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Endocrinology, doi:10.1210/en.2005-0250
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Endocrinology Vol. 146, No. 11 4878-4886
Copyright © 2005 by The Endocrine Society

Regulation of the Endothelin/Endothelin Receptor System by Interleukin-1ß in Human Myometrial Cells

Michelle Breuiller-Fouché, Catherine Morinière, Emmanuelle Dallot, Stéphanie Oger, Régis Rebourcet, Dominique Cabrol and Marie-Josèphe Leroy

Institut National de la Santé et de la Recherche Médicale Unité 427 Paris (M.B.-F., C.M., E.D., S.O., D.C., M.-J.L.), Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, F-75270 Paris cedex 06, France; and Institut National de la Santé et de la Recherche Médicale Unité 709 Paris (R.R.) and Maternité Port-Royal (D.C.), Hôpital Cochin Assistance Publique-Hôpitaux de Paris, Université René Descartes, F-75014 Paris, France

Address all correspondence and requests for reprints to: Michelle Breuiller-Fouché, Institut National de la Santé et de la Recherche Médicale Unité 427 Paris, F-75270 cedex 06 France; or Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, 4 Avenue de l’Observatoire, F-75006 Paris, France. E-mail: breuiller-fouche{at}cochin.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proinflammatory cytokines produced at the fetomaternal interface, such as IL-1ß, have been implicated in preterm and term labor. The present study was performed to evaluate the influence of IL-1ß on the endothelin (ET)/ET receptor system in human myometrial cells. We report that myometrial cells under basal conditions not only respond to but also secrete ET-1, one of the main regulators of uterine contractions. Prolonged exposure of the cells to IL-1ß led to a decrease in prepro-ET-1 and ET-3 mRNA correlated with a decrease in immunoreactive ET-1 and ET-3 levels in the culture medium. Whereas ETA receptor expression at both protein and mRNA levels was not affected by IL-1ß treatment, we demonstrated an unexpected predominance of the ETB receptor subtype under this inflammatory condition. Whereas the physiological function of ETB remains unclear, we confirmed that only ETA receptors mediate ET-1-induced myometrial cell contractions under basal conditions. By contrast, prolonged exposure of the cells to IL-1ß abolished the contractile effect induced by ET-1. Such a regulation of IL-1ß on the ET release and the balance of ETA to ETB receptors leading to a loss of ET-1-induced myometrial cell contractions suggest that complex regulatory mechanisms take place to constraint the onset of infection-induced premature contractions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MECHANISMS OF human parturition are not fully understood because of the multiplicity of factors involved (hormonal, mechanical, genetic, and/or environmental), but a role for a local inflammatory process seems certain, (for a review, see Ref.1). At the end of pregnancy, leukocytes, mostly neutrophils and macrophages, are known to infiltrate uterine tissues concomitantly with the increased production of proinflammatory cytokines, chiefly IL-1ß, IL-6, and TNF{alpha}, at the fetomaternal interface. Such an inflammatory response can be initiated prematurely by a maternal ascending infection and evidence has emerged over the last decades that an infection of the decidua, the fetal membranes, or the amniotic fluid is associated with preterm labor. The cytokine-mediated inflammatory process is directly involved in the release of uterotonic agents. One example is the up-regulation of cyclooxygenase-2 (2) and prostaglandin (PG) production, which have been shown to induce cervical ripening (3), premature rupture of the fetal membranes, and myometrial contractions (4, 5) leading to delivery. In contrast to PGs, the expected role of inflammatory mediators on activation of the oxytocin (OT)/OT receptor system is less clear. Some data have confirmed an up-regulation of OT receptors after treatment of human myometrial cells with proinflammatory cytokines, whereas other data did not support this conclusion (for a review see Ref.6). Another peptide, endothelin (ET)-1, has been shown to cause potent in vitro myometrial contractions in humans (7). An increased concentration of ET-1 was found in maternal plasma during labor and delivery as well as human amniotic fluid (8). Furthermore, because women with preterm labor and positive amniotic fluid cultures for microorganisms have higher amniotic fluid concentrations of ET-1 than those without microbial invasion of the amniotic cavity, it has been hypothesized that ET-1 may play a role in the regulation of myometrial contractility in infection-induced preterm labor (9). Although in the last few years ET-1 has attracted considerable attention in the myometrium, interactions between the cytokine network and ET-1 in this tissue remain unknown.

ET-1 belongs to a family of three 21-amino acid isopeptides, each encoded by three separate genes at distinct chromosomal loci (10). Two distinct endothelin receptors denoted ETA, which is ET-1 selective (11), and ETB, which exhibits similar affinity for all three ET isopeptides and sarafotoxin 6c (S6c) (12), have been identified in human myometrium (13). We and others have demonstrated that only the ETA receptors mediate the contractile effect of ET-1 in this tissue (14, 15) and human cultured myometrial cells (16, 17, 18). Moreover, an increase in the density of myometrial ETA receptors (19, 20) accompanies the increase of uterine contractile responsiveness to ET-1 at the end of pregnancy (21). By contrast, the physiological role of ETB receptors in myometrial tissue is still to be determined.

In light of this information, the present study was conducted to assess whether the ET/ET receptor system participates in the regulation of the inflammatory response. For this purpose, we used a model of inflammation by exposing human myometrial cells to a proinflammatory cytokine IL-1ß, as designed by Oger et al. (22), to investigate whether such treatment affects gene and protein expressions of ET and the two subtypes of ET receptors as well as their binding properties. Furthermore, the consequences of IL-1ß treatment on the contractile properties of ET-1 were evaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
DMEM, trypsin-EDTA, penicillin-streptomycin mixture, PBS with and without calcium, and fetal calf serum (FCS) were obtained from In Vitrogen Life Technologies (Cergy-Pontoise, France). Collagen was purchased from Becton Dickinson Biosciences (Bedford, MA). [125I]ET-1 (specific activity 81.4 TBb/mmol) was obtained from PerkinElmer (Boston, MA). ET-1, ET-3, S6c, and the ET receptor antagonists FR139317 and BQ788 were from Neosystem Laboratoire (Strasbourg, France). Antibody against ETA and ETB receptors were from Chemicon (Temecula, CA). They were raised in rabbits against peptides corresponding to amino acid sequences 413–426 and 298–314 of rat ETA and ETB receptors, respectively. A donkey antirabbit IgG conjugated to horseradish peroxidase was from Amersham International (Chalfont, Buckinghamshire, UK). Electrophoresis reagents were obtained from Bio-Rad Laboratories (Richmond, CA). Hybond-C membranes, enhanced chemiluminescence detection system, and x-ray film were obtained from Amersham International.

Myometrial cell culture
Biopsies of myometrium were obtained from nonpregnant cycling women undergoing hysterectomy for benign gynecological indications. Tissues samples were excised from normal muscle in areas free of macroscopically visible abnormalities. This study was approved by the Comité Consultatif de Protection des Personnes pour la Recherche Biomédicale (Paris-Cochin, France). After collection, the myometrial biopsies were placed in DMEM supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. Human myometrial cells were obtained by the explant method as previously described (23). Cells were cultured in DMEM supplemented with antibiotic solution and 10% FCS and routinely passaged when 90–95% of cells were confluent. Confluent cells between their fourth and fifth passages were used 72 h after serum deprivation. No noticeable difference between results were obtained with cells from individual passages and cells obtained from different uteri. Each population of myometrial cells studied has been taken from a different patient. Confluent myometrial cells were identified by their typical hill-and-valley microscopic appearance and their positive reaction to a monoclonal antibody raised against smooth muscle {alpha}-actin.

Cell stimulation and membrane preparation
Confluent myometrial cells were incubated in the absence or the presence of 10 ng/ml IL-1ß for the indicated times. At the end of incubation, cells were lysed and scraped into a protein lysis buffer [1.5 mM Tris HCl (pH 7.5), 2 mM MgCl2, 0.3 mM EDTA, and protein inhibitor cocktail], sonicated, and centrifuged twice at 50,000 x g for 20 min as previously described (16). The final pellet referred as membrane preparation was suspended in either Laemmli buffer (for Western blot) or 50 mM Tris-HCl (pH 7.4) (for binding studies) at a protein concentration of about 2 mg/ml. The membrane preparation was stored at –80 C until use.

RT-PCR analysis
Total RNA was extracted from myometrial cells using the TRIzol reagent method (Life Technologies, Inc., Cergy-Pontoise, France). Briefly, scraped cells (107 cells) were resuspended in 1 ml TRIzol and homogenized by repeated pipetting. RNA preparations were recovered by phenol/chloroform extraction, isopropanol precipitation, and ethanol wash, according to the manufacturer’s instructions. The first strand of cDNA was generated from 8 µg total RNA using random hexamers to prime the reverse transcription in a total reaction volume of 50 µl. Total RNA was denatured by heating at 72 C for 10 min and cooling immediately on ice. The preparation was then incubated with 800 U murine reverse transcriptase (Life Technologies) in the presence of 10 mM dithiothreitol, 20 µM random hexamers, and 20 U RNasin ribonuclease inhibitor (Promega Corp., Lyon, France) for 60 min at 39 C. The reaction was stopped by heating at 95 C for 5 min, followed by cooling. Reverse transcription products were stocked at –20 C. Preparations achieved without reverse transcriptase were routinely used as a control for each RNA sample. No PCR product was detected in the absence of reverse transcriptase during the reverse transcription step, indicating that the RNA preparations were free of genomic DNA. Amplification was performed in 1x PCR buffer [50 mM KCl and 20 mM Tris-HCl (pH 8.3)] in a 25-µl total reaction volume. This contained 200 µM of each deoxy-NTP and 1–2 mM MgCl2 together with 1 µM of each primer, sense and antisense, 1.25 U Taq DNA polymerase (Life Technologies,), and 3 µl reverse transcription product (480 ng cDNA). Primers for ET-1, ET-3, ETA, and ETB receptors and PCR conditions were previously described (19, 24). We confirmed that the number cycle products were within the linear logarithmic phase of the amplification curve (data not shown).

After amplification, a 15-µl aliquot from each reaction mixture was resolved by electrophoresis on a 3% NuSieve agarose gel (TEBU, Le Perray en Yvelines, France) and visualized by ethidium bromide staining under UV light. To check the size of PCR products, a DNA molecular mass standard ladder (123-bp DNA ladder; Life Technologies) was concomitantly subjected to electrophoresis. Additional control of validity was carried out using Southern blot analysis of the PCR products with specific internal oligonucleotides as previously described (19). Amplification of an endogenous marker, the human ß2-microglobulin cDNA, was used as an internal control.

Measurement of ET in supernatants of myometrial cells in culture
ET-1 and ET-3 secretions were measured in supernatants, collected from cultures of myometrial cells untreated and treated by 10 ng/ml IL-1ß for 18 h, using an ELISA kit assay after an HPLC separation as previously described (25). Briefly, reverse-phase HPLC was performed using a Symmetry 300 C18 HPLC column (Waters, Saint-Quentin-en-Yvelines, France), and the two isoforms of ET were eluted at a flow rate of 0.7 ml/min with the following linear gradient: solvent A = 0.1% trifluoroacetic acid (TFA); solvent B = 60% acetonitrile, 0.1% TFA. The gradient was A for 10 min and then B in A (vol/vol) from 0 to 50% for 10 min, 50 to 65% for 30 min, and to 100% for 5 min. After extensive washing, elution profiles were determined by UV adsorption at 214 nm. Fractions (0.7 ml) were collected, evaporated to dryness, and subjected to ELISA kit assay (Endothelin-1 EIA kit; Cayman Chemical Co., Ann Arbor, MI), according to manufacturer’s instructions. The cross-reactivity of ET-1 and ET-3 was 100% and the lower limit of detection was 1.5 pg/ml.

Western blot analysis
Equal amounts of protein from membrane preparations of myometrial cells were separated by SDS-PAGE on 10% gels and transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked by incubating the membranes with 5% fat-free dried milk in 10 mM Tris HCl (pH 7.5), 0.15 M NaCl, and 0.1% Tween 20. A rabbit polyclonal antibody raised against ETA or ETB was added at the appropriate concentration (1:200) and incubated for suitable times at room temperature. Membranes were washed with 10 mM Tris HCl (pH 7.5), 0.15 M NaCl, and 0.1% Tween 20 and incubated with the secondary antibody, a donkey antirabbit IgG (dilution 1:5000) conjugated with horseradish peroxidase. The blots were developed with enhanced chemiluminescence reagents and visualized on x-ray films (Kodak, Rochester, NY). Molecular-weight markers were run in parallel and human umbilical vein endothelial cells (HUVECs) were used as positive control for ETB receptors. The specificity of the immunoreactive band of ETA and ETB was demonstrated by specific blocking in the presence of the respective antigen peptide against which the antibody has been raised. The membranes were stripped with a buffer containing 62.5 mM Tris (pH 6.2), 2% sodium dodecyl sulfate, and 100 mM ß-mercaptoethanol at 50 C for 30 min and reprobed with a goat polyclonal antibody raised against ß-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Binding assay
Binding studies were performed at 30 C for 60 min in a volume of 0.25 ml containing the membrane preparation (8 µg protein), 50 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 0.1% BSA, and [125I]ET-1 from to 5 to 200 pM (saturation analysis) or 50 pM (competition studies) as previously reported (13). Briefly, bound [125I]ET-1 was separated from free [125I]ET-1 by rapid filtration through glass fiber filters (GF/C; Whatman, Middlesex, UK) presoaked in 0.1% BSA in 50 mM Tris-HCl (pH 7.4). The filters were washed with an additional 15 ml of 50 mM Tris-HCl (pH 7.4), dried, and counted on a Packard {gamma}-counter with 85% efficiency. The specific binding was defined as the difference between the amounts of ET-1 bound in the absence (total binding) and the presence (nonspecific binding) of 1 µM unlabeled ET-1. Estimates of maximal binding capacity (Bmax), dissociation constant (Kd), and IC50 values were calculated with the nonlinear curve-fitting program GraphPad Prism (version 4.01, GraphPad Software Inc., San Diego, CA). Inhibition constant values (Ki) were calculated from IC50 values using the Cheng and Prusoff equation: Ki = IC50/(1 + [L]/Kd). Results are expressed as the mean ± SEM.

Preparation of three-dimensional hydrated collagen lattices
Human myometrial cells were included into collagen lattices as described by Dallot et al. (17). Briefly, confluent cells harvested with trypsin 0.05%-EDTA 0.02% were centrifuged at 1000 x g for 5 min and resuspended in 10% FCS-DMEM at the required cell density. A type I collagen solution (4.1 mg/ml in 0.2 N acetic acid) was adjusted to pH 7.2 with 0.1 N NaOH. The final concentration of collagen was 1.5 mg/ml. The appropriate concentration of myometrial cells (150,000 cells/well) was then added to the neutralized collagen solution. Collagen gel-cell suspensions were incubated in untreated culture dishes (35 mm diameter) for 2 h at 37 C to allow gelling, and then 2 ml of fresh DMEM supplemented with 10% FCS were added over the cell-collagen lattice. Two days later, the culture medium was replaced. The lattices were then gently detached from the sides and lifted off the bottom of the well containing 2 ml of serum-free medium. When tested, IL-1ß was introduced at this step. The myometrial cells in the lattice were then exposed to ET-1 (50 nM) or S6c (100 µM). We checked that the growth rate of myometrial cells cultured into collagen gels as well as cell viability was similar to that of cells cultured on plastic dishes. Images of the floating gels were captured and digitized using a scanner (Studio Scan IISI; Agfa, New York, NY) before adding the test agents and after incubating for 24 h. The lattice was assimilated to an ellipse and the area calculated after measuring the major and minor diameters of the gel. Collagen contraction was expressed as percentage contraction ± SEM of triplicate determinations from five to seven separate experiments, in which the percentage of contraction is the percentage of decrease of the original surface area.

Statistical analysis
Results were expressed as the mean ± SEM. Groups of data were evaluated by ANOVA. A Bonferroni correction was performed to adjust for multiple comparisons of gel areas. Differences among groups in binding experiments were determined by Student’s t test for unpaired data or by ANOVA as appropriated. Values of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of IL-1ß treatment on the mRNA expression of prepro-ET isoforms and on ET release
To determine whether IL-1ß modulates prepro-ET-1 and ET-3 gene expression, mRNA from myometrial cells untreated and treated with 10 ng/ml IL-1ß for 5, 12, and 18 h were extracted and reverse transcribed for PCR analysis. After amplification using external oligonucleotides specific for the prepro-ET-1 sequence, a product of the predicted size (442 bp) was detected in untreated and treated cells at each time point (Fig. 1Go). When myometrial cells were treated with IL-1ß, the intensity of the signal for prepro-ET-1 decreased at 5, 12, and 18 h. The expression of prepro-ET-3 mRNA was investigated in the same myometrial cell preparations, and a DNA fragment of 479 bp as predicted was obtained. IL-1ß treatment led to a slight decrease in the intensity of this signal.



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  		FIG. 1. Effect of IL-1ß treatment on the mRNA expression of prepro-ET isoforms in human myometrial cells. A, Total RNA was prepared from myometrial cells that were cultured for 5, 12, and 18 h with either vehicle or 10 ng/ml IL-1ß. RT-PCR was conducted using specific primers for prepro ET-1 and ET-3. RNA extracts from treated or untreated cells were matched in each instance and gave identical signals for amplification of the internal control, ß2-microglobulin. The ethidium bromide-stained gel pictures are representative of three to five separate experiments with cells from explants of different myometria. DNA molecular mass standards appear in the far left lane.

 
ET release was measured in the conditioned medium of myometrial cells untreated and treated with 10 ng/ml IL-1ß for 18 h. Two isoforms of ET were separated and recovered after HPLC separation as shown in Fig. 2Go. Using an ELISA for ET determination, we found that ET-1 was at a significant lower concentration in the conditioned medium recovered after IL-1ß treatment (545 ± 270 pg/3 x 106 cells) than that recovered from untreated cells (2729 ± 1237 pg/3 x 106 cells) (P < 0.05). In the conditioned medium of IL-1ß-treated cells, there was a tendency of lower ET-3 content (24 ± 12 pg/3 x 106 cells), compared with the content in untreated cells (96 ± 68 pg/3 x 106 cells) without achieving the level of significance.



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  		FIG. 2. Effect of IL-1ß treatment on the release of ir-ET from human myometrial cells. Cells were incubated for 18 h with either vehicle or 10 ng/ml IL-1ß. A, HPLC separation of synthetic ET-1 and ET-3 peptides. Detection was performed by UV absorbance at 214 nm. The continuous line denotes the linear gradient of acetonitrile (33–37.5%) + TFA. B, Fractions (0.7 ml) were collected, evaporated to dryness, and subjected to ELISA kit. The data show ET-1 and ET-3 release into the supernatant of cells treated or not by IL-1ß and were expressed as picograms per 3 x 106 cells. Data are representative of three independent experiments for each condition with cells from explants of different myometria.

 
Effect of IL-1ß treatment on the expression of ETA and ETB receptors at both mRNA and immunoreactive protein levels
As illustrated in Fig. 3Go, electrophoresis on agarose gel stained with ethidium bromide revealed PCR products of the correct predicted sizes, 626 bp for ETA and 564 bp for ETB receptors, in both untreated myometrial cells and cells treated with 10 ng/ml IL-1ß for various times. No difference was found in the intensity of the signal for ETA receptor mRNA in untreated cells comparatively with cells treated by IL-1ß. By contrast, IL-1ß treatment induced a clear increase in the intensity of the signal for the ETB transcripts after 5, 12, and 18 h of incubation.



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  		FIG. 3. Effect of IL-1ß treatment on the mRNA expression of ETA and ETB receptors in human myometrial cells. A, Total RNA was prepared from myometrial cells that were cultured for 5, 12, and 18 h with either vehicle or 10 ng/ml IL-1ß. RT-PCR was conducted using specific primers for ETA and ETB receptors. RNA extracts from treated or untreated cells were matched in each instance and gave identical signals for amplification of the internal control, ß2-microglobulin. The ethidium bromide-stained gel pictures are representative of three to five separate experiments with cells from explants of different myometria. DNA molecular mass standards appear in the far left lane.

 
Immunoblot analyses were performed to confirm at the protein level the up-regulation of ETB receptor gene product observed in IL-1ß-treated cells. As illustrated in Fig. 4Go, two bands with molecular masses at 34 and 48 kDa were labeled with the ETB-specific antibody in membrane extracts from untreated and treated myometrial cells and were also detected when using the HUVEC-positive control cells. The signal intensity of the two labeled immunoreactive bands was decreased in the presence of the corresponding blocking peptide. The 48-kDa band likely represents the mature, nonglycosylated proteins, whereas the protein at 34 kDa may be the product of proteolytic cleavage as reported by Takasuka et al. (26). Upon treatment of myometrial cells with 10 ng/ml IL-1ß for 18 h, the intensity of the 34- and 48-kDa signals was stronger than that of the signals obtained with the untreated cells.



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  		FIG. 4. Effect of IL-1ß treatment on the expression of ETA and ETB immunoreactive proteins in human myometrial cells. Cells were incubated for 18 h with either vehicle or 10 ng/ml IL-1ß. Aliquots of membrane preparations (10 µg protein) were subjected to SDS-PAGE and immunoblotted with specific ETA (top panel) or ETB antibody (bottom panel) as described in Materials and Methods. HUVEC extract was used as a positive control for ETB receptors (left panel). The specificity of staining was confirmed by preincubating the ETB receptor primary antibody with the appropriate antigen peptide (right panel). ß-Actin was used as internal loading control. Arrows indicate the positions of the molecular mass markers (kilodaltons). These two immunoblots are representative of six separate experiments with cells from explants of different myometria.

 
Immunoblot analysis also indicated the presence of ETA immunoreactive protein in myometrial cells. ETA receptor gives a single immunoreactive band at approximately 50 kDa. Whatever the studied conditions, no modification in the intensity of the immunoreactive ETA protein was detected.

Effect of IL-1ß treatment on the expression of ET-1 binding sites
Binding of [125I]ET-1 to membrane preparation of untreated and IL-1ß-treated myometrial cells was saturable (data not shown). Nonlinear analysis of the saturation curve demonstrated the appearance of a one-site plot, indicating a high-affinity site with an apparent Kd of 84 ± 8 pM and a Bmax of 206 ± 17 fmol·mg–1 protein in untreated myometrial cells (Table 1Go). After 5 and 18 h of incubation with IL-1ß, the Kd values did not significantly differ from that of untreated cells. The Bmax of treated cells was markedly higher than that of control cells (318 ± 31 fmol·mg–1 protein at 5 h incubation and 381 ± 28 fmol·mg–1 protein at 18 h incubation), but we observed a significant increase in the Bmax values only at 18 h of IL-1ß treatment (P < 0.05).


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  		TABLE 1. Effect of IL-1ß treatment on the [125I]ET-1 binding parameters in human myometrial cells

 
In untreated myometrial cells, ET-1 was the most potent in displacing [125I]ET-1 binding, as judged by its Ki value (110 ± 40 pM), whereas the selective ETB receptor agonist S6c caused only a 40% inhibition even at 1 µM (Fig. 5AGo). The conventional ETB antagonist BQ788 was much weaker than the ETA antagonist FR139317 in competing for [125I]ET-1 binding. The Ki values were 215 ± 29 and 2.9 ± 1.2 nM, respectively. These data are consistent with our previous results (16) and indicate that human myometrial cells possess predominantly ETA receptors in these conditions.



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  		FIG. 5. Competitive inhibition of specific [125I]ET-1 binding by ET-1, ET-3, S6c, FR139317, and BQ788 in human myometrial cells treated without (A) or with 10 ng/ml IL-1ß for 18 h (B). Crude membrane cell preparation was incubated with [125I]ET-1 (50 pM) and increasing concentrations of unlabeled ligands. Binding is expressed as percentage of specific binding in the absence of competitors. The data shown are representative of a typical experiment carried out in duplicate from one patient. Similar data have been obtained in eight independent series of experiments.

 
When myometrial cells were cultured for 18 h in the presence of IL-1ß, competition curves changed, and significant different Ki values were observed for these ET receptor ligands, indicating that IL-1ß treatment resulted in a shift of ET receptor subtypes (Fig. 5BGo). Under these conditions, S6c was able to displace [125I]ET-1 binding with an apparent Ki value of 9.0 ± 0.9 nM. The ET-1 and FR139317 competition curves were shifted to the right, and the Ki values (2.0 ± 0.3 nM and 12.4 ± 1.9 nM, respectively) were significantly different from those obtained in untreated cells (P < 0.05). When myometrial cells were treated with IL-1ß, the BQ788 competition curve was shifted to the left with a significant lower Ki value than the one determined for the untreated cells (66.0 ± 9.0 nM and 215 ± 29 nM, respectively) (P < 0.05).

The proportion of ET receptor subtype(s) in myometrial cells treated with IL-1ß for 18 h was further estimated by performing binding experiments with [125I]ET-1 in the presence of S6c to block all ETB receptors. The Bmax value for [125I]ET-1 binding to membranes prepared from IL-1ß-treated cells in the absence of S6c was 401 ± 24 fmol·mg–1 protein. In the presence of 0.5 µM S6c, [125I]ET-1 binding was significantly decreased, and the Bmax value was 147 ± 39 fmol·mg–1 protein (P < 0.05). In addition, this latter Bmax was not significantly different from that observed in control myometrial cells (206 ± 17 fmol·mg–1 protein), suggesting that the increase of ET binding sites induced by IL-1ß treatment is predominantly attributable to an ETB receptor subtype. The Kd values between binding experiments without (Kd = 156 ± 40 pM) or with S6c (Kd = 113 ± 23 pM) were not significantly different. In the presence of S6c and FR139317, [125I]ET-1 binding was totally abolished (data not shown).

Effect of IL-1ß on ET-1-mediated collagen lattice contraction
The effect of IL-1ß on the ability of ET-1 to contract myometrial cells is illustrated in Fig. 6Go. We first confirmed that when myometrial cells were cultured in a three-dimensional collagen gel and detached from the underlying surface, cells contracted the gel over 24 h in serum-free DMEM, demonstrating their basal contractile tone (control: 17.6 ± 1.7% contraction). When ET-1 (50 nM) was added to the medium of untreated cells, a subsequent contraction of the lattice was observed. We found that treatment of myometrial cells with IL-1ß alone at 10 ng/ml for 24 h resulted in less decrease of the size of the gels than untreated cells. The inefficiency of S6c to induce contractile activity in untreated cells was preserved after IL-1ß treatment for 24 h. In contrast, we observed that the presence of IL-1ß blocked the ability of ET-1 to promote myometrial cell contraction.



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  		FIG. 6. Effect of IL-1ß on ET-1-promoted myometrial cell contraction. A, Myometrial cell collagen lattices were incubated without or with 10 ng/ml IL-1ß for 24 h before the addition of 50 nM ET-1 or 100 µM S6c. Results are expressed as the mean percentage contraction of collagen lattices ± SEM of triplicate determinations from seven separate experiments, each performed on a different population of myometrial cultured cells. *, P < 0.05 vs. basal; °, P < 0.05 vs. ET-1 alone. B, Photographs shown are of free-floating collagen lattices in culture dishes (diameter, 35 mm). Collagen lattices are shown at time = 0 min (upper panel) and after incubating for 24 h in serum-free DMEM alone (basal) or containing indicated drugs (lower panel). These data are typical experiments and are representative of the seven experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because intrauterine infection has been recognized as an important etiological factor of preterm labor and IL-1ß is thought to have a major role in the signaling cascade leading to the onset of labor, the present study was designed to evaluate whether IL-1ß can up-regulate uterine stimuli such as ET-1 to induce preterm myometrial contractions. This concept has relevance in vivo because an elevated concentration of ET-1 was found in the amniotic fluid of women with preterm labor associated with microbial invasion of the amniotic cavity (9).

In the present study, we originally demonstrated that human myometrial cells not only are a paracrine target for ET-1 but also represent a source of production. To support this, we established that under control culture conditions, quiescent human myometrial cells express prepro ET-1 mRNA and release immunoreactive (ir) ET-1 into the supernatant. Our results seem to contradict another study reporting an absence of immunopositive cells for ET-1 in the human nonpregnant myometrium (18).

From our present data, it is tempting to suggest that ET-1 was undetectable by immunohistochemistry methods rather than that it was not expressed in myometrium. This apparent discrepancy may also be explained by the differentiation state of the myometrial cells in culture. Nevertheless, our study was conducted on an appropriate cell model that retained most of its in vitro cellular characteristics (biochemical features and contractile properties) (17, 23).

It is noteworthy that ET-1 has also be reported to be produced by other human smooth muscle cells such as cells originating from the penis (27) or pulmonary artery (28). Interestingly, when applied to myometrial cells, IL-1ß induces a decrease in prepro-ET-1 mRNA and ET-1 mature peptide production, indicating that this proinflammatory cytokine can act either at the transcriptional level or by increasing mRNA degradation. On the contrary, Sagawa et al. (29) previously reported that addition of 100 ng/ml IL-1ß to cultured amnion cells induced a secretion of ET-1. Such a discrepancy may be ascribed to the difference in cell species and the concentration of IL-1ß used. Indeed, our results are supported by the more recent data of Wort et al. (30) demonstrating that IL-1ß inhibited ET-1 release by human pulmonary artery smooth muscle cells. These authors have shown that cAMP-elevating agents such as PGI2 mimetic and forskolin, which activate adenylate cyclase, or Rolipram, which inhibits type 4 phosphodiesterases, all decreased ET-1 production. In a model of inflammation of human myometrial cells, we and others have found that IL-1ß induces an accumulation of cAMP after 18 h of incubation (22, 31). It remains to be determined whether cAMP pathway acts as an endogenous constraint on ET-1 release by human myometrial cells during such an inflammatory process. Regarding ET-3 production, to our knowledge, the possibility that myometrial cells represent an autocrine source of this isopeptide has never been demonstrated. Our data demonstrate that untreated myometrial cells express prepro-ET-3 gene and release detectable ir-ET-3 in the conditioned medium. However, the decrease of prepro-ET-3 mRNA observed after IL-1ß-treatment is not accompanied by a significant decrease of ir-ET-3 release in the culture medium.

ET-1 remains the most studied among the ET family, and its function and location in myometrium is now well documented. ET-1 is a potent stimulant of uterine contractions. Its action is mediated by ETA receptors (15, 14) that are highly expressed in human pregnant term myometrium (19, 20). Whereas ETA and ETB receptors coexist in human myometrial tissue (13), only ETA receptors were detected in cultured myometrial cells: a major component classically sensitive to the conventional ETA antagonists FR139317 and BQ123 and a minor component sensitive to BQ123 (16). Evidence for the lack of ETB receptors was based on the absence of specific [125I]ET-3 binding and on analysis of competition [125I]ET-1 binding experiments yielding an order of potency of ET-1 >>S6c, which is consistent with the interaction of ETA receptor (16). In this study, we confirmed this finding by additional evidence showing that FR139317 displaced [125I]ET-1 binding with a higher affinity than the ETB antagonist BQ788. Although BQ788 may be considered a potent and selective ETB receptor antagonist (IC50, 1.2 nM), it was not devoid of affinity for ETA receptor. Indeed, the Ki value reported here (215 nM) was almost comparable with the affinity reported for an ETA receptor on vascular smooth muscle (IC50, 280 nM) (32). However, despite the observation that untreated myometrial cells exclusively possess ETA-like binding activity, we demonstrated both ETA and ETB mRNA expression.

It then seems possible that these cells express low level of ETB mRNA that do not result in expression of functional ETB binding sites. The expression of ETA receptor was unchanged when human myometrial cells were treated with IL-ß for 18 h. Interestingly, such treatment results in an increase in Bmax. This increase of ET receptors was accompanied by a predominance of the ETB receptors as observed by the shift in selectivity of BQ788 toward FR139317 to displace [125I]ET-1 binding. In support of this concept, we also found that IL-1ß affects only ETB mRNA and ETB immunoreactive protein expressions. These compelling data suggest that the prolonged presence of the proinflammatory mediator IL-1ß affects preferentially the ETB receptor. Whether the selective increase in ETB receptors is a specific feature of myometrial cells treated by IL-1ß remains to be clarified. Activation of ETB receptors may elicit a variety of responses dependent on the target cell on which they are expressed, but the role of ETB receptors in myometrium is still unknown. In blood vessels, ETB receptors located on endothelial cells mediate vasodilatation through the generation of nitric oxide and prostacyclin (PGI2) (33), whereas in vascular smooth muscle cells (VSMCs), ETB receptors mediate vasoconstriction via an increase in intracellular Ca2+ (34, 35, 36).

Many of the pathological conditions in which the ET/ET receptor system appears to be involved are associated with elevation of proinflammatory cytokine concentrations in the plasma. For example, in patients with coronary artery diseases such as atherosclerosis, a chronic inflammatory disease in which the cytokine network has a central role, a stimulation of the production of ir-ET-1 as well as an up-regulation of ETB receptors was observed in VSMCs of the patients (37, 38). White et al. (39) proposed that proinflammatory cytokines could affect latent ETB receptor-mediated contraction of human temporal artery and thereby contribute to pathological situations. High levels of ETB receptor expression are observed in myometrium from preeclamptic women (40), another inflammatory disease specific to pregnancy from placental origin (for a review, see Ref.41). However, because of the absence of in situ hybridization or immunohistochemistry studies, it is difficult to conclude whether these ETB receptors are localized on endothelial cells of the small intramyometrial arteries or myometrial smooth muscle cells. Whether modulations of the ET-1/ETB response may be triggered by particular cytokines also remain to be explored. Like other authors, we do not have any explanation concerning the absence of a functional role for ETB receptors in untreated myometrial cells in culture.

As previously published, we confirmed that exogenous ET-1 induces myometrial contractions via selective activation of the ETA receptors. Additional evidence for this was provided by the lack of contractile effect of the ETB-selective agonist S6c to modify myometrial cell tension in a conventional collagen gel retraction assay (17). However, long-term exposure of myometrial cells with IL-1ß was shown to abolish the contractile effect induced by ET-1. Such observation could not be explained by a loss of ETA receptors; one possible explanation is that ETB receptors, which appear to be up-regulated after IL-1ß treatment, act as clearance receptors (42). Because IL-1ß leads to the decrease of ET-1 release in addition to up-regulate postulated ETB clearance receptors, it may not be surprising to observe a decrease of myometrial cell contractions under these inflammatory conditions. In vascular diseases such as arteriosclerosis, it is well known that many inflammatory cytokines, including IL-1ß, affect VSMC differentiation (for a review, see Ref.43). Thus, an alternative explanation is that the increase in ETB receptors observed after IL-1ß treatment is associated with dedifferentiation of the myometrial cells. There is much evidence indicating that phenotypic changes of VSMCs in culture can be associated with the changes in ET receptor properties (44). Eguchi et al. (45) found that the ETB receptor subtype is related to phenotypic modulation in cultured VSMCs and may in part contribute to the potentiation of mitogenic activity by ET isopeptides. Whether ETB activation after IL-1ß treatment results in myometrial cell proliferation will need further investigations. However, our previous findings of a selective ETA-mediated stimulation of myometrial cell growth (46) is in apparent contrast with this hypothesis.

Given that proinflammatory cytokines at the fetomaternal interface during infection occupy a crucial position in the regulation of uterine contractions leading to preterm delivery, these results appear to be paradoxical. However, we cannot rule out that alternative proinflammatory cytokines, other than IL-1ß, such as IL-6 and/or TNF{alpha}, did or did not affect ET-1 and its receptors in cultured myometrial cells. Thus, down-regulation of the OT receptors was also found under IL-1ß in myometrial cells (47, 48), whereas opposite results reported by Rauk et al. (49) demonstrated that IL-6 up-regulates OT receptors in the same cells. However, Schmid et al. (50) proposed that both IL-1ß and IL-6 have a negative role in the transcriptional regulation of OT receptor gene in human immortalized myometrial cells in culture. These data support the clinical observations that a causal relationship often exists between high-virulence bacteria in the amniotic fluid and labor abnormalities, an insensitiveness to OT being observed in patients at risk for the development of intrapartum infection (51).

In summary, the decrease of ET-1 release and the shift of ETA to ETB receptors leading to a loss of ET-1-induced human myometrial cell contraction under our inflammatory conditions are compelling arguments to suggest that complex regulatory mechanisms take place against the onset of premature contractions. But when an intrauterine infection was clinically asserted, we can speculate that reorganization of the cascade of events including uterotonic agent production and propagation of myometrial contractions leading to preterm birth occurs. Such data have significant implications for both understanding the physiopathology of parturition and directing future therapies.


   Acknowledgments
 
We are very grateful to Dr. Céline Méhats for her constructive discussion.


   Footnotes
 
This work was supported by grants from the Fondation de la Recherche Médicale.

First Published Online August 18, 2005

Abbreviations: Bmax, Maximal binding capacity; ET, endothelin; FCS, fetal calf serum; HUVEC, human umbilical vein endothelial cell; ir, immunoreactive; Kd, dissociation constant; Ki, inhibition constant value; OT, oxytocin; PG, prostaglandin; S6c, sarafotoxin 6c; TFA, trifluoroacetic acid; VSMC, vascular smooth muscle cell.

Received March 2, 2005.

Accepted for publication August 10, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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