Estrogen metabolites in the release of inflammatory mediators from human amnion-derived cells

Life Sciences 88 (2011) 551–558

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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Estrogen metabolites in the release of inflammatory mediators from human amnion-derived cells Barbara Pavan a, Guglielmo Paganetto b, Alessandro Dalpiaz c,⁎, Carla Biondi a, Laura Lunghi a a b c

Department of Biology, General Physiology sect., University of Ferrara, via L. Borsari, 46 – 44121 Ferrara, Italy Department of Biology and Evolution, University of Ferrara, Corso Ercole I d'Este, I-44121 Ferrara, Italy Department of Pharmaceutical Chemistry, University of Ferrara, Via Fossato di Mortara 19, I-44121 Ferrara, Italy

a r t i c l e

i n f o

Article history: Received 23 July 2010 Accepted 6 January 2011 Keywords: Amniotic cells Prostaglandins Arachidonic acid

a b s t r a c t Aims: Human amnion-derived cells have been used as in vitro models to test the release of inflammatory mediators, such as arachidonic acid (AA) and prostaglandin E2 (PGE2). We compared estrogen metabolites for their ability to induce AA release, to influence PGE2 production and to interact toward intracellular estrogen receptors (ERs). Main methods: Metabolite effects on AA and PGE2 release were examined by radiolabelled substrate incorporation and by colorimetric enzyme immunoassays, respectively. [3H]17-β-estradiol binding displacements were performed on Ro-20-1724 treated whole cells. Key findings: In WISH cells, estrone, 2-hydroxy-estrone and estriol induced a rapid dose dependent release of AA that was not inhibited by cycloheximide. Estrone and 2-hydroxy-estrone showed biphasic dose–response curves of PGE2, whereas estriol and 16-α-hydroxy-estrone increased PGE2 levels at high concentrations. 2-methoxyestrone, 4-hydroxy-estradiol and 4-hydroxy-estrone did not significantly affect PGE2 release. 2-methoxyestradiol and 2-hydroxy-estradiol decreased the PGE2 release. Effects of metabolites on PGE2 were inhibited by cycloheximide and by the ER antagonist tamoxifen. In AV3 cells PGE2 production was poorly detectable. On Ro20-1724 treated WISH cells the Ki of 17-β-estradiol was 29.2± 5.4 nM. Estrone, 2-methoxy-estrone and 2methoxy-estradiol showed similar affinity values. The hydroxyl substituent at position 2, 4 and 16 decreased or markedly increased the affinity for estradiol or estrone derivatives, respectively. Significance: The estrogen metabolites induced nongenomic effects on AA release from WISH cells. The influence on PGE2 release was detectable only on WISH cells. These effects appeared genomic and mediated by intracellular ERs, whose properties seemed strongly dependent on intracellular cAMP levels. © 2011 Elsevier Inc. All rights reserved.

Introduction During pregnancy amnion cells produce inflammatory mediators including cytokines, arachidonic acid (AA) and prostaglandins (PGs) in response to autocrine, paracrine and endocrine signals (Myatt and Sun 2010). PGs released by amnion cells represent a key event to trigger uterine contractions (Blickstein 2009). Besides, striking analogies between the inflammatory response and the onset of myometrium activity during labor are recognized (Straub 2007; Gotsch et al. 2008; Østensen and Skomsvoll 2004). Concerning the mechanisms responsible for the regulation of PG synthesis, some agents act by influencing protein synthesis, while others exert their effects through the production of intracellular second messengers (Pavan et al. 2003; Ackerman et al. 2005). These interactions have been analyzed in WISH cells (Ackerman et al. 2005), a human amnion-derived cell line, constituting a model for in vitro studies of

⁎ Corresponding author. Tel.: +39 0532 455274; fax: +39 0532 455953. E-mail address: [email protected] (A. Dalpiaz). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.01.018

amnion functions, particularly for AA and PGE2 production and release (Pavan et al. 2003). WISH cells can be also employed to evaluate the mechanisms linking the regulation of immune function and AA metabolism (Pavan et al. 2003). Furthermore, WISH cells and fetal membranes are known not only as a site of estrogen synthesis and release, but also as a target for such hormones. As an example, they secrete estradiol (Kniss et al. 2002) and express estrogen receptor beta (ERβ) (Fiorini et al. 2003). Therefore, WISH cells could be useful for in vitro studies about the molecular effects exerted during pregnancy by steroid hormones and their metabolites, that show important biological activity in inflammatory processes (Straub 2007). It is indeed known that cortisol, estrogens, progesterone, as well as endogenous estrogen metabolite levels increase in amniotic fluid during pregnancy (Myatt and Sun 2010). Catechol estrogens can moreover participate in the process of implantation and in the initiation of labor, being able to stimulate the synthesis of prostaglandins (Biswas et al. 1991a,b). The complex role of downstream metabolites of estrone and 17-βestradiol in inflammation has been comprehensively reviewed (Straub 2007). Actually, the intracellular metabolism of estrogens leads to

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important biologically active metabolites with quite different anti- and pro-inflammatory functions. The effects of steroid hormones can be genomic or nongenomic, being delayed (N10 min), or rapid (b10 min), respectively (Wehling 1997). In WISH cells the genomic responses appear mediated by classical intracellular estrogen receptors (ERs) (Pavan et al. 2001), but other rapid effects raised by concentrations of estradiol ranging from 10 nM to 1 μM are related to a supposed estradiol membrane binding site (Fiorini et al. 2003). To the best of our knowledge, the effects of estrogen metabolites have not yet been investigated on WISH cells. Therefore, the main objective of this study was to investigate on both eventual nongenomic and genomic actions induced by several

Materials and methods Materials [2,4,6,7,-3H]17-β-estradiol (87.0 Ci/mmol) and [5,6,8,9,11,12,14,153H]AA (205 Ci/mmol) were purchased from Amersham Biosciences

OH

H

H

17 16

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metabolites of estradiol (Fig. 1) on WISH cells. In this aim, the prototypical AA and PGE2 inflammation pathways have been assayed. To match up results obtained in WISH cells, the human amnion-derived AV3 cells have been also utilized. Finally, the ability of the metabolites to interact with intracellular ERs has been investigated.

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Fig. 1. Chemical structures of estradiol and its metabolites investigated here.

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(Milan, Italy). 17-β-estradiol and the A-ring metabolites 2-hydroxyestrone, 2-methoxy-estrone, 2-hydroxy-estradiol, 2-methoxy-estradiol, 4-hydroxy-estrone, 4-methoxy-estrone, 4-hydroxy-estradiol, and 4methoxy-estradiol as well as the D-ring metabolites estrone, estriol and 16-α-hydroxy-estrone were purchased from Sigma (Milan, Italy). The steroids were dissolved in ethanol, which did not exceed the final concentration of 1% (v/v) in the medium of cell culture wells. Tamoxifen, BSA (bovine serum albumin), Ro 20-1724 (4-(3-Butoxy-4methoxyphenyl)methyl-2-imidazolidone), AACOCF3 (1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one), cycloheximide and diethylstilbestrol were obtained from the Sigma Chemical Co. (St. Louis, MO). All cell culture reagents and media were acquired from Invitrogen (Milan, Italy).

Cell culture Human amnion epithelial cell lines (WISH and AV3 cells; ATCC CCL-25 and ATCC CCL-21, respectively; American Type Culture Collection, Manassas, VA) were grown at 37 °C in an atmosphere of 5% CO2/95% air, in DMEM/F12 (1:1 vol/vol) medium, supplemented with 10% fetal bovine serum, 30 μg/ml gentamicin and 0.25 μg/ml amphotericin B. Planned for all assays, cells were seeded into 24-well plates at 2 × 105 cells per well, and grown to about 70% confluence (2– 3 days). Then, the medium was changed to a serum-free one and the test substances were added for 60 min at 37 °C. Basal or control values were determined by addition of vehicle alone.

Measurement of [3H]AA The AA released from the cells was determined as described previously by Fiorini et al. (2003). Briefly, WISH cells (2 × 105 cells per well) were radiolabeled with 0.5 μCi/well [3H]AA in serum-free medium 18 h before assay, achieving maximal radioactivity incorporated at this time. Cells were then washed three times with pseudoamniotic fluid (PAF), i.e. 20 mM Hepes/Tris, pH 7.0, containing 125 mM NaCl, 4.0 mM KCl, 2.5 mM CaCl2, 1.15 mM KH2PO4, 1.15 mM MgSO4, and 25.0 mM NaHCO3, supplemented with 2.0 mM glucose, 6.0 mM urea, and 0.2% BSA (pH 7.0), gassed with a 95% O2/5% CO2 mixture. Cells were supplied with PAF at a constant flow rate of 0.3 ml/min by a four-channel peristaltic pump (Gilson, Villier Le Bel, France), then perifused with PAF for 1 h before treatment to obtain a stable [3H]AA basal release. The test substances were infused into the wells by means of the same pump. Fractions of perifusate were collected every 3 min, and radioactivity of the perifusate solution was determined by means of a Beckman LS 6500 scintillation spectrometer. Basal release ranged from 90 to 150 dpm/3-min fraction among the different cell cultures, and it remained substantially unchanged throughout the entire experiment. Experiments were performed in triplicate, using different cell cultures. Ethanol and DMSO, as vehicle used, did not interfere with the assay.

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Binding assay Whole cell ligand binding assays were performed as previously reported (Pavan et al. 2001). Briefly, WISH cells were plated in 24well plates (2 × 105 cells per well) and 70% confluent cells were preincubated for 1 h with 10 μM Ro 20-1724. Cells were washed with PBS, then incubated in the presence of ten different concentrations of [3H]estradiol ranging from 5 to 250 nM for saturation experiments. For the competitive binding assays, cells were incubated in the presence of 10 nM [3H]estradiol alone to determine specific binding, or in combination with nine different concentrations of estrogen metabolites ranging from 0.1 nM to 10 μM. Nonspecific binding was determined by adding 10 μM diethylstilbestrol. All incubations were carried out at 37 °C for 30 min, in a final volume of 0.5 ml of serumfree medium containing 20 mM NaMoO4. The unbound ligand was removed by washing the cells twice with PBS supplemented with 20 mM NaMoO4 and 1 mg/ml BSA, and once with normal PBS. Cells were disrupted with 1 N NaOH (0.25 ml) and collected from the cluster dishes. Bound radioactivity was measured by scintillation spectrometry (LS 6500; Beckman Instruments, Palo Alto, CA). Data of the saturation experiments (binding dissociation constant KD and maximum binding capacity BMAX values) were obtained by computer analysis of saturation curves and of the corresponding Scatchard plots. Competitor binding was expressed as a percentage of maximum specific binding. The metabolite concentrations displacing 50% of [3H] estradiol (IC50 values) were obtained by computer analysis of displacement curves. Inhibitory binding constants (Ki values) were derived from the IC50 values according to the Cheng and Prusoff (1973) equation Ki = IC50/(1 + [*C]/KD*) were [C*] is the concentration of [3H]estradiol and KD* its dissociation constant. In this respect care was taken so that total binding never exceeded 10% of the total amount of radioligand added. The relative binding affinity (RBA) with respect to estradiol was calculated according to the following equation: RBA =

KD for E2 × 100: Ki for the test compound

All binding data were analyzed using the non-linear regression curve fitting computer program PRISM, version 4.0 (Graph Pad Inc., San Diego, CA, USA). Statistical analysis All results were expressed as means ± SD. Estrogens treatments, concentrations, and their interaction were tested by one- and twoway ANOVA. A value of p b 0.05 was considered statistically significant. Bonferroni's test was applied for post hoc comparison. Data were analyzed using the software PRISM, version 4.0 (Graph Pad Inc., San Diego, CA). Results

PGE2 immunoassay

Effects of estrogens on arachidonic acid release

Collected media were centrifuged at 2000 ×g for 10 min. After centrifugation, the clear incubation medium was used for measurements of PGE2 release. In particular, PGE2 levels were detected in WISH and AV3 cells using the high sensitivity colorimetric enzyme immunoassay (EIA) kit (Cayman Chemical, Inalco, Milano, Italy) according to the manufacturer's instructions. All measurements were run in triplicate for each sample. PGE2 concentration was determined spectrophotometrically by Victor3v microplate reader (Perkin-Elmer, Norwalk, CT, USA). Assay sensitivity was 15 pg per tube, and the intraor inter-assay coefficients of variations were b 10%. Data were expressed as ng of PGE2 produced per 106 cells.

The effect of increasing doses of the most active estrogens on prostanoid release from WISH cells was evaluated by analyzing the AA release induced by estrone, estriol and 2-hydroxy-estrone, whose chemical structures are reported in Fig. 1. As shown in Fig. 2, the tested compounds exerted a dose-dependent stimulatory effect. The stimulatory action became statistically significant at 10 nM reaching the maximum (+507% for estrone, +302% for estriol and +349% for 2hydroxy-estrone) at the highest dose employed (1 μM). This dose was used in all the further experiments. Firstly, we have investigated the effect of AACOCF3 on the AA output induced by estrone, estriol and 2-OH estrone. AACOCF3 is a trifluoromethylketone derivative of arachidonic

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acid, able to act as a specific phospholipase (PL)A2 inhibitor, with IC50 values in the order of magnitude of 10− 5 M (10 μM) (Riendeau et al. 1994). As reported in Fig. 3, 1 μM AACOCF3 was able to completely block the action of the tested drugs, even if this concentration was per se ineffective. We have afterwards verified that 30 min pre-incubation of WISH cells with the protein synthesis inhibitor cycloheximide (5 μg/ml) did not modify the AA release evoked by estrone, estriol and 2-hydroxyestrone (data not shown).

In the above described experiments, WISH cells were perfused with PAF containing 0.2% BSA. Since it is well known that this protein stimulates per se the AA outflow (Beck et al. 1998) and prevents estrogen cell penetration by binding these hormones (Wehling 1997), we performed another set of experiments using PAF without BSA. In these experimental conditions, both basal and estrone-,

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fractions Fig. 2. Changes in [3H]arachidonic acid release from WISH cells upon addition of increasing concentrations (from 1 nM to 1 μM) of estrone (A), estriol (B) and 2-hydroxy-estrone (2OH-estrone) (C). Extent of treatments is indicated by shaded bars. Graphs are representative of three independent experiments performed on different cell preparations. Standard deviations were less than 15% of the average. Basal values ranged from 90 to 150 dpm for fraction. *Pb 0.05 compared with basal value, Bonferroni's post-test. **P b 0.01 compared with basal value, Bonferroni's post-test.

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fractions Fig. 3. Changes in [3H]arachidonic acid release from WISH cells upon addition of 1 μM estrone (A), 1 μM estriol (B) and 1 μM 2-hydroxy-estrone (2-OH-estrone) (C) in combination with PLA2 inhibitor AACOCF3 (in frame). Extent of treatments is indicated by shaded bars. Graphs are representative of three independent experiments performed on different cell preparations. Standard deviations were less than 15% of the average. Basal values ranged from 90 to 150 dpm for a fraction. **P b 0.01 compared with basal value, Bonferroni's post-test.

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estriol- and 2-OH estrone-stimulated AA release was similar to that observed when the cells were perfused with PAF + BSA (Fig. 4). As expected, 0.2% BSA increased AA release when assayed both alone and in combination with estrone, estriol and 2-hydroxy-estrone. The AA release from WISH cells, obtained in all the experimental conditions described above, occurred within a few minutes following

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the stimulus (Figs. 2–4). This behavior could be therefore related to nongenomic responses mediated by the estrogen metabolites. Effects of estrogens on PGE2 release WISH cells produced high PGE2 levels into the medium (1.5 ± 0.15 ng/106 cells) under normal culture conditions (60 min, 37 °C). The release of PGE2 following the 60 min addition of estrogen metabolites was significantly affected to a different extent (Fig. 5). Two-way ANOVA indicated that a variation in the PGE2 release significantly depended on cell treatment (pb 0.0001) and metabolite concentrations (pb 0.0001). 2hydroxy-estrone and estrone were both characterized by biphasic dose– response curves (pb 0.001–0.001, Bonferroni's test, two-way ANOVA; Fig. 5) showing the most potency and efficacy (EC50 =0.30 ±0.02 nM, IC50 = 1.5 ±0.2 μM for 2-hydroxy-estrone and EC50 =0.41 ±0.02 nM, IC50 = 89 ± 10 μM for estrone). 16-α-hydroxy-estrone and estriol showed a slight stimulation of PGE2 secretion until 100 nM, whereas higher concentrations increased the prostanoid outflow significantly, (pb 0.001–0.001, Bonferroni's test, one-way ANOVA; Fig. 5) reaching a 3fold enhancement over the basal at 10 μM. 2-hydroxy-estradiol and 4hydroxy-estrone showed a slight inhibitory effect on prostanoid release at higher concentrations although not significant for 4-hydroxy-estrone (pN 0.05, Bonferroni's test; one-way ANOVA; Fig. 5). No significant effects were also registered for 4-hydroxy-estradiol (pN 0.05, Bonferroni's test; one-way ANOVA; Fig. 5). Equally, 2-methoxy-estrone was ineffective at all the concentrations tested (p N 0.05, Bonferroni's test; one-way ANOVA; Fig. 5). Noteworthy, the parent hormone 17-β-estradiol had the smallest effect on PGE2 release in comparison to own and estrone metabolites (pb 0.05, Bonferroni's test; one-way ANOVA; Fig. 5). In particular, it exerted a significant stimulatory effect on PGE2 release at the concentration range of 10 nM to 1 μM, but revoked it to the basal level at the highest concentration of 10 μM (pb 0.05, Bonferroni's test; two-way ANOVA; Fig. 5). Finally, 2-methoxy-estradiol showed inhibiting effects on PGE2 release at the lower concentrations tested (pb 0.001, Bonferroni's test; one-way ANOVA; Fig. 5). Otherwise, all estrogen-induced effects on the basal PGE2 production were avoided in WISH cells after 30 min pre-incubation with the protein synthesis inhibitor cycloheximide (5 μg/ml) or with the ER antagonist tamoxifen (100 μM), as well as after the hormones were adsorbed to bovine serum albumine (BSA, 1 mg/ml), hindering their permeation through the cell membrane (data not shown). The influence of estradiol and its metabolites on PGE2 release from WISH cells therefore appears related to genomic effects. Among the downstream products of AA cascade, PGE2 is known to play a major role and to be generated in substantial amounts at sites of inflammation (Portanova et al. 1996). Therefore, we compared the effects of estrogen metabolites on PGE2 release obtained in WISH cells 2-OH-E1

*§

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fractions Fig. 4. Changes in [3H]arachidonic acid release from WISH cells upon addition of 1 μM estrone (A), 1 μM estriol (B) and 1 μM 2-hydroxy-estrone (2-OH-estrone) (C) compared to 0.1% BSA and their combination. Extent of treatments is indicated by shaded bars. Graphs are representative of three independent experiments performed on different cell preparations. Standard deviations were less than 15% of the average. Basal values ranged from 90 to 150 dpm for a fraction. *Pb 0.05 compared with basal value, Bonferroni's posttest. **P b 0.01 compared with basal value, Bonferroni's post-test. **§Pb 0.01 compared with BSA alone, Bonferroni's post-test.

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Table 1 Ki values of several estrogens and their relative binding affinity (RBA) values against [3H]17-β-estradiol. The RBA values are compared with those obtained by Zhu et al. (2006) with the employment of human purified β-ERs. Estrogens

Ki (nM)a

RBAb

RBAc

17-β-estradiol (E2) 2-methoxy-estradiol (2-Met-E2) 2-hydoxy-estradiol (2-OH-E2) 4-hydroxy-estradiol (4-OH-E2) Estriol (E3) Estrone (E1) 2-methoxy-estrone (2-Met-E1) 2-hydroxy-estrone (2-OH-E1) 4-hydroxy-estrone (4-OH-E1) 16-α-hydroxy-estrone (16-α-OH-E1)

29.2 ± 5.4 21.8 ± 3.3 148 ± 14 96.0 ± 14.5 291 ± 26 53.5 ± 3.5 30.7 ± 3.8 3.1 ± 0.3 4.6 ± 1.9 4.5 ± 0.3

98% 131% 19% 30% 9.8% 54% 93% 924% 623% 637%

100 1% 35% 56% 2% 2% ≪1% 0,4% 1% 35%

a The Ki of the various estrogens was calculated using the Cheng and Prusoff equation. Data are reported as mean ± SD of three independent experiments. b Relative binding affinity against estradiol. The values were obtained with respect to the KD of [3H]17-β-estradiol of 28.65 nM (Pavan et al. 2001). c Data reported from Zhu et al., 2006.

A

E2 100 E3 2-OH-E2 75 4-OH-E2

β bound

556

2-Met-E2 50

25

0 -10

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-8

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-5

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Steroid-binding specificity

E2 E1 75 16-α-OH-E1

β bound

with another human amnion-derived cell line, AV3 cells. Under the same experimental conditions used for WISH cells, AV3 cells released basal levels of PGE2 very near to our limit of detection (15 pg/mg protein/1 h). Consequently, all effects of metabolites on PGE2 production in AV3 cells became poorly measureable.

2-OH-E1 50

4-OH-E1 2-Met-E1

3

Our previous data reported the presence of a single class of [ H]17-βestradiol specific binding sites in whole WISH cells, showing a KD value of 28.65 ± 3.5 nM and a BMAX of 1432 ± 26 fmol/mg protein (Pavan et al. 2001). These data were obtained by treating the cells with Ro 201724, an inhibitor of cyclic nucleotide phosphodiesterase (Soderling et al. 1998). In the study reported in this paper, we have then assessed several estrogens for their potential interaction with the same binding sites of [3H]17-β-estradiol on Ro 20-1724 treated WISH cells, by means of competitive binding assay experiments. All steroids tested caused competitive displacement of [3H]17-β-estradiol binding at concentrations up to 10 μM (Fig. 5). Ki values are generally assumed to be equivalent to the dissociation equilibrium constants (Borea et al. 1995) and are reported in Table 1, where the relative binding affinity values (RBA) of the tested compounds against the affinity of [3H]17-β-estradiol are included. In Fig. 6A the displacement curves of 17-β-estradiol and its derivatives are shown, whereas the curves referred to estrone and its derivatives are reported in Fig. 6B, which include the data of 17-βestradiol as a term of comparison. The Ki of 17-β-estradiol was 29.2 ± 5.4 (RBA = 98%), a value not significantly different from the KD of the radiolabelled homologous. The presence of the methoxyl substituent in position 2 of estradiol was not able to sensibly influence its binding, as indicated by the Ki and RBA values of 2-methoxy-estradiol (21.8 ± 3.3 nM and 131%, respectively). The hydroxyl substituent in position 2 or 4 of estradiol induced a decrease of the binding. In particular, the Ki values of 2hydroxy-estradiol and 4-hydroxy-estradiol were found to be 148 ± 14 (RBA = 19%) and 96.0 ± 14.5 nM (RBA = 34%), respectively. A relatively marked decrease of binding was observed with estriol, characterized by the presence of an hydroxyl group at position 16 of estradiol. Indeed, the estriol affinity appeared reduced by one order of magnitude with respect to its parent compound (Ki = 291 ± 26 nM, RBA = 10%). Differently from estriol, estrone showed an affinity similar to that of estradiol (Ki = 53.5 ± 3.5 nM, RBA = 54%) and also in this case the presence of a methoxyl substituent in position 2 was not able to sensibly influence its binding (Ki of 2-methoxyestrone = 30.7 ± 3.8 nM, RBA = 93%). On the other hand, the presence of the hydroxyl substituent at position 2, 4 or 16 appeared efficacious in enhancing the estrone affinity of about an order of magnitude (the

25

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Log [compound] Fig. 6. Representative competition of the binding of [3H]17-β-estradiol to treated Ro 201724 treated whole WISH cells by estradiol and several its derivatives (A) and by estrone and several its derivatives (the estradiol curve is reported as term of comparison) (B). Each data point was the mean of triplicate measurements. Standard deviations were less than 15% of the average.

Ki value of 2-hydroxy-estrone, 4-hydroxy-estrone and 16α-hydroxyestrone were found 3.1 ± 0.3 nM with RBA = 924%, 4.6 ± 1.9 nM with RBA = 623% and 4.5 ± 0.3 with RBA = 637%, respectively). Discussion Many studies have suggested that estrogen metabolites may act in target tissues as local mediators of estrogen activity, or may activate their own receptors or effectors, even at a concentration exceeding several times those of their parent substance (Zhu and Conney 1998; Salama et al. 2009). Although certain estrogen metabolites regulate target cells through traditional nuclear mechanisms, other metabolites appear to elicit unique biological responses not associated with activation of intracellular receptors (Salama et al. 2009). The classical action of the estrogen-bound receptors as ligand-dependent transcription factors (genomic effect) occurs after a lag of many minutes and persists over hours, whereas several rapid effects observed indicate that estrogens operate also at a site close to the cell surface (nongenomic effects). Reports of membrane binding sites for estrogen have been already reported since 1977 (Pietras and Szego 1977) and recently reviewed by Manavathi and Kumar (2006). Although a longago study suggested a membrane receptor site of binding for 2hydroxy-estradiol (Schaefer et al. 1980), the binding affinity of many other estrogen metabolites towards membrane receptor is still not known.

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The first part of our experimental work involved estrone, estriol and 2-hydroxy-estrone for AA release studies from WISH cells. E1 and E3 were chosen as representative estrogens, being the major estrogens circulating in a woman under different physiological conditions; estriol, in particular, is a quantitatively predominant estrogen metabolite produced during pregnancy (Zhu et al. 2006). The oxidative metabolite of estrone, 2-hydroxy-estrone, is predominant in a nonpregnant woman together with its precursor. We used WISH cells as a human amnion epithelial cell model (Pavan et al. 2003), useful for studying key aspects of inflammation, such as the AA release and PGE2 formation, as well as in the evaluation of mechanisms linking regulation of immune function and arachidonic acid (AA) metabolism (Harris et al. 1988; Pavan et al. 2003). AA release constitutes a key regulatory step in the endogenous production of PGs as second messengers in human gestational tissues (Farina et al. 2007). AA is released as a PG precursor from membrane phospholipids through the action of phospholipase A2 (PLA2) whose activity enhances towards the end of gestation (Farina et al. 2007). Our results indicate that the AA release induced in WISH cells by estrone, 2-hydroxy-estrone and estriol was a rapid and concentration-dependent response significantly detectable at 10− 8 M, an order of magnitude normally found in human pregnancy plasma for these compounds (Berg and Kuss 1992). The rapid response suggests a nongenomic signaling phenomena. This hypothesis appears corroborated by the inability of the protein synthesis inhibitor cycloheximide to modify the AA release evoked by estrogen metabolites. Moreover, given the completely abolished AA release by the specific PLA2 inhibitor AACOCF3, a direct action of estrogen metabolites on PLA2 activity may be also supposed, as previously indicated just in amniotic membranes (Bonney and Franks 1987). This action may be mediated by membrane receptors, as previously suggested in WISH cells for the parent compound 17-β-estradiol, which induced the same AA release pattern of the metabolites (Fiorini et al. 2003). Then, we have investigated the effects of estrone, 2-OH-estrone and estriol on PGE2 secretion from WISH cells. Our results indicate that estrone and 2-hydroxy-estrone induce a biphasic pattern of PGE2 secretion. A bimodal role in influencing the PGE2 secretion from WISH cells was shown also by 17-β-estradiol, even if its effects appeared strongly reduced both in potency and activity with respect to estrone and 2-hydroxy-estrone. The behavior of estrone and 2-hydroxy-estrone was consistent with data reported by Calabrese (2001), indicating a bimodal role of estrogen metabolites on inflammatory pathways in several cell types, where estrogens at high doses inhibited many inflammatory mechanisms, whereas at low concentrations no effects or even opposite effects were observed. According to our results, 2hydroxy-estrone and estrone seem to act as pro-inflammatory signals at lower concentrations (up to 10− 8 M) and as anti-inflammatory signals when their concentrations are higher, such as at pregnancy levels which in plasma have been found up to 4 × 10− 8 M (Straub 2007). Although 2hydroxy-estrone is the major circulating catechol metabolite, it is known that it exhibits the highest metabolic clearance rate occurring for a natural steroid, due to the action of cell enzyme catechol-Omethyltransferase to form the metabolically stable derivative 2methoxy-estrone (Merriam et al. 1980). As a consequence, it is important to take into account that the clinical significance of 2-OHestrone can be strongly influenced by its pharmacokinetics, an aspect not evaluated by our measurements in WISH cells. Differently from estrone and 2-hydroxy-estrone, estriol worked ineffectively at lower concentrations and displayed a strong stimulatory effect at the highest concentrations tested, expressing a potential proinflammatory behavior. We have then investigated other estrogen metabolites in the aim to better investigate the role of their substituents in influencing the PGE2 secretion from WISH cells. Interestingly, 16-αhydroxy-estrone showed the same identical behavior of estriol. Both these compounds are characterized by an –OH group at position 16 (Fig. 1). Their effects on PGE2 secretion could be significant when referred to pharmacological dosages (Dubey et al. 2004). On the other hand, 2-

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methoxy-estrone was totally ineffective in the range of concentrations investigated, suggesting that the methylation of the 2-hydroxy-estrone completely impairs its bimodal effect on PGE2 release. 2-methoxy-estrone has been hypothesized acting as a pro-hormone able to stimulate estrogen target tissues, which possess demethylating enzymes (Martucci 1983). Similarly, the presence of the –OH group in the position 4 of estrone and estradiol was able to inhibit their bimodal effects on PGE2 release. We remark that the potential clinical significance of our results should be evaluated on the basis of new data coming from experimental and theoretical studies oriented to the quantifications of the level of concentration of estradiol metabolites in human tissues. The estrogen metabolites 2-methoxy-estradiol and 2-hydroxyestradiol were shown able to inhibit the PGE2 release from WISH cells at the lower and higher concentrations tested, respectively, implying potential anti-inflammatory effects in an amnion-like tissue. In this regard, it is important to underline that 2-methoxy-estradiol has been recognized as an anticancer and anti-angiogenic agent not only on several cell culture lines, but also in animal and human clinical studies (Mooberry 2003). It is indeed well established that tumor promoting microenvironments are largely orchestrated by inflammatory processes (Coussen and Werb 2002). Our results about the influence of estrogen metabolites on PGE2 release from WISH cells appear therefore in agreement with these phenomena, not only in the case of 2-methoxyestradiol, but also in the case of other estradiol derivatives tested by us. In particular, the biphasic pattern obtained by the influence of estrone and 2-hydroxy-estrone on PGE2 secretion from WISH cells was also previously displayed on breast cancer cell line MCF-7 proliferation (Lippert et al. 2003). Moreover, the ability of estriol and 16-α-hydroxyestrone to stimulate the PGE2 secretion at high amounts appears related to the ability of these compounds to stimulate the cell line MCF-7 proliferation at high dosages (Lippert et al. 2003). Differently from the AA release, the effects of estrogen metabolites on PGE2 production were strongly inhibited by the synthesis inhibitor cycloheximide, or by the intracellular ER antagonist tamoxifen, or in the presence of BSA. These results clearly indicate that the influence of estrogen metabolites on PGE2 release are related to genomic phenomena mediated by intracellular ERs. PGE2 output was also tested in human amnion-derived AV3 cell lines, but it appeared poorly appreciable in our experimental conditions. Similarly, very low basal production of PGE2 was found by Potter et al. (1999) in this cell line. To the best of our knowledge, a notable use of AV3 cells for their PGE2 production has never been reported. For all these reasons, we focused our attention on WISH cells, that appeared functionally adapted to generate substantial amounts of PGE2 in a relatively short time. We have therefore analyzed the ability of the estrogen metabolites to interact with WISH intracellular ERs. The binding studies were performed on cells treated with Ro 20–174, necessary to obtain a measurable binding of [3H]17-β-estradiol that allowed to identify a single class of β-ERs characterized by KD = 28.65 nM (Pavan et al. 2001; Fiorini et al. 2003), here confirmed by the estradiol Ki = 29.2 nM. The presence of both the C = O substituent at position 17 and the methoxy group at position 2 did not influenced sensibly the estradiol binding to β-ERs, being the Ki values of 2-methoxyestradiol, 2-methoxy-estrone and estrone ranging from 21.8 nM to 53.5 nM, and the related RBA (relative binding affinity against estradiol) values from 54% to 131%. The presence of the hydroxyl group at position 2, 4 or 16 of estradiol induced its affinity decrease towards β-ERs, being the Ki values of 2-hydroxy-estradiol, 4-hydroxyestradiol and estradiol ranging from 96.0 nM to 291 nM and the related RBA values from 9.8% to 30%. On the other hand, the same type of substitutions performed on the estrone structure induced a relatively high affinity enhancement towards β-ERs. Indeed, the Ki values of 2-hydroxy-estrone, 4-hydroxy-estrone and 16-α-hydroxyestrone ranged from 3.1 nM to 4.6 nM and the related RBA values from 623% to 924%.

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The affinity values and SAR obtained here are generally different from those normally detected in most cells or referred to human ERs. In particular, the estriol affinity detected on Ro 20–174 treated WISH cells (Ki = 29.2 ± 5.4 nM) appears reduced with respect to those normally registered with Ki or KD values around 0.1–1 nM (Pavan et al. 2001; Zhu et al. 2006). Moreover, as a term of comparison, Table 1 reports the RBA values obtained by Zhu et al. (2006) for estradiol metabolite interaction towards human purified β-ERs. In this case it can be evidenced that estrone and its derivatives do not maintain or gain affinity in comparison with estradiol as we found out, but they drastically reduce their ability to interact toward β-ERs. Indeed the RBA values of estrone, 2-methoxy-estrone, 2-hydroxy-estrone, 4-hydroxy-estrone and 16-αhydroxy-estrone were found to range from a value ≪ 1% to 35%. Similarly, the methoxy group at position 2 of estradiol appeared detrimental for its β-ER affinity (RBA= 1%). These differences underline that the treatment of WISH cells with Ro 20–1724 induces a strong intracellular increase of cAMP levels that have been demonstrate to sensibly influence the intracellular ER activity (Coleman et al. 2003; Zhang and Trudeau 2006). Therefore, a comparison of the RBA values between Ro 20–1724 treated WISH cells and human purified β-ERs suggests that changes of cAMP intracellular levels can induce important changes on the role of estradiol and its metabolites toward intracellular ERs. Conclusions In conclusion, these data show that several derivatives of estradiol can influence human amnion-like WISH cell function by acting on AA and PGE2 production. The proposed mechanism for these actions is through intracellular and cell-surface estrogen receptors, respectively. The identification of an amnion estradiol membrane receptor could not only provide a mechanistic explanation for the observed effect of estradiol, but also indicates a potential additional site of estrogens in human reproductive function. Furthermore, considering WISH cells as a suitable model for human amnion cells, our results suggest that active estrogen metabolites may finely modulate the onset of human labor. Yet, looking on recent reports, by which WISH cells should be more meaningfully be likened to neoplastic cells (Kniss et al., 2002; Ross and Wang, 2003), our results could support estrogen metabolite relevance in neoplastic competence via inflammation pathway activation. Conflict of interest statement We have nothing to declare.

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