| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Biology, Section of General Physiology, University of Ferrara, 44100-I Ferrara, Italy
b Montell Italia, Centro Ricerche "G. Natta," 44100-I Ferrara, Italy
ABSTRACT
In human amnion-derived WISH cells [3H]estradiol-17ß binding sites are not detectable, but they become measurable in cells exposed to cAMP elevating agents such as forskolin or Ro 20-1724. In cells unexposed to these drugs, 17ß-estradiol stimulates prostaglandin (PG)E2 release but exerts an evident inhibitory effect in cells exposed to Ro 20-1724. Both stimulatory and inhibitory actions are inhibited by the estrogen receptor antagonist, tamoxifen, by cell pretreatment with cycloheximide, or when the hormone is bound to BSA. Our data demonstrate for the first time that 1) 17ß-estradiol modulates PGE2 release from WISH cells, interacting with specific intracellular receptors and probably evoking new protein synthesis, and 2) WISH cell responsiveness to 17ß-estradiol seems to be modulated by cAMP, whose levels are significantly increased by the steroid hormone in the presence of Ro 20-1724. The nucleotide is presumably responsible for the enhacement of hormone receptor availability and for the inhibition of PGE2 release observed in the presence of Ro 20-1724.
estradiol, estradiol receptor, mechanisms of hormone action, pregnancy, signal transduction
INTRODUCTION
The mechanisms responsible for the initiation of human parturition are still poorly understood. Several factors are likely involved, among these oxytocin and prostaglandins, because they are able to evoke myometrial contractions. Fetal membranes and maternal decidua are sources for these compounds, whose release increases during the progression of pregnancy [1, 2].
Human intrauterine tissues also synthesize estrogens that are at higher levels around the time of parturition, and concomitantly, a decrease of progesterone occurs [3]. The consequent enhancement in the local estrogen/progesterone ratio may exert several actions aimed at preparing the uterus for delivery. An increase of both gap junction formation and oxytocin receptor number is indeed observed in myometrial cells [4, 5], together with the stimulation of oxytocin gene expression in human choriodecidua [6]; in addition, estrogens modulate prostaglandin (PG) production by intrauterine tissues [7]. As for PGE2 output from amnion cells, in 1983 Olson et al. [8] demonstrated that 17ß-estradiol induces a stimulatory action in cells obtained after spontaneous labor and vaginal delivery but no effect in nonlaboring amnion cells. To our knowledge, no recent report exists about the influence of 17ß-estradiol on PGE2 release from amnion cells; moreover, similar studies have never been carried out on human amnion-derived WISH cells. This cell line is considered a good model for analysis of the physiological functions of amnion cells, as well as for the characterization of PGE2 release modulation by different agonists. As a matter of fact, PGE2 has been found to be the main prostanoid produced by fetal tissues as well as by WISH cells; moreover, PGE2 output from these cells is evoked by agonists, including growth factors, cytokines, and oxytocin [9–11] that induce the same effect in amnion cells [12–14].
Because WISH cells in certain aspects appear to behave like nonlaboring amnion tissue, they represent the ideal candidate for an in vitro study of events triggering the mechanism of delivery. Thus, with the aim of improving our knowledge regarding the role of estrogen in human parturition, in this paper we describe our investigations into the presence of hormone receptors in WISH cells, as well as the characterization of the cellular response, in terms of PGE2 release, evoked by hormone-receptor interaction.
MATERIALS AND METHODS
Chemicals
[5,6(n)-3H]PGE2 (181 Ci/mmol), [G-3H]adenosine 3'-5'-monophosphate (27 Ci/mmol) and [2,4,6,7-3H]estradiol (87.0 Ci/mmol) were purchased from Amersham Italia Srl (Milan, Italy). PGE2, antiserum anti-PGE2-BSA, cAMP, forskolin, diethylstilbestrol, tamoxifen, cycloheximide, BSA, and Ro 20-1724 were from Sigma Chemical Co. (St. Louis, MO). 17ß-Estradiol was purchased from Calbiochem-Novabiochem (Inalco Milan, Italy). Tissue culture media and sera were purchased from Gibco/BRL (Paisley, Scotland). All other chemicals were the highest reagent grades commercially available. BSA was dissolved in serum-free culture medium; 17ß-estradiol, cycloheximide, tamoxifen, and Ro 20-1724 were dissolved in ethanol and diluted with medium.
Cell Culture and Treatment
Amnion-derived WISH cells (ATCC CCL-25) were grown at 37°C in an atmosphere of 5% CO2/95% air, in a mixture of Ham F12 and Dulbecco modified Eagle medium (F12/DMEM) (1:1 vol/vol) supplemented with 10% fetal bovine serum (10% FBS), 30 µg/ml gentamicin, and 0.25 µg/ml amphotericin B. The cells were seeded into 24-well plates at 2 x 105 cells/well in F12/DMEM + 10% FBS and grown to confluence (2–3 days).
For PGE2 determination, the medium was removed and replaced with fresh serum-free F12/DMEM, containing test substances. Ro 20-1724, when requested, was added 30 min before the addition of agonists. After incubation of samples for 30 min, the media were collected and stored at -80°C until PGE2 RIA was performed. In order to test the effect of cycloheximide on prostanoid output, WISH cells were exposed to the drug (5 µg/ml) for 1 h before the preincubation time. When indicated, 17ß-estradiol was diluted in a serum-free medium containing BSA (1 mg/ml) and added at the start of incubation. Tamoxifen, when present, was added 30 min before 17ß-estradiol.
For intracellular cAMP level determination, cells were preincubated for 30 min in fresh serum-free F12/DMEM in the absence or in the presence of 10-5 M Ro 20-1724 and incubated for 30 min with test substances. The reaction was terminated by rapid removal of the medium and the addition of 0.5 ml of ice-cold 5% trichloroacetic acid to each well; cells were then scraped, collected, and frozen at -80°C until cAMP assay. In order to test the effect of cycloheximide on the nucleotide production, WISH cells were exposed to the drug (5 µg/ml) for 1 h before the preincubation time with Ro 20-1724.
Binding Assay
Whole cell ligand binding assays were performed following the method of Stoica et al. [15]. Briefly, WISH cells were plated in 24-well plates (2 x 105 cells/well) and, at a 70% confluence, cells were preincubated for 1 h in the absence or in the presence of 10-5 M Ro 20-1724 or 10-6 M forskolin. Cells were washed with PBS, then incubated in the presence of 10 different concentrations of [3H]estradiol ranging from 5 to 250 nM. Nonspecific binding was determined by adding 10-5 M diethylstilbestrol. All incubations were carried out at 37°C for 30 min, in a final volume of 0.5 ml of serum-free 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 culture dishes. Bound radioactivity was measured by scintillation spectrometry (LS 6500; Beckman Instruments, Palo Alto, CA).
Data from saturation experiments (KD and Bmax values) were obtained by computer analysis of saturation curves and of the corresponding Scatchard plots, using the computer program Prism (Graph Pad, San Diego, CA).
Prostaglandin E2 RIA
The amount of PGE2 was assayed in the collected media by an RIA procedure, as previously described [11]. Assay sensitivity was 40 pg/106 cells, and the intraassay or interassay coefficients of variations were <10%. Data are expressed as ng PGE2 produced/106 cells.
Assay for cAMP Levels
Cyclic AMP levels were assayed by the method of Brown et al. [16], as previously described [11]. Assay sensitivity was 0.5 pmol/106 cells, and the intraassay or interassay coefficients of variations were <10%. Data are expressed as pmol cAMP/106 cells/30 min.
Adenylyl Cyclase Assay
Adenylyl cyclase activity was assayed on WISH cell membrane preparations, as previously described [11]. Data are expressed as pmol cAMP/mg protein/10-min.
Protein Determination
Protein concentration was determined according to the method of Lowry et al. [17], with BSA as standard.
Calculations and Statistics
Statistical significance was assessed by one-way ANOVA followed by Dunnett multiple comparison test, using the computer program Prism (version 2.0, Graph Pad Inc.).
RESULTS
Binding Experiments
In whole untreated WISH cells, [3H]estradiol-17ß binding sites were not detectable, but they became measurable in cells pretreated for 1 h with 10-5 M Ro 20-1724 (Fig. 1A) or 10-6 M forskolin (Fig. 1B). Saturation curves of [3H]estradiol-17ß obtained in cells pretreated with both compounds demonstrate that saturable binding sites were present; moreover, the linearity of Scatchard plots (r 
0.98) in the inserts suggested the presence, in both cases, of a single class of binding sites. Data from saturation experiments were KD = 28.65 ± 2.0 nM and Bmax = 1432 ± 26 fmol/mg protein or 36 654 dpm, in cells pretreated with Ro 20-1724; KD = 32.81 ± 2.8 nM and Bmax = 1455 ± 10 fmol/mg protein or 31 923 dpm, in cells pretreated with forskolin. Percent specific binding was around 45%. In WISH cells pretreated with 0.1% ethanol, i.e., the maximal concentration of vehicle employed, [3H]estradiol binding sites were not detectable.
|
Effect of 17ß-Estradiol on PGE2 Release
Figure 2 shows that 17ß-estradiol dose-dependently increased PGE2 production from WISH cells up to 10-6 M; the hormone effect became statistically significant (P < 0.05) at 10-8 M, reaching the maximum (1.6-fold, with respect to the basal value) at 10-6 M. In the presence of higher 17ß-estradiol concentrations a further increase in PGE2 output was not observed. Cycloheximide treatment of WISH cells (5 µg/ml for 60 min)—which does not influence cell viability, as determined by the trypan blue exclusion test—completely blocked PGE2 output evoked by 17ß-estradiol, without significantly modifying the basal prostanoid release. The stimulation of PGE2 output was also prevented by administering 17ß-estradiol bound to BSA (1 mg/ml), as reported in the same figure, and significantly reduced by the estrogen receptor antagonist tamoxifen (Tmx, 10-5 M), which, per se, was ineffective on basal PGE2 release (Table 1).
|
|
The same experiments were then carried out on WISH cells exposed to the cAMP phosphodiesterase inhibitor Ro 20-1724 (10-5 M) (Fig. 3). The drug alone slightly stimulated PGE2 output (118%); upon the addition of increasing doses of 17ß-estradiol, a reduction of PGE2 release was observed. A statistically significant (P < 0.05) effect was obtained at 10-8 M, then inhibition rose at higher doses (about 50%, at 10-7 M). Cycloheximide treatment of WISH cells and the presence of BSA (Fig. 3), or the addition of 10-5 M tamoxifen (Table 1) again blocked 17ß-estradiol action. The inhibitory effect of 10-7 M 17ß-estradiol plus Ro 20-1724 was almost unchanged when the latter drug was administered at concentrations varying from 10-7 to 10-4 M (Table 2). The maximal concentration (0.3%) of vehicle employed did not significantly influence PGE2 release from WISH cells.
|
|
17ß-Estradiol Effect on cAMP System
Figure 4 shows that 17ß-estradiol (10-9–10-5 M) slightly increased (P < 0.05 at 10-6 M) cAMP levels in WISH cells preincubated in the absence of Ro 20-1724. When the hormone was added in combination with the cAMP phosphodiesterase inhibitor (10-5 M), which per se significantly enhanced the nucleotide levels (139%, P < 0.05), a dose-dependent cAMP increase was observed; this effect reached statistical significance at 10-7 M 17ß-estradiol. The same figure shows that cycloheximide treatment of WISH cells (5 µg/ml for 60 min) did not significantly change the cAMP enhancement induced by Ro 20-1724, either alone or in the presence of 17ß-estradiol. The maximal concentration (0.3%) of vehicle employed did not significantly influence cAMP levels in WISH cells. We finally assayed the adenylyl cyclase activity in WISH cell membrane preparations. The enzyme was greatly stimulated by general adenylyl cyclase activators, such as sodium fluoride and forskolin [18, 19], as well as by the receptor agonist epinephrine; on the contrary, the enzyme was completely unaffected by 17ß-estradiol, tested at 10-6 M (not shown).
|
DISCUSSION
Chibbar et al. [6] were the first to demonstrate the presence of mRNA for estrogen receptors in human amnion, even if in lower amounts with respect to choriodecidua. In previous studies estrogen receptors could not be detected in human term amnion, probably due to the relatively low sensitivity of the methods employed [20]. In this paper, we report that also in WISH cells no detectable [3H]estradiol-17ß binding is present, but it becomes measurable when the cells are exposed to Ro 20-1724 or forskolin. The effect of these compounds is probably mediated by cAMP enhancement. In fact, it has been demonstrated that the nucleotide modulates estrogen receptor expression in several cell lines [21, 22], and that forskolin or cholera toxin exert similar effects in rat endometrial cells [23]; moreover, we here demonstrate that Ro 20-1724 and forskolin increase WISH cell cAMP concentration.
In most cells, the estrogen receptor affinity toward its ligand (0.1–1 nM) [24, 25] is higher than that measured in WISH cells. However, our studies have been carried out in cells different from those utilized until now to measure the affinity of estrogens, and we used different preparations for binding assays (whole cells versus cytosol). Another reason for the different behavior, perhaps the most important, is that estrogen receptors are not greatly expressed in naive WISH cells (i.e., cells unexposed to Ro 20-1724 or forskolin), but they became available after exposure to these drugs; only cells naturally expressing estrogen receptors have been utilized in the studies in which the affinity of estrogens has been measured. All these differences in the experimental conditions may contribute to the different results obtained.
Because 17ß-estradiol stimulates PGE2 release from naive WISH cells in a dose-dependent manner, and this response is counteracted by the estrogen receptor antagonist tamoxifen, it must be assumed that estrogen binding sites, although undetectable, are present. When prostanoid output is measured in cells exposed to Ro 20-1724, 17ß-estradiol inhibits PGE2 output and, also in this condition, tamoxifen counteracts the hormonal effect. Both stimulatory and inhibitory actions of 17ß-estradiol are prevented by the protein synthesis blocker cycloheximide, or else when the hormone is bound to BSA, to avoid its diffusion through the cellular membrane. 17ß-Estradiol thus modulates PGE2 release from WISH cells by interacting with specific intracellular receptors and probably evoking new protein synthesis. Intriguing is our demonstration that, in the presence of Ro 20-1724, 17ß-estradiol inhibits PGE2 release: an enhancement of prostanoid output would rather be expected because, in similar experimental conditions, Ro 20-1724 induces an increase of 17ß-estradiol receptors.
Based on our data, it can therefore be hypothesized that naive WISH cells possess few constitutive 17ß-estradiol receptors, whose activation leads to an enhancement of PGE2 release; when exposed to Ro 20-1724 these cells express new receptors, functionally prevailing over the preexisting ones that are predisposed to inhibit prostanoid release. Alternatively, both the constitutive and induced receptors could undergo a conformational change responsible for the inhibitory effect on PGE2 release, possibly due to the enhanced intracellular cAMP concentration. Obviously, the slight nucleotide increase evoked by 17ß-estradiol in the absence of Ro 20-1724 (condition in which the hormone stimulates prostanoid release) may be too small to provoke new receptor expression or the switch in receptor functionality. A direct effect of Ro 20-1724, not secondary to its action on cAMP phosphodiesterase, seems improbable because the extent of inhibition of prostanoid output by the combination Ro 20-1724 plus 17ß-estradiol is independent of the Ro 20-1724 dose but dependent on the hormone concentration.
Cyclic AMP enhancement evoked by 17ß-estradiol and Ro 20-1724, alone or in combination, is unaffected by cycloheximide, suggesting that nongenomic mechanisms may be responsible for the nucleotide elevation: nongenomic actions of 17ß-estradiol are extensively described [26]. However, we can exclude a direct effect of the hormone on a membrane receptor coupled to adenylyl cyclase.
In conclusion, our data provide the first evidence for the presence of functional 17ß-estradiol binding sites in WISH cells and demonstrate that their expression is enhanced by forskolin and Ro 20-1724, probably through an increase of cAMP levels. Because these receptors influence PGE2 release in a complex way, they could contribute to the modulation of uterine contractions, both during pregnancy and at the beginning of labor. Whether cAMP enhancement is also responsible for the reversion of the 17ß-estradiol effect on PGE2 release observed in the presence of Ro 20-1724 still remains an open question. If this were the case, we could speculate that WISH cell responsiveness to 17ß-estradiol may be modulated at different steps depending on the intracellular cAMP concentration. This, in turn, is influenced by the various agonists that directly regulate the enzymes of cAMP metabolism, as well as by compounds that modulate the nucleotide levels through indirect mechanisms.
ACKNOWLEDGMENTS
We are grateful to Mrs. Linda Bruce, a qualified mother tongue English teacher, for English revision of the text.
FOOTNOTES
First decision: 12 September 2000.
1 This work was supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (60%). 
2 Correspondence: Maria Enrica Ferretti, Department of Biology, Section of General Physiology, University of Ferrara, via Luigi Borsari 46, 44100-I Ferrara, Italy. FAX: 0532 207143; clm{at}dns.unife.it
Accepted: January 16, 2001.
Received: August 3, 2000.
REFERENCES
in human amnionic WISH cells by various stimuli occurs through distinct intracellular mechanisms. J Pharmacol Exp Ther 1997; 280:1065–1074
-phosphate of GTP. EMBO J 1987; 6:2907–2913[Medline]This article has been cited by other articles:
|
|
 |
|
  L. W. Chang, Y.-C. Chang, C.-C. Ho, M.-H. Tsai, and P. Lin Increase of carcinogenic risk via enhancement of cyclooxygenase-2 expression and hydroxyestradiol accumulation in human lung cells as a result of interaction between BaP and 17-beta estradiol Carcinogenesis, July 1, 2007; 28(7): 1606 - 1612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Korita, H. Itoh, N. Sagawa, S. Yura, M. Yoshida, K. Kakui, M. Takemura, and S. Fujii 17{beta}-Estradiol Up-Regulates Prostacyclin Production in Cultured Human Uterine Myometrial Cells Via Augmentation of Both Cyclooxygenase-1 and Prostacyclin Synthase Expression Reproductive Sciences, October 1, 2004; 11(7): 457 - 464. [Abstract] [PDF]
|
|
|
|
 |
|
 S. Fiorini, M. E. Ferretti, C. Biondi, B. Pavan, L. Lunghi, G. Paganetto, and L. Abelli 17{beta}-Estradiol Stimulates Arachidonate Release from Human Amnion-Like WISH Cells through a Rapid Mechanism Involving a Membrane Receptor Endocrinology, August 1, 2003; 144(8): 3359 - 3367. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Concentration at which the effect of 17ß-estradiol plus Ro 20-1724 becomes statistically different (P < 0.05) with respect to Ro 20-1724 alone