Molecular Human Reproduction, Vol. 9, No. 1, 1-8,
January 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Interleukin-1ß induces glycosaminoglycan synthesis via the prostaglandin E2 pathway in cultured human cervical fibroblasts
Submitted on February 7, 2002; resubmitted on August 7, 2002. accepted on October 31, 2002
1 INSERM U 361, Université René Descartes, Paris and 2 Maternité Port-Royal, Hopital Cochin, AP-HP, Université René Descartes, Paris, France 3 To whom correspondence should be addressed at: INSERM U 361, Pavillon Baudelocque, 123, Bd de Port-Royal, F-75014 Paris, France. e-mail: tsn{at}club-internet.fr
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ABSTRACT |
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The aim of this study was to identify, in cultured human cervical fibroblasts, the mechanisms by which interleukin (IL)-1ß induces the synthesis of glycosaminoglycans (GAG) and to explore the putative role of prostaglandin E2 (PGE2) in this process. Exposure of the cells for 24 h to IL-1ß induced a significant (P < 0.05) dose-dependent increase in GAG synthesis. IL-1ß (1 ng/ml) induced the expression of cyclooxygenase-2 (COX-2) protein 6 h after treatment, accompanied by a 7.5-fold increase in PGE2 production. We confirmed that NS398, a selective COX-2 inhibitor, dose-dependently blocked PGE2 augmentation following IL-1ß treatment. AH23848, the selective EP4 receptor antagonist, completely abolished IL-1ß-induced GAG synthesis, whereas AH6809, an EP2 receptor antagonist, had no effect on the stimulatory effects of IL-1ß. Furthermore, we demonstrated that 6 h exposure to IL-1ß induced a notable increase in EP4 receptor mRNA expression and a decrease in EP1 receptor mRNA but had no effect on the expression of EP2 and EP3 receptor transcripts. In conclusion, these findings indicate that IL-1ß not only induced GAG synthesis by increasing COX-2 protein expression and the subsequent PGE2 production but also enhanced the responsiveness of cervical fibroblasts to PGE2 by selectively up-regulating EP4 receptor mRNA expression. These results suggest that PGE2 may regulate human cervical ripening in an autocrine/paracrine manner via EP4 receptors.
Key words: cervical ripening/EP receptors/glycosaminoglycan/IL-1ß/prostaglandin E2
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Introduction |
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Cervical ripening is an active process, independent from uterine contractions (Uldbjerg et al., 1983a), which ensures that the cervix becomes a soft and compliant tissue able to dilate to permit the passage of the fetus during labour. Softening of the cervix involves a complex combination of biochemical and structural changes affecting the cervical stroma, and leading to an extensible organ (Uldbjerg et al., 1983a). Cervical ripening is characterized by an oedema, a dispersion of the collagenic network and an increase in total glycosaminoglycans (GAG) (Uldbjerg et al., 1983b). This increase in GAG results mainly from an augmentation of the concentration of cervical hyaluronic acid (HA) (Danforth et al., 1974; Von Maillot et al., 1979; Osmers et al., 1993), a hydrogenated GAG that plays a major role in oedema constitution.
Prostaglandin E2 (PGE2) has been identified as a central mediator in the cervical ripening process. PGE2 induces GAG synthesis by cervical fibroblasts, both in vitro (Carbonne et al., 1996, 2000) and in vivo (Norström, 1982; Cabrol et al., 1987). PGE2 transduces its signal via seven transmembrane domain G-protein coupled receptors, named EP receptors (Narumiya et al., 1999), found in the human cervix (Adelantado et al., 1988). The EP receptor family has been further classified into four subtypes: EP1, EP2, EP3 and EP4 (Coleman et al., 1994b). EP1 and EP3 are linked to the Ca2+/phospholipase C pathway (Funk et al., 1993; An et al., 1994), whereas EP2 and EP4 are coupled to the cAMP/adenylyl cyclase cascade (Bastien et al., 1994; Regan et al., 1994). We have already demonstrated that exogenous PGE2 induces GAG production via the cAMP pathway (Carbonne et al., 1996; Schmitz et al., 2001), and that only EP4 receptors were implicated in this effect in cultured human cervical fibroblasts (Schmitz et al., 2001).
Cervical ripening is now widely considered to be an inflammatory reaction (Liggins, 1981), because of the similarity between the biochemical features involved in the cervix at the end of pregnancy with those observed during tissue inflammation, i.e. leukocyte infiltration (Junqueira et al., 1980) and increased local production of prostanoids and cytokines (Ito et al., 1988; Barclay et al., 1993; Sennström et al., 2000). Particular significance has been attributed to one of these cytokines, IL-1ß. Local application of IL-1ß induces cervical ripening in guinea-pigs (Chwalisz et al., 1994) and rabbits (El Maradny et al., 1995) with biochemical and morphological effects that are indistinguishable from those observed during spontaneous cervical ripening at term. Although it has already been demonstrated that IL-1ß enhances GAG synthesis in cultured human cervical fibroblasts (Ogawa et al., 1998), the mechanisms by which IL-1ß induces cervical ripening remain largely unknown. IL-1ß is one of the most potent inducers of cyclooygenase-2 (COX-2) (Maier et al., 1990; Bartlett et al., 1999; Rauk and Chiao, 2000), the inducible form of COX implicated in the rise of PGE2 production at parturition. Knowing the important role played by PGE2 in GAG synthesis during cervical ripening (Bukowski et al., 2001), we have explored the potential involvement of PGE2 in IL-1ß-induced HA synthesis in human cervical fibroblasts in culture, as well as the effect of IL-1ß on the expression of EP receptors in this model.
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Materials and methods |
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Culture of human cervical fibroblasts
Cervical biopsies were obtained after hysterectomy in pre-menopausal women. This procedure was aproved by the Consultative Commitee for Protection of Persons in Biomedical Research (CCPPRB) of Paris–Cochin. All patients gave informed consent. Special care was taken in removing exo- and endo-cervical epithelia. Cell cultures were prepared as previously described (Cavaillé et al., 1996; Carbonne et al., 1996, 2000; Schmitz et al., 2001). Briefly, biopsies were minced and plated out in 60 mm diameter plastic dishes in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% inactived fetal calf serum (FCS) (Life Technologies, Cergy-Pontoise, France), 2 mmol/l glutamine and penicillin (100 U/ml)–streptomycin (100 µg/ml) (Life Technologies). Dishes were placed in a 5% CO2/95% air humidified incubator at 37°C. After 7 days, cells started growing out from the biopsies and the medium was then replaced by DMEM containing only 10% FCS. Cells became confluent
3 weeks after tissue collection and were then passaged every 7 days in DMEM containing 10% FCS. All experiments were performed on cells from five different explants between passages 3 and 6. There was no significant difference between the results obtained from cells at different passages, or with cells obtained from different cervices. The viability of the cells was checked by trypan blue exclusion. Viability was >95%.
Determination of glycosaminoglycan synthesis by human cervical fibroblasts
Cervical cells were subcultured on 24-well plastic culture plates at a density of 5x104 cells per well and allowed to grow to confluence. Cells were rinsed with phosphate-bufered saline (PBS) (Life Technologies) and incubated for 3, 6, 12, 24 or 48 h in serum-free DMEM without phenol red in the presence of IL-1ß (0.1, 1 and 10 ng/ml) (R&D Systems, Abingdon, UK) or vehicle alone as control. If necessary, cells were preincubated with EP receptor antagonists, AH6809 or AH23848 (gift from GlaxoWellcome, Stevenage, UK), or with a COX-2 inhibitor, NS398 (SPI-BIO, France), for 30 min before adding IL-1ß. The radiolabelled precursor, 2.5 µCi/ml [3H]glucosamine (Amersham, Little Chalfont, UK) was then added for 24 h. GAG were extracted as previously described (Wasteson et al., 1973) and modified (Redini et al., 1991). Briefly, at the end of the labelling period, monolayer cells were washed with PBS and digested with pronase (1 mg/ml) (Boehringer Mannheim, Meylan, France) in 100 mmol/l Tris–HCl (pH 7.5)/5 mmol/l CaCl2. Proteolysis was continued for 24 h at 56°C. GAG were precipitated at 37°C with 1% w/v cetylpyridinium chloride (CPC) (Sigma, St Louis, MO, USA), in the presence of carriers (hyaluronic acid 1 mg/ml, chondroitin-4-sulphate and chondroitin-6-sulphate 0.5 mg/ml) (Sigma). The GAG–CPC complex was recovered by centrifugation and dissolved in 2 mol/l MgCl2. GAG were then precipitated overnight at 4°C with cold ethanol. The final pellet was dissolved in 75 mmol/l NaCl. The radioactivity incorporated into GAG was measured by scintillation counting (Beckman LS 6000IC). The assays were performed in quadruplicate for each experiment. The intra-assay and inter-assay coefficients of variance were 6.5 and 25% respectively. The detection limit of the assay was 0.3 µg.
Western blot analysis
After 1, 3, 6, 12 or 24 h of treatment with vehicle or IL-1ß (1 ng/ml), the media were removed, and cells (20x106/75 cm2 culture flask) were harvested by scraping in ice-cold homogenization buffer. The homogenization buffer consisted of 100 mmol/l Tris–HCl (pH 7.4), 2 mmol/l MgSO4, 2 mmol/l EDTA, 10% glycerol and 1 mmol/l ß-mercaptoethanol and was supplemented with a cocktail of protease inhibitors: leupeptin (1 µmol/l), aprotinin (10 µg/ml), pefabloc (25 µg/ml), benzamidine (130 µg/ml) and soybean trypsin inhibitor (50 µg/ml). All protease inhibitors were from Sigma except pefabloc which was from Interchim, Montluçon, France. After sonification, samples were immediately stored at –20°C until use. Samples (3 µg of proteins/lane) were dissolved (vol/vol) in Laemmli buffer x2 and boiled for 5 min before analysis by electrophoresis on a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). After migration, proteins were transferred to Hybond-P PVDF membrane (Amersham) in a Bio-Rad Transplot apparatus. Blots were dried overnight and blocked in 10% non-fat dried milk powder in TBS-T (Tris 10 mmol/l, NaCl 150 mmol/l, and Tween-20 0.1%, pH 7.6) at room temperature for 1 h. The blocked membranes were washed three times in TBS-T. For immunodetection, the blots were then incubated for 90 min at room temperature with antihuman COX-2 polyclonal IgG (sc-1745; SantaCruz Biotechnologies, Santa Cruz, CA, USA) at a dilution of 1:1000 in TBS-T containing 1% nonfat dried milk powder. Control experiments were performed by incubating membranes with goat anti-COX-2 primary antibody pre-adsorbed on the related blocking peptide (sc-1745p; Santa Cruz Biotechnologies). After three washes in TBS-T, the blots were incubated for 45 min at room temperature with horse-radish peroxidase-linked donkey antigoat secondary IgG antibody (sc-2020; SantaCruz Biotechnologies) diluted 1:2000 in TBS-T containing 1% non-fat dried milk powder and washed five times with TBS-T. Immunoreactive proteins were detected by chemiluminescence (Amersham ECL reagents) following the manufacturer’s instructions. The molecular mass of the COX-2 protein was 72 kDa, as deduced from the relative mobility on SDS–PAGE compared with the molecular mass standard. The 72 kDa immunoreactive bands were quantified densitometrically using a computer-linked scanner and Adobe Photoshop 6.0 software package (Mountain View, CA, USA). The results are expressed in arbitrary density units (ADU) as the mean ± SEM.
Determination of PGE2 release from human cervical fibroblasts
Cells were subcultured on 6-well plastic culture plates at a density of 105 cells per well and allowed to grow to confluence. Cells were incubated for 1, 3, 6, 12 or 24 h in 1 ml serum-free DMEM without phenol red in the presence of IL-1ß (1 ng/ml) or vehicle. When necessary, cells were preincubated with NS398 (1 µmol/l) 30 min before adding IL-1ß. PGE2 produced by human cervical fibroblasts was measured in the culture medium by radioimmunoassay. The assay was conducted using [3H]PGE2 (NEN, Boston, MA, USA) and an anti-PGE2 antibody purchased from Pasteur-Productions (Paris, France). The concentration of PGE2 was determined in duplicate in unextracted medium samples. Briefly, to 5 ml tubes were added 0.1 ml [3H]PGE2 (2000 cpm), 0.1 ml of PGE2 standard (Cayman, Ann Arbor, MI, USA) used in increasing concentrations in order to establish a standard curve (5–2000 pg/ml) or 0.1 ml of culture medium, and 0.1 ml of a dilution of the anti-PGE2 antibody such that the initial binding in the absence of standard was 40% of the total radioactivity. [3H]PGE2, anti-PGE2 antibody, PGE2 standards and samples were diluted in 0.1 mol/l phosphate buffer, pH 7.25 containing 0.1% gelatin and 0.01% sodium azide. Samples were incubated overnight at 4°C. Free and bound radioactivities were separated by adding 2 ml of 0.1 mol/l phosphate buffer containing 0.25% activated charcoal and 0.025% dextran, and then centrifugating at 4°C for 40 min at 1700 g. The radioactive supernatant was counted by scintillation counting (Beckman LS 6000IC). Calculation of the standard curve and unknown samples was by computer-generated logit method. The sensitivity of the assay was defined as the quantity of PGE2 necessary to give 20% displacement of B0 (zero standard) and was 2 pg/tube. The intra-assay CV was 9.5% and the inter-assay CV was 14%.
Ribonucleic acid preparation and RT
Total RNA from human cultured cervical cells, exposed to IL-1ß (1 ng/ml) or vehicle for 6 or 24 h, was extracted using the Trizol reagent method (Life Technologies). Briefly, scraped cells were resuspended in 1 ml Trizol reagent and homogenized by repeated pipetting. Total RNA preparations were recovered by phenol chloroform extraction, isopropanol precipitation, and ethanol washing, according to the manufacturer’s instructions. RNA samples were then treated with DNAse I (Life Tecnologies) following the manufacturer’s protocol. Reverse transcription was performed by using random hexanucleotides (20 µmol/l) as primers on 4 µg of total RNA in the presence of 200 IU of Mo-MLV reverse transcriptase (Life Technologies). The cDNA products were stored at –20°C until required for PCR.
PCR
To define the optimal amplification conditions, a series of pilot studies was performed by using varying quantities of RT products from 80 to 650 ng and from 20 to 42 cycles of PCR amplification. On the basis of these initial experiments, the linear part of the amplification was determined for the four EP receptor genes. Amplification was performed in a 25 µl total reaction volume in 1xPCR buffer (50 mmol/l KCl and 20 mmol/l Tris–HCl, pH 8.3) containing 200 µmol/l of each deoxy-NTP and 0.75–1 mmol/l MgCl2 together with 1 µmol/l of each primer, sense and antisense, 1.25 IU Taq polymerase (Life Technologies), and RT–products from 360 ng of RNA. The amplification profile consisted of denaturation at 94°C for 1 min, annealing for 1 min at the specific temperature (Table I), and extension at 72°C for 1 min, with a final extension cycle at 72°C for 10 min. The generic primers for EP2, EP3 and EP4 receptor (Table I) were those from Belley and Chadee (1999). A set of generic primers for EP1 receptor were designed to amplify a 3' fragment of the described human EP1 receptor (Funk et al., 1993). After amplification, a 15 µl aliquot from each reaction mixture was resolved by electrophoresis on a 3% Nusieve agarose gel and visualized under UV light after ethidium bromide staining. The DNA molecular mass standard ladder consists of fragments that are multiples of 123 bp (123 bp DNA ladder; Life Technologies). To ensure that there was no genomic DNA contamination, a control reaction containing total RNA without reverse transcriptase was performed in all experiments. An endogenous marker, human ß2-microglobulin cDNA, was used as an internal control because its related protein is found on the surface of nearly all nucleated cells (Suggs et al., 1981; Güssow et al., 1987). The intensities of the bands on Polaroid images were quantified densitometrically using a computer-linked scanner and Adobe Photoshop 6.0 software package (Mountain View, CA, USA). The results are expressed in arbitrary densitometric units (ADU) as the mean ± SEM.
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Southern blot analysis
The PCR products were further analysed by Southern blot analysis. DNA fragments were transferred to Hybond-N+ membranes (Amersham) in 2xSSC. Hybridization was performed using a specific internal oligonucleotide of each EP receptor subtype (Table I) labelled with fluorescein-11-desoxy-uridine triphosphate by using an ECL 3' oligolabelling and detection system kit (Amersham) according to the manufacturer’s instructions.
Statistical analysis
The non-parametric Wilcoxon–Mann–Whitney test for paired samples was applied to compare GAG synthesis, COX-2 and PGE2 concentrations, and EP receptor mRNA steady state level. The results are expressed as the mean ± SEM. P < 0.05 was considered significant.
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Results |
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Effect of IL-1ß on GAG synthesis
In an attempt to determine the biochemical mechanisms by which IL-1ß induces cervical ripening, we exposed human cervical fibroblasts in culture to IL-1ß to see whether this treatment modified GAG synthesis, usually considered to be a marker of the ripeness of the cervix. As illustrated in Figure 1A and B, IL-1ß induces a time- and dose-dependent increase in the incorporation of [3H]glucosamine into GAG (P < 0.05). The effect of IL-1ß was maximum after 24 h treatment and peaked at a concentration of 1 ng/ml (1.70-fold increase, P < 0.05).
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Effect of IL-1ß on COX-2 expression and PGE2 production
We investigated whether the effect of IL-1ß on GAG synthesis could result from an increased expression of COX-2 protein and the subsequent production of PGE2. We first demontrated that IL-1ß (1 ng/ml), the concentration at which IL-1ß had the greatest impact on GAG synthesis, induced a time-dependent expression of an anti-COX-2 immunoreactive band of
72 kDa, as detected by Western blot analysis. Pre-adsorbing the primary antibody onto the related blocking peptide completely abolished the 72 kDa signal (Figure 2B), confirming the specificity of detection of the 72 kDa COX-2 protein in cervical cells. The expression of COX-2 increased up to 6 h and then decreased after 12 and 24 h of treatment (Figure 2A). As shown in Figure 2C, we confirmed the induction of COX-2 protein in cervical cells from four other patients. To further investigate the involvement of PGE2 in IL-1ß-stimulated GAG synthesis, we first examined the effect of IL-1ß on the time-course of PGE2 synthesis. We found that IL-1ß (1 ng/ml) induced a time-dependent increase in PGE2 production by cervical fibroblasts. This effect was maximum after 12 h of treatment (Figure 3A). PGE2 production was significantly increased 7.5-fold (P < 0.05) greater in IL-1ß-treated cells compared with control cells (Figure 3B). To explore the role of COX-2 in IL-1ß-induced PGE2 production, cells were preincubated for 30 min with NS398 (0.01–1000 nmol/l), the selective COX-2 inhibitor (Barnett et al., 1994). As shown in Figure 3B, NS398 dose-dependently blocked IL-1ß-stimulated PGE2 production, this inhibition being complete at 100 nmol/l.
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Effect of EP2 and EP4 receptor antagonists on IL-1ß-stimulated GAG synthesis
We previously reported that exogenous PGE2 induced GAG synthesis in cervical fibroblasts via the cAMP/adenylyl cyclase pathway (Carbonne et al., 1996; Schmitz et al., 2001). To further examine the mechanims by which PGE2 could mediate IL-1ß-induced GAG synthesis, we looked at the effects of antagonists of EP receptors linked to adenylate cyclase, i.e. EP2 and EP4, on IL-1ß-stimulated GAG synthesis. As illustrated in Figure 4, AH6809, an EP2 receptor antagonist (Woodward et al., 1995), had no effect on IL-1ß-induced GAG production at any of the concentrations tested. Conversely, AH23848, the selective EP4 receptor antagonist (Coleman et al., 1994b), induced a significant dose-dependent inhibition of IL-1ß- induced GAG production (P < 0.05) (Figure 5).
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Effect of IL-1ß on EP receptor mRNA expression
To establish whether IL-1ß could induce a selective up-regulation of the expression of the EP receptor transcripts, mRNA from treated and untreated cells was extracted and reverse transcribed for PCR analysis. As shown in Figure 6A, using specific primers for each EP receptor subtype, PCR amplification in control and treated cells yielded fragments of the appropriate size, as assessed by migration of the molecular weight standard. We checked normalization of RNA input by obtaining equivalent signal intensity for the endogenous marker, ß2-microglobulin, in control versus treated cells (Figure 6A). The specificity of each amplification product was checked by Southern blot analysis using four different internal oligonucleotides specific for each EP receptor (Figure 6B). When reverse transcriptase was omitted from the reaction mixture (RT–) no amplification product was observed (Figure 6A, B). After incubating the cells with IL-1ß (1 ng/ml) for 6 h, there was a notable increase in the PCR signal for the EP4 receptor (P < 0.05) and a decrease for the EP1 receptor signal (P < 0.05), whereas the intensity of the PCR signal for the EP2 and EP3 receptors was not modified by the treatment (Figure 6A, B). After exposure to IL-1ß for 24 h, the PCR signal for EP1, EP2, EP3 and EP4 returned to basal levels (data not shown).
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Discussion |
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In order to clarify the mechanisms by which an inflammatory reaction can induce cervical ripening, we explored the effects of a proinflammatory cytokine, IL-1ß, on GAG synthesis, commonly considered to be a biochemical marker of cervical maturation (Danforth et al., 1974; Von Maillot et al, 1979; Osmers et al., 1993). We first confirmed that IL-1ß induced a significant time- and dose-dependent increase in GAG synthesis as shown previously (Ogawa et al., 1998). The molecular mechanisms by which IL-1ß mediates this effect remain completely unknown.
A likely signal transduction pathway for IL-1ß involves COX-2, the enzyme responsible for PGE2 synthesis. In support of this, an elevation of COX-2 expression in fetal membranes (Teixeira et al., 1994; Slater et al., 1995; Fuentes et al., 1996) and in myometrium (Slater et al., 1999; Erkinheimo et al., 2000) has been shown to be correlated with the onset of labour. This increased expression of COX-2 and the resulting augmentation in PGE2 production (Rauk and Chiao, 2000) at the time of parturition may contribute to the myometrial activation observed at term pregnancy. IL-1ß is a well-known inducer of COX-2 and is implicated in the COX-2 up-regulation observed at the time of parturition via the NF-
B pathway (Belt et al., 1999; Allport et al., 2001). In contrast to the considerable literature on the expression and regulation of COX-2 in fetal membranes and myometrium, little is known about the implication of COX-2 in human cervical ripening. In the present study, we found that exposing cultured human cervical fibroblasts to IL-1ß induces COX-2 expression and that this phenomenon is accompanied by a 7.5-fold increase in PGE2 production.
PGE2 is a central mediator of cervical ripening and a well-known inducer of GAG synthesis during this process. We speculated that IL-1ß induces GAG synthesis via the PGE2 pathway. To confirm this hypothesis, we investigated whether IL-1ß could induce COX-2 induction and the subsequent PGE2 production and which EP receptors were implicated in IL-1ß-mediated GAG synthesis. The increase in PGE2 production following IL-1ß treatment was completly blocked by a specific COX-2 inhibitor, NS398. This strongly suggests that COX-2 is involved in this effect. The sequence of events, i.e. IL-1ß-induced COX-2 expression/PGE2 synthesis/GAG production, determined by the time-course experiments, further supports the role of PGE2 in this process. These data indicate that the results of Sato et al. (2001) showing COX-2 induction and subsequent PGE2 production after IL-1ß treatment in rabbit cervical fibroblasts are also relevant to humans.
Although the precise EP receptor by which PGE2 stimulated GAG synthesis, when cervical fibroblasts were treated with IL-1ß, remains unclear, it seems likely that EP4 receptors are potential candidates. Indeed, we have already demonstrated that exogenous PGE2 enhances GAG synthesis in human cervical fibroblasts via the EP4 receptor subtype (Schmitz et al., 2001). Since PGE2-stimulated GAG synthesis is dependent on the cAMP pathway (Carbonne et al., 1996; Schmitz et al., 2001) and since we have already shown that the EP2 receptor subtype is functional in this model (Schmitz et al., 2001) in terms of cAMP production, the possibility remains that the EP2 receptor may also be implicated in IL-1ß-induced GAG synthesis. However, the lack of inhibition of IL-1ß-stimulated GAG synthesis by the EP2 receptor antagonist excludes this possibility. Conversely, the complete inhibition of GAG synthesis by the selective EP4 receptor antagonist strongly supports the fact that PGE2 released after IL-1ß treatment mediates IL-1ß-induced GAG synthesis via its EP4 receptor subtype. It is of note that two receptors with the same second messager, cAMP, do not have the same effect on GAG synthesis. It is possible that the pathways downstream of cAMP could differ. cAMP is a well-known activator of protein kinase A (PKA) and EP2 activation in myometrium has been demonstrated to induce relaxation via a cAMP/PKA-dependent pathway (Asboth et al., 1997–98). In previous studies, we demonstrated that PGE2 increases GAG synthesis in cultured human cervical fibroblasts via a cAMP-dependent pathway [using 8-Br-cAMP (Carbonne et al., 1996) and a specific cAMP-phosphodiesterase-4 inhibitor (Schmitz et al., 2001)] and the EP4 receptor. Surprisingly, this effect was PKA independent (Schmitz et al., 2001). Such data could be explained by the fact that cAMP can act independently of PKA and can activate other protein kinases, as has been demonstrated in other cellular systems. For example, in vascular smooth muscle, cAMP activates cGMP-dependent protein kinase (Han et al., 1999). PGE2-stimulated cAMP production enhances protein kinase C in chondrocytes (Schwartz et al., 1998). Furthermore, cAMP has been identified as an activator of small GTPase proteins, such as Rap-1 and Ras in thyroid and brain (de Rooij et al., 1998; Tsygankova et al., 2000). From a clinical point of view, any attempt to control cervical ripening must take on board these new findings that only EP4 receptors appear to mediate PGE2-stimulated GAG synthesis.
To investigate whether IL-1ß-induced GAG synthesis required transcriptional regulation of EP receptors, we analysed mRNA expression of each EP receptor subtype by RT–PCR followed by Southern blot analysis. The four PGE2 receptor subtypes are constitutively expressed in unstimulated cervical fibroblasts. We found that IL-1ß selectively increased EP4 receptor mRNA expression, and decreased EP1 receptor mRNA levels, whereas the levels of EP2 and EP3 transcripts were not modified. Such data are in accordance with the transcriptional regulation of the EP4 receptor by IL-1ß previously reported in myometrial and ovarian cells (Erkinheimo et al., 2000; Narko et al., 2001). Thus, IL-1ß enhancement of GAG synthesis requires COX-2 induction and PGE2 production on the one hand, and a selective up-regulation of EP4 receptor expression to increase the responsiveness of human cervical fibroblasts to PGE2 on the other. Taken together, these results suggest that PGE2 may act as an autocrine/paracrine factor, as demonstrated in cervix carcinoma and endometrium (Milne et al., 2001; Sales et al., 2001).
Besides this increased expression of EP4 receptor mRNA, we found that IL-1ß also decreases the expression of EP1 receptor mRNA. Conversely, Spaziani et al. (1999) highlighted an increase in EP1 receptor transcripts and related proteins after IL-1ß treatment of amnion WISH cells. Therefore regulation of EP receptor expression might be tissue-dependent, since IL-1ß had opposing effects in these two different cell types. This hypothesis requires further investigation.
Recently, it has been demonstrated that the expression of EP2 receptor mRNA decreases in the baboon cervix during labour (Smith et al., 2001), whereas the mRNA level of the EP1, EP3 and EP4 receptor subtypes is not modified. These authors concluded that loss of EP2 receptors at parturition may be related to cervical dilatation. Due to the presence of functional EP2 receptors coupled to the cAMP pathway in human cervical fibroblasts (Schmitz et al., 2001), we cannot rule out the implication of the EP2 receptor subtype in PGE2-stimulated collagen breakdown, the other fundamental biochemical aspect of cervical ripening. Experiments are now in progress in our laboratory to clarify the involvement of the previously reported funtional EP2 receptor in the cervical ripening process and to establish a link with the decrease in expression of EP2 receptor mRNA observed in the baboon cervix at parturition (Smith et al., 2001).
In conclusion, our data strongly indicate that IL-1ß induces COX-2 protein expression and subsequent PGE2 production that acts on EP4 receptors to stimulate GAG synthesis, but also enhances the responsiveness of cervical fibroblasts to PGE2 by selectively up-regulating EP4 receptor mRNA levels. These findings suggest that PGE2 may regulate the human cervical ripening process in an autocrine/paracrine manner via EP4 receptors. The fact that the EP4 receptor mediates both endogenous and exogenous PGE2-induced GAG synthesis, enhances the clinical relevance of the development of EP4 receptor agonists and antagonists for controlling cervical ripening through stimulation or inhibition of the EP4 receptor.
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Acknowledgements |
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Thomas Schmitz was the recipient of a grant from the Institut National de la Santé et de la Recherche Médicale (INSERM). We are grateful to Dr Lister (GlaxoWellcome) for kindly provinding AH6809 and AH23848. We are indebted to Dr Gilles Charpigny, Pascale Roux and Eric Bastier for expert technical assistance.
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