2 was found to be the most potent known inducer of aromatase activity in endometriotic stromal cells (52). In fact, this PGE2 effect was found to be mediated by the cAMP-inducing Ep2 receptor subtype (Fig. 2 and our previously unpublished observations). Moreover, estrogen was reported to increase PGE2 formation by stimulating the cyclooxygenase type-2 enzyme in endometrial stromal cells in culture (62, 63). Thus, a positive feedback loop for continuous local production of estrogen and PGs is established, favoring the proliferative and inflammatory characteristics of endometriosis (Fig. 3).
It would be naive to propose that aberrant aromatase expression is the only important molecular mechanism in the development and growth of pelvic endometriosis. Endometriosis seems to be a polygenically inherited disease with a multifactorial etiology. There may be many important molecular mechanisms that favor the development of endometriosis, such as abnormal expression of proteinase-type enzymes that remodel tissues or their inhibitors (matrix metalloproteinases, tissue inhibitor of metalloproteinase-1), certain cytokines (IL-6, RANTES), and growth factors (epidermal growth factor) (19 –21, 23, 64, 65).
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FIGURE 3 Origin of estrogen in endometriotic lesions. Estradiol in an endometriotic lesion arises from several body sites. In an ovulatory woman, estradiol is secreted directly from the ovary in a cyclic fashion. In the early follicular phase and after menopause, peripheral tissues (adipose and skin) are the most important sources of circulating estradiol. Finally, estradiol is produced locally in the endometriotic implant itself in both ovulatory and postmenopausal women. The most important precursor, androstenedione of adrenal origin, becomes converted to estrone, which is in turn reduced to estradiol-17 in the peripheral tissues and endometriotic implants. We demonstrated significant levels of 17-hydroxysteroid dehydrogenase type-1 expression in endometriosis, which catalyzes the conversion of estrone to estradiol-17 (27). Estradiol-17 both directly and indirectly (through cytokines) induces PG synthase-2 (cyclooxygenase-2), which gives rise to elevated concentrations of PGE2 in endometriosis (62, 63). PGE2, in turn, is the most potent known stimulator of aromatase in endometriotic stromal cells (52). Therefore, a positive feedback loop in favor of continuous estrogen formation is established in endometriosis.
comparable to those in the placental syncytiotrophoblast (52). No significant stimulation was observed with various cytokines (IL-1, IL-2, IL-6, IL-11, oncostatin M, IL-15, or TNF-␣), growth factors (basic fibroblast growth factor or platelet-derived growth factor), or steroids (E2-P agonist R5020, or dexamethasone). As emphasized before, PGE2 was found to be the most potent known inducer of aromatase activity by increasing cAMP levels in endometriotic stromal cells. On the other hand, neither cAMP analogues nor PGE2 was capable of stimulating any detectable aromatase activity in eutopic endometrial stromal cells in culture. The obvious question concerned the molecular differences that induce aromatase expression in endometriosis and its inhibition in eutopic endometrium. To address this, we first determined that the cAMP-inducible promoter II was used for in vivo aromatase expression in endometriotic tissues and in cAMP- or PGE2stimulated endometriotic stromal cells. Then, we found that a stimulatory transcription factor, steroidogenic factor-1 (SF-1), and an inhibitory factor, chicken ovalbumin upstream promoter transcription factor (COUP-TF), competed for the same binding site in the aromatase promoter II. COUP-TF was expressed ubiquitously in both eutopic endometrium and endometriosis, whereas SF-1 was expressed specifically in endometriosis but not in eutopic endometrium. SF-1 binds to the aromatase promoter more avidly than COUP-TF. Thus, SF-1 and other transcription factors (e.g., CREB) bind to promoter II and interact to initiate transcription of the aromatase gene in endometriosis, whereas COUP-TF, which occupies the same DNA site in eutopic endometrium, inhibits this process (18) (Fig. 4).
Zeitoun. Pathophysiology of endometriosis. Fertil Steril 1999.
Alternatively, a defective immune system that fails to clear the peritoneal surfaces of the retrograde menstrual efflux has been proposed in the development of endometriosis (14 –16, 25, 66 –70). The development of endometriosis in an individual woman probably requires the coexistence of a threshold number of these genetic aberrations. Nonetheless, aberrant aromatase expression is clinically relevant because aromatase inhibitors suppress postmenopausal endometriosis (71).
REGULATION OF AROMATASE EXPRESSION IN ENDOMETRIOTIC STROMAL CELLS Aromatase activity in endometriotic stromal cells was stimulated by cAMP analogues to strikingly high levels 964
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In summary, one of the molecular alterations leading to local aromatase expression in endometriosis but not in normal endometrium is the aberrant production of SF-1 in endometriotic stromal cells, which overcomes the protective inhibition of transcription of the aromatase gene maintained normally by COUP-TF in the eutopic endometrium.
INTERCONVERSIONS OF E1 AND E2 IN ENDOMETRIOSIS The primary substrate for aromatase activity in endometriosis is A of adrenal and ovarian origin in premenopausal women and adrenal A in postmenopausal women. Because the concentration of T in circulating blood is extremely low in women (approximately one-tenth the level of A) and in vivo peripheral aromatization of this steroid takes place in much lower quantities compared with that of A, aromatization of T in endometriosis is probably not significant (36, 38, 40, 72–75). The major product of aromatase activity in endometriosis, namely E1, is only weakly estrogenic and must be converted to the potent estrogen E2 to exert a full estrogenic effect. We demonstrated that the enzyme 17-HSD type 1, Vol. 72, No. 6, December 1999
FIGURE 4 Proposed mechanism of regulation of aromatase (P450arom) expression by SF-1 and COUP-TF in eutopic endometrium and endometriosis. (A), Binding of COUP-TF (dimer) to a specific DNA site (nuclear receptor half-site) upstream of aromatase promoter II in eutopic endometrial stromal cells. In the eutopic endometrium, COUP-TF binds to nuclear receptor half-site practically in the absence of any competition by SF-1 because SF-1 expression is not detected in the majority of endometrial samples. Thus, COUP-TF exerts its inhibitory effect on the complex of general transcription factors (GTFs) that bind to the TATA box. (B), In the endometriotic stromal cell, where SF-1 is also present, SF-1 binds as a monomer to the nuclear receptor half-site with a higher affinity than COUP-TF, which binds to this site relatively loosely as a dimer. Upon replacing COUP-TF, SF-1 synergizes with cAMP response element binding protein (CREB, bound to upstream CRE) to activate transcription of the aromatase gene in response to cAMP (18).
17-HSD type 2 in endometrial glands and its enzymatic activity have been viewed as protective against E2 action in eutopic endometrium. Because P stimulates 17-HSD type-2 expression in the endometrium, inactivation of E2 to E1 may be considered as one of the antiestrogenic properties of P (81). The expression of 17-HSD type 2 is deficient in endometriotic glandular cells (27). This was confirmed in paired samples of eutopic endometrium and pelvic endometriosis obtained simultaneously during the luteal phase. No detectable mRNA or protein could be shown in endometriosis, whereas abundant 17-HSD type-2 expression was demonstrated in eutopic endometrium. Thus, this protective mechanism, which lowers E2 levels, is lost in endometriosis (27). The aberrant expression of aromatase, the presence of 17HSD type 1, and the absence of 17-HSD type 2 in endometriosis collectively give rise to elevated local levels of E2 compared with eutopic endometrium. Besides the direct mitogenic effects of E2, these events may be viewed as a defective action of P, which fails to induce 17-HSD type 2 in endometriosis (Fig. 5). This is important because P antagonizes the formation of endometriosis, and administration of progestins is an important treatment modality in this disease. Other evidence of resistance to P is shown in studies on dioxin, an environmental toxin associated with the development of endometriosis. In dioxin-treated rat models of endometriosis, the effect of dioxin seems to involve blockage of the inhibitory effect of P on lesion development in association with defective transforming growth factor- expression (23, 25).
EPITHELIAL-STROMAL INTERACTIONS IN ENDOMETRIUM AND ENDOMETRIOSIS
Zeitoun. Pathophysiology of endometriosis. Fertil Steril 1999.
which catalyzes the conversion of E1 to E2, is expressed in endometriosis (27, 76). In contrast to 17-HSD type 1, the enzyme 17-HSD type 2 (encoded by a separate gene) catalyzes the conversion of E2 to E1 (77–79). In other words, it inactivates E2. One of the major tissue sites of 17-HSD type-2 expression is endometrial glandular cells during the luteal phase (80). In fact, induction of the activity of this enzyme by P in endometrial glands had been demonstrated by a series of earlier studies (81, 82). The expression of FERTILITY & STERILITY威
Compartmentalization of steroidogenic enzyme expression, i.e., aromatase in stromal cells of endometriosis and 17-HSD type 2 in secretory glandular epithelial cells of endometrium, points to important epithelial-stromal interactions. In fact, other epithelial-stromal interactions in endometrium and endometriosis were reported previously. For example, when experimental endometriotic lesions were induced in rodents by transplanting endometrium to the peritoneal cavity, implantation was successful only when both glandular and stromal components were present in the transplanted tissue (83, 84). This finding indicated a critical role for the stromal component in supporting the growth of the glands, and vice versa. Other experiments using estrogen receptor (ER)-␣ knockout mice demonstrated that the ER in the stromal compartment of the eutopic endometrium is critical for stimulation of growth of the glandular epithelial cells of this tissue. The growth of ER-negative glandular epithelium was stimulated by paracrine factors secreted from ER-positive endometrial 965
FIGURE 5 Defective inactivation of estradiol in endometriosis. Estradiol (E2) reaches the endometriotic lesions through the bloodstream (and possibly the peritoneal fluid). Additionally, aromatase (P450arom) in the stromal cell catalyzes the conversion of androstenedione (A) to estrone (E1), which is further reduced to E2 by 17-HSD type 1 in the endometriotic tissue. (At this time, the cell type that expresses 17-HSD type 1 in endometriotic lesions is not known.) E2 is normally inactivated by conversion to E1 by 17-HSD type 2 in epithelial cells of the eutopic endometrium during the secretory phase. In endometriotic tissue, however, E2 is not metabolized because of the lack of 17-HSD type 2, giving rise to increased local concentrations of this potent estrogen. Elevated E2, in turn, promotes the growth of endometriotic tissue and local PGE2 formation in stromal cells (62, 63). Because PGE2 is the most potent known inducer of aromatase in endometriosis, this will complete the positive feedback cycle that favors increased levels of E2 in endometriosis through enhanced biosynthesis and deficient metabolism.
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stroma stimulated with estrogen. ERs in the glandular epithelial cells were also found to have important functions during these elegant experiments (85, 86). Similar models were characterized in other tissues, including endometrial and breast carcinomas, where aromatase was expressed only in the stromal component whereas 17HSD type 1 was detected in the glandular cells (55). These two-cell models for estrogen biosynthesis represent cellular and molecular mechanisms that serve to protect eutopic endometrium from glandular hyperplasia and to promote growth in endometriosis. For example, the lack of aromatase in endometrium precludes local estrogen biosynthesis in this tissue. E2 that arrives in the endometrium through the circulation is inactivated to E1 by glandular 17-HSD type 2. On the other hand, aberrant aromatase expression in endometriotic stroma induces the conversion of circulating A 966
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to E1. The conversion of E1 to E2 in endometriotic cells is then catalyzed by 17-HSD type 1. Finally, the deficiency of 17HSD type 2 in endometriotic glandular cells allows the accumulation of local concentrations of E2 in this tissue (Fig. 5).
RATIONALE FOR USING AROMATASE INHIBITORS TO TREAT ENDOMETRIOSIS Endometriosis is successfully suppressed by estrogen deprivation using danazol or GnRH analogues, or by inducing surgical menopause. Danazol is no longer used because of its high rate of masculinizing side effects. Control of pelvic pain using GnRH agonists (GnRH-a) is usually successful during and immediately after the treatment, but pain associated with endometriosis returns in up to 75% of these treated women (87, 88). There are various potential explanations for the failure of GnRH-a treatment of endometriosis. First, suppression of ovarian estrogen secretion is transient (Fig. 6). Upon completion of a 6-month treatment, ovarian E2 secretion resumes and stimulates the growth of residual endometriosis. Another likely explanation for the failure of GnRH-a treatment is the presence of multiple extraglandular sites of estrogen biosynthesis in women (Fig. 6). In other words, significant E2 production continues in the adipose tissue, skin, and endometriotic implant per se during GnRH-a treatment. Therefore, blockage of aromatase activity in these extraovarian sites using an aromatase inhibitor may keep a larger number of patients in remission for a longer time. The most striking evidence for the importance of extraovarian estrogen production is the recurrence of endometriosis after successful hysterectomy and bilateral salpingo-oophorectomy in a number of women (71, 89). Endometriotic tissue in one such aggressive case was reported to express much higher levels of aromatase than premenopausal endometriosis (71). We recently reported the treatment of a 57-year-old overweight woman who had recurrence of severe endometriosis after hysterectomy and bilateral salpingo-oophorectomy (71). Two additional laparotomies were performed because of persistent severe pelvic pain and bilateral ureteral obstruction, leading to left renal atrophy and right hydronephrosis. Treatment for 4 months with megestrol acetate was ineffective. Levels of gonadotropins were within the postmenopausal range. A large (3-cm) vaginal endometriotic lesion contained unusually high levels of aromatase mRNA. The patient was given anastrozole (an aromatase inhibitor) orally at 1 mg/d together with elemental calcium at 1.5 g/d and alendronate (a nonsteroidal inhibitor of bone resorption) at 10 mg/d for 9 months. We observed dramatic relief of the pain and regression of the vaginal endometriotic lesion within the first month of treatment. At the same time, E2 Vol. 72, No. 6, December 1999
FIGURE 6 Site of action of GnRH agonists and aromatase inhibitors to treat endometriosis. This figure depicts the origins of estrogen in women with endometriosis: [1] delivery from the ovary and adipose/skin through the circulation and [2] local biosynthesis in endometriosis. GnRH agonists eliminate estradiol secreted by the ovary by down-regulating the pituitary hypothalamic unit. In cases resistant to treatment with GnRH agonists or in postmenopausal endometriosis, the use of aromatase inhibitors to block estrogen formation in the skin and adipose tissue as well as in endometriotic stromal cells may be critical in controlling the growth of endometriotic tissue. Recurrent endometriosis, especially after surgical removal of the ovaries, may represent lesions that are sensitive to extremely low levels of estradiol, and thus suppression of estradiol production in the periphery (adipose and skin) and in endometriotic tissue may be mandatory for successful treatment of endometriosis.
stimulate local biosynthesis of PGE2, which, in turn, stimulates aromatase expression (Fig. 3). In summary, the recently developed potent aromatase inhibitors are candidate drugs in the treatment of endometriosis that is resistant to standard regimens. In fact, aromatase inhibitors may be the only available treatment for aggressive postmenopausal endometriosis. The occurrence of substantial bone loss despite the addition of alendronate to the treatment regimen in this particular case should be studied further in large clinical trials. It remains to be seen whether aromatase inhibitors, alone or together with current means of therapy, in premenopausal women will increase the pain-free interval and time to recurrence after discontinuation of therapy (Fig. 6).
CONCLUSIONS The development and growth of endometriotic lesions are estrogen dependent. The mechanisms and effectiveness of hormonal treatments for endometriosis should be reevaluated in view of the new advances that have increased our understanding of the body sites of estrogen production in women with endometriosis. In addition to ovarian secretion of E2, E2 is produced in peripheral sites (skin and adipose tissue) and in the endometriotic lesion per se. We suggest that the intracrine and paracrine effects of E2 produced in the target tissue amplify estrogenic action in comparison with steroid hormones delivered through the circulation. Additionally, defective inactivation of E2 in endometriosis in contrast to eutopic endometrium may enhance this further.
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levels were reduced to 50% of baseline values. Markedly high pretreatment levels of aromatase mRNA in the endometriotic tissue became undetectable in a repeat biopsy 6 months later, and the lesion nearly disappeared after 9 months of therapy. We observed, however, a decrease in bone density of 6.2% in the lumbar spine. In this case, there were at least two potential mechanisms that accounted for the strikingly successful treatment of postmenopausal endometriosis using an aromatase inhibitor. First, there was evidence of suppression of peripheral (i.e., skin and adipose tissue) aromatase activity, causing a significant decrease in the serum E2 level. Second, unusually high levels of aromatase expression in the endometriotic lesion disappeared after treatment with the aromatase inhibitor anastrozole. Besides the expected direct inhibition of aromatase activity in endometriosis by anastrozole, the disappearance of aromatase mRNA expression in the lesion may be due to the denial of estrogen, which is known to FERTILITY & STERILITY威
Aberrant aromatase activity and defective E2 metabolism in endometriosis are consequences of specific molecular defects, such as inappropriate expression of a stimulatory transcription factor or resistance to P in this tissue. The clinical relevance of these findings was shown recently by the successful treatment of a severe case of recurrent postmenopausal endometriosis with use of an aromatase inhibitor. Future treatment strategies may be designed to target the signal transduction for aromatase expression in endometriosis or to enhance the action of P in this tissue.
Acknowledgments: The authors thank Rosemary Bell for skilled editorial assistance and Tom Ames for assistance with the figures.
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73. MacDonald PC. Determinants of the rate of estrogen formation in postmenopausal women. Eur J Obstet Gynecol Reprod Biol 1979;9: 187–9. 74. MacDonald PC, Grodin JM, Siiteri PK. The utilization of plasma androstenedione for estrone production in women. In: Excerpta Medica International Congress series no. 184, progress in endocrinology. Proceedings of the Third International Congress of Endocrinology (Mexico). 1968:770 – 6. 75. Gurpide E, MacDonald PC, Chapdelaine A, VandeWiele RL, Lieberman S. Studies on the secretion and interconversion of the androgens. II. Methods for estimation of rates of secretion and of metabolism from specific activities of urinary metabolites. J Clin Endocrinol Metab 1965;25:1537–56. 76. Labrie F, Luu-The V, Labrie C, Be´rube´ D, Couet J, Zhao H-F, et al. Characterization of two mRNA species encoding human estradiol 17dehydrogenase and assignment of the gene to chromosome 17. J Steroid Biochem 1989;34:189 –97. 77. Labrie Y, Durocher F, Lachance Y, Turgeon C, Simard J, Labrie C, et al. The human type II 17-hydroxysteroid dehydrogenase gene encodes two alternatively spliced mRNA species. DNA Cell Biol 1995;14:849 – 61. 78. Wu L, Einstein M, Geissler WM, Chan HK, Elliston KO, Andersson S. Expression cloning and characterization of human 17-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20␣-hydroxysteroid dehydrogenase activity. J Biol Chem 1993;268:12964 –9. 79. Andersson S, Moghrabi N. Physiology and molecular genetics of 17hydroxysteroid dehydrogenases. Steroids 1997;62:143–7. 80. Casey ML, MacDonald PC, Andersson S. 17-Hydroxysteroid dehydrogenase type 2: chromosomal assignment and progestin regulation of gene expression in human endometrium. J Clin Invest 1994;94:2135– 41. 81. Satyaswaroop PG, Wartell DJ, Mortel R. Distribution of progesterone receptor, estradiol dehydrogenase, and 20␣-dihydroprogesterone dehydrogenase activities in human endometrial glands and stroma: progestin induction of steroid dehydrogenase activities in vitro is restricted to the glandular epithelium. Endocrinology 1982;111:743–9. 82. Tseng L, Gurpide E. Estradiol and 20␣-dihydroprogesterone dehydrogenase activities in human endometrium during the menstrual cycle. Endocrinology 1974;94:419 –23. 83. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44:684 –94. 84. Zamah NM, Dodson MG, Stephens LC, Buttram VC Jr, Besch PK, Kaufman RH. Transplantation of normal and ectopic human endometrial tissue into athymic nude mice. Am J Obstet Gynecol 1984;149: 591–7. 85. Cooke PS, Buchanan DL, Young P, Setiawan T, Brody J, Korach KS, et al. Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. Proc Natl Acad Sci USA 1997;94:6535– 40. 86. Cooke PS, Buchanan DL, Lubahn DB, Cunha GR. Mechanism of estrogen action: lessons from the estrogen receptor-␣ knockout mouse. Biol Reprod 1998;59:470 –5. 87. Henzl MR, Corson SL, Moghissi K, Buttram VC, Berqvist C, Jacobson J. Administration of nasal nafarelin as compared with oral danazol for endometriosis. N Engl J Med 1988;318:485–9. 88. Waller KG, Shaw RW. Gonadotropin-releasing hormone analogues for the treatment of endometriosis: long-term follow-up. Fertil Steril 1993; 59:511–5. 89. Metzger DA, Lessey BA, Soper JT, McCarty KS Jr, Haney AF. Hormone-resistant endometriosis following total abdominal hysterectomy and bilateral salpingo-oophorectomy: correlation with histology and steroid receptor content. Obstet Gynecol 1991;78:946 –50.
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MODERN TRENDS
Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Edward E. Wallach, M.D. Associate Editor
Aromatase: a key molecule in the pathophysiology of endometriosis and a therapeutic target Khaled M. Zeitoun, M.D.,* and Serdar E. Bulun, M.D. Department of Obstetrics and Gynecology, The University of Texas Southwestern Medical Center, Dallas, Texas
Received April 23, 1999; revised and accepted May 11, 1999. Supported in part by an unrestricted research grant from the American Society for Reproductive Medicine– Organon, National Cancer Institute Grant CA-67167 and USAMRMC Grant DAMD17-97-1-7025 (to S.E.B.), and by an American Association of Obstetricians and Gynecologists Foundation Fellowship Award (to K.M.Z.). Reprint requests and present address: Serdar E. Bulun, M.D., Department of Obstetrics-Gynecology, University of Illinois at Chicago, 820 South Wood Street M/C 808, Chicago, Illinois 60610 (FAX: 312996-4238; E-mail: sbulun@uic .edu). * Present address: Department of Obstetrics and Gynecology, Columbia-Presbyterian Medical Center, 622 W. 168th Street, New York, New York 10032-3702. 0015-0282/99/$20.00 PII S0015-0282(99)00393-3
Objective: To provide a clinically useful model illustrating the molecular aberrations affecting estrogen biosynthesis and metabolism in endometriosis and to discuss the therapeutic role of aromatase inhibitors. Design: Literature review. Result(s): Several molecular aberrations were found in endometriotic lesions (in contrast to eutopic endometrium) that favor increased local concentrations of E2. Endometriotic stromal cells aberrantly express aromatase, which converts C19 steroids to estrogens. Aromatase activity in these cells is stimulated by prostaglandin (PG)E2. Estrogen stimulates cyclooxygenase-2, giving rise to increased PGE2 formation. Thus, this positive feedback loop produces increasing quantities of E2 and PGE2 in endometriosis. The lack of aromatase expression in eutopic endometrium is maintained by binding of an inhibitory transcription factor, COUP-TF, to the aromatase promoter. In endometriosis, however, an aberrantly expressed factor, SF-1, displaces COUP-TF to bind to this same promoter and activates aromatase expression and thus local estrogen biosynthesis. Additionally, endometriotic glandular cells are deficient in 17-hydroxysteroid dehydrogenase type 2, which converts E2 to estrone in the eutopic endometrium in response to P. Deficiency of this enzyme in endometriosis impairs the inactivation of E2 and may be a consequence of insensitivity to P. Conclusion(s): Molecular aberrations that increase local E2 concentrations may be important in the etiology of endometriosis. These molecules may be targeted to develop novel therapeutic strategies. The clinical relevance of aromatase expression in endometriosis was shown recently by the successful treatment of an unusually aggressive case of postmenopausal endometriosis with use of an aromatase inhibitor. (Fertil Steril威 1999;72:961–9. ©1999 by American Society for Reproductive Medicine.) Key Words: Aromatase, endometriosis, endometrium, estrogen, progesterone, 17-hydroxysteroid dehydrogenase type 2, aromatase inhibitor
Endometriosis is characterized by the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites and is linked to pelvic pain and infertility. It is estimated to affect 2%–10% of women in the reproductive age group (1–5). Although endometriosis is traditionally considered a disease of women in the reproductive age period, the natural history of this disorder, including onset and progression, has not been well established. For example, the incidences of endometriosis in teenagers and postmenopausal women are probably underestimated (6 –10). Endometriosis is considered a polygenically inherited disease with a complex, multifactorial etiology (11). Sampson’s theory (12, 13) of transplantation of endometrial tissue on the pelvic peritoneum through retrograde menstru-
ation is the most widely accepted explanation for the development of pelvic endometriosis because of convincing circumstantial and experimental evidence. Because retrograde menstruation is observed in almost all cycling women, endometriosis is postulated to develop as a result of a coexisting defect in clearance of the menstrual efflux from the pelvic peritoneal surfaces, possibly involving the immune system (14 –16). Alternatively, intrinsic molecular aberrations in pelvic endometriotic implants were proposed to contribute significantly to the development of endometriosis. Some of these molecular defects include aberrant expression of aromatase (17, 18), certain cytokines (19), and tissue metalloproteinases (20 –26); deficiency of 17-hydroxysteroid dehydrogenase 961
(17HSD) type 2 (27); and resistance to the protective action of P (21, 23, 25, 26). Because endometriosis is an estrogen-dependent disorder, aromatase expression and deficiency of 17-HSD type 2 are of paramount importance in the pathophysiology. In this article, we review the aberrant mechanisms of estrogen biosynthesis and metabolism in women with endometriosis, with an emphasis on identifying targets for new treatment strategies.
ESTROGEN BIOSYNTHESIS AND METABOLISM IN HUMANS
FIGURE 1 Extraovarian estrogen synthesis in women. Estradiol in women is either directly secreted by the ovary or produced in the extraglandular sites (adipose tissue and skin). The principal substrate for extraglandular aromatase activity in ovulatory women is androstenedione of adrenal and ovarian origin. In women receiving GnRH agonists or postmenopausal women, the adrenal gland remains the primary source of androstenedione. Androstenedione is converted by aromatase to estrone in adipose and skin fibroblasts. Estrone is further converted to estradiol by 17-HSD type 1 activity in peripheral tissues. Thus, peripheral aromatization is the major source of circulating estradiol in the postmenopausal period or during ovarian suppression.
The conversion of androstenedione (A) and T to estrone (E1) and E2 is catalyzed by aromatase P450 (P450arom), which is expressed in a number of human tissues and cells, such as ovarian granulosa cells, placental syncytiotrophoblast, adipose and skin fibroblasts, and the brain. In the reproductive-age woman, the ovary is the most important site of estrogen biosynthesis, and this takes place in a cyclic fashion. There is no marked E2 secretion from the ovary in the beginning of the menstrual cycle (during menstruation), but as the dominant follicle matures, granulosa cells replicate and express increasing levels of aromatase under the influence of FSH (28 –32). Aromatase catalyzes the conversion of A to E1, which is further converted to the potent estrogen E2 by the enzyme 17-HSD type 1 in the granulosa cell. At midcycle, peak serum E2 levels are attained just before ovulation, and ovarian E2 secretion from the corpus luteum then continues at lower levels. Upon binding of FSH to its G-protein– coupled receptor in the granulosa cell membrane, levels of intracellular cyclic adenosine monophosphate (cAMP) rise, which gives rise to the binding of two critical transcription factors, i.e., steroidogenic factor-1 (SF-1) and cAMP response element binding protein (CREB), to the classically located proximal promoter II of the aromatase gene (33, 34). This, in turn, activates aromatase mRNA and protein formation and is responsible for the striking stimulation of aromatase activity and, consequently, estrogen secretion from the preovulatory follicle (34, 35). In postmenopausal women, on the other hand, estrogen formation takes place in extraglandular tissues such as adipose and skin (36 –39) (Fig. 1). In contrast to cAMP regulation of aromatase expression in the ovary, this is controlled primarily by cytokines (interleukin [IL]-6, IL-11, tumor necrosis factor [TNF]-␣) and glucocorticoids through the alternative use of promoter I.4 in adipose and skin fibroblasts (35). It should be pointed out here that the major substrate for adipose/skin aromatase is A of adrenal origin. In postmenopausal women, approximately 2% of circulating A is converted to E1, which is further converted to E2 in peripheral tissues. This gives rise to pronounced serum levels of E2 962
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capable of causing endometrial hyperplasia or even carcinoma (36, 39 – 45).
AROMATASE EXPRESSION IN ¨ LLERIAN-DERIVED TISSUES MU Until recently, estrogen action was classically viewed to occur only through an “endocrine” mechanism. In other words, it was thought that only circulating E2, whether secreted by the ovary or formed in the adipose tissue, could exert an estrogenic effect after delivery to target tissues through the bloodstream. Studies on aromatase expression in breast cancer have demonstrated that paracrine mechanisms play an important role in estrogen action in this tissue (46 – 49). Estrogen produced by aromatase activity in breast adipose fibroblasts was demonstrated to promote the growth of adjacent malignant breast epithelial cells (50). Finally, we demonstrated a possible “intracrine” effect of estrogen in uterine leiomyomas and endometriosis (17, 51, 52). Estrogen produced by aromatase activity in the cytoplasm of leiomyoma smooth muscle cells or endometriotic stromal cells can exert its effects by readily binding to its nuclear receptor within the same cell. Vol. 72, No. 6, December 1999
Mu¨llerian tissues are known targets of estrogen action. Thus, the presence or absence of aromatase expression (i.e., estrogen biosynthesis) in the endometrium and myometrium has important physiologic and clinical consequences. Disease-free endometrium and myometrium lack aromatase expression (51, 53). On the other hand, neoplastic counterparts of these tissues display high levels of aromatase expression and activity (51, 54 –56). The expression of aromatase mRNA and protein was demonstrated in stromal cells of endometrial cancer and in smooth muscle cells of uterine leiomyomas (51, 55, 57, 58). Aromatase expression in both of these tumors is regulated by cAMP through the ovariantype promoter II.
FIGURE 2 Aromatase activity in endometriosis-derived stromal cells. Confluent stromal cells in primary culture were maintained for 24 hours in serum-free medium. Treatments consisted of the following: [1] Bt2cAMP (0.5 mmol/L); [2] sulprostone (10⫺6 mol/L), an Ep3 (Ep1, Ep2, Ep3 are PGE2 receptor subtypes) receptor agonist; [3] 11-deoxy-16,16-dimethyl prostaglandin E2 (10⫺6 mol/L), an Ep2 receptor agonist; and [4] 17-phenyl trinor prostaglandin E2 (10⫺6 mol/L), an Ep1 receptor agonist. The stimulation of Ep2 receptors has been demonstrated to increase intracytoplasmic cAMP levels.
In the normal ovary, aromatase expression is limited to granulosa cells and is absent in theca and other stromal cells (30, 59). It should also be pointed out that aromatase expression was demonstrated in the stromal cells of epithelial ovarian neoplasms (55, 59, 60). At present, the importance of aromatase expression (i.e., estrogen biosynthesis) in epithelial ovarian tumors is not known, but it is of interest that most of these aromatase-positive ovarian tumors were of the endometrioid type. Because endometrioid ovarian tumors are proposed to arise from foci of endometriosis, aromatase expression in these epithelial ovarian cancers and endometriosis may share common origins (55, 60, 61). In fact, the growth of endometrioid carcinomas arising from foci of endometriosis in the ovary may be stimulated by estrogen produced by local aromatase activity. Zeitoun. Pathophysiology of endometriosis. Fertil Steril 1999.
THE ROLE OF AROMATASE EXPRESSION IN ENDOMETRIOSIS Despite its role in other estrogen-responsive pelvic disorders, aromatase expression has been studied in greatest detail in endometriosis (17, 18, 52, 56). First, extremely high levels of aromatase mRNA were found in extraovarian endometriotic implants and endometriomas. Second, endometriosis-derived stromal cells in culture incubated with a cAMP analogue displayed extraordinarily high levels of aromatase activity comparable to that in placental syncytiotrophoblast (52). These exciting findings led us to test a battery of growth factors, cytokines, and other substances that might induce aromatase activity through a cAMP-dependent pathway in endometriosis.
Additionally, aromatase mRNA was detected in the eutopic endometrial samples of women with moderate to severe endometriosis but not in those of disease-free women, albeit in much smaller quantities compared with endometriotic implants (17). This might suggest a genetic defect in women with endometriosis, which is manifest by this subtle finding in the eutopic endometrium. We propose that when defective endometrium with low levels of aberrant aromatase expression reaches the pelvic peritoneum by retrograde menstruation, it causes an inflammatory reaction that exponentially increases local aromatase activity, i.e., estrogen formation, induced directly or indirectly by PGs and cytokines (52, 62). This may be a major contributing factor in the formation of endometriotic implants.
PGE2 was found to be the most potent known inducer of aromatase activity in endometriotic stromal cells (52). In fact, this PGE2 effect was found to be mediated by the cAMP-inducing Ep2 receptor subtype (Fig. 2 and our previously unpublished observations). Moreover, estrogen was reported to increase PGE2 formation by stimulating the cyclooxygenase type-2 enzyme in endometrial stromal cells in culture (62, 63). Thus, a positive feedback loop for continuous local production of estrogen and PGs is established, favoring the proliferative and inflammatory characteristics of endometriosis (Fig. 3).
It would be naive to propose that aberrant aromatase expression is the only important molecular mechanism in the development and growth of pelvic endometriosis. Endometriosis seems to be a polygenically inherited disease with a multifactorial etiology. There may be many important molecular mechanisms that favor the development of endometriosis, such as abnormal expression of proteinase-type enzymes that remodel tissues or their inhibitors (matrix metalloproteinases, tissue inhibitor of metalloproteinase-1), certain cytokines (IL-6, RANTES), and growth factors (epidermal growth factor) (19 –21, 23, 64, 65).
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FIGURE 3 Origin of estrogen in endometriotic lesions. Estradiol in an endometriotic lesion arises from several body sites. In an ovulatory woman, estradiol is secreted directly from the ovary in a cyclic fashion. In the early follicular phase and after menopause, peripheral tissues (adipose and skin) are the most important sources of circulating estradiol. Finally, estradiol is produced locally in the endometriotic implant itself in both ovulatory and postmenopausal women. The most important precursor, androstenedione of adrenal origin, becomes converted to estrone, which is in turn reduced to estradiol-17 in the peripheral tissues and endometriotic implants. We demonstrated significant levels of 17-hydroxysteroid dehydrogenase type-1 expression in endometriosis, which catalyzes the conversion of estrone to estradiol-17 (27). Estradiol-17 both directly and indirectly (through cytokines) induces PG synthase-2 (cyclooxygenase-2), which gives rise to elevated concentrations of PGE2 in endometriosis (62, 63). PGE2, in turn, is the most potent known stimulator of aromatase in endometriotic stromal cells (52). Therefore, a positive feedback loop in favor of continuous estrogen formation is established in endometriosis.
comparable to those in the placental syncytiotrophoblast (52). No significant stimulation was observed with various cytokines (IL-1, IL-2, IL-6, IL-11, oncostatin M, IL-15, or TNF-␣), growth factors (basic fibroblast growth factor or platelet-derived growth factor), or steroids (E2-P agonist R5020, or dexamethasone). As emphasized before, PGE2 was found to be the most potent known inducer of aromatase activity by increasing cAMP levels in endometriotic stromal cells. On the other hand, neither cAMP analogues nor PGE2 was capable of stimulating any detectable aromatase activity in eutopic endometrial stromal cells in culture. The obvious question concerned the molecular differences that induce aromatase expression in endometriosis and its inhibition in eutopic endometrium. To address this, we first determined that the cAMP-inducible promoter II was used for in vivo aromatase expression in endometriotic tissues and in cAMP- or PGE2stimulated endometriotic stromal cells. Then, we found that a stimulatory transcription factor, steroidogenic factor-1 (SF-1), and an inhibitory factor, chicken ovalbumin upstream promoter transcription factor (COUP-TF), competed for the same binding site in the aromatase promoter II. COUP-TF was expressed ubiquitously in both eutopic endometrium and endometriosis, whereas SF-1 was expressed specifically in endometriosis but not in eutopic endometrium. SF-1 binds to the aromatase promoter more avidly than COUP-TF. Thus, SF-1 and other transcription factors (e.g., CREB) bind to promoter II and interact to initiate transcription of the aromatase gene in endometriosis, whereas COUP-TF, which occupies the same DNA site in eutopic endometrium, inhibits this process (18) (Fig. 4).
Zeitoun. Pathophysiology of endometriosis. Fertil Steril 1999.
Alternatively, a defective immune system that fails to clear the peritoneal surfaces of the retrograde menstrual efflux has been proposed in the development of endometriosis (14 –16, 25, 66 –70). The development of endometriosis in an individual woman probably requires the coexistence of a threshold number of these genetic aberrations. Nonetheless, aberrant aromatase expression is clinically relevant because aromatase inhibitors suppress postmenopausal endometriosis (71).
REGULATION OF AROMATASE EXPRESSION IN ENDOMETRIOTIC STROMAL CELLS Aromatase activity in endometriotic stromal cells was stimulated by cAMP analogues to strikingly high levels 964
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In summary, one of the molecular alterations leading to local aromatase expression in endometriosis but not in normal endometrium is the aberrant production of SF-1 in endometriotic stromal cells, which overcomes the protective inhibition of transcription of the aromatase gene maintained normally by COUP-TF in the eutopic endometrium.
INTERCONVERSIONS OF E1 AND E2 IN ENDOMETRIOSIS The primary substrate for aromatase activity in endometriosis is A of adrenal and ovarian origin in premenopausal women and adrenal A in postmenopausal women. Because the concentration of T in circulating blood is extremely low in women (approximately one-tenth the level of A) and in vivo peripheral aromatization of this steroid takes place in much lower quantities compared with that of A, aromatization of T in endometriosis is probably not significant (36, 38, 40, 72–75). The major product of aromatase activity in endometriosis, namely E1, is only weakly estrogenic and must be converted to the potent estrogen E2 to exert a full estrogenic effect. We demonstrated that the enzyme 17-HSD type 1, Vol. 72, No. 6, December 1999
FIGURE 4 Proposed mechanism of regulation of aromatase (P450arom) expression by SF-1 and COUP-TF in eutopic endometrium and endometriosis. (A), Binding of COUP-TF (dimer) to a specific DNA site (nuclear receptor half-site) upstream of aromatase promoter II in eutopic endometrial stromal cells. In the eutopic endometrium, COUP-TF binds to nuclear receptor half-site practically in the absence of any competition by SF-1 because SF-1 expression is not detected in the majority of endometrial samples. Thus, COUP-TF exerts its inhibitory effect on the complex of general transcription factors (GTFs) that bind to the TATA box. (B), In the endometriotic stromal cell, where SF-1 is also present, SF-1 binds as a monomer to the nuclear receptor half-site with a higher affinity than COUP-TF, which binds to this site relatively loosely as a dimer. Upon replacing COUP-TF, SF-1 synergizes with cAMP response element binding protein (CREB, bound to upstream CRE) to activate transcription of the aromatase gene in response to cAMP (18).
17-HSD type 2 in endometrial glands and its enzymatic activity have been viewed as protective against E2 action in eutopic endometrium. Because P stimulates 17-HSD type-2 expression in the endometrium, inactivation of E2 to E1 may be considered as one of the antiestrogenic properties of P (81). The expression of 17-HSD type 2 is deficient in endometriotic glandular cells (27). This was confirmed in paired samples of eutopic endometrium and pelvic endometriosis obtained simultaneously during the luteal phase. No detectable mRNA or protein could be shown in endometriosis, whereas abundant 17-HSD type-2 expression was demonstrated in eutopic endometrium. Thus, this protective mechanism, which lowers E2 levels, is lost in endometriosis (27). The aberrant expression of aromatase, the presence of 17HSD type 1, and the absence of 17-HSD type 2 in endometriosis collectively give rise to elevated local levels of E2 compared with eutopic endometrium. Besides the direct mitogenic effects of E2, these events may be viewed as a defective action of P, which fails to induce 17-HSD type 2 in endometriosis (Fig. 5). This is important because P antagonizes the formation of endometriosis, and administration of progestins is an important treatment modality in this disease. Other evidence of resistance to P is shown in studies on dioxin, an environmental toxin associated with the development of endometriosis. In dioxin-treated rat models of endometriosis, the effect of dioxin seems to involve blockage of the inhibitory effect of P on lesion development in association with defective transforming growth factor- expression (23, 25).
EPITHELIAL-STROMAL INTERACTIONS IN ENDOMETRIUM AND ENDOMETRIOSIS
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which catalyzes the conversion of E1 to E2, is expressed in endometriosis (27, 76). In contrast to 17-HSD type 1, the enzyme 17-HSD type 2 (encoded by a separate gene) catalyzes the conversion of E2 to E1 (77–79). In other words, it inactivates E2. One of the major tissue sites of 17-HSD type-2 expression is endometrial glandular cells during the luteal phase (80). In fact, induction of the activity of this enzyme by P in endometrial glands had been demonstrated by a series of earlier studies (81, 82). The expression of FERTILITY & STERILITY威
Compartmentalization of steroidogenic enzyme expression, i.e., aromatase in stromal cells of endometriosis and 17-HSD type 2 in secretory glandular epithelial cells of endometrium, points to important epithelial-stromal interactions. In fact, other epithelial-stromal interactions in endometrium and endometriosis were reported previously. For example, when experimental endometriotic lesions were induced in rodents by transplanting endometrium to the peritoneal cavity, implantation was successful only when both glandular and stromal components were present in the transplanted tissue (83, 84). This finding indicated a critical role for the stromal component in supporting the growth of the glands, and vice versa. Other experiments using estrogen receptor (ER)-␣ knockout mice demonstrated that the ER in the stromal compartment of the eutopic endometrium is critical for stimulation of growth of the glandular epithelial cells of this tissue. The growth of ER-negative glandular epithelium was stimulated by paracrine factors secreted from ER-positive endometrial 965
FIGURE 5 Defective inactivation of estradiol in endometriosis. Estradiol (E2) reaches the endometriotic lesions through the bloodstream (and possibly the peritoneal fluid). Additionally, aromatase (P450arom) in the stromal cell catalyzes the conversion of androstenedione (A) to estrone (E1), which is further reduced to E2 by 17-HSD type 1 in the endometriotic tissue. (At this time, the cell type that expresses 17-HSD type 1 in endometriotic lesions is not known.) E2 is normally inactivated by conversion to E1 by 17-HSD type 2 in epithelial cells of the eutopic endometrium during the secretory phase. In endometriotic tissue, however, E2 is not metabolized because of the lack of 17-HSD type 2, giving rise to increased local concentrations of this potent estrogen. Elevated E2, in turn, promotes the growth of endometriotic tissue and local PGE2 formation in stromal cells (62, 63). Because PGE2 is the most potent known inducer of aromatase in endometriosis, this will complete the positive feedback cycle that favors increased levels of E2 in endometriosis through enhanced biosynthesis and deficient metabolism.
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stroma stimulated with estrogen. ERs in the glandular epithelial cells were also found to have important functions during these elegant experiments (85, 86). Similar models were characterized in other tissues, including endometrial and breast carcinomas, where aromatase was expressed only in the stromal component whereas 17HSD type 1 was detected in the glandular cells (55). These two-cell models for estrogen biosynthesis represent cellular and molecular mechanisms that serve to protect eutopic endometrium from glandular hyperplasia and to promote growth in endometriosis. For example, the lack of aromatase in endometrium precludes local estrogen biosynthesis in this tissue. E2 that arrives in the endometrium through the circulation is inactivated to E1 by glandular 17-HSD type 2. On the other hand, aberrant aromatase expression in endometriotic stroma induces the conversion of circulating A 966
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to E1. The conversion of E1 to E2 in endometriotic cells is then catalyzed by 17-HSD type 1. Finally, the deficiency of 17HSD type 2 in endometriotic glandular cells allows the accumulation of local concentrations of E2 in this tissue (Fig. 5).
RATIONALE FOR USING AROMATASE INHIBITORS TO TREAT ENDOMETRIOSIS Endometriosis is successfully suppressed by estrogen deprivation using danazol or GnRH analogues, or by inducing surgical menopause. Danazol is no longer used because of its high rate of masculinizing side effects. Control of pelvic pain using GnRH agonists (GnRH-a) is usually successful during and immediately after the treatment, but pain associated with endometriosis returns in up to 75% of these treated women (87, 88). There are various potential explanations for the failure of GnRH-a treatment of endometriosis. First, suppression of ovarian estrogen secretion is transient (Fig. 6). Upon completion of a 6-month treatment, ovarian E2 secretion resumes and stimulates the growth of residual endometriosis. Another likely explanation for the failure of GnRH-a treatment is the presence of multiple extraglandular sites of estrogen biosynthesis in women (Fig. 6). In other words, significant E2 production continues in the adipose tissue, skin, and endometriotic implant per se during GnRH-a treatment. Therefore, blockage of aromatase activity in these extraovarian sites using an aromatase inhibitor may keep a larger number of patients in remission for a longer time. The most striking evidence for the importance of extraovarian estrogen production is the recurrence of endometriosis after successful hysterectomy and bilateral salpingo-oophorectomy in a number of women (71, 89). Endometriotic tissue in one such aggressive case was reported to express much higher levels of aromatase than premenopausal endometriosis (71). We recently reported the treatment of a 57-year-old overweight woman who had recurrence of severe endometriosis after hysterectomy and bilateral salpingo-oophorectomy (71). Two additional laparotomies were performed because of persistent severe pelvic pain and bilateral ureteral obstruction, leading to left renal atrophy and right hydronephrosis. Treatment for 4 months with megestrol acetate was ineffective. Levels of gonadotropins were within the postmenopausal range. A large (3-cm) vaginal endometriotic lesion contained unusually high levels of aromatase mRNA. The patient was given anastrozole (an aromatase inhibitor) orally at 1 mg/d together with elemental calcium at 1.5 g/d and alendronate (a nonsteroidal inhibitor of bone resorption) at 10 mg/d for 9 months. We observed dramatic relief of the pain and regression of the vaginal endometriotic lesion within the first month of treatment. At the same time, E2 Vol. 72, No. 6, December 1999
FIGURE 6 Site of action of GnRH agonists and aromatase inhibitors to treat endometriosis. This figure depicts the origins of estrogen in women with endometriosis: [1] delivery from the ovary and adipose/skin through the circulation and [2] local biosynthesis in endometriosis. GnRH agonists eliminate estradiol secreted by the ovary by down-regulating the pituitary hypothalamic unit. In cases resistant to treatment with GnRH agonists or in postmenopausal endometriosis, the use of aromatase inhibitors to block estrogen formation in the skin and adipose tissue as well as in endometriotic stromal cells may be critical in controlling the growth of endometriotic tissue. Recurrent endometriosis, especially after surgical removal of the ovaries, may represent lesions that are sensitive to extremely low levels of estradiol, and thus suppression of estradiol production in the periphery (adipose and skin) and in endometriotic tissue may be mandatory for successful treatment of endometriosis.
stimulate local biosynthesis of PGE2, which, in turn, stimulates aromatase expression (Fig. 3). In summary, the recently developed potent aromatase inhibitors are candidate drugs in the treatment of endometriosis that is resistant to standard regimens. In fact, aromatase inhibitors may be the only available treatment for aggressive postmenopausal endometriosis. The occurrence of substantial bone loss despite the addition of alendronate to the treatment regimen in this particular case should be studied further in large clinical trials. It remains to be seen whether aromatase inhibitors, alone or together with current means of therapy, in premenopausal women will increase the pain-free interval and time to recurrence after discontinuation of therapy (Fig. 6).
CONCLUSIONS The development and growth of endometriotic lesions are estrogen dependent. The mechanisms and effectiveness of hormonal treatments for endometriosis should be reevaluated in view of the new advances that have increased our understanding of the body sites of estrogen production in women with endometriosis. In addition to ovarian secretion of E2, E2 is produced in peripheral sites (skin and adipose tissue) and in the endometriotic lesion per se. We suggest that the intracrine and paracrine effects of E2 produced in the target tissue amplify estrogenic action in comparison with steroid hormones delivered through the circulation. Additionally, defective inactivation of E2 in endometriosis in contrast to eutopic endometrium may enhance this further.
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levels were reduced to 50% of baseline values. Markedly high pretreatment levels of aromatase mRNA in the endometriotic tissue became undetectable in a repeat biopsy 6 months later, and the lesion nearly disappeared after 9 months of therapy. We observed, however, a decrease in bone density of 6.2% in the lumbar spine. In this case, there were at least two potential mechanisms that accounted for the strikingly successful treatment of postmenopausal endometriosis using an aromatase inhibitor. First, there was evidence of suppression of peripheral (i.e., skin and adipose tissue) aromatase activity, causing a significant decrease in the serum E2 level. Second, unusually high levels of aromatase expression in the endometriotic lesion disappeared after treatment with the aromatase inhibitor anastrozole. Besides the expected direct inhibition of aromatase activity in endometriosis by anastrozole, the disappearance of aromatase mRNA expression in the lesion may be due to the denial of estrogen, which is known to FERTILITY & STERILITY威
Aberrant aromatase activity and defective E2 metabolism in endometriosis are consequences of specific molecular defects, such as inappropriate expression of a stimulatory transcription factor or resistance to P in this tissue. The clinical relevance of these findings was shown recently by the successful treatment of a severe case of recurrent postmenopausal endometriosis with use of an aromatase inhibitor. Future treatment strategies may be designed to target the signal transduction for aromatase expression in endometriosis or to enhance the action of P in this tissue.
Acknowledgments: The authors thank Rosemary Bell for skilled editorial assistance and Tom Ames for assistance with the figures.
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