Biological mechanisms underlying the clinical effects of mifepristone (RU 486) on the endometrium.

The abortifacient and menstrual effects of the potent antiprogestin, RU 486 (mifepriston) are associated with both endometrial hemorrhage and extracellular matrix (ECM) degradation. Such processes reflect reduced perivascular decidual cell hemostatic and increased ECM-degrading protease activity. In this review, we summarize the effects of RU 486 on different proteases involved in these processes and expressed by in vitro decidualized endometrium stromal cells. The expression of tissue factor (TF), the primary initiator of hemostasis; urokinase-type plasminogen activator (uPA); tissue-type plasminogen activator (tPA); plasminogen activator inhibitor-1 (PAI-1) as well as the potent matrix metalloprotease, MMP-3 was assessed. These endpoints of decidualization are regulated by progesterone. It was observed, that RU 486 blocks and reverses progestin-enhanced stromal cell TF protein and mRNA expression and PAI-1 protein and mRNA expression, whereas blocks and reverses progestin-inhibited stromal cell uPA, tPA and MMP-3 protein and mRNA expression. These coordinate enhancement of plasminogen activator and MMP-3 expression promotes proteolysis of the decidual ECM, which leads to endometrial sloughing during menstruation. Moreover, destabilization of endometrial microvessels resulting from degradation of their surrounding ECM is consistent with the heavy bleeding after RU 486 administration. On the other hand, with blocking the expression of TF and PAI-1, RU 486 creates a haemorrhagic and fibrinolytic milieu around the endometrial vessels, suggesting a mechanism for RU 486-induced endometrial hemorrhage. The steroid antagonist RU 486 (mifepristone) causes menstrual bleeding when given during the luteal phase of the menstrual cycle (1) and induces abortion in 64-85% of pregnant patients when administered before the 50. postmenstrual days (2, 3). These clinical actions are thought to reflect the antiprogestational effects of RU 486. Pathologic studies showed, that the effects of RU 486 on primate and human luteal phase endometrium include reduced stromal edema, increased venular diameter. Erythrocyte and leukocyte diapedesis, focal hemorrhage, degeneration of the stromal extracellular matrix, and eventual disruption of the superficial layer of the endometrium (4, 5). This antihormone acts at the receptor level and possibly also at the postreceptor level(s) (6). The most important mechanism of action is to compete with progesterone at the level of their respective binding site in the ligand binding domain of the progesterone receptors. The binding of RU 486 to the receptor leads to conformational changes in the DNA-binding site of the progesterone-receptor (7). As a consequnce of these changes, the interaction between the receptor and the progesterone-response elements in the promoter region of progesterone-responsive genes is altered (7). The menorrhagic and abortifacient properties of RU 486 are associated with the induction of endometrial hemorrhage. The physiological mechanisms by which human endometrium permits menstrual hemorrhage in the absence of pregnancy yet maintains hemostasis during endovascular trophoblast invasion (avoiding early abortion) has been investigated in our laboratory by evaluating endometrial expression of different proteins that play role in the process of hemostasis. Besides the endometrial haemostasis, we also examined the proteolytic processes leading extracellular matrix (ECM) degradation, which is also an integral part of menstruation. In this review we sought to summarize the biological mechanisms underlying the clinical effects of RU 486 on endometrial haemorrhage/haemostasis and on ECM degradation.

Implications of decidualization-associated protease expression in implantation and menstruation.

During progesterone-induced decidualization of estradiol (E2)-primed human endometrial stromal cells (HESCs), the interstitial-type extracellular matrix (ECM) of the follicular phase endometrium is transformed in the luteal phase to a mixture of residual interstitial- and new basal laminar-type components. This transformation is accelerated by reduced proteolytic activity of HESCs undergoing decidualization (DZ). In cultured HESCs, progestins, but not E2, induce the expression of several DZ markers, and E2 enhances these effects despite the lack of response to E2 alone. Using this well-characterized in vitro DZ model we evaluated the expression of plasminogen activators (PAs), which degrade ECM components that undergo rapid turnover, and matrix metalloproteinases (MMPs), which degrade the bulk of ECM components. Medroxyprogesterone acetate (MPA) inhibited the catalytic activity of urokinase-type PA (uPA) and tissue-type PA (tPA) as well as the expression of such MMPs as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3). Moreover, E2 + MPA elicited greater inhibitory effects on the expression of all of these proteases. Progestin inhibition of PA activities reflected reciprocal upregulation in the output of the PA inhibitor PAI-1, which produced large molar excesses of PAI-1 compared with the PAs in HESC-conditioned medium. By contrast, the tissue inhibitor of the MMPs, TIMP1, as well as gelatinase A (MMP-2), was constitutively expressed by the HESCs. In the absence of implantation, menstruation-associated degradation of the functional endometrial ECM is triggered by withdrawal of circulating ovarian steroids. This process was evaluated in cultured HESCs that were first decidualized during 10 days of exposure to E2 + MPA, and then withdrawn to steroid-free medium with and without the antiprogestin RU 486. As expected, steroid withdrawal reversed progestin-inhibited PA activity as well as the expression of MMP-1 and MMP-3 and progestin-enhanced PAI-1; much greater reversal was observed in medium supplemented with RU 486. Unlike the changes in PAI-1, neither TIMP1, nor MMP-2 expression was affected by withdrawal to steroid-free or to RU 486-medium. By altering the composition of the ECM of the luteal phase endometrium, progestin-elicited inhibition of the PAs, uPA and tPA, as well as that of the MMPs, MMP-1 and MMP-3, modulates trophoblast adhesion, migration and differentiation. Conversely, steroid withdrawal elicited increases in uPA, MMP-1 and MMP-3 activities would promote endometrial sloughing by degrading the mixture of decidual cell-derived basement membrane-like proteins and interstitial components that comprise the stromal ECM of the perimenstrual endometrium.

Matrix metalloproteinase and matrix metalloproteinase inhibitor expression in endometrial stromal cells during progestin-initiated decidualization and menstruation-related progestin withdrawal.

Estradiol (E) primes human endometrial stromal cells (HESCs) for the decidualizing effects of progesterone in vivo and in vitro. Matrix metalloproteinase (MMP) expression was evaluated in confluent HESCs incubated in control medium, and in medium supplemented with either E, or the synthetic progestin medroxyprogesterone acetate (P), or E + P. Measurements with a specific ELISA indicated that basal pro-MMP-1 output was unaffected by E, whereas E + P, which induces the expression of several decidualization-related markers, produced a time-dependent inhibition in HESC-secreted levels of pro-MMP-1. Consistent with progestin inhibition of MMP-1 protein expression in the HESCs, P but not E, reduced steady state levels of MMP-1 messenger RNA (mRNA) as determined by Northern analysis. By contrast, mRNA levels for MMP-2 and the MMP inhibitor TIMP-1 were not altered by either P or E. Steroid withdrawal studies indicated that after MMP-1 expression was suppressed by incubation of the HESCs with E + P, 4 days of exposure to the antiprogestin RU 486 (mifepristone) significantly up-regulated MMP-1 levels in the conditioned medium by severalfold compared with cultures maintained in E + P. The change to steroid-free control medium required a more prolonged period of withdrawal to attain up regulatory effects that were comparable with those evoked by RU 486. The ELISA measurements were validated by immunoblot analysis with a specific MMP-1 antibody, which showed corresponding changes in a band at the expected mobility of about 50 kDa. Moreover, Northern analysis revealed parallel changes in MMP-1 mRNA levels, whereas neither MMP-2 nor TIMP-1 mRNA levels were modulated by adding or withdrawing steroids. The contrast between regulated MMP-1 expression and constitutive MMP-2 expression observed in the cultured HESCs is consistent with the demonstrated presence on the MMP-1 promoter of regulatory elements such as AP-1 and PEA-3 that are absent from the MMP-2 promoter. Extrapolation of these in vitro changes in HESCs to in vivo endometrial events suggests that: 1) inhibition of MMP-1 expression by E and progesterone would stabilize the perivascular endometrial ECM to prevent local hemorrhage during endovascular invasion by the implanting trophoblast; 2) enhanced expression of MMP-1 evoked by steroid withdrawal would mediate endometrial ECM degradation leading to sloughing of the functional layer during menstruation.

Decidual cell regulation of hemostasis during implantation and menstruation.

Progesterone stimulation of the estradiol (E2)-primed human endometrium initiates DZ of the stromal cells around the spiral arterioles. Under continued steroid stimulation, DZ spreads wave-like to establish the decidual cell as a major cell type of the luteal phase and pregnant endometrium. Because of their widespread distribution throughout the endometrium and concentration at perivascular sites, decidual cells are spatially and temporally positioned to mediate the opposing requirements of maintaining hemostasis during endovascular trophoblast invasion, yet promoting menstrual hemorrhage in the absence of implantation. The experimental results summarized in this review indicate that the paradoxical properties manifested by endometrial stromal/decidual cells are controlled by several proteins with either hemostatic or ECM-degrading or vasoactive activity, and that their expression is altered in response to changes in levels of circulating ovarian steroids during the menstrual cycle. These conclusions are drawn primarily from studies with a well-characterized in vitro model of DZ using monolayers of stromal cells derived from specimens of predecidualized endometrium. Thus, progestins modify the expression of several DZ-related markers in the cultured stromal cells, and E2 enhances these effects despite the lack of response to E2 alone. These responses are consistent with the differential actions displayed by E2 and progesterone in vivo, by which E2 primes the endometrium for the decidualizing effects of progesterone by elevating progesterone receptor levels. Accordingly, during steroid-induced in vitro DZ, a marked increase in the expression of stromal cell TF and PAI-1 and reciprocal inhibition of tPA activity suggest mechanisms to account for the absence of hemorrhage during invasion of the endometrial vasculature by implanting trophoblasts. In contrast to steroid-induced DZ, the events of menstruation are initiated in response to a decline in circulating levels of ovarian steroids. Accordingly, subjecting in vitro decidualized stromal cells to steroid withdrawal results in pronounced reversal in the expression of all of the end points listed above. Consequently, the local hemostatic environment is transformed into a hemorrhage-promoting milieu. Taken together with vascular injury resulting from ischemia induced by spiral artery vasoconstriction, the net effect is attainment of two prerequisites for menstrual hemorrhage, vascular injury and inadequate hemostasis.

Biological mechanisms underlying the clinical effects of RU 486: modulation of cultured endometrial stromal cell stromelysin-1 and prolactin expression.

During in vitro decidualization of human endometrial stromal cells (HESCs), medroxyprogesterone acetate (MPA) inhibits expression of the potent extracellular matrix (ECM)-degrading protease stromelysin-1 (MMP-3), but enhances PRL expression. Consistent with its priming role in vivo, estradiol (E2) augments these effects. In the current study, immunoblot analysis revealed that coincubation with 10(-6) M RU 486 blocked the inhibition in HESC-secreted MMP-3 levels (50,000 mol wt) evoked by 10(-8) M E2 + 10(-7) M MPA. Although MPA can act as a glucocorticoid, the HESCs were refractory to 10(-7) M dexamethasone added alone or with E2. Because E2 elevates progesterone but not glucocorticoid receptor levels, MPA and RU 486 control MMP-3 expression as a progestin and antiprogestin, respectively. To study RU 486 involvement in steroid withdrawal leading to menstruation, HESCs were decidualized during 10 days incubation with E2 + MPA, and parallel cultures were kept in E2 + MPA or withdrawn to either control or RU 486-containing medium. Compared with E2 + MPA-suppressed HESCs, increases in levels of secreted MMP-3 (2.0-fold), and its 2.1-kilobase messenger RNA (10-fold) were observed in HESCs after 4 days of withdrawal to control medium, with much greater increases seen in RU 486-containing medium (10-fold protein, 100-fold messenger RNA). Previously, we showed that RU 486 up-regulated E2 + MPA-inhibited plasminogen activator expression in the cultured HESCs. Extrapolation of these in vitro observations to endometrial events following RU 486 administration suggests that coordinate enhancement of MMP-3 and plasminogen activator expression promotes proteolysis of the stromal/decidual ECM, which leads to endometrial sloughing. Moreover, destabilization of endometrial microvessels resulting from degradation of their surrounding ECM is consistent with the heavy menstrual bleeding stemming from RU 486 administration. However, in contrast to the marked RU 486-initiated reversal of MMP-3 expression, RU 486 did not significantly reverse E2 + MPA-enhanced PRL secretion by the cultured HESCs. Interestingly, decidual PRL, unlike decidual MMP-3, does not appear to play a role in menstruation. Interleukin-1 beta counteracted E2 + MPA-mediated inhibition of secreted MMP-3 levels, implying that leukocyte/trophoblast-derived cytokines can modulate steroid-regulated MMP-3 expression by stromal/decidual cells during menstruation and pregnancy.

Human endometrial decidual cell-associated 11 beta-hydroxysteroid dehydrogenase expression: its potential role in implantation.

During pregnancy excess corticosteroid exposure can disturb the normal pattern of growth and differentiation of the primate fetus. This is normally prevented by the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which converts cortisol to its biologically inactive 11-oxo form, thereby ensuring that little or no cortisol is transferred to the fetus. During implantation, extravillous trophoblasts breech uterine vessels that are embedded in a decidual cell matrix. Through this invasive process the embryo gains requisite access to the maternal blood supply, while risking exposure to high circulating glucocorticoid levels. Thus, the expression of 11 beta-HSD by the decidual cell layer may be essential in regulating cortisol exposure of the developing embryo prior to placentation. In order to investigate the potential contribution of decidual cells to glucocorticoid metabolism, we evaluated the expression of both known 11 beta-HSD isoforms, 11 beta-HSD1, whose catalytic activity is NADP(+)-dependent, and NAD(+)-dependent 11 beta-HSD2, during decidualization of monolayers of human endometrial stromal cells. The differential actions of ovarian steroids on human endometrium are simulated in this in vitro model. Thus, progestins induce the expression of several decidualization markers in the cultured stromal cells, and consistent with its priming action in vivo, estradiol augments this expression. The results of our studies established a link between in vitro decidualization and enhanced glucocorticoid metabolizing capacity. Accordingly, the catalytic activities of both 11 beta-HSD isoforms were enhanced by incubation of the precursor stromal cells with medroxyprogesterone acetate, and further enhanced by estradiol, despite a lack of response to estradiol alone. This differential response to estradiol and progestin was reflected in parallel changes in steady state levels of 11 beta-HSD1 messenger RNA. The role of glucocorticoid metabolizing activity of the decidual cell is discussed in terms of its implications in determining the exposure of the implanting embryo to biologically active glucocorticoids.

Plasminogen activator activity during decidualization of human endometrial stromal cells is regulated by plasminogen activator inhibitor 1.

Progesterone acts on the estradiol (E2)-conditioned human endometrium to induce decidualization of stromal cells. Consistent with these differential hormone actions in vivo, progestins regulate several end points of decidualization in human endometrial stromal cell monolayers, and E2 augments the effects of progestin. This study shows that in vitro decidualization of the stromal cells is accompanied by diminished plasminogen activator (PA) expression. Polyacrylamide gel electrophoretic separation after immunoprecipitation of biosynthetically labeled PAs revealed that medroxyprogesterone acetate (MPA) lowered levels of secreted tissue type PA (tPA) at 67 kilodaltons and urokinase type PA (uPA) at 55 kilodaltons. These levels were reduced further by E2 plus MPA despite a lack of response to E2 alone. Although tPA activity was readily measured by a chromogenic assay, detection of uPA activity required prior activation, indicating that uPA is released as the pro-uPA zymogen. Comparisons of levels of immunogenic PAs, as measured by specific enzyme-linked immunosorbent assays, with the corresponding catalytic activities revealed selective progestational inhibition of PA activity vs. antigen after 3 days of experimental incubation. Thus, 10(-7) mol/L MPA produced about a 2-fold greater reduction of levels of PA activity than that of its corresponding antigen. More strikingly, 10(-8) mol/L E2 plus 10(-7) mol/L MPA virtually eliminated both tPA activity (99% inhibition; P < 0.005) and uPA activity (93% inhibition; P < 0.005); the reductions in levels of the corresponding antigens were only about 50% of the control levels and did not attain statistical significance. Only after 3-6 days of incubation with E2 plus MPA was statistically significant inhibition achieved for immunogenic levels of both tPA (P < 0.05) and uPA (P < 0.005). Preferential inhibition of levels of PA activities compared with those of the corresponding PA antigens reflects the action of the potent PA inhibitor PAI-1. Thus, the concentration of PAI-1 in the stromal cell-conditioned medium at the end of 0-3 days exceeded those of tPA and uPA, respectively, by 28- and 12-fold in response to MPA and by 52- and 25-fold in response to E2 plus MPA. Extrapolation of these in vitro results to the events of the luteal phase, whose steroidal milieu is mimicked by E2 plus MPA, indicates that decidual cell-derived PAI-1 is a key regulator of proteolytic degradation of extracellular matrix and fibrinolysis during implantation and menstruation.

The role of progestationally regulated stromal cell tissue factor and type-1 plasminogen activator inhibitor (PAI-1) in endometrial hemostasis and menstruation.

The physiologic mechanisms whereby the human endometrium maintains hemostasis during endovascular trophoblast invasion, yet permits menstrual hemorrhage, are unknown. This paradoxical relationship was investigated by evaluating endometrial expression of tissue factor (TF), the primary initiator of hemostasis, and plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of fibrinolysis. We observed increased immunostaining for TF and PAI-1 in sections of decidualized stromal cells from luteal phase and gestational endometrium. To determine whether TF and PAI-1 expression are directly linked to decidualization, both endpoints were monitored in a well described in vitro model of decidualization. Thus, confluent stromal cell cultures were exposed to vehicle control, 10(-8) M estradiol (E2), 10(-8) to 10(-6) M medroxyprogesterone acetate (MPA) or both E2 + MPA for 2-24 days in serum-containing or defined media. The progestin enhanced the content of stromal cell-associated immunoreactive and functionally active TF and PAI-1 released into the medium and elevated levels of stromal cell TF and PAI-1 mRNA. While E2 alone was ineffective, it greatly augmented MPA-enhanced TF and PAI-1 protein and mRNA content. Dose-dependent effects on TF and PAI-1 content were observed between 10(-8) to 10(-6) M MPA +/- E2. Similar results were observed for decidual cells derived from first trimester endometrium and cultured in type 1 collagen gels. Following optimal induction of TF and PAI-1 expression by E2 + MPA in stromal cell cultures, removal of these steroids greatly reduced levels of both TF and PAI-1 protein and mRNA within 4 days. These studies suggest a mechanism whereby endometrial hemostasis is maintained during trophoblast invasion yet reduced at the end of nonfertile cycles to permit menses.

Steroid-modulated stromal cell tissue factor expression: a model for the regulation of endometrial hemostasis and menstruation.

This study examined steroidal regulation of tissue factor expression by cultured endometrial stromal cells. Confluent stromal cell cultures derived from cycling human endometria were exposed to vehicle control, 10(-8) mol/L estradiol (E2), 10(-8)-10(-6) mol/L medroxyprogesterone acetate (MPA), or both E2 and MPA for 2-24 days in serum-containing medium. The progestin enhanced immunoreactive and functionally active stromal cell tissue factor content, achieving peak effects by 8-12 days of culture. Although E2 alone was ineffective, it augmented MPA-enhanced tissue factor content by 8 days of culture, with continued increases beyond 20 days. Dose-dependent effects on tissue factor protein content were observed between 10(-8)-10(-6) mol/L MPA added alone or together with E2. The content of tissue factor mRNA was also increased by MPA and synergistically increased by E2 plus MPA. Similar steroidal effects on stromal cell tissue factor protein and mRNA content were observed using a defined medium. After optimal induction of tissue factor expression by E2 plus MPA, removal of these steroids reduced levels of stromal cell tissue factor mRNA and protein, with virtually complete reversal by day 7 of withdrawal. These time-course and dose-response relationships establish in vitro conditions with which to dissect factors controlling endometrial hemostasis, whereas the observed effects of steroid withdrawal establish a novel model for the study of mechanisms regulating normal and abnormal uterine bleeding.

Progestational regulation of human endometrial stromal cell tissue factor expression during decidualization.

Decidualized endometrial stromal cells from mid- to late secretory phase and decidual cells from gestational human endometrium displayed prominent immunohistochemical staining for tissue factor, a primary initiator of hemostasis. Consistent with the regulation by progesterone of the decidualization process in vivo, medroxy-progesterone acetate elevated the tissue factor content of primary stromal cell cultures 8-fold over basal values. This increase paralleled the release of immunoreactive PRL, a marker of decidualization. The induced, as well as basal, tissue factor displayed full functional activity and the expected electrophoretic mobility (46 kilodaltons). Moreover, Northern analysis of RNA from cultured stromal cells indicated that medroxyprogesterone acetate increased tissue factor mRNA levels approximately 10-fold relative to control levels. In contrast, cultured stromal cell tissue factor protein content and mRNA levels were unaffected by exogenous estradiol. These findings indicate that enhancement of endometrial stromal cell tissue factor content is associated with progesterone induction of the decidualization process. In humans, trophoblastic invasion of the endometrial vasculature during blastocyst implantation risks hemorrhage. Therefore, increases in perivascular decidual cell tissue factor expression could serve to promote periimplantational endometrial hemostasis.

In vitro metabolism of C19 steroids in human endometrium.

In order to quantitate the extent of intracellular metabolic conversions of C19 steroids in human endometrium, specimens of proliferative and secretory tissue were superfused at a constant rate with several pairs of labeled compounds at low concentrations. About 16% of dehydroepiandrosterone sulfate interacting with endometrial cells was converted to dehydroepiandrosterone and about 3% of this compound was converted to androstenedione. Androstenedione was reversibly reduced to testosterone and the extent of this conversion was shown to be several-fold higher in secretory than in proliferative tissue. About 1% of testosterone entering the cells was reduced to 5 alpha-dihydrotestosterone. These results demonstrate that the conversion of the main circulating C19 steroids in women, i.e. dehydroepiandrosterone sulfate and androstenedione, to 5 alpha-dihydrotestosterone, the compound considered to be the true intracellular androgen, is very small. In contrast, formation of testosterone from androstenedione is extensive and increases during the luteal phase under the influence of progesterone, a hormone known to stimulate the activity of 17 beta-hydroxysteroid dehydrogenase in human endometrium.

Receptors, enzymes, and hormonal responses of endometrial cells.

Studies of hormone-related biochemical parameters of human endometrium are facilitated by the availability of a variety of types of endometrial tissue resulting from changes in hormonal environment during the menstrual cycle or from decidual, hyperplastic, and neoplastic transformations. Estrogen and progesterone receptors levels, rates of metabolism of ovarian hormones, enzymatic activities regulated by hormones or affecting intracellular concentrations of steroids, rates of production of prolactin and prostaglandins, and endogenous levels of hormones have been measured in samples of endometrial tissue and in separated glands and stroma. These studies have also included an evaluation of effects of estrogens and progestins administered to patients or added to the medium during in vito incubations of endometrium under organ culture conditions. Epithelial cells derived from isolated glands and stromal cells have been grown in primary cultures and also subcultured. These studies have revealed two mechanisms by which progestins exert an antiestrogenic effect on the endometrial tissue: viz, they were found to provoke a decline in estrogen receptor levels and to increase the metabolism of E2 through stimulation of estradiol 17 beta-dehydrogenase activity. In addition, studies of cells in culture have led to the unexpected finding of wide and rapid fluctuations in the levels of estrogen receptors, thus providing an opportunity to investigate factors involved in the regulation of concentrations of specific E2-binding sites.