| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Article |
INSERM, U-361, Maternité Port Royal Cochin, Université Paris V, René Descartes, 75014 Paris, France
Address all correspondence and requests for reprints to: Dr. Marie-Josèphe Leroy, INSERM, U-361, Pavillon Baudelocque, 123 boulevard Port Royal, 75014 Paris, France. E-mail: leroy-zamia{at}cochin.inserm.fr.
Abstract
Intrauterine infections are important etiological factors of preterm labor. They trigger an increase in proinflammatory cytokines, in particular IL-1ß, that induces a cascade of events resulting in the production of potent effectors of myometrial contractility, such as the prostaglandin E2 (PGE2). Within the smooth muscle cells, contractility is under the control of cAMP content, partly regulated by cAMP-phosphodiesterase 4 (PDE4), the predominant family of PDEs expressed in human myometrium. In the present study, using a model of inflammation of human myometrial cells in culture, we demonstrated that exposing the cells to IL-1ß resulted in a significant up-regulation of PDE4 activity through an increase in PDE4B2 mRNA and protein levels. The IL-1ß-induced PDE4 activity occurs after an increase in PGE2 production and subsequent cAMP augmentation. Pretreatment with indomethacin or NS 398 completely blocked this long-term effect of IL-1ß, revealing a PGE2-dependent pathway. Accordingly, our results demonstrated that the PDE4B2 variant can participate in the regulation of the inflammatory reaction that occurs at term or in preterm labor and leads to myometrial contractions. Knowing the myorelaxant effect of PDE4 inhibitors and the implication of the PDE4B2 in the inflammatory process, this isoform may be an appropriate target for discovering antiinflammatory drugs to manage infection-induced preterm deliveries.
DURING PREGNANCY, THE quiescence of the uterine smooth muscle is essential to support and accommodate the development of the fetus. The reversal of this quiescence and the adequate response to uterotonics lead to parturition. At present, the exact factors initiating human parturition remain unknown, but a role for a local inflammatory process seems certain. Leukocytes invade the myometrium, placenta, cervix, and fetal membranes at or immediately after the onset of labor (1). Coincident with these events is the production of proinflammatory cytokines, particularly IL-1ß and TNF
in the reproductive tissues of the feto-maternal interface. Such an inflammatory response can be initiated prematurely by an intrauterine infection. After stimulation by an infectious agent, cytokines such as IL-1ß are released in elevated concentrations by damaged tissues and act to promote the inflammatory response. Activation of the cytokine cascade ultimately results in the production of prostaglandins (PGs) such as PGE2, an important path in the onset and propagation of myometrial contractility and subsequent labor (2, 3).
Human myometrial contractility is under the control of a wide variety of hormones, neurotransmitters, and other chemical substances that act through several major second messengers, the cyclic nucleotides cAMP and cGMP, and the calcium ions. Compelling evidence demonstrates that elevation of the cAMP content, resulting from agonist-mediated stimulation of the receptor adenylyl cyclase complex, is involved in maintaining the quiescent state of the myometrium during pregnancy (4). The magnitude of cAMP elevation is critically influenced by the rate of its hydrolysis by phosphodiesterase (PDE) isoenzymes.
To date, 11 different PDE families have been characterized (5). Multiple related genes and additional mRNA splicing create various isoforms that are differentially expressed and regulated in individual cell types. From a pharmacological perspective, this complexity implies that specific PDEs are potential targets for therapeutic intervention in diseases involving cyclic nucleotide signaling (6). The usefulness of cyclic nucleotide PDE inhibitors, especially selective inhibitors of the cAMP-specific PDE4 family, has been recently emphasized for the treatment of several inflammatory disorders such as asthma. Indeed, their therapeutic values are derived from a combination of antiinflammatory and myorelaxant properties (7). A recent human study has shown that a new PDE4 inhibitor, roflumilast, was efficacious in exercise-induced asthma and led to the suppression of lipopolysaccharides (LPS)-stimulated TNF ex vivo (8).
In human myometrium we have previously shown that among the five PDE families identified, the PDE4 family, which specifically hydrolyzes cAMP, is predominantly expressed (9). PDE4 isoforms are also involved in the contraction/relaxation process of the myometrium, as rolipram, a PDE4-selective inhibitor, exerts a complete relaxant effect on spontaneous contraction of myometrial strips (10, 11, 12). Four PDE4 genes, 4A, 4B, 4C, and 4D, have been isolated in mammals. PDE4 genes produce the so-called long, short, and super-short isoforms. These various isoforms are derived from the use of distinct promoters and alternative mRNA splicing (13, 14). The four PDE4 genes are all expressed in human pregnant and nonpregnant myometrium, but, interestingly, an increase in the PDE4B2 transcript, a short-form product of the PDE4B gene, and a concomitant accumulation of the related protein were detected in near-term pregnant myometrium compared with levels in nonpregnant tissue (9, 12, 15). Consequently, at the end of pregnancy this overexpression of a PDE4B variant might result in an increased PDE4 participation in myometrial cAMP degradation favorable to contraction.
The PDE4B2 isoform is now described as a specific variant involved in the regulation of inflammatory responses. In human monocytes, LPS specifically induces PDE4B gene transcription (16), and PDE4B2 is the only detectable molecular species of PDE4B in these cells. PDE4B2 is also the predominant PDE4 isoform in human neutrophils (17). In addition, selective PDE4 inhibitors, but not selective inhibitors of other PDE families, significantly inhibit TNF and IL-1ß release by LPS-stimulated macrophages (18).
From these data we hypothesized that PDE4B2 participates in the regulation of the inflammatory process that occurs during preterm labor induced by intrauterine infection and leads to myometrial contractions. To validate this concept, we established a model of inflammation by exposing human myometrial cells to IL-1ß, and we investigated whether such treatment affected PDE4 activity and PDE4B2 expression. The consequences of such treatment on the cellular signaling cascade, i.e. PGE2 and cAMP productions, were evaluated.
Materials and Methods
Cell culture and drug treatments
Biopsies of myometrium were collected from nonpregnant cycling women undergoing hysterectomies for benign gynecological indications. For such biopsies ethical approval was obtained by the "comité consultatif de protection des personnes pour la recherche biomédicale" (CCPPRB, Paris-Cochin, France). Tissue samples were excised from normal muscle in areas free of macroscopically visible abnormalities, at some distance from the endometrium, and free of serosa. Myometrial cells were prepared by the explant method as previously described by Cavaillé et al. (19). Cells were cultured in DMEM supplemented with antibiotic solutions and 10% fetal calf serum (Life Technologies, Inc., Cergy-Pontoise, France). After four subcultures, cells at confluence were placed in a serum-free medium for 72 h, allowing the expression of smooth muscle markers: -smooth muscle actin, myosin heavy chain isoforms (SM1 and SM2), and desmin (19).
For the experimental procedure cells were incubated with IL-1ß or vehicle (PBS supplemented with 0.1% BSA). When used, actinomycin D or cycloheximide was dissolved in DMEM. Rolipram, a selective PDE4 inhibitor, was dissolved in DMSO, and indomethacin was dissolved in ethanol (0.3% final concentration). The presence of ethanol and DMSO had no effect on PDE4 activity, and the presence of ethanol had no effect on cAMP and PGE2 productions (data not shown). Actinomycin D, cycloheximide, rolipram, and indomethacin were added 30 min before the beginning of the incubation with IL-1ß.
cAMP-PDE assays
After treatments, myometrial cells (5 x 105 cells/35-mm dish) were washed twice in cold PBS and harvested by scraping in ice-cold homogenization buffer [100 mM Tris-HCl (pH 7.4), 2 mM MgSO4, 2 mM EDTA, 10% glycerol, 1 mM ß-mercaptoethanol, 1 µM leupeptin, 10 µg/ml aprotinin, 25 µg/ml Pefabloc, 130 µg/ml benzamidine, and 50 µg/ml soybean trypsin inhibitor]. After sonication, samples were immediately stored at -20 C until use.
cAMP-PDE activity was determined using the Kincaid and Manganiello method (20). Specific activities were measured under high affinity conditions with 1 µM [3H]cAMP as substrate (Amersham International, Little Chalfont, UK). All assays were carried out under linearity conditions with respect to time and protein concentration. PDE4 activity was gauged as the fraction of total cAMP PDE activity that was inhibited by 10 µM rolipram; non-PDE4 activity was estimated as the remaining cAMP PDE activity. Protein concentrations were determined using a modified Bradford protein assay (21) with BSA as a standard (Bio-Rad Laboratories, Inc., Richmond, CA). PDE-specific activity was expressed as picomoles per minute per milligram of protein.
cAMP assay
To measure cAMP content, treated cells (5 x 105/35-mm dish) were exposed to 10% ice-cold trichloroacetic acid to stop the reaction (22). Cells were then scraped and stored at -20 C. At the time of the cAMP assay, cell preparations were thawed on ice, and the precipitated proteins were separated from the soluble extract by centrifugation at 300 x g for 10 min at 4 C. Trichloroacetic acid was removed from the sample by three successive extractions with water-saturated ethyl ether. cAMP content was measured using a commercially available RIA kit (Biotrak, Amersham International). cAMP content was expressed as nanomoles of cAMP per 5 x 105 cells.
PGE2 assay
The production of PGE2 by myometrial cells (5 x 105cells/35-mm dish) was measured in the culture medium by RIA (23). The assay was conducted using [3H]PGE2 (NEN Life Science Products, Boston, MA) and an anti-PGE2 antibody purchased from Pasteur Productions (Paris, France). Briefly, 0.1 ml [3H]PGE2 (2000 cpm) and either 0.1 ml PGE2 standard (Cayman Chemicals, Ann Arbor, MI) used in increasing concentrations to establish a standard curve (5–2000 pg/ml) or 0.1 ml culture medium sample and 0.1 ml of the anti-PGE2 antibody were mixed in a 5-ml tube. [3H]PGE2, anti-PGE2 antibody, PGE2 standards, and samples were diluted in 0.1 mol/liter phosphate buffer (pH 7.25) containing 0.1% gelatin and 0.01% sodium azide. Incubations were conducted overnight at 4 C. Free and bound radioactivity were separated by adding 2 ml 0.1 mol/liter phosphate buffer containing 0.25% activated charcoal and 0.025% dextran and centrifuging at 4 C for 40 min at 3000 rpm. The interassay coefficient of variation was 14%, and the intraassay coefficient of variation ranged from 8–12%. PGE2 content was expressed as picograms per 5 x 105 cells.
RT-PCR analysis
Total RNA was extracted from myometrial cells using the TRIzol reagent method (Life Technologies, Inc., Cergy-Poutoise, France). Briefly, scraped cells (
107) 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 RT 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, Inc.) 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. RT 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 RT 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, Inc.), and 3 µl RT product (480 ng cDNA). The amplification profile consisted of denaturation at 94 C for 1 min, annealing for 1 min at 58 C, and extension at 72 C for 1 min, with a final extension at 72 C for 10 min. The primers for PDE4B2 were designed from the reported primary sequences (forward, AAATAATGAAGGAGCACG; reverse, GAGAATATCCAGCCACAT) (15). After 30 cycles of denaturation and extension, 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. The DNA molecular mass standard ladder consists of fragment multiples of 123 bp (123-bp DNA ladder; Life Technologies, Inc.). Additional validity control was achieved by appropriate-sized digestion with specific restriction endonucleases and Southern blot analysis of the PCR product as previously described (15) (data not shown).
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 (24, 25).
The intensities of the bands on Polaroid (European Imap Scientist, Massy, France) pictures of the ethidium bromide-stained gels were analyzed densitometrically using a computer-linked scanner and the NIH Image 1.60 software package (NIH, Bethesda, MD). The results are expressed in arbitrary densitometric units as the mean ± SEM.
Immunodetection of PDE4 isozymes
Immunoblotting was carried out using rabbit polyclonal antibodies (donated by Dr. H. Tenor, Byk Gulden Pharmaceuticals, Konstanz, Germany) that recognize a specific peptide sequence selected from the C-terminal ends of the PDE4B protein (26).
Samples of myometrial cells (40 µg protein) were dissolved (vol/vol) in Laemmli buffer and boiled for 5 min before electrophoresis on 10% SDS-PAGE. After electrophoretic separation, proteins were transferred to a nitrocellulose membrane (Amersham International) using a Transblot apparatus (Bio-Rad Laboratories, Inc.). Blots were dried and then blocked for 1 h in 10% nonfat dried milk in 10 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (pH 7.6; TBS-T), at room temperature. Blocked membranes were washed three times with TBS-T. The blots were then incubated overnight at 4 C with the primary antibody (1:2000 dilution in TBS-T containing 1% nonfat dried milk powder). After three washes with TBS-T, blots were incubated for 45 min with the appropriate horseradish peroxidase-linked secondary antibody and washed five additional times with TBS-T. Immunoreactive proteins were detected by chemiluminescence (enhanced chemiluminescence reagents, Amersham International).
Another set of antibodies (donated by Prof. M. Conti, Stanford University, Stanford, CA) was used for additional controls (K118, rabbit polyclonal antibodies raised against PDE4B). They were used as described above.
Materials
Rolipram was a gift from Schering Health Care Ltd. (Burgess Hill, UK). All compounds, except Pefabloc and NS 398, were purchased from Sigma (St. Louis, MO). Pefabloc was purchased from Interchim (Montluçon, France), and NS 398 was purchased from Cayman Chemicals.
Statistical analysis
The t test for paired samples was applied for the comparison between IL-1ß-treated cells and nontreated cells. All results were expressed as the mean ± SEM. The difference was considered significant at P < 0.05.
Results
IL-1ß is able to up-regulate cAMP PDE4 activity in human myometrial cells
To determine whether PDE4 activity could over the long term be modulated by a proinflammatory cytokine, human cultured myometrial cells were treated from 3–24 h with increasing concentrations of IL-1ß.
IL-1ß led to a significant increase in the total cAMP PDE activity of the cells (Table 1
). Interestingly, only PDE4 activity was selectively modified, whereas rolipram-resistant PDE activity (non-PDE4 activity) remained identical in control vs. treated cells.
|
). After exposing the cells to 10 ng/ml IL-1ß, PDE4 activity significantly rose after 18 h of incubation, and a 1.7-fold induction was reached after 24 h of treatment.
|
, incubation of the cells for 18 h with a range of 0.15–10 ng/ml IL-1ß led to a dose-dependent increase in PDE4 activity. We observed a significant rise in PDE4 activity from 0.15 ng/ml IL-1ß, and a maximal 1.75-fold increase was obtained with 10 ng/ml IL-1ß compared with untreated cells.
|
). When used alone, actinomycin D or cycloheximide did not modify PDE4 activity.
|
To establish whether IL-1ß induces a selective up-regulation of PDE4B2 transcripts, mRNA from nontreated cells and cells treated with 10 ng/ml IL-1ß for various times were extracted and reverse transcribed for PCR analysis.
Using primers designed to discriminate the short-form product of PDE4B, namely PDE4B2, a signal, i.e. a fragment of 567 bp, was detected in untreated and treated cells at each time point (Fig. 3, A and B). IL-1ß treatment led to a noticeable increase in the intensity of the signal for the short-form transcript PDE4B2 after 5, 12, and 18 h of incubation. The increase appeared more important after 12 h of incubation. Successful normalization of RNA amounts was verified by amplification of ß2-microglobulin as an internal standard, given equal signal intensity in control and treated cells.
|
, one band was labeled with the PDE4B-specific antibody in extracts from untreated and treated myometrial cells. This band that migrated at 80 ± 3 kDa may correspond to the PDE4B2 form because it has the same migration in SDS-PAGE as the PDE4B2 form detected in the U937 monocytic cell line (27). Upon treatment of the cells with 10 ng/ml IL-1ß for 18 h, the intensity of the 80-kDa signal was stronger than that of the signal obtained with the control cells. This increasing signal of PDE4B2 protein by IL-1ß has been confirmed when using polyclonal PDE4B-specific K118 antibodies raised against a different PDE4B-specific epitope (data not shown).
|
The ability of IL-1ß to stimulate PGE2 production and to induce a rise in the cAMP content was assessed in cultured myometrial cells. As shown in Fig. 5, 10 ng/ml IL-1ß caused a time-dependent increase in PGE2 production. A significant rise started after 5 h of treatment, and IL-1ß produced a 70-fold increase in PGE2 production after 24 h of incubation.
|
). cAMP production remained significantly different from the control value up to 18 h despite the observation that the elevation of cAMP production, compared with that at 12 h, was lower and thereafter declined to become insignificantly different at 24 h.
|
Pretreatment of myometrial cells with indomethacin, a nonselective inhibitor of PGH synthase, significantly reduced the increase in cAMP and PGE2 production and completely blocked the rise in PDE4 activity induced by 10 ng/ml IL-1ß in 18 h (Fig. 7). The effect of a PGH synthase-2 (PGHS-2)-selective inhibitor, NS 398, on IL-1ß-induced PDE4 activity was also evaluated. As shown in the Table 3, NS 398 was as potent as indomethacin at blocking the induction of PDE4 activity after IL-1ß treatment. We insured that indomethacin and NS 398 pretreatment did not modify the basal levels of cAMP and PGE2 or the basal PDE4 activity.
|
|
The last trimester of human pregnancy is crucial to maturation of the fetus. The interruption of this process due to early delivery constitutes the leading cause of neonatal morbidity and mortality. Intrauterine infection has been recognized as an important etiological factor of preterm labor, and multiple lines of evidence now support the idea that proinflammatory cytokines, such as IL-1ß, play a major role in the onset of labor. Such an infection-inflammatory reaction triggers a cascade of events resulting in the production of PGs, such as PGE2, a potent effector of uterine contractions. At the cellular level, myometrial contractile activity depends on cAMP content partly regulated by cAMP-PDE4, the predominant PDE family expressed in human myometrium. In this way, selective PDE4 inhibitors, rolipram and RP 73401, by increasing cAMP levels, completely abolish the spontaneous contractions of human myometrial strips (10, 11, 12). The participation of the PDE4 family in the inflammatory response that occurs in the uterus at term and in preterm labor is completely unknown. However, a rise in the cAMP level caused by the inhibition of PDE4 has been shown to modify the activation of a variety of cells involved in the inflammatory process. To our knowledge, the present study provides the first evidence that a short-form product of the PDE4B gene, PDE4B2, is up-regulated by the proinflammatory cytokine IL-1ß in human smooth muscle myometrial cells through a PGE2- and cAMP-dependent pathway.
We first established that treatment of myometrial cells with IL-1ß was associated with a significant increase in selective PDE4 activity. The PDE4 gene is remarkably versatile, in that it provides two general types of regulation that adjust PDE4 activity throughout the time course of hormone stimulation (13). The first is a short-term regulation that involves phosphorylation of a preexisting PDE protein and occurs within minutes of addition of the hormonal stimulus. The second, termed long-term regulation, requires protein synthesis and develops in hours or days (28). In our study IL-1ß stimulates PDE4 activity by a long-term mechanism of regulation because exposure of the cells with IL-1ß for at least 18 h was necessary to observe a significant increase in PDE4 activity. Furthermore, this augmentation of PDE4 activity was blocked in the presence of either actinomycin or cycloheximide, suggesting that IL-1ß regulates PDE4 activity by a transcriptional and translational mechanism.
Among the different PDE4 isoforms, we have previously shown that the PDE4B2 isoform is more abundant in the myometrium of pregnant women near term than in the myometrium of nonpregnant women (15). Moreover, we recently reported that PGE2, a potent uterine effector whose production by intrauterine tissues dramatically increases during preterm and term labor, led to a significant increase in PDE4B2 expression in myometrial cells in culture (29). Our results demonstrated that IL-1ß led to an accumulation of mRNA for the PDE4B2 variant after at least 5 h of treatment, followed by detectable augmentation of the respective protein after 18 h. Such data indicate that the transcript requires at least 6 h to be translated into protein product. Little is known about how the time course of transcription and translation of PDE4 genes is regulated. The hormonal induction of the PDE4B and PDE4D short forms has been observed in a number of cells, and the time of PDE4 activity induction differs depending on the cell type concerned (6). However, it has been shown, in a model of Sertoli cells exposed to FSH, that there is a time lag of 6 h between the maximal increase in PDE4 transcripts and the induction of PDE4 activity (30).
The up-regulation of PDE4B2 by IL-1ß that we observed in our model is in agreement with study results obtained for other cells that highlight the importance of the PDE4B2 isoform in inflammatory processes. In cultured airway smooth muscle cells, treatment with an combination of IL-1ß and TNF led to a significant increase in mRNA expression of PDE4B2 (31). In human monocytes, LPS specifically induced PDE4B gene expression, and only the PDE4B2 variant was detectable in these cells (16) as it was in neutrophils (17). In human osteoarthritis chondrocytes, IL-1ß up-regulated PDE4 activity and was associated with an augmentation of the PDE4B2 protein (26). Furthermore, it has been reported that PDE4B may play a major role in regulating human inflammatory cell function, as the inhibition of LPS-stimulated TNF release from monocytes was closely correlated to the inhibition of PDE4A and/or PDE4B (32). Finally, a recent study using mice deficient in the PDE4B gene has demonstrated that the induction of the PDE4B was essential for the LPS-elicited innate immune response in monocytes and macrophages (33).
Our results together with these previous observations strongly suggest the participation of the PDE4B2 isoform in the inflammatory response that occurs in human myometrium during the early stage of labor and also during infection-induced preterm deliveries.
The second aim of this study was to examine the transducing mechanisms by which IL-1ß induced PDE4 activity. Earlier studies have focused on the action of proinflammatory cytokines in the myometrial smooth muscle. Stimulation of human myometrial cells in culture with IL-1ß was shown to provoke an important release of PGE2 and cAMP (34, 35). In our study the production of PGE2 and cAMP increased after 5 h of treatment with IL-1ß, before the augmentation of PDE4 activity was detectable. The use of indomethacin, a nonselective blocker of PGHS, prevented the IL-1ß-stimulated PGE2 and cAMP syntheses. It also blocked IL-1ß-stimulated PDE4 activity. To further examine the PGHS isoform involved in this process, we used a selective inhibitor of PGHS-2, NS 398 (36). We previously determined by Western blotting that IL-1ß treatment induced PGHS-2 protein as early as 5 h, and this induction was still observed after 18 h of treatment (data not shown). This selective PGHS-2 inhibitor is of similar potency to indomethacin at inhibiting the up-regulation of PDE4 activity. Therefore, the long-term effect of IL-1ß on PDE4 activity appears to be PGE2 dependent, and the main isoenzyme form of PGHS involved in IL-1ß induced PDE4 activity is PGHS-2. The four subtypes of PGE2 receptors, EP1, EP2, EP3, and EP4, have been identified in human myometrial cells (37). Among them, EP2 and EP4 have the capacity to trigger elevation of intracellular cAMP. We suspect that the cAMP elevation observed in our cellular model can be mediated by EP2 and/or EP4 receptors. In a large variety of cells, it has been well described that induction of PDE4 occurred by elevation of its own substrate, cAMP (38, 39, 40, 41). In myometrial cells we know that forskolin, a direct activator of adenylate cyclase, or 8-bromo-cAMP, a cell-penetrating analog of cAMP, induces an increase in PDE4B2 isoform expression and leads to an augmentation of PDE4 activity (42). We have also shown that PGE2 treatment leads to an increase in PDE4 activity through a rise in cellular cAMP content (29). Such activation of the mRNA short-form variant, PDE4B2, by cAMP can be explained in part by the presence of a cAMP response element consensus site localized on the promoter of the PDE4B2 gene (43).
In light of these observations, we strongly suggest that the increase in cAMP levels that begins after 5 h of IL-1ß treatment and reaches a maximum at 12 h, consecutively with the stimulation of PGE2 release, is responsible for the activation of mRNA PDE4B2 expression that is also at its highest at 12 h. This increase in the mRNA level leads to the augmentation of PDE4 activity, detectable only after 18 h of treatment. As the PDE4 family specifically hydrolyzes cAMP, this up-regulation of PDE4 activity at 18 h of treatment might be responsible for the decrease in cAMP production observed at the same time point. cAMP levels would no longer be sufficient to up-regulate PDE4B2 mRNA. This is in complete accordance with the observation that transcript production decreases at 18 h to return to levels similar to those observed at 5 h. Such noteworthy data reflect the complexity of the positive and negative mechanisms of regulation that take place between the PDE4 family and its own substrate, cAMP. Such up-regulation of PDE4 activity by IL-1ß can therefore contribute to an extended mechanism of attenuated cAMP accumulation. Other regulations that act in synergy participate in the diminution of the cAMP level. In human uterine smooth muscle cells, we know that IL-1ß stimulates the production of PGE2, but also leads to an increase in oxytocin secretion, a peptide that plays a significant role in contracting the uterus during labor (44). Both PGE2, via the EP3 receptor, and oxytocin inhibit adenylyl cyclase activity through Gi protein activation (45, 46, 47). These different mechanisms might together regulate uterine smooth muscle motility and consequently lead to labor as well as trigger infection-induced preterm contractions.
To conclude, this study demonstrates that IL-1ß increases PDE4 activity associated with an up-regulation of PDE4B2 expression in human myometrial cells through a PGE2- and cAMP-dependent pathway. This PDE4B2 variant, by influencing the rate of cAMP hydrolysis, may participate in regulation of the contractility of human pregnant myometrium during the inflammatory reaction. Based on these findings and our knowledge of the utero-relaxant effect of PDE4 inhibitors (10, 11, 12), selective inhibition of the PDE4B2 isoform could represent a new therapeutic strategy for the clinical management of preterm labor.
Acknowledgments
We thank Dr. H. Tenor (Byk Gulden Pharmaceuticals, Konstanz, Germany) for generously donating polyclonal PDE4B antibodies. We acknowledge Prof. M. Conti (Stanford University) for kindly providing the K118 antibodies. We are grateful to Drs. G. Charpigny and P. Roux for their technical assistance with the PGE2 assay. We are indebted to the medical staff of the Department of Gynecology of Cochin-Port Royal for assistance in obtaining uterine tissues. We thank C. Spencer for editorial work on the manuscript.
Footnotes
Abbreviations: LPS, Lipopolysaccharides; PDE, phosphodiesterase; PGE2, prostaglandin E2; PGHS-2, prostaglandin H synthase-2.
Received April 11, 2002.
Accepted August 15, 2002.
References
ex vivo. J Clin Pharmacol 42:297–303[Abstract]
and IL-6 release by endotoxin-stimulated macrophages. J Immunol 155:4909–4916[Abstract]
-induced glucocorticoid-sensitive changes in multiple gene expression and altered responsiveness in airway smooth muscle. Am J Respir Cell Mol Biol 25:761–771 responses. Proc Natl Acad Sci USA 99:7628–7633This article has been cited by other articles:
 |
|
 |
|
  D. Markovic, M. Vatish, M. Gu, D. Slater, R. Newton, H. Lehnert, and D. K. Grammatopoulos The Onset of Labor Alters Corticotropin-Releasing Hormone Type 1 Receptor Variant Expression in Human Myometrium: Putative Role of Interleukin-1{beta} Endocrinology, July 1, 2007; 148(7): 3205 - 3213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Kossintseva, S. Wong, E. Johnstone, L. Guilbert, D. M. Olson, and B. F. Mitchell Proinflammatory cytokines inhibit human placental 11{beta}-hydroxysteroid dehydrogenase type 2 activity through Ca2+ and cAMP pathways Am J Physiol Endocrinol Metab, February 1, 2006; 290(2): E282 - E288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Belmonte, C. Ticconi, S. Dolci, M. Giorgi, A. Zicari, A. Lenzi, E. A. Jannini, and E. Piccione Regulation of Phosphodiesterase 5 Expression and Activity in Human Pregnant and Non-pregnant Myometrial Cells by Human Chorionic Gonadotropin Reproductive Sciences, December 1, 2005; 12(8): 570 - 577. [Abstract] [PDF]
|
|
|
|
|
|
M. Breuiller-Fouche, C. Moriniere, E. Dallot, S. Oger, R. Rebourcet, D. Cabrol, and M.-J. Leroy Regulation of the Endothelin/Endothelin Receptor System by Interleukin-1{beta} in Human Myometrial Cells Endocrinology, November 1, 2005; 146(11): 4878 - 4886. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. G. Tilley and D. H. Maurice Vascular Smooth Muscle Cell Phenotype-Dependent Phosphodiesterase 4D Short Form Expression: Role of Differential Histone Acetylation on cAMP-Regulated Function Mol. Pharmacol., September 1, 2005; 68(3): 596 - 605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. El-Haroun, D. Bradbury, A. Clayton, and A. J. Knox Interleukin-1{beta}, Transforming Growth Factor-{beta}1, and Bradykinin Attenuate Cyclic AMP Production by Human Pulmonary Artery Smooth Muscle Cells in Response to Prostacyclin Analogues and Prostaglandin E2 by Cyclooxygenase-2 Induction and Downregulation of Adenylyl Cyclase Isoforms 1, 2, and 4 Circ. Res., February 20, 2004; 94(3): 353 - 361. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Oger, C. Mehats, M. S. Barnette, F. Ferre, D. Cabrol, and M.-J. Leroy Anti-Inflammatory and Utero-Relaxant Effects in Human Myometrium of New Generation Phosphodiesterase 4 Inhibitors Biol Reprod, February 1, 2004; 70(2): 458 - 464. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
