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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0055
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3534-3543
Copyright © 2005 by The Endocrine Society

15-Deoxy-{Delta}12,14-Prostaglandin J2 Inhibits Interleukin-1ß-Induced Nuclear Factor-{kappa}B in Human Amnion and Myometrial Cells: Mechanisms and Implications

Tamsin M Lindström and Phillip R Bennett

Parturition Research Group, Institute of Reproductive and Developmental Biology, Hammersmith Campus, Imperial College, London NW12 0NN, United Kingdom

Address all correspondence and requests for reprints to: Tamsin M. Lindström, 3rd Floor IRDB, Hammersmith Campus, Imperial College, Du Cane Road, London W12 0NN, United Kingdom. E-mail: t.lindstrom{at}imperial.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Proinflammatory cytokines and prostaglandins play key roles in term and preterm human labor. The expression of the prostaglandin synthetic enzyme cyclooxygenase (COX)-2 and cytokines IL-1ß and IL-8 increases within the uterus at the time of labor, and each is regulated by the transcription factor nuclear factor-{kappa}B (NF-{kappa}B). In addition to its role in driving inflammation, COX-2 may also synthesize 15-deoxy-{Delta} (12, 14)-prostaglandin J2 (15d-PGJ2), an antiinflammatory cyclopentenone prostaglandin (cyPG), which acts in some cells as an agonist of peroxisome proliferator-activated receptors (PPARs).

We found that PPAR{alpha} and -{gamma} proteins are expressed in both amnion epithelial and myometrial cells, but synthetic PPAR agonists could not inhibit NF-{kappa}B activity or COX-2 expression. 15d-PGJ2 inhibited NF-{kappa}B activity and COX-2 expression in both cell types. This was unaffected by a PPAR antagonist and could be mimicked by the cyPG PGA1 but not 9,10-dihydro-15d-PGJ2 in which the cyclopentenone ring is disrupted. This shows that, in amnion and myometrium, inhibition of NF-{kappa}B activity and COX-2 expression by 15d-PGJ2 is independent of PPARs and requires the cyclopentenone ring. We further show that 15d-PGJ2 acts at multiple levels in the NF-{kappa}B pathway: blocking inhibitor of {kappa}B{alpha} degradation by repressing inhibitor of {kappa}B kinase activation and the 26S proteasome and also repressing NF-{kappa}B DNA binding and phosphorylation.

Our data suggest that PPARs are unlikely to play a role in the regulation of either NF-{kappa}B or COX-2 in human amnion and myometrium. Targeting of NF-{kappa}B is a potential therapeutic strategy in preterm labor. PPAR agonists are unlikely to be effective in this context, but cyPGs may have potential.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
HUMAN PARTURITION IS associated with an inflammatory response, accumulating evidence pointing to a pivotal role for proinflammatory cytokines and prostaglandins (PGs) (1). Elevated levels of TNF{alpha}, IL-1ß, and IL-6 are detected in the amniotic fluid of women with infection-associated preterm labor (2, 3, 4). In normal term labor, there are elevated IL-1ß levels in amniotic fluid (5), gestational membranes (6, 7), and the lower uterine segment (8). Proinflammatory cytokines are thought to contribute to the onset of labor by stimulating IL-8 and PG synthesis (9, 10). PGs stimulate uterine contractions (11, 12), indirectly increase fundally dominant myometrial contractility by up-regulation of oxytocin receptor and synchronization of contractions (13) and act in concert with IL-8 to remodel the cervix (14).

Nuclear factor-{kappa} B (NF-{kappa}B) is a transcription factor classically associated with inflammation, which is activated in response to bacterial infection and proinflammatory cytokines, such as those elevated during labor. In resting cells, NF-{kappa}B dimers are sequestered in the cytoplasm by association with the inhibitor of {kappa}B (I{kappa}B) proteins. In the canonical NF-{kappa}B pathway (15), inflammatory stimuli activate the I{kappa}B kinase (IKK) complex consisting of IKK{alpha}, IKKß, and NF-{kappa}B essential modulator (NEMO or IKK{gamma}). Activated IKKß phosphorylates I{kappa}B{alpha}, resulting in its polyubiquitination and targeting it for degradation by the 26S proteasome. The degradation of I{kappa}B{alpha} exposes the nuclear localization signal of NF-{kappa}B resulting in translocation of the p50/p65 NF-{kappa}B dimer to the nucleus in which it can bind to NF-{kappa}B recognition elements ({kappa}Bs) in the promoter of target genes and promote transcription.

NF-{kappa}B is a key positive regulator of proinflammatory cytokines in gestational tissues (16). Cytokine-induced NF-{kappa}B may therefore precipitate a positive feed-forward loop resulting in amplification of cytokine production and further NF-{kappa}B activation. Cyclooxygenase (COX)-2 is the inducible rate-limiting enzyme responsible for the increased PG synthesis seen in the uterus with labor onset (17, 18). {kappa}B binding sites are essential for both IL-8 (6) and COX-2 (19) expression in amnion cells. These elements are also critical to COX-2 expression in myometrial cells (20), and Soloff et al. (21) demonstrated the in situ binding of NF-{kappa}B proteins to the promoters of the IL-8 and COX-2 genes in primary myometrial cells using chromatin immunoprecipitation. Human labor is associated with increased NF-{kappa}B activity in the amnion in which, in addition to its proinflammatory actions, it may serve as a physiological antagonist of the progesterone receptor (22). In the mouse uterus, NF-{kappa}B signaling is activated by surfactant protein-A and plays a critical role in the initiation of labor (23). In the human myometrium, there is an increase in DNA binding of the transactivating p50/p65 NF-{kappa}B dimer (24). Thus, NF-{kappa}B is a key regulator of multiple pathways involved in both intrauterine inflammation and normal spontaneous labor and may therefore constitute a therapeutic target in the management of preterm labor.

In addition to catalyzing the synthesis of proinflammatory PGs, COX-2 can also play an antiinflammatory role. In a pleurisy model of inflammation in rats, Gilroy et al. (25) showed COX-2 expression to be biphasic. The early peak was associated with increasing inflammation and PGE2 production, whereas a later, stronger peak in expression was associated with resolution of the inflammation, with reduced PGE2 synthesis but increased levels of PGD2 and its metabolite, 15-deoxy-{Delta} (12, 14)-prostaglandin J2 (15d-PGJ2). 15d-PGJ2 is spontaneously produced from PGD2 via a nonenzymatic dehydration reaction (26). The human placenta produces substantial amounts of PGD2 (27), and both PGD2 and 15d-PGJ2 are present in human amniotic fluid (28). 15d-PGJ2 possesses potent antiinflammatory properties: it represses the production of proinflammatory mediators in a number of cell types (29, 30), including those from placenta and fetal membranes (31). Ricote et al. (32) reported the suppression of proinflammatory transcription factors, including NF-{kappa}B, activator protein-1 (AP-1), and signal transducers and activators, in macrophages by 15d-PGJ2. COX-2-mediated production of 15d-PGJ2 could therefore form a negative feedback mechanism ensuring inflammatory homeostasis and pregnancy maintenance.

Unlike conventional prostaglandins, which bind to cell surface prostanoid receptors and act through second messenger systems, PGD2 metabolites may be actively transported into cells by a specific carrier and accumulate in the nucleus (33, 34) in which they may engage nuclear receptors. 15d-PGJ2 is a ligand for the peroxisome proliferator-activated receptors (PPARs) (35, 36). In addition to their well-established roles in lipid metabolism, recent work has highlighted an emerging role of PPARs in the regulation of inflammatory responses (37). PPAR{alpha}, PPAR{delta}, and PPAR{gamma}, encoded by distinct genes, are detected at mRNA level in human intrauterine tissues (38, 39). In addition to their ability to act as transcriptional activators through peroxisome proliferator response elements (PPREs), PPAR{gamma} can also repress gene transcription by negatively interfering with the NF-{kappa}B-, AP-1-, signal transducer and activator-, and CCAAT/enhancer-binding protein-mediated signaling pathways (40, 41, 42, 43).

Both PPAR-dependent and -independent antiinflammatory actions of 15d-PGJ2 have been identified, depending on cell type and promoter context. Dunn-Albanese et al. (44) recently reported a decrease in PPAR{gamma} and an increase in COX-2 expression in intrauterine tissues in association with labor, and PPAR{gamma} has been proposed as a candidate for therapeutic intervention in preterm labor (31). However, the mechanism of the antiinflammatory actions of 15d-PGJ2 within the uterus has not been conclusively determined. In this study, we investigated the mechanism of 15d-PGJ2 modulation of NF-{kappa}B and COX-2 in human amnion epithelial and myometrial cells.


   Patients and Methods
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 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Reagents

Antibodies against p65, p50, I{kappa}B{alpha}, COX-2, c-jun, phospho-c-jun, IKKß, and PPAR{alpha} were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho-IKK{alpha}/ß were from Cell Signaling Technology (Beverly, MA). PPAR{gamma} antibodies and {alpha}-smooth muscle actin antibodies were from Upstate Cell Signaling Solutions (Lake Placid, NY). ß-Actin antibodies were purchased from Abcam (Cambridge, UK) and lamin B1 antibodies from Oncogene Research Products (San Diego, CA). Ubiquitin antibodies were from Chemicon (Temecula, CA). 15d-PGJ2, PGA1, troglitazone, GW1929, and GW9662 were from Alexis Biochemicals (San Diego, CA). WY-14643 and MG-132 were from Calbiochem (La Jolla, CA). Recombinant IL-1ß was from R&D Systems (Minneapolis, MN).

Primary human amnion epithelial and myometrial cells

Institutional Ethics Committee approval was obtained for the collection of placental and myometrial tissues and all patients gave informed consent. Myometrial biopsies were collected at term from the upper margin of uterine incision at the time of lower segment caesarean section. Myometrial tissue was dissected, rinsed in PBS, and digested in serum-free DMEM containing 15 mg/ml collagenase 1A (Sigma, St. Louis, MO), 15 mg/ml collagenase X (Sigma), and 50 mg/ml BSA for 45 min at 37 C. The cell suspension was filtered through a cell strainer, centrifuged at 400 x g for 5 min and the pellet resuspended and plated out in DMEM, 10% fetal calf serum (FCS) (Helena BioScience, Sunderland, Tyne, and Wear, UK), 2 mM L-glutamine, and 100 U penicillin-streptomycin. Cells were used between passage numbers 1 and 5. Placentae were obtained from patients at term either at elective cesarean section before labor or after spontaneous labor onset and vaginal delivery. The amnion was washed in PBS, cut into strips, and incubated in 0.5 mM EDTA in PBS for 15 min. The strips were washed in PBS and digested with 2.5 mg/ml dispase in serum-free DMEM for 35 min at 37 C. The amnion was then shaken vigorously in DMEM, 10% FCS to dissociate the cells and the cell suspension pelleted at 2000 rpm for 10 min, resuspended, filtered through a cell strainer, and cultured in DMEM, 10% FCS (Sigma), 2 mM L-glutamine, and 1% penicillin-streptomycin. Each experiment was performed at least three times using cell preparations from three different patients.

Western immunoblots

Confluent cell monolayers were rinsed in PBS and lysed in a buffer containing 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM dithiothreitol (DTT), 1% (vol/vol) Nonidet P-40 (NP-40) and complete protease inhibitor tablets (Roche Diagnostics, Mannheim, Germany). Cell lysates were incubated on ice for 10 min and NP-40 added to a final concentration of 1% (vol/vol). Lysates were vortexed and centrifuged for 30 sec at 4 C, 12,000 x g. The supernatants were retained as the cytosolic protein fraction. The pellets were resuspended in a buffer containing 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM DTT, 400 mM NaCl, 1% NP-40 (vol/vol), and complete protease inhibitor tablets and shaken on ice for 15 min. The nuclear protein extracts were obtained in the supernatant after a 5-min centrifugation at 4 C, 12,000 x g. For whole-cell lysates, cells were lysed in radioimmunoprecipitation assay/sodium dodecyl sulfate (SDS) buffer [1% NP-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 2 mM NaF], incubated on ice for 15 min, and the supernatant collected after a 5-min centrifugation at 4 C, 12,000 x g. For the detection of phosphorylated proteins, sodium orthovanadate was added fresh to the extraction buffers at a final concentration of 1 mM. Protein concentrations of cell lysates were determined using DC protein assay reagents (Bio-Rad Laboratories, Hercules, CA). Then 20–70 µg proteins were resolved in SDS-PAGE gels and transferred onto nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK). The membrane was blocked in buffer containing 5% (wt/vol) milk powder, PBS, and 0.1% Tween 20 for 30 min, and immunoblotted with primary antibody for 1–16 h in 1% milk buffer followed by secondary antibody for 45 min. Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at 1:3000 were used with ECL Plus (Amersham Biosciences) or Pico Supersignal (Pierce, Rockford, IL) chemiluminescent reagents for signal detection. To detect multiple signals from a single membrane, the blot membrane was treated with a stripping buffer [2% SDS, 62.5 mM Tris-HCl (pH 6.7), and 100 mM 2-mercaptoethanol] for 30 min at 50 C, washed in PBS and Tween 20, and then preblocked and reprobed with a different primary antibody. The levels of cellular actin were used as a control for intersample variability, and the expression of lamin B1 was used to confirm the integrity of nuclear/cytosolic fractionation.

EMSA

Consensus double-stranded oligonucleotides were obtained from Promega Life Science (Madison, WI): NF-{kappa}B, 5'-AGT TGA GGG GAC TTT CCC AGG C-3', and octamer transcription factor-1 (Oct-1), 5'-TGT CGA ATG CAA ATC ACT AGA A-3'. Oct-1 consensus oligonucleotides were used as a control for intersample variability. Oligonucleotides were end labeled with 32P({gamma}ATP) by incubating for 30 min at 37 C with T4 polynucleotide kinase. Labeled probes were purified on MicroSpin G-25 Sephadex columns (Amersham Biosciences). Then 3- to 5-µg nuclear protein extracts were incubated on ice for 1 h in a binding buffer [20% (vol/vol) glycerol, 5 mM MgCl2, 2 mM EDTA, 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 2 mM DTT, 1 µg poly(dI-dC)], followed by a 45-min incubation with labeled probes. The resulting protein/DNA complexes were separated in a 4% nondenaturing acrylamide gel and the gel dried under vacuum at 80 C and exposed to x-ray film.

Transfections

Cells at 70–80% confluence in 24-well plates were transfected using the liposome reagent Transfast (Promega Life Sciences). Cells were transfected with 0.4 µg of luciferase reporter construct per well ± 0.2 µg of expression construct in serum-free DMEM for 1 h. Cells were cotransfected with a ß-galactosidase vector as an internal control for transfection efficiencies. Empty expression vectors were used as filler where appropriate. DMEM and 10% FCS were then added and the cells incubated at 37 C for 16 h. The medium was replaced with DMEM and 2% FCS for a further 24 h and the cells treated with various agonists/inhibitors or vehicle for 6–24 h. Transfections were analyzed in a firefly luciferase (Promega Life Sciences)/ß-galactosidase (Galacto-Light Plus, Tropix, Foster City, CA) assay. The reporter construct used to determine NF-{kappa}B-mediated transcriptional activity was pGL3.6{kappa}B.BG.luc, containing three copies of the decameric {kappa}B binding site upstream of a minimal ß-globin promoter in a pGL3 vector. Empty pGL3-basic and mutated NF-{kappa}B-LUC plasmids were used as controls to confirm NF-{kappa}B-mediated transactivation. The reporter construct TK.pGL3.3PPRE.luc, comprising three copies of the consensus PPAR binding motif in a TK-pGL3 vector, was used to assess PPAR-mediated transcriptional activity; TK-pGL3 reporter was used as a negative control. Expression constructs used were pcDNA3.1/PPAR{gamma}, pcDNA3.1/PPAR{alpha}, pcDNA3.1/PPAR{delta}, and pSG5/p65.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
15-dPGJ2 inhibits NF-{kappa}B activity in primary amnion epithelial and myometrial cells

To investigate the molecular mechanisms by which 15d-PGJ2 may exert its antiinflammatory effects in amnion epithelial and myometrial cell cultures, we initially performed transient transfection experiments with a NF-{kappa}B-dependent luciferase reporter. In myometrial cells, preincubation with 15d-PGJ2 dose-dependently inhibited IL-1ß-induced reporter activity, reducing it to basal levels at 15- and 30-µM concentrations (Fig. 1AGo). 15d-PGJ2 repressed both IL-1ß-induced and background NF-{kappa}B transcriptional activity in amnion cells to below basal levels (Fig. 1BGo). EMSA was used to determine the effects of 15d-PGJ2 on the DNA-binding activity of NF-{kappa}B. 15d-PGJ2 inhibited IL1-ß-induced NF-{kappa}B DNA binding in myometrial cells with an EC50 of approximately 8 µM, completely abolishing binding at 32 µM (Fig. 1CGo). In amnion cells, 15d-PGJ2 inhibited both basal and IL1-ß-induced NF-{kappa}B DNA binding, with complete inhibition achieved at 16 µM (Fig. 1DGo). 15d-PGJ2 had no effect on protein binding to a consensus Oct-1 probe (Fig. 1Go, C and D), nor did it affect binding to consensus specificity protein-1 or AP-1 probes (data not shown).



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  		FIG. 1. 15d-PGJ2 inhibits NF-{kappa}B DNA binding and transcriptional activity in myometrial and amnion epithelial cells. Myometrial (A) and amnion (B) epithelial cells were transiently transfected with a NF-{kappa}B-luciferase reporter and pretreated with vehicle or 15d-PGJ2 for 2 h, followed by IL-1ß stimulation (1 ng/ml) for 6 h. Cells were cotransfected with a ß-galactosidase reporter plasmid as an internal control, and luciferase activity normalized for ß-galactosidase reporter readout. Values are presented as the fold induction relative to reporter activity in cells treated with vehicle only. Results are the mean ± SEM obtained for each treatment done in triplicate. NF-{kappa}B DNA binding was measured by EMSA in nuclear protein extracts from myometrial (C) and amnion (D) epithelial cells pretreated with vehicle or 15d-PGJ2 for 2 h followed by a 15-min stimulation with IL-1ß (1 ng/ml). Binding of proteins to a consensus Oct-1 probe was used as a control.

 
PPAR expression and activation

15d-PGJ2 has been shown to exert its antiinflammatory effects via PPARs in several cell types (32, 45), particularly through PPAR{gamma}, which has been suggested to play a role in repressing inflammatory mediators in the uterus (31, 44). The EC50 for 15d-PGJ2 required to block NF-{kappa}B activity in our studies is similar to that required to induce adipogenesis (36), suggesting action through PPAR. The expression and activation of PPARs in human amnion epithelial and myometrial cells was therefore examined. PPAR{gamma} protein was found to be expressed in the nucleus and the cytoplasm of both amnion epithelial and myometrial cells (Fig. 2AGo). PPAR{alpha} protein was expressed in the nucleus of both cell types, with myometrial cells expressing relatively greater amounts and also containing substantial cytosolic PPAR{alpha} (Fig. 2Go, A and B).



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  		FIG. 2. PPAR{alpha} and PPAR{gamma} proteins are expressed in myometrial and amnion epithelial cells. Western blot analysis of nuclear and cytoplasmic protein extracts from unstimulated or IL-1ß-stimulated cells (1 ng/ml, 12 h). Membranes were sequentially probed with antibodies against PPAR{alpha}, PPAR{gamma}, lamin B1, and ß-actin. Lamin B1 expression was used to confirm the integrity of nuclear/cytosolic fractionation and ß-actin expression was used to confirm equal protein loading. A, Myometrial and amnion cells, 40 µg protein loaded. B, Amnion cells, 70 µg protein loaded. Myometrial (C) and amnion (D) epithelial cells were transiently transfected with a PPRE-dependent reporter construct as well as a ß-galactosidase reporter plasmid as an internal control for transfection variability. Cells were cotransfected with expression plasmids for PPAR{gamma}, PPAR{alpha}, PPAR{delta}, or empty vector and incubated with vehicle or 15d-PGJ2 for 2 h, followed by IL-1ß (1 ng/ml) for 20 h. Luciferase activity was normalized for ß-galactosidase reporter readout. Values are presented as the fold induction relative to reporter activity in cells treated with vehicle only. Results are the mean ± SEM obtained for each treatment done in triplicate.

 
The ability of 15d-PGJ2 to act as a functional PPAR ligand was confirmed in transient transfection experiments using a PPRE-dependent luciferase reporter. 15d-PGJ2 was able to transactivate the reporter through PPAR{gamma} in both amnion epithelial and myometrial cells and additionally through PPAR{alpha} in amnion cells (Fig. 2CGo). In myometrial cells, overexpression of PPAR{alpha} transactivated the PPRE reporter independently of ligand, and 15d-PGJ2 could not further increase reporter activity (Fig. 2DGo). PPAR{delta} did not induce PPRE reporter activity in the presence or absence of 15d-PGJ2.

15d-PGJ2 inhibits NF-{kappa}B activity independently of PPARs

Because the ability of 15d-PGJ2 to engage PPAR{gamma} and PPAR{alpha} was confirmed and amnion epithelial and myometrial cells expressed these receptors, we next examined the ability of synthetic PPAR agonists to mimic the inhibitory effects of 15d-PGJ2. Troglitazone, a thiazolidinedione PPAR{gamma} agonist, had no effect on either NF-{kappa}B DNA binding or nuclear levels of p65 at 10–50 µM (Fig. 3AGo), concentrations that have been shown to be sufficient to inhibit NF-{kappa}B in other cell types (46, 47) and in excess of those required to stimulate the activity of PPAR{gamma} (data not shown). The potent PPAR{gamma} agonist, GW1929, which lacks the thiazolidinedione moiety, also failed to inhibit NF-{kappa}B DNA binding (Fig. 3BGo). The synthetic PPAR-{alpha} agonist WY-14643, which can transactivate PPAR{alpha} at 5- to 25-µM concentrations (48), had no effect on NF-{kappa}B DNA binding, even at 100-µM concentrations (Fig. 3CGo). 15d-PGJ2 can inhibit NF-{kappa}B phosphorylation (see below and Fig. 5Go, D and E). However, troglitazone, GW1929, and WY-14643 had no effect on IL-1ß-induced phosphorylation of p65 at serine 536 or on the phosphorylation of p50 (Fig. 3DGo), and PPAR{alpha} and -{gamma} agonists failed to inhibit the activity of a NF-{kappa}B-dependent reporter in amnion epithelial and myometrial cells when used under the same conditions as 15d-PGJ2 (Fig. 3EGo).



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  		FIG. 3. PPAR agonists do not inhibit NF-{kappa}B activation. Myometrial cells were pretreated with vehicle or PPAR agonists for 2 h, followed by stimulation with IL-1ß (1 ng/ml) for 15 min. A, NF-{kappa}B DNA binding was measured by EMSA in nuclear protein extracts from cells treated with troglitazone, and nuclear p65 expression was assessed by Western blotting. EMSA analysis of NF-{kappa}B DNA binding was measured in nuclear protein extracts from cells treated with GW1929 (B) or WY-14643 (C). Antibodies against p65 and p50 were used for supershift analysis. D, Western blot analysis of whole-cell lysates from cells treated with troglitazone, GW1929 or WY-14643. Membranes were probed with antibodies against Ser536-phosphorylated p65, p50, or ß-actin. E, Myometrial cells were transiently transfected with a NF-{kappa}B-luciferase reporter and pretreated with vehicle, troglitazone, or WY-14643 for 2 h, followed by IL-1ß stimulation (1 ng/ml) for 6 h. Cells were cotransfected with a ß-galactosidase reporter plasmid as an internal control, and luciferase activity normalized for ß-galactosidase reporter readout. Values are presented as the fold induction relative to reporter activity in cells treated with vehicle only. Results are the mean ± SEM obtained for each treatment done in triplicate. Similar results were obtained in amnion epithelial cells.

 


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  		FIG. 5. The cyclopentenone ring is essential for cyPG inhibition of NF-{kappa}B. A, NF-{kappa}B-DNA binding was measured by EMSA in nuclear protein extracts from myometrial cells pretreated with vehicle, 15d-PGJ2, or PGA1 for 2 h, followed by stimulation with IL-1ß (1 ng/ml) for 15 min. Antibodies against p50 and p65 were used for supershift analysis. B, Myometrial cells were transiently transfected with a NF-{kappa}B-luciferase reporter and a ß-galactosidase reporter plasmid, pretreated with vehicle or PGA1 for 2 h, and stimulated with IL-1ß (1 ng/ml) for 6 h. Luciferase activity was normalized for ß-galactosidase reporter readout. Values are presented as the mean ± SEM obtained for each treatment done in triplicate. Western blot analysis of nuclear p65 and p50 expression in myometrial cells treated with PGA1 (C) or 15d-PGJ2 (D) for 2 h, followed by stimulation with IL-1ß (1 ng/ml) for 15 min. E, Western blot analysis of whole-cell lysates from myometrial cells treated with 15d-PGJ2 or 9,10-dihydro-15d-PGJ2 for 2 h, followed by stimulation with IL-1ß (1 ng/ml) for 15 min. Membranes were probed with antibodies against p65 and Ser 536-phosphorylated p65. Similar results were obtained in amnion epithelial cells.

 
To more directly exclude a role for PPAR{gamma} in mediating the inhibitory effects of 15d-PGJ2, NF-{kappa}B DNA binding was assessed in cells treated with 15d-PGJ2 in the presence of the PPAR{gamma} inhibitor GW-9662, which binds irreversibly to PPAR{gamma} through covalent modification of Cys285 in the ligand binding domain (49) and effectively reverses the inhibitory effects of both synthetic PPAR{gamma} ligands and 15d-PGJ2 (50, 51). Whereas GW-9662 was able to repress troglitazone-induced activation of PPAR{gamma}-mediated transcription, it failed to alleviate 15d-PGJ2 inhibition of NF-{kappa}B (Fig. 4Go, A and B). 15d-PGJ2 was able to inhibit NF-{kappa}B transcription in the absence of exogenous PPAR{gamma} (Fig. 1Go). Because endogenous levels of PPARs were not sufficient for 15d-PGJ2 to drive the PPRE reporter in the transfection system used, cells transfected with a NF-{kappa}B-dependent reporter were cotransfected with PPAR expression plasmids and treated with 15d-PGJ2. Neither PPAR{gamma} nor PPAR{alpha} overexpression was able to promote the inhibition of IL-1ß-stimulated NF-{kappa}B activity by 15d-PGJ2 in either cell type (Fig. 4CGo). PPAR{gamma} overexpression also had no effect on the 15d-PGJ2 inhibition of basal NF-{kappa}B activity in amnion cells (data not shown). Taken together, these findings indicate that 15d-PGJ2 acts independently of PPARs to inhibit NF-{kappa}B in amnion and myometrial cells.



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  		FIG. 4. Inhibition or overexpression of PPAR{gamma} does not affect repression of NF-{kappa}B by 15d-PGJ2. A, Myometrial cells were transiently cotransfected with a PPRE-luciferase reporter and a PPAR{gamma} expression plasmid or empty vector and incubated with vehicle or GW9662 for 1 h, followed by treatment with vehicle or troglitazone (30 µM) for 20 h. B, NF-{kappa}B-DNA binding was measured by EMSA in nuclear protein extracts from myometrial cells treated with vehicle or GW9662 for 1 h, followed by vehicle or 15d-PGJ2 for 2 h, and stimulation with IL-1ß (1 ng/ml) for 15 min. C, Myometrial cells were transiently cotransfected with a NF-{kappa}B-luciferase reporter and expression plasmids for PPAR{gamma} or PPAR{alpha} and incubated with vehicle or 15d-PGJ2 for 2 h, followed by IL-1ß (1 ng/ml) for 6 h. Luciferase activity was normalized for ß-galactosidase reporter readout. Values are presented as the mean ± SEM obtained for each treatment done in triplicate. Similar results were obtained in amnion epithelial cells.

 
15d-PGJ2 inhibits NF-{kappa}B via its cyclopentenone ring

15d-PGJ2 belongs to a class of PGs termed cyclopentenone PGs (cyPGs) due to the presence of an electrophilic cyclopentenone ring containing an {alpha},ß-unsaturated carbonyl group. This ring can react covalently with nucleophiles such as the free sulfhydryls in cysteine residues of cellular proteins. Because receptor-independent actions of 15d-PGJ2 have been attributed to its cyclopentenone ring, amnion epithelial and myometrial cells were treated with PGA1, a cyPG that does not act as a PPAR ligand (52) but, in common with 15d-PGJ2, contains a reactive cyclopentenone ring. PGA1 was able to inhibit IL-1ß-stimulated NF-{kappa}B DNA binding and NF-{kappa}B transcriptional activity in both cell types (Fig. 5Go, A and B), albeit at higher concentrations than 15d-PGJ2, possibly because PGA1 has one reactive electrophilic carbon, whereas 15d-PGJ2 contains two. In contrast, PGE2, which does not contain a cyclopentenone ring (35), was unable to inhibit IL-1ß-induced NF-{kappa}B DNA binding (data not shown). Treatment of cells with PGA1 was found to decrease the IL-1ß-induced nuclear translocation of p65 (Fig. 5CGo). Similarly, 15d-PGJ2 decreased p65 nuclear expression to basal levels and also inhibited the nuclear expression of a phosphorylated form of the p50 subunit (Fig. 5DGo) [which is important for NF-{kappa}B DNA binding (53)]. Furthermore, 15d-PGJ2 was shown to inhibit the phosphorylation of p65 at serine 536 in amnion epithelial and myometrial cells. Phosphorylation at this residue is mediated by IKKß and/or IKK{alpha} (54, 55) and is required for efficient NF-{kappa}B-mediated transcription (56). Cells were incubated in parallel with 9,10-dihydro-15d-PGJ2, an analog of 15d-PGJ2, which retains PPAR{gamma} agonist activity but in which the cyclopentenone ring has been disrupted. 9,10-Dihydro-15d-PGJ2 could not reproduce the effects of 15d-PGJ2 (Fig. 5EGo). Taken together, these findings indicate that the inhibitory effects of 15d-PGJ2 on NF-{kappa}B in amnion epithelial and myometrial cells can be attributed to its electrophilic ring.

15d-PGJ2 inhibits multiple steps in the IKK/NF-{kappa}B pathway

Having demonstrated the PPAR-independent inhibition of NF-{kappa}B by 15d-PGJ2, we next sought to determine which event(s) in the NF-{kappa}B signaling pathway are targeted by this cyPG. Treatment of amnion epithelial and myometrial cells with either 15d-PGJ2 (Fig. 6AGo) or PGA1 (data not shown) prevented the degradation of I{kappa}B{alpha} by IL-1ß, indicating that cyPG inhibition of NF-{kappa}B activity is mediated, at least in part, by retention of NF-{kappa}B in the cytoplasm of the cell, complexed to its inhibitor. The IKK complex is responsible for the phosphorylation of I{kappa}B{alpha}, a necessary step in the stimulus-induced degradation of I{kappa}B{alpha}. Therefore, we examined the effect of 15d-PGJ2 on the activation of the IKKs. IL-1ß treatment induced the rapid phosphorylation, and therefore activation, of IKKß and IKK{alpha}, and 15d-PGJ2 inhibited this phosphorylation (Fig. 6BGo).



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  		FIG. 6. 15d-PGJ2 inhibits multiple steps in the NF-{kappa}B pathway. Myometrial cells were treated with 15d-PGJ2 or 20 µM MG132 for 2 h, followed by stimulation with IL-1ß (1 ng/ml) for 15 min. Western blot analysis was carried out on cytoplasmic protein extracts using antibodies against I{kappa}B{alpha} (A and C), phosphorylated IKK{alpha}/IKKß and total IKKß (B), and ubiquitin (D). Similar results were obtained in amnion epithelial cells. E, Amnion epithelial cells were transiently cotransfected with a NF-{kappa}B-luciferase reporter and a p65 expression plasmid or empty vector and treated with vehicle or 15d-PGJ2. Luciferase activity was normalized for ß-galactosidase reporter readout. Values are presented as the mean ± SEM obtained for each treatment done in triplicate. Similar results were obtained in myometrial cells but with no effect of 15d-PGJ2 on basal NF-{kappa}B-luciferase reporter.

 
At high concentrations 15d-PGJ2 inhibited the phosphorylation of IKK{alpha}/ß and the degradation of I{kappa}B{alpha}. At lower concentrations, which did not abolish IKK phosphorylation, 15d-PGJ2 treatment led to the accumulation of phosphorylated I{kappa}B{alpha} (Fig. 6AGo). This was similar to the accumulation of phosphorylated I{kappa}B{alpha} seen after treatment of cells with MG132, a compound that inhibits the proteasome (Fig. 6CGo). Treatment with either MG132 or 15d-PGJ2 resulted in increased accumulation of ubiquitinated proteins (Fig. 6DGo), lending further support to the notion that 15d-PGJ2 is able to inhibit the 26S proteasome responsible for the degradation of I{kappa}B{alpha}.

Myometrial and amnion epithelial cells were cotransfected with a NF-{kappa}B-dependent luciferase reporter and a p65 expression plasmid (Fig. 6EGo). p65 overexpression activated the reporter that was inhibited by 15d-PGJ2, showing that, in addition to repressing NF-{kappa}B translocation, 15d-PGJ2 can also directly inhibit NF-{kappa}B by targeting the p65 subunit, independent of the release of NF-{kappa}B from I{kappa}B{alpha}.

15d-PGJ2 inhibits COX-2 expression

IL-1ß-induced COX-2 expression in amnion epithelial and myometrial cells was inhibited by 15-dPGJ2 at concentrations that repressed NF-{kappa}B activity (Fig. 7AGo). In contrast, synthetic PPAR{gamma} or PPAR{alpha} agonists had no effect on COX-2 expression. Furthermore, pretreatment of the cells with the GW9662 PPAR{gamma} antagonist did not reverse the 15d-PGJ2-mediated inhibition (Fig. 7BGo).



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  		FIG. 7. 15d-PGJ2 inhibits COX-2 expression independently of PPARs. COX-2 expression in cytoplasmic protein extracts from myometrial cells pretreated with vehicle or GW9662 for 1 h, followed by treatment with vehicle or 30 µM 15d-PGJ2 for 2 h and addition of IL-1ß (1 ng/ml) for 6 h (A), or treated with 30 µM 15d-PGJ2, 180 µM PGA1, 30 µM troglitazone, 30 µM GW1929 or 30 µM WY-14643, or vehicle for 2 h, followed by stimulation with IL-1ß (1 ng/ml) for 6 h (B). Similar results were obtained in amnion epithelial cells.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In the present study, we report the expression, localization, and activation of PPARs in primary amnion epithelial and myometrial cells; demonstrate the inhibition of NF-{kappa}B activity and COX-2 expression by 15d-PGJ2; and show this inhibition to be independent of PPARs and likely to be a direct effect of the cyclopentenone ring of 15d-PGJ2 acting at multiple levels in the NF-{kappa}B pathway: inhibition of IKK activation, the 26S proteasome, and NF-{kappa}B DNA binding and phosphorylation.

PPAR{gamma} expression has previously been reported at mRNA and protein level in human term placenta and fetal membranes, and PPAR{alpha} has been detected at mRNA level in these tissues (39, 44, 57). Our data show that PPAR{gamma} and PPAR{alpha} are also expressed in the myometrium. We also confirmed the ability of 15d-PGJ2 to induce PPAR{gamma}- and PPAR{alpha}- but not PPAR{delta}-mediated transcription, in agreement with the studies by Forman et al. (35). In myometrial cells, overexpression of PPAR{alpha} transactivated the PPRE reporter independently of ligand. This may be because of high levels of endogenous PPAR-{alpha} ligands (e.g. fatty acids and eicosanoids) in these cells.

Reciprocal expression of COX-2 and PPAR{gamma} was recently reported in fetal membranes and placenta in association with labor (44), and PPAR{gamma} has been proposed as a candidate for therapeutic intervention in preterm labor (31). Therefore, it seemed possible that 15d-PGJ2, a putative endogenous PPAR ligand detectable in amniotic fluid (28), could modulate inflammatory mediators by binding to PPARs. However, our data suggest that PPARs do not regulate either NF-{kappa}B or COX-2 in the human myometrium or amnion and that 15d-PGJ2 acts in a receptor-independent manner to inhibit NF-{kappa}B activity and COX-2 expression.

Inhibition of COX-2 expression by 15d-PGJ2 has been reported in other cell types, but this interaction appears to be cell type specific: Janabi (58) demonstrated 15d-PGJ2 inhibition of COX-2 in astrocytes but not brain macrophages, whereas inhibition of COX-2 by 15d-PGJ2 was mediated through AP-1 repression in cervical cancer cells (59) and through PPAR{gamma} activation in both cervical cancer cells and differentiated macrophages (45, 59). Chawla et al. (60), using mice chimeric for PPAR{gamma}-deficient alleles, reported that 15d-PGJ2 inhibition of TNF{alpha} and IL-6 is not impaired in PPAR{gamma}-deficient macrophages and urge caution in ascribing functions to PPAR{gamma} on the basis of the action of PPAR ligands at high concentrations. In PPAR{gamma}-negative HeLa cells, 15d-PGJ2 inhibited NF-{kappa}B activity in the absence of PPAR{gamma}, with overexpression of PPAR{gamma} reducing the concentration of 15d-PGJ2 required for inhibition (61), indicating the possibility of both PPAR{gamma}-dependent and -independent mechanisms of 15d-PGJ2 inhibition even within the same cell type. Our data show that 15d-PGJ2 inhibition of NF-{kappa}B and COX-2 in amnion epithelial and myometrial cells is independent of PPARs because the response to 15d-PGJ2 was neither mimicked by synthetic PPAR agonists nor modified by a PPAR{gamma} antagonist. When exogenous PPARs were overexpressed in amnion epithelial and myometrial cells, we still did not observe any contribution of these receptors to 15d-PGJ2-mediated inhibition of NF-{kappa}B.

15d-PGJ2 is characterized by the presence of a cyclopentenone ring with an electrophilic carbon that is able to form Michael adducts with free sulfhydryls in cysteine residues. 15d-PGJ2 may exert some of its receptor-independent effects through such covalent interactions. Our data indicate that 15d-PGJ2 inhibits NF-{kappa}B and COX-2 in amnion epithelial and myometrial cells by virtue of its cyclopentenone ring because another cyPG, which also contains a reactive cyclopentenone ring but is not a PPAR agonist (52), could mimic the effects of 15d-PGJ2, whereas a 15d-PGJ2 analog in which the electrophilic carbon has been lost could not.

15d-PGJ2 inhibited multiple steps in the NF-{kappa}B pathway. The degradation of I{kappa}B{alpha} was blocked by 15d-PGJ2, thereby sequestering NF-{kappa}B in the cytoplasm as an inactive complex. This was achieved, in part, by repressing the activation of the IKK complex. 15d-PGJ2 also blocked I{kappa}B{alpha} degradation through another mechanism. Whereas at high concentrations 15d-PGJ2 inhibited the activation of IKK{alpha}/ß and thereby attenuated I{kappa}B{alpha} phosphorylation, at lower concentrations I{kappa}B{alpha} phosphorylation was maintained but did not lead to I{kappa}B{alpha} degradation. 15d-PGJ2 can thus inhibit both the phosphorylation of I{kappa}B{alpha}, which targets it for degradation, and the machinery by which I{kappa}B{alpha} is degraded, the cellular proteasome. Recent studies in macrophages show that inhibiting the proteasome during lipopolysaccharide signaling results in a conversion to an antiinflammatory phenotype (62).

In addition to repressing NF-{kappa}B translocation, we demonstrated that 15d-PGJ2 can directly inhibit NF-{kappa}B by targeting the p65 subunit, independent of the release of NF-{kappa}B from I{kappa}B{alpha}. There may be differential targeting of the NF-{kappa}B pathway by 15d-PGJ2 in amnion epithelial cells, compared with myometrial cells, because 15d-PGJ2 inhibited basal NF-{kappa}B activity in amnion cells but not myometrial cells. It is possible that, in amnion cells, inhibition of NF-{kappa}B by 15d-PGJ2 may occur largely through direct inhibition of a constitutively nuclear NF-{kappa}B subunit, independently of the IKK/I{kappa}B{alpha} pathway, whereas in myometrial cells, 15d-PGJ2 may predominantly target IKK activation. One possible mechanism for the direct inhibition of NF-{kappa}B is the modification of the DNA-binding properties of p65 through a direct interaction. NF-{kappa}B proteins contain a conserved cysteine residue in their DNA-binding domain and alkylation of this cysteine impairs DNA binding (63). Mutation of Cys (62) in the DNA-binding domain of p50 has been shown to confer resistance to 15d-PGJ2-mediated inhibition of NF-{kappa}B in HeLa cells (64). Another possible receptor-independent mechanism for the inhibitory effects of 15d-PGJ2 includes changes in the redox status of the cell (65, 66). Such alterations in the oxidative state of cellular proteins could affect either phosphorylation events in the NF-{kappa}B signaling pathway (67) or the DNA binding of NF-{kappa}B (68).

Targeting of NF-{kappa}B is a potential therapeutic strategy in preterm labor. However, whereas PPAR agonists are unlikely to be effective in this context, cyPGs may have potential. 15d-PGJ2 has been used in vivo to attenuate the development of inflammation-associated disorders as diverse as arthritis (69, 70), bacterially induced brain abscess (71), renal failure (72), fever resolution (73), septic shock (74), and allergic asthma (75). Endogenous synthesis of 15d-PGJ2 by COX-2 within the uterus may be important in the regulation of NF-{kappa}B activity and could form a negative feedback loop on COX-2 expression, as proposed for other cell types (45, 59, 76). This may explain why prophylactic use of COX-2 specific nonsteroidal antiiflammatory drugs increases, rather than decreases the risk of preterm labor (77).


   Footnotes
 
This work was supported by Wellbeing.

First Published Online March 8, 2005

Abbreviations: AP-1, Activator protein-1; COX, cyclooxygenase; cyPG, cyclopentenone prostaglandin; 15d-PGJ2, 15-deoxy-{Delta} (12 14 )-prostaglandin J2; DTT, dithiothreitol; FCS, fetal calf serum; I{kappa}B, inhibitor of {kappa}B; IKK, I{kappa}B kinase; NF-{kappa}B, nuclear factor-{kappa}B; NP-40, Nonidet P-40; Oct-1, octamer transcription factor-1; PG, prostaglandin; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; SDS, sodium dodecyl sulfate.

Received January 10, 2005.

Accepted March 2, 2005.


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