Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print April 20, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.039032
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/3/527    most recent
biolreprod.104.039032v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ackerman, W. E.
Right arrow Articles by Kniss, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ackerman, W. E., IV
Right arrow Articles by Kniss, D. A.
Agricola
Right arrow Articles by Ackerman, W. E.
Right arrow Articles by Kniss, D. A.
BIOLOGY OF REPRODUCTION 73, 527–535 (2005)
DOI: 10.1095/biolreprod.104.039032
© 2005 by the Society for the Study of Reproduction, Inc.

Modulation of Cytokine-Induced Cyclooxygenase 2 Expression by PPARG Ligands Through NF{kappa}B Signal Disruption in Human WISH and Amnion Cells1

William E. Ackerman, IV 3, Xiaolan L. Zhang 5, Brad H. Rovin 5, and Douglas A. Kniss 2, 3,4 

Department of Obstetrics and Gynecology, Laboratory of Perinatal Research and Division of Maternal-Fetal Medicine,3 Center for Biomedical Engineering,4 Dorothy M. Davis Heart and Lung Research Institute and Department of Internal Medicine,5 The Ohio State University, Columbus, Ohio 43210


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase (COX) activity increases in the human amnion in the settings of term and idiopathic preterm labor, contributing to the generation of uterotonic prostaglandins (PGs) known to participate in mammalian parturition. Augmented COX activity is highly correlated with increased COX2 (also known as prostaglandin-endoperoxide synthase 2, PTGS2) gene expression. We and others have demonstrated an essential role for nuclear factor {kappa}B (NF{kappa}B) in cytokine-driven COX2 expression. Peroxisome proliferator-activated receptor gamma (PPARG), a member of the nuclear hormone receptor superfamily, has been shown to antagonize NF{kappa}B activation and inflammatory gene expression, including COX2. We hypothesized that PPARG activation might suppress COX2 expression during pregnancy. Using primary amnion and WISH cells, we evaluated the effects of pharmacological (thiazolidinediones) and putative endogenous (15-deoxy-{Delta}12,14-prostaglandin J2, 15d-PGJ2) PPARG ligands on cytokine-induced NF{kappa}B activation, COX2 expression, and PGE2 production. We observed that COX2 expression and PGE2 production induced by tumor necrosis factor alpha (TNF) were significantly abrogated by 15d-PGJ2. The thiazolidinediones rosiglitazone (ROSI) and troglitazone (TRO) had relatively little effect on cytokine-induced COX2 expression except at high concentrations, at which these agents tended to increase COX2 abundance relative to cells treated with TNF alone. Interestingly, treatment with ROSI, but not TRO, led to augmentation of TNF-stimulated PGE2 production. Mechanistically, we observed that 15d-PGJ2 markedly diminished cytokine-induced activity of the NF{kappa}B transcription factor, whereas thiazolidinediones had no discernable effect on this system. Our data suggest that pharmacological and endogenous PPARG ligands use both receptor-dependent and -independent mechanisms to influence COX2 expression.

cytokines, gene regulation, parturition, placenta, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandins (PGs) that elicit myometrial contractions and cervical maturation are pivotal to the onset and maintenance labor in humans and all other mammalian species that have been investigated [1]. The exact triggers for the onset of parturition at term are incompletely understood, but there is a growing consensus that cytokines, such as interleukin 1ß (IL1B) and tumor necrosis factor alpha (TNF), are instrumental in unleashing vigorous biosynthesis of PGE2 and PGF2{alpha} within intrauterine tissues, leading to active labor and birth [2]. In addition, it appears that many of the fundamental biological forces governing labor at term are also evoked during episodes of preterm labor elicited by inflammatory insults to the mother [3].

While the majority of parturition research has focused on the feed-forward mechanisms through which uterine stimulants are generated, a relevant question remains unanswered: What biological inputs keep the uterus from contracting and the cervix from ripening during the vast majority of pregnancy? It is reasonable to hypothesize that tonically active signals prohibit myometrial activation before term and that these biomolecules are of fundamental importance for a thorough understanding of the mechanisms governing parturition. In this regard, it has recently been reported that PGs of the J2 series may exert anti-inflammatory activity in a variety of circumstances [4]. Peroxisome proliferator-activated receptor gamma (PPARG) is a nuclear receptor whose ligands include PGs of the J2 series (particularly 15-deoxy-{Delta}12,14-prostaglandin J2, 15d-PGJ2) in addition to polyunsaturated fatty acids, products of lipoxygenase metabolism, and oral hypoglycemic agents (thiazolidinediones) [5, 6]. Several groups have reported that PPARG antagonizes the expression of inflammatory signals in a broad array of experimental contexts [7, 8], including human gestational tissues [9]. Moreover, some PPARG ligands inhibit the expression of inflammatory gene products, such as inducible nitric oxide synthase (also known as nitric oxide synthase 2A, NOS2A), cyclooxygenase 2 (COX2, also known as prostaglandin-endoperoxide synthase 2, PTGS2), and microsomal prostaglandin E synthase-1 (also known as prostaglandin E synthase, PTGES) by mechanisms that may be PPARG-dependent and/or -independent [7, 8, 10, 11].

A common theme among the proinflammatory enzymes cited above is their regulation at the transcriptional level by nuclear factor {kappa}B (NF{kappa}B) [12, 13]. The genes encoding these inflammatory proteins harbor one or more copies of a {kappa}B-binding motif through which transcription may be enhanced [12]. For example, cytokines use NF{kappa}B within intrauterine cells to stimulate expression of the COX2 gene [14–17], whose product catalyzes the committing and rate-limiting step in uterotonic PG formation [18]. Recent evidence suggests that COX2-mediated synthesis of PGD2 metabolites (including the PPARG ligand, 15d-PGJ2) may provide a mechanism for feedback control of PG biosynthesis [4, 19]. Furthermore, we recently reported that a reciprocal relationship exists between the expression of COX2 and PPARG proteins in fetal membranes obtained from women before the onset of labor compared with tissues collected following delivery [20]. Thus, in the present study, we examined the mechanism by which known PPARG ligands govern COX2 expression in WISH cells and primary cultures of human amnion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Recombinant human TNF was purchased from R&D Systems (Minneapolis, MN). Antibodies against inhibitory factor {kappa}B{alpha} (I{kappa}B{alpha}, also known as nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, NFKBIA), I{kappa}B kinase {alpha} (IKK{alpha}, also known as conserved helix-loop-helix ubiquitous kinase, CHUK), I{kappa}B kinase ß (IKKß, also known as inhibitor of kappa light polypeptide gene enhancer in B-cells kinase beta, IKBKB), COX2, and NF{kappa}B subunits p65 (also known as reticuloendotheliosis viral oncogene homolog A, RELA), p50 (also known as nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, NFKB1), p52 (also known as nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, NFKB2), cRel (also known as reticuloendotheliosis viral oncogene homolog, REL), and RelB (also known as reticuloendotheliosis viral oncogene homolog B, RELB) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPD) antibody, which is cross-reactive with the human isoform, was purchased from Chemicon International (Temecula, CA). Antibodies recognizing phosphorylated I{kappa}B{alpha} (Ser32) and IKK{alpha} (Ser180)/IKKß (Ser181) were from Cell Signaling Technology (Beverly, MA), as were rabbit anti-human PPARG antibodies. A second PPARG antibody was purchased from Affinity Bioreagents (Golden, CO). The 1.8-kilobase (kb) cDNA fragment used for COX2 mRNA Northern blotting was a kind gift from Dr. Timothy Hla (University of Connecticut, Farmington, CT). Arachidonic acid, all PGs, rosiglitazone, and PGE2 ELISA kits were obtained from Cayman Chemical (Ann Arbor, MI). Troglitazone was from BIOMOL (Plymouth Meeting, PA). DIG Nucleic Acid Detection and DIG-High Prime kits were purchased from Roche Diagnostics (Indianapolis, IN). Assays-on-Demand gene expression target assay mix (Hs00153133_m1), 18S rRNA assay mix, and TaqMan Universal Master Mix were obtained from Applied Biosystems (Foster City, CA). SuperSignal chemiluminescent detection reagents were obtained from Pierce Biotechnology (Rockford, MA). Prolong antifade mounting reagent and Alexa Fluor-594-conjugated goat anti-rabbit antibodies were purchased from Molecular Probes (Eugene, OR). The NF{kappa}B consensus oligonucleotide was obtained from Promega (Madison, WI). All other reagents, unless otherwise specified, were obtained from Sigma (St. Louis, MO).

Cell Cultures

Human WISH cells were obtained from the American Type Culture Collection (CCL-25) and maintained in Ham F-12/Dulbecco modified Eagle medium (F-12/DMEM, Invitrogen, Carlsbad, CA) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% (v/v) newborn calf serum. Cells were grown at 37°C in a humidified atmosphere of 95% air/ 5% CO2 and used for experiments between the 3rd and 25th passages.

Primary cultures of human amnion cells were obtained at the time of uncomplicated scheduled cesarean delivery at term in the absence of labor or membrane rupture. There were no clinical signals of infection in any of these subjects. Informed consent was obtained in all cases following a protocol approved by the Institutional Review Board of The Ohio State University. Cultures were established according to the method of Okita et al. [21], with modifications. Briefly, amnion tissue was dissected from the choriodecidua within minutes of delivery, placed in sterile Hanks buffered salt solution (HBSS), and transported to our laboratory for aseptic processing. Amnion tissue was washed in ice-cold HBSS until free of blood, minced into ~1-cm2 fragments, and digested for 30 min in HBSS containing 0.4% (w/v) trypsin at 37°C with constant rocking. Undigested material was recovered and fresh trypsin solution was added. Following an additional 30-min incubation, cells were strained through 60 mesh and the flow-through was collected. This process was repeated using the remaining undigested tissue. The flow-through from the second and third digestions was pooled and cells were collected by centrifugation at 600 x g for 10 min. Cells were then washed, plated into tissue culture dishes, and grown in F-12/DMEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamicin sulfate, and 10% (v/v) fetal bovine serum. Within 7–10 days, cultures had attained confluence and were used for experiments.

Northern Blot Analysis

To assess COX2 mRNA expression in WISH cells following treatments, total RNA was extracted using TRIZOL according to manufacturer's specifications (Invitrogen). Total RNA (20 µg/lane) was fractionated on a 1% (w/v) agarose gel containing 2% (v/v) formaldehyde, transferred to a nylon membrane, and immobilized using ultraviolet irradiation. Membranes were then probed with digoxigenin (DIG)-containing cDNA probes labeled using the DIG-High Prime kit. A 1.8-kb cDNA fragment encoding human COX2 and a 0.6-kb cDNA fragment encoding human GAPD were used for detection. Blots were hybridized overnight at 42°C in 7% (w/v) SDS, 50% (v/v) formamide, 325 mM sodium chloride, 32.5 mM sodium citrate, 50 mM sodium phosphate (pH 7.0), 0.1% (w/v) N-lauroylsarcosine, 50 µg/ml sheared salmon sperm DNA, and 2% (w/v) DIG Blocking Reagent (Roche). Bound probes were identified using the DIG Nucleic Acid Detection kit (Roche). Chemiluminescent signals were detected using the VersaDoc Imaging System and analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA).

Real-Time Reverse Transcriptase-Polymerase Chain Reactions

Following treatments, total RNA was extracted from amnion cell cultures using TRIzol (Invitrogen). Because total RNA yields from amnion cells were typically ~10% that of the WISH cells, quantities were insufficient to allow for detection by Northern blotting using the relatively insensitive DIG-labeled cDNA probes. Therefore, relative abundance of COX2 mRNA was performed by real-time reverse transcriptase-polymerase chain reactions (RT-PCR) detection using an ABI PRISM 7700 sequence detector (Applied Biosytems, Foster City, CA). Two µg/reaction of total RNA were reverse transcribed to cDNA using random hexamer primers and SuperScript II reverse transcriptase (Invitrogen), according to instructions provided by the manufacturer. Human COX2 expression was detected using the Assays-on-Demand gene expression target assay mix (Hs00153133_m1; Applied Biosystems), which contained a mixture of specific primers and a hydrolysis probe containing the fluorescent dye 6-FAM. As the reporter probe sequence was designed to detect a region spanning multiple intron-exon junctions, target cDNA was detected selectively, irrespective of the presence of contaminant genomic DNA. A primer/VIC-labeled probe set for detecting 18S rRNA (Applied Biosystems) was used as an internal control in each reaction. Amplification mixtures contained 2.5 µl 20x target assay mix (Applied Biosystems), 1 µl first-strand cDNA synthesis mixture (corresponding to 50 ng reverse transcribed RNA), 0.5 µl of 18S rRNA control mix (Applied Biosystems), 25 µl of 2x TaqMan Universal Master Mix (Applied Biosystems), and nuclease-free distilled deionized water to a total volume of 50 µl. Amplification was performed over 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min.

For each treatment condition, duplicate amplifications were performed. Relative abundance of COX2 mRNA was normalized to the 18S rRNA internal standard and calculated as 2, where {Delta}CT denotes difference between the sample CT (cycle threshold) and that of the reference message present in cells under nonstimulated conditions. Amplification curves were analyzed using software provided by the manufacturer (Applied Biosystems).

Electrophoretic Mobility Shift Assay

Cells were treated with the test substances indicated and nuclear protein extracts were prepared [22]. Nuclear protein extracts (10 µg/reaction) were then mixed with an oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC; Promega) representing the consensus sequence of the {kappa}B-binding motif (underlined) labeled with [32P]-ATP (approximately 104 cpm/ tube) as previously described [22]. Control incubations contained either no nuclear extracts, a 20-fold excess of an unlabeled, irrelevant oligonucleotide (5'-GGCGAAACTTCTGGAATATTCCCGAACTTTCAG, a sequence from the human heat shock 70-kDa protein 1 [HSPA1A] gene promoter [23]), or a 20-fold excess of unlabeled {kappa}B oligonucleotide. After a 20-min incubation at room temperature, the extracts were fractionated on nondenaturing 5% (w/v) polyacrylamide gels in 0.25 M Tris-boric acid-EDTA buffer. Gels were then dried and exposed to x-ray film at –80°C.

To conduct supershift assays, samples were prepared for electrophoretic mobility shift assay (EMSA) as described above and to each tube 2 µg of anti-p65, anti-p50, anti-p52, anti-RelB, or anti-cRel was added for 30 min at room temperature. Control incubations contained the same concentration of isotype-specific IgG. Samples were fractionated on nondenaturing gels and autoradiographed as described above.

Immunoblot Analysis

Cellular proteins were extracted as previously described [24]. Proteins (30 µg/lane) were resolved by SDS-PAGE and transferred to nitrocellulose. Immunoblotting was performed using antibodies directed against native and phosphorylated I{kappa}B{alpha}, native and phosphorylated IKK{alpha}/ß, PPARG, COX2, and GAPD in Tris-buffered saline (pH 8.0) containing 0.05% (v/v) Tween-20 and 5% (w/v) BSA. Following exposure to horseradish peroxidase-conjugated secondary antibodies, chemiluminescent signals were revealed using SuperSignal chemiluminescent detection reagents (Pierce). Immunoreactive proteins were visualized using the VersaDoc Imaging System and analyzed using Quantity One software (Bio-Rad Laboratories).

Immunofluorescence

Cells were seeded onto flame-sterilized glass coverslips placed in a 24-well tissue culture plate. Following treatments, cells were fixed for 1 h in 4% (w/v) paraformaldehyde/PBS, made permeable by the addition of 0.2% (v/v) Triton X-100/PBS for 15 min, and blocked in 5% (v/v) horse serum/ PBS overnight at 4°C before the addition of antibodies. Monoclonal antibodies directed against p65 (sc-8008; Santa Cruz) were then applied. In control experiments, an equivalent concentration of isotype-specific normal mouse IgG was substituted for the primary antibody. After stringent washing in PBS, the coverslips were exposed to fluorochrome-conjugated secondary antibodies (Molecular Probes). Nuclei were counterstained with 5 µg/ml 4',6-diamidino-2-phenylindole (DAPI, Sigma). Cells were mounted using the ProLong Antifade kit (Molecular Probes) and visualized using an epifluorescence microscope (Nikon Instruments, Melville, NY).

Prostaglandin E2 ELISA

Cells were plated in 48-well tissue culture plates in serum-free culture medium supplemented with 5 µM arachidonic acid. Following 6-h incubations with test substances, the media were collected and frozen at –80°C until assays were performed. Media were analyzed for PGE2 content using commercially available ELISA kits (Cayman Chemical). Cells were solubilized with 1 N NaOH, total protein was measured, and the data were expressed as nanograms of PGE2 produced/mg protein. In all ELISA experiments, 4–6 replicates were performed per assay and the experiments were repeated a minimum of twice. The intra- and interassay coefficients of variation of this assay were <10% and the limit of detection was 15 pg/ml (corresponding to approximately 12.5 pg/mg when adjusted for the average protein content/well).

Statistical Analysis

ELISA and densitometric data were assessed by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparisons post hoc test; P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PPARG Ligands Modulate PGE2 Biosynthesis

Among several potential endogenous ligands for PPARG, 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2), a nonenzymatically-derived terminal metabolite of PGD2, has been extensively studied [19, 23, 25–28]. We examined the effects of this cyclopentenone in relation to pharmacological PPARG agonists of the thiazolidinedione class (rosiglitazone [ROSI] and troglitazone [TRO]). WISH cells were preincubated for 30 min in the absence or presence of 15d-PGJ2, ROSI, or TRO and then challenged with TNF (25 ng/ml) for 6 h. Control cultures were incubated with 0.1% (v/v) ethanol (vehicle for the thiazolidinediones) or 0.8% (v/v) methyl acetate (MeAc, vehicle for 15d-PGJ2). To exclude the possibility of cross-reactivity between the antibodies and the reagents, in some cases, we spiked aliquots of test medium with the highest experimental concentrations of 15d-PGJ2, ROSI, or TRO and subjected these to ELISA. None of these compounds were found to interfere with the assay system. Expression of the PPARG receptor protein in WISH and primary amnion cells was confirmed by immunoblotting (Fig. 1A).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 1. Effects of PPARG ligands on TNF-induced PGE2 production. A) Expression of PPARG (57 kDa) in WISH and primary amnion cells was confirmed by immunoblotting using polyclonal antibodies purchased from Cell Signaling Technology (2492, top panel) and Affinity Bioreagents (PA3-821, bottom panel). B) WISH cells were preincubated for 30 min with 15d-PGJ2 (0.3–30 µM), ROSI (0.1–10 µM), TRO (0.1–10 µM), or vehicle (0.8% [v/v] MeAc) and then treated for 6 h in the absence or presence of 25 ng/ml of TNF. The media were collected and subjected to PGE2 ELISA. PGE2 content was normalized to the total protein content for each sample and has been represented graphically (mean ± SEM for quadruplicate determinations, n = 2 individual experiments at the concentration ranges shown, which were confirmatory of two additional experiments in which single concentrations [30 µM for 15-dPGJ2, and 10 µM each for ROSI and TRO] were used). It should be noted that the effect of a second vehicle (0.1% [v/v] ethanol, not shown) on stimulated PGE2 production was similar to that of MeAc. * P < 0.001 vs. TNF (ANOVA)

In the presence of 30 µM 15d-PGJ2, TNF-stimulated PGE2 production was diminished compared with cells preincubated with vehicle alone (Fig. 1B and data not shown). At 3 µM of 15d-PGJ2, this response was less marked, and no effect was evident when 0.3 µM of 15d-PGJ2 was used (Fig. 1B). By contrast, preincubation with ROSI or TRO did not abrogate PG generation (Fig. 1B). In fact, in preliminary experiments, it was found that preincubation with 10 µM ROSI led to a significant enhancement of cytokine-induced PGE2 release, whereas 10 µM TRO had no discernable effect (data not shown). When tested at an expanded range of concentrations (0.1–10 µM), it was discovered that cytokine-mediated PGE2 synthesis was consistently enhanced in the presence of ROSI at concentrations of 1 µM or greater (Fig. 1B). There was no enhancement of basal PGE2 production when these cells were incubated with ROSI or TRO in the absence of TNF (data not shown). These data indicated that, while both 15d-PGJ2 and thiazolidinediones are activators of PPARG, they exert differential effects on PGE2 formation in this model system.

15-deoxy-{Delta}12,14-PGJ2 Prohibits TNF-Induced COX2 Accumulation

The mechanisms through which 15d-PGJ2 exerts its anti-inflammatory effects remain controversial, as both PPARG-dependent and -independent actions have been reported [10, 28, 29]. Like the other PGD2 derivatives (PGJ2 and {Delta}12-PGJ2), 15d-PGJ2 has a cyclopentenone ring structure that is characterized by one or two {alpha},ß-unsaturated carbonyl moieties with electrophilic carbons that are very susceptible to nucleophilic addition reactions (reviewed in [30], also see Fig. 2A). Many of the PPARG-independent effects of 15d-PGJ2 appear to be similar to those seen with other cyclopentenone PGs, and may arise through the ability of these compounds to form thiol conjugates with cysteinyl residues in a variety of cellular proteins [30, 31].



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 2. Effects of cyclopentenone PGs and thiazolidinediones on TNF-elicited COX2 protein expression. A) Chemical structures of PGD2 and the cyclopentenone PGs used in this study are shown in relation to that of the parent compound, cyclopentenone. Electrophilic carbons are denoted by gray asterisks. B, C) Representative immunoblots prepared from WISH cell lysates 4 h following challenge with TNF (25 ng/ml) alone or in combination with PGD2 (30 µM), cyclopentenone PGs (PGA1, PGJ2, {Delta}12-PGJ2, 15d-PGJ2, 15d-PGA1, 15d-PGA2, and 15d-PGD2, all at 30 µM) or thiazolidinediones (ROSI and TRO, 0.1–10 µM). Control cells received either 0.8% (v/v) MeAc (B) or 0.1% (v/v) ethanol (C). D, E) Immunoreactive COX2 (72 kDa) was quantified by densitometric scanning. Data were normalized to GAPD (34 kDa) protein levels for each treatment group and have been represented graphically as ratios of arbitrary densitometric units (mean ± SEM, n = 2 [D] or 3 [E] individual experiments). * P < 0.01 vs. TNF (ANOVA). ** P < 0.05 vs. TNF (ANOVA)

To determine whether diminished PGE2 biosynthesis was due to decreased COX2 protein following cytokine stimulation, we preincubated WISH cells either with 15d-PGJ2 (30 µM), ROSI (0.1–10 µM), TRO (0.1–10 µM), or vehicles and then challenged the cells with TNF for 4 h. Additionally, to compare the effects of 15d-PGJ2 with other cyclopentenone PGs, cells were also treated with equivalent concentrations (30 µM) of the following: PGA1, PGD2, PGJ2, {Delta}12-PGJ2, 15d-PGA1, 15d-PGA2, and 15d-PGD2. Cell extracts were subjected to immunoblotting with anti-COX2 antibodies. In the absence of cytokine treatment, immunoreactive COX2 was barely detectable. By contrast, treatment with TNF elicited a dramatic increase in COX2 protein (Fig. 2, B and C, lane 2), which was consistent with an earlier report from our group [24]. When cells were preincubated with 15d-PGJ2, TNF did not stimulate an increase in COX2 production (Fig. 2B, compare lanes 2 and 7). Interestingly, among cells pretreated with related cyclopentenones (PGA1, PGJ2, {Delta}12-PGJ2) or their 15-deoxy derivatives (15d-PGA1, 15d-PGA2, 15d-PGD2), only {Delta}12-PGJ2 inhibited COX2 protein expression, and this effect was modest (Fig. 2D). Neither ROSI nor TRO inhibited TNF-induced COX2 protein expression (Fig. 2C, in which it should be noted that the apparent diminution of immunoreactive COX2 in lane 4 was not a consistent finding). Rather, for both ROSI and TRO at 10 µM, we observed a consistent trend toward increased COX2 protein (Fig. 2E).

15-Deoxy-{Delta}12,14-PGJ2 and Thiazolidinediones Differentially Regulate COX2 mRNA Expression

After demonstrating that 15d-PGJ2 inhibits TNF-stimulated COX2 protein expression in WISH cells, we determined whether 15d-PGJ2 or thiazolidinediones attenuate COX2 mRNA upregulation following cytokine exposure. In Northern blots, COX2 mRNA was characteristically upregulated following a 60-min challenge with TNF (Fig. 3A, lane 2). When WISH cells were preincubated with 15d-PGJ2 for 30 min before cytokine stimulation, we noted nearly complete abrogation of COX2 mRNA accumulation (Fig. 3A, compare lanes 1, 2, and 7). A similar, but less substantial, effect was noted for {Delta}12-PGJ2, a second PGD2 metabolite that has been shown to exhibit both PPARG-dependent and -independent activities [31, 32]. With respect to the other PGs, PGA1, 15d-PGA1, and 15d-PGA2 all exhibited similar effects in attenuating TNF-elicited COX2 mRNA expression (Fig. 3, A and B). By contrast, neither PGD2, PGJ2, nor 15d-PGD2 produced a discernable effect on COX2 mRNA levels in response to cytokine challenge, despite the presence of electrophilic carbons on PGJ2 and 15d-PGD2 (Fig. 3, A and B). RNA from WISH cells that had been pretreated with ROSI or TRO followed by TNF showed no diminution in COX2 mRNA (Fig. 3A). Instead, at thiazolidinedione concentrations of 10 µM, there was a modest but reproducible enhancement of cytokine-mediated COX2 mRNA accumulation (Fig. 3A, lanes 11 and 13).



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 3. Effects of cyclopentenone PGs and thiazolidinediones on cytokine-induced COX2 mRNA expression. A) Representative Northern blot demonstrating COX2 mRNA levels in WISH cells 1 h following treatment with TNF (25 ng/ml) in the absence or presence of PGD2 (30 µM), cyclopentenone PGs (PGA1, PGJ2, {Delta}12-PGJ2, 15d-PGJ2, 15d-PGA1, 15d-PGA2, and 15d-PGD2, all at 30 µM) or thiazolidinediones (ROSI and TRO, 1– 10 µM). Control cells received either 0.8% (v/v) MeAc or 0.1% (v/v) ethanol, with similar effects. B) Chemiluminescent detection of the major COX2 mRNA band (4.5 kb) was quantified by densitometric scanning. Data were normalized to GAPD mRNA levels in each treatment group and have been represented graphically as a ratio of arbitrary densitometric units (mean ± SEM, n = 3 individual experiments). C) Real-time RT-PCR was used to assess COX2 mRNA expression in primary amnion cells following treatment with vehicle alone, vehicle with TNF (25 ng/ml), or TNF in the presence of 15d-PGJ2 (30 µM), ROSI (10 µM), or TRO (10 µM). The threshold cycle (CT) at which COX2 mRNA was detected was normalized to that of the 18S rRNA internal control. The ratios of COX2 mRNA/18S rRNA have been expressed graphically as the fold increase in relation to control cells, for which the level of COX2 mRNA expression was set to 1 (mean ± SEM for duplicate determinations, n = 2 individual experiments). * P < 0.01 vs. TNF (ANOVA). ** P < 0.05 vs. TNF (ANOVA)

To examine whether the observations in WISH cells were also seen in amnion cells cultured from full-term fetal membranes, we preincubated amnion cell monolayers for 30 min with vehicle, 15d-PGJ2, ROSI, or TRO and then challenged cells for 1 h with TNF. Total RNA was extracted and assayed by real-time RT-PCR using a human COX2 primer pair/probe set. Figure 3C demonstrates that TNF caused a nearly fivefold increase in COX2 mRNA expression following cytokine treatment. In contrast, amnion cells preincubated with 15d-PGJ2 manifested an approximately 40% decrease in COX2 mRNA when challenged with cytokine (Fig. 3C). When amnion cells were preincubated with ROSI or TRO and then TNF, there was an enhancement of cytokine-induced COX2 mRNA accumulation (Fig. 3C). Thus, both WISH and primary amnion cells exhibited decreased expression of COX2 mRNA when preincubated with 15d-PGJ2, while there was no such diminution when cells were pretreated with thiazolidinediones. In fact, thiazolidinedione pretreatment elicited a modest and consistent elevation in COX2 mRNA expression following cytokine stimulation in both cell types.

Cyclopentenones Inhibit TNF-Stimulated Activation of NF{kappa}B

Proinflammatory cytokines use the NF{kappa}B pathway as a principal means to elicit COX2 expression within intrauterine cells [15–17]. Under basal conditions, NF{kappa}B dimers (which can be composed of the subunits p65, p50, p52, cRel, and RelB) are bound to an inhibitor (I{kappa}B) [12]. In the presence of I{kappa}B, the net intracellular distribution of NF{kappa}B is cytoplasmic; however, when cells are stimulated, I{kappa}B is phosphorylated by I{kappa}B kinases (IKKs), which results in the destruction of this inhibitor by the 26 S proteasome complex [12]. Once this occurs, the NF{kappa}B complex translocates to the nucleus, binds to response elements within the promoters of target genes, and activates transcription.

To examine the biochemical mechanisms that underlie cyclopentenone inhibition of cytokine signaling via NF{kappa}B, WISH cells were preincubated either with PGD2, cyclopentenones, thiazolidinediones, or vehicles and then stimulated with TNF (25 ng/ml) for 15 min (a time point at which the signaling events responsible for NF{kappa}B activation were at their peak, as established previously [17]). Immunoblots were probed with antibodies recognizing the native and phosphorylated forms of IKK{alpha}, IKKß, and I{kappa}B{alpha}.

TNF elicited phosphorylation of IKK{alpha} and IKKß, which was followed by phosphorylation and rapid degradation of I{kappa}B{alpha} (data not shown). This resulted in the loss of I{kappa}B{alpha} immunoreactivity observed by 15 min poststimulation (Fig. 4A, compare lanes 1 and 2). As previously demonstrated [17], this loss in I{kappa}B{alpha} immunoreactivity could be blocked with MG-132, an inhibitor of protein degradation by the 26 S proteasome complex (data not shown). Neither PGD2, the cyclopentenones, nor the thiazolidinediones were found to affect signaling events leading to TNF-mediated phosphorylation of IKK{alpha} or IKKß (Fig. 4, A and B). However, in the presence of 15d-PGJ2, cytokine-induced phosphorylation of I{kappa}B{alpha} was completely blocked, and there was no evidence of lost I{kappa}B{alpha} immunoreactivity (Fig. 4A, lane 7). Several other cyclopentenones, including PGA1, PGJ2, {Delta}12-PGJ2, 15d-PGA1, and 15d-PGA2, caused retention of phosphorylated I{kappa}B{alpha} following TNF stimulation, consistent with disruption or delay in the events leading to NF{kappa}B activation (Fig. 4A). The findings of inhibited I{kappa}B{alpha} phosphorylation and/or degradation were congruous with prior reports, in which the activities of both IKK and the proteasome complex were found to be directly inhibited by the cyclopentenone PGs [28, 33–35]. By contrast, neither PGD2, 15d-PGD2, nor the thiazolidinediones blocked the phosphorylation/degradation of I{kappa}B{alpha}.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 4. Effects of cyclopentenone PGs and thiazolidinediones on cytokine-stimulated NF{kappa}B activation. A, B) WISH cells were challenged for 15 min with TNF (25 ng/ml) in the absence or presence of PGD2 (30 µM), cyclopentenone PGs (PGA1, PGJ2, {Delta}12-PGJ2, 15d-PGJ2, 15d-PGA1, 15d-PGA2, and 15d-PGD2, all at 30 µM) or thiazolidinediones (ROSI and TRO, 0.1–10 µM). Control cells received either 0.8% (v/v) MeAc (A) or 0.1% (v/v) ethanol (B). Activation of NF{kappa}B was assessed using antibodies detecting native and phosphorylated forms of IKK{alpha} (85 kDa), IKKß (87 kDa), and I{kappa}B{alpha} (39 kDa). These blots are representative of the results of two independent experiments. C) Intracellular localization of NF{kappa}B subunit p65 was detected by immunofluorescence in WISH (panels 1–5) or primary amnion (panels 6–12) cells treated with or without 25 ng/ml of TNF for 15 min in the absence (vehicle only) or presence of ROSI (10 µM, panels 3 and 8), 15d-PGJ2 (30 µM, panels 4 and 9), or PGA1 (30 µM, panels 5 and 10). Arrows indicate cell nuclei. A representative control experiment is shown (panel 11) in which an equivalent amount of normal mouse IgG1 was substituted for the primary antibody; DAPI staining within this same field demonstrates the location of cell nuclei (panel 12). Bars = 20 µm

We next conducted immunolocalization studies to evaluate whether 15d-PGJ2 attenuated cytosolic to nuclear translocation of the NF{kappa}B subunit p65. WISH cells were preincubated for 30 min either with vehicle, ROSI, or 15d-PGJ2. To test the potential specificity of 15d-PGJ2, we also preincubated some cultures with a second cyclopentenone, PGA1. The cells were then challenged with TNF and fixed for immunostaining with anti-p65 at various intervals from 5 to 60 min. As previously observed with IL1B [17], TNF stimulation resulted in translocation of p65 from cytosol to nucleus within 15 min (Fig. 4C, arrows in panel 2). ROSI had no effect on the movement of p65 to the nuclear compartment (Fig. 4C, arrows in panel 3). 15d-PGJ2, however, completely abrogated the ability of TNF to elicit p65 translocation to the nucleus, even through the 60-min time period tested (the effect of this agent at 15 min poststimulation is shown in Fig. 4C, panel 4). PGA1, though somewhat less efficacious, was able to delay p65 nuclear localization (data not shown). By 15 min following TNF challenge in the presence of PGA1, nuclear p65 immunoreactivity was observed in a minority of cells (Fig. 4C, panel 5). Similar to the data obtained in WISH cells, we also noted in primary amnion cells that 15d-PGJ2, but not ROSI, inhibited TNF-stimulated p65 nuclear translocation, while the effects of PGA1 were incomplete (Fig. 4C, panels 6–10).

We next examined whether 15d-PGJ2 could prevent binding of NF{kappa}B to radiolabeled oligonucleotides representing the consensus {kappa}B motif, similar to those found in the 5'-promoter region of the human COX2 gene [36]. In initial EMSA experiments, it was observed that nuclear extracts prepared from WISH cells stimulated for 30 min with TNF led to the restricted mobility of two specific bands (I and II in Fig. 5, A and B). A third specific band migrating above band II was noted in some cases (Fig. 5B). Supershifting with polyclonal antibodies directed against NF{kappa}B subunits p65 and p50 demonstrated that the DNA binding activity of band I likely consisted of p50/p50 homodimers, while that of band II likely consisted of p65/p50 heterodimers. We ruled out the likely participation of other NF{kappa}B members using antibodies to p52, cRel, and RelB (data not shown). Following a 30-min preincubation with 30 µM of 15d-PGJ2 or 10 µM of ROSI, we observed that TNF failed to elicit protein binding to the {kappa}B motif in the presence of 15d-PGJ2 only (Fig. 5C). Collectively, these data demonstrated that 15d-PGJ2, like some of the other cyclopentenones (PGA1, PGJ2, {Delta}12-PGJ2, 15d-PGA1, and 15d-PGA2), but in contrast with the pharmacological PPARG ligands, was able to attenuate cytokine-stimulated NF{kappa}B activation.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 5. Modulation of cytokine-induced NF{kappa}B DNA binding by cyclopentenone PGs and thiazolidinediones. A) The conditions for EMSA were verified by incubating a [32P]-labeled oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC) containing the {kappa}B consensus site (underlined) in the presence of nuclear extract prepared from TNF-stimulated WISH cells. As a control for probe specificity, companion extracts were prepared in the absence of nuclear extract (Probe Only) or in the presence of a 20-fold excess of an unlabeled competitor probe containing either an irrelevant sequence (HSF Comp) or the {kappa}B binding motif ({kappa}B Comp). B) Nuclear extracts from WISH cells treated for 30 min with media alone (Veh) or with TNF (25 ng/ml) were prepared for supershift EMSA using polyclonal antibodies directed against NF{kappa}B subunits p65 ({alpha}p65) or p50 ({alpha}p50). C) Nuclear extracts were prepared from WISH cells 30 min following stimulation with TNF (25 ng/ml) in the absence or presence of 15d-PGJ2 (30 µM) or ROSI (10 µM) and subjected to EMSA. Control cells were treated with vehicle only (0.8% [v/v] MeAc). The blots are representative of two independent experiments. Arrows denote the locations of specific bands (I and II)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this investigation, we evaluated the ability of PPARG ligands to downregulate the COX2 gene and subsequent formation of PGE2 in WISH and primary amnion cells. Our data demonstrated an interesting dichotomy between 15d-PGJ2, a cyclopentenone PG, and the thiazolidinediones, which are pharmacological PPARG activators. Whereas 15d-PGJ2 treatment of amnion cells in primary culture or WISH cells almost completely abrogated TNF-driven COX2 expression, the thiazolidinediones partially upregulated COX2 expression. These data render a straightforward mechanism through ligation of nuclear PPARG unlikely and instead invoke a more complex level of COX2 regulation by these agonists.

In this study, we employed WISH cells in many experiments, as these cells are easily manipulated genetically and manifest many phenotypic features of amnion epithelial cells [37–39]. Although WISH cells have been used as reliable and reproducible facsimile for amnion epithelial cells for many years [14, 17, 24, 27, 40], it must be acknowledged that these cells contain HeLa chromosome markers, as we have recently reported [41]. We verified the applicability of our data by repeating key experiments using primary amnion cell cultures, which in these cases showed similar results. Thus, to a degree, WISH cells remain a very realistic surrogate for molecular mechanistic studies in amnion cells.

In the case of 15d-PGJ2, suppression of cytokine-elicited COX2 expression appears to have arisen to a large extent through interference with NF{kappa}B activation. In amnion cell models, the NF{kappa}B signaling cascade appears to be the predominant mechanism through which cytokines stimulate COX2 gene expression [14, 16, 17]. Moreover, our group has reported that NF{kappa}B is a prominent transcription factor governing COX2 gene expression in ED27 cells [22]. Like other cyclopentenone PGs, 15d-PGJ2 has been shown to impede NF{kappa}B signaling at multiple steps: it may interfere with the phosphorylation of I{kappa}B by suppressing IKK activity [33], it may interfere with I{kappa}B degradation by inhibiting activity of the 26 S proteasome complex [34], and it may interfere directly with the ability of NF{kappa}B subunits to bind to their cognate DNA response elements [28]. These effects are thought to occur independently of PPARG, through the formation of covalent adducts between 15d-PGJ2 and critical cysteinyl residues within these proteins [28, 35]. Consistently, we observed that other cyclopentenone PGs bearing {alpha},ß-unsaturated ketone groups also interfered with cytokine-mediated NF{kappa}B activation, albeit far less potently, whereas thiazolidinediones did not alter this signaling pathway. Although prior reports have lead to the speculation that PPARG might interfere with NF{kappa}B activity through direct protein-protein interactions [8] or through competition for limiting amounts of coactivator proteins [42], our data suggest that, in WISH and in amnion cells cultured in vitro, PPARG may not be involved directly in the antagonism of NF{kappa}B activation. In support of this, Lindstrom and Bennett have recently reported that antagonism of cytokine-elicited NF{kappa}B activation by 15d-PGJ2 occurs independently of PPARG in amnion and myometrial cells [43].

In addition to inhibiting NF{kappa}B, cyclopentenone PGs have also been shown to suppress the activity of other transcription factors involved in cytokine-stimulated COX2 expression, such as activator protein-1 (AP-1) [8, 44]. By impeding such signaling cascades, it is likely 15d-PGJ2 and other cyclopentenones suppress TNF-stimulated COX2 expression. In general, the ability of the cyclopentenones to attenuate COX2 induction was correlated with the number of electrophilic ß-carbons present within the molecules. Both 15d-PGJ2 and {Delta}12-PGJ2, with cross-conjugated {alpha},ß-unsaturated ketones, exhibited greater potency relative to the cyclopentenones having only a simple {alpha},ß-unsaturated carbonyl moiety, which is consistent with prior observations [35]. However, given that 15d-PGJ2 suppressed cytokine-induced COX2 expression to a greater degree than did {Delta}12-PGJ2, our results suggest that the number of reactive ß-carbons alone is insufficient to explain the effects of these agents. It is therefore noteworthy that 15d-PGD2, which bears an exocyclic {alpha},ß-unsaturated ketone, was unable to suppress either cytokine-mediated NF{kappa}B activation or COX2 expression. Although we do not yet have a suitable explanation for this observation, we speculate that endocyclic ß-carbons might exhibit greater reactivity than exocyclic ß-carbons for the formation of cyclopentenone protein conjugates.

Many previous studies have demonstrated proapoptotic actions of cyclopentenones such as 15d-PGJ2 in several cell types [45, 46]. Keelan et al. [27] reported that 15d-PGJ2 elicited programmed cell death in WISH cells. However, in the present study, we treated WISH cells and primary amnion cells for relatively brief periods (<12 h) and observed little evidence of apoptotic cell death (data not shown). Thus, it is unlikely that cytotoxicity is an explanation for the observed decreases in COX2 gene expression and subsequent PGE2 biosynthesis reported here. We cannot exclude the possibility that longer incubations may lead to apoptosis, however.

In contrast with 15d-PGJ2 and other cyclopentenones, we observed a tendency for thiazolidinediones, when given at high concentrations, to augment TNF-stimulated COX2 expression. This effect was particularly evident when low concentrations of TNF (5 ng/ml) were used (data not shown). While this finding was unexpected, there is precedence that PPARG might positively regulate COX2 gene expression in certain cell types [47]. The human COX2 gene contains a PPAR response element located approximately 3900 bp upstream of the translational start site, through which ligand-bound PPARG may promote transcriptional activation [47]. However, in numerous experiments, we failed to detect any stimulation in basal COX2 expression by thiazolidinediones (including troglitazone, rosiglitazone, and pioglitazone, data not shown) in the absence of cytokine stimulation. Furthermore, despite the fact that TRO and ROSI enhanced cytokine-elicited COX2 expression to similar degrees, only ROSI was found to augment TNF-stimulated PGE2 production. In ongoing experiments, we are examining whether this effect might have occurred through differential regulation of an enzyme downstream of COX2 in the PGE2 metabolic pathway (such as PTGES), or through a more global change in cellular arachidonic acid utilization. It is noteworthy that these effects were seen only when high concentrations of these agents were employed. While it is not unusual for thiazolidinediones to be used at concentrations of 10 µM (or higher) in cell culture experiments [9, 10, 48], it is nevertheless true that such amounts are often in excess of what is required for saturation of the PPARG receptor (which, in the case of ROSI, is below 1 µM [49]). Therefore, we speculate that some of the pharmacological effects observed may have occurred independently of PPARG. We are currently conducting experiments to discern the PPARG dependency of these effects in a comprehensive manner, but the present data provide a vital backdrop for these investigations.

The true physiological significance of these data awaits further experimental clarification. While it has been demonstrated that cells of the fetal membranes, including the amnion, express the PPARG receptor [20], the existence of endogenous PPARG ligands in these tissues remains unclear. It has been established that the placenta is the major source of PGD2, the precursor for PGs of the J series (PGJ2, {Delta}12-PGJ2, and 15d-PGJ2) within the uterus [50]. Furthermore, amniotic fluid contains PGD2 [51], and there is some evidence to suggest that 15d-PGJ2 also might be present [52], although this latter finding is controversial. Therefore, it is not unreasonable to suggest that substances such as the PGs of the J2 might serve a physiological role during gestation. However, there is a growing body of literature suggesting that 15d-PGJ2 is not the only potential endogenous PPARG ligand; polyunsaturated fatty acids (PUFAs) and products of lipoxygenase metabolism have also been shown to activate PPARG [6, 53], and a novel, as yet uncharacterized, endogenous PPARG ligand has recently been discovered [54]. These and potentially other unidentified ligands may comprise a mileau of anti-inflammatory biomolecules that are present endogenously throughout normal human gestation, as suggested by the recent findings of Waite and colleagues [55]. Members of our laboratory are currently developing screening systems to allow for the detection and identification of such PPARG ligands within amniotic fluid, maternal plasma, and gestational tissues.

In summary, we have demonstrated in WISH and primary amnion cells that reported ligands for PPARG (cyclopentenone PGs and thiazolidinediones) exert differential effects on COX2 gene expression. Inasmuch as these two classes of PPARG agonists do not act in precisely the same manner, we have proposed a working model to delineate the potential mechanisms through which these agents govern COX2 expression and PG biosynthesis (Fig. 6).



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 6. Working model for the effects of 15d-PGJ2 and thiazolidinediones on TNF-stimulated NF{kappa}B activation and COX2 expression. Depicted in this schematic diagram is the NF{kappa}B signaling pathway, through which TNF and other cytokines stimulate COX2 gene expression in WISH and amnion cells [17]. Upon binding to its cellular receptor, TNF invokes a signaling cascade whereby the I{kappa}B kinase (IKK) complex becomes activated. IKK acts to phosphorylate I{kappa}B, leading to the destruction of this protein by the 26 S proteasome complex. Once this occurs, NF{kappa}B can migrate to the nucleus, bind to response elements within the 5'-promoter region of the COX2 gene, and activate transcription. Independently of PPARG, 15d-PGJ2 may act at multiple steps in this pathway to impede NF{kappa}B activation, either by inhibiting of IKK activity (1), or by attenuating destruction of I{kappa}B by the 26 S proteasome complex (2). The effects of 15d-PGJ2 through activation of the PPARG receptor are unclear (3); however, our current data indicate that pharmacological activation of PPARG by thiazolidinediones (TZDs) might lead to enhanced COX2 transcription


   ACKNOWLEDGMENTS
 
We thank Dr. Timothy Hla for his generous donation of the human COX2 cDNA probe.


   FOOTNOTES
 
1 Supported in part by NIH grant RO1 HD35881 (D.A.K.) and The Ohio State University Perinatal Research and Development Fund. Portions of this work were presented at the 51st annual meeting of the Society for Gynecologic Investigation, Houston, Texas, March 24–27, 2004. Back

2 Correspondence: Douglas A. Kniss, Laboratory of Perinatal Research, Department of Obstetrics and Gynecology, The Ohio State University, 5th Floor Means Hall, 1654 Upham Drive, Columbus, OH 43210. FAX: 614 293 5728; kniss.1{at}osu.edu Back

Received: 13 December 2004.

First decision: 17 January 2005.

Accepted: 18 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Olson DM, Mijovic JE, Sadowsky DW. Control of human parturition. Semin Perinatol 1995 19:52-63[CrossRef][Medline]
  2. Kniss DA, Iams JD. Molecular effectors of human preterm parturition. In: Seifer DB, Samuels P, Kniss DA (eds.), Physiologic Basis of Gynecology and Obstetrics. Philadelphia, PA: Lippincott Williams and Wilkins; 2001:442–455
  3. Gibb W. The role of prostaglandins in human parturition. Ann Med 1998 30:235-241[Medline]
  4. Colville-Nash PR, Gilroy DW. COX-2 and the cyclopentenone prostaglandins—a new chapter in the book of inflammation?. Prostaglandins Other Lipid Mediat 2000 62:33-43[CrossRef][Medline]
  5. Murphy GJ, Holder JC. PPAR-gamma agonists: therapeutic role in diabetes, inflammation and cancer. Trends Pharmacol Sci 2000 21:469-474[CrossRef][Medline]
  6. Huang JT, Welch JS, Ricote M, Binder CJ, Willson TM, Kelly C, Witztum JL, Funk CD, Conrad D, Glass CK. Interleukin-4-dependent production of PPAR-gamma ligands in macrophages by 12/15-lipoxygenase. Nature 1999 400:378-382[CrossRef][Medline]
  7. Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 1998 391:82-86[CrossRef][Medline]
  8. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 1998 391:79-82[CrossRef][Medline]
  9. Lappas M, Permezel M, Georgiou HM, Rice GE. Regulation of proinflammatory cytokines in human gestational tissues by peroxisome proliferator-activated receptor-gamma: effect of 15-deoxy-delta(12,14)-PGJ(2) and troglitazone. J Clin Endocrinol Metab 2002 87:4667-4672[Abstract/Free Full Text]
  10. Cheng S, Afif H, Martel-Pelletier H, Pelletier J-P, Li X, Farrajota K, Lavigne M, Fahmi H. Activation of peroxisome proliferator-activated protein {gamma} inhibits interleukin-1ß-induced membrane-associated prostaglandin E2 synthase-1 expression in human synovial fibroblasts by interfering with Egr-1. J Biol Chem 2004 279:22057-22065[Abstract/Free Full Text]
  11. Petrova TV, Akama KT, Van Eldik LJ. Cyclopentane prostaglandins suppress activation of microglia: downregulation of inducible nitric oxide syntase by 15-deoxy-delta12,14-prostaglandin J2. Proc Natl Acad Sci U S A 1999 96:4668-4673[Abstract/Free Full Text]
  12. Baeuerle PA. IkappaB-NF-kappaB structures: at the interface of inflammation control. Cell 1998 95:729-731[CrossRef][Medline]
  13. Poligone B, Baldwin AS. Positive and negative regulation of NF-kappaB by COX-2: roles of different prostaglandins. J Biol Chem 2001 276:38658-38664[Abstract/Free Full Text]
  14. Allport VC, Slater DM, Newton R, Bennett PR. NF-kappaB and AP-1 are required for cyclo-oxygenase 2 gene expression in amnion epithelial cell line (WISH). Mol Hum Reprod 2000 6:561-565[Abstract/Free Full Text]
  15. Belt AR, Baldassare JJ, Molnar M, Romero R, Hertelendy F. The nuclear transcription factor NF-kappaB mediates interleukin-1beta-induced expression of cyclooxygenase-2 in human myometrial cells. Am J Obstet Gynecol 1999 181:359-366[CrossRef][Medline]
  16. Yan X, Wu XC, Sun M, Tsang BK, Gibb W. Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta. Biol Reprod 2002 66:1667-1671[Abstract/Free Full Text]
  17. Ackerman WE IV, Rovin BH, Kniss DA. Epidermal growth factor and interleukin-1beta use divergent signaling pathways to synergistically upregulate cyclooxygenase-2 gene expression in human amnion-derived WISH cells. Biol Reprod 2004 71:2079-2086[Abstract/Free Full Text]
  18. Kniss DA. Cyclooxygenases in reproductive medicine and biology. J Soc Gynecol Invest 1999 6:285-292[Medline]
  19. Cuzzocrea S, Wayman NS, Mazzon E, Dugo L, Di Paola R, Serraino I, Britti D, Chatterjee PK, Caputi AP, Thiemermann C. The cyclopentenone prostaglandin 15-deoxy-{Delta}12,14-prostaglandin J2 attenuates the development of acute and chronic inflammation. Mol Pharmacol 2002 61:997-1007[Abstract/Free Full Text]
  20. Dunn-Albanese LR, Ackerman WE 4th, Xie Y, Iams JD, Kniss DA. Reciprocal expression of peroxisome proliferator-activated receptor-gamma and cyclooxygenase-2 in human term parturition. Am J Obstet Gynecol 2004 190:809-816[CrossRef][Medline]
  21. Okita JR, Sagawa N, Casey ML, Snyder JM. A comparison of human amnion tissue and amnion cells in primary culture by morphological and biochemical criteria. In Vitro 1983 19:117-126[Medline]
  22. Kniss DA, Rovin B, Fertel RH, Zimmerman PD. Blockade NF-kappaB activation prohibits TNF-alpha-induced cyclooxygenase-2 gene expression in ED27 trophoblast-like cells. Placenta 2001 22:80-89[CrossRef][Medline]
  23. Zhang X, Lu L, Dixon C, Wilmer W, Song H, Chen X, Rovin BH. Stress protein activation by the cyclopentenone prostaglandin 15-deoxy-delta12,14-prostaglandin J2 in human mesangial cells. Kidney Int 2004 65:798-810[CrossRef][Medline]
  24. Perkins DJ, Kniss DA. Tumor necrosis factor-alpha promotes sustained cyclooxygenase-2 expression: attenuation by dexamethasone and NSAIDs. Prostaglandins 1997 54:727-743[CrossRef][Medline]
  25. Hinz B, Brune K, Pahl A. 15-Deoxy-{Delta}12,14-prostaglandin J2 inhibits the expression of proinflammatory genes in human blood monocytes via a PPAR-{gamma}-independent mechanism. Biochem Biophys Res Commun 2003 302:415-420[CrossRef][Medline]
  26. Janabi N. Selective inhibition of cyclooxygenase-2 expression by 15-deoxy-{Delta}12,14-prostaglandin J2 in activated human astrocytes, but not in human brain macrophages. J Immunol 2002 168:4747-4755[Abstract/Free Full Text]
  27. Keelan JA, Helliwell RJA, Nijmeijer BE, Berry EBE, Sato TA, Marvin KW, Mitchell MD, Gilmour RS. 15-Deoxy-{Delta}12,14-prostaglandin J2-induced apoptosis in amnion-like WISH cells. Prostaglandins Other Lipid Mediat 2001 66:265-282[CrossRef][Medline]
  28. Straus DS, Pascual G, Li M, Welch JS, Ricote M, Hsiang CH, Sengchanthalangsy LL, Ghosh G, Glass CK. 15-Deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway. Proc Natl Acad Sci U S A 2000 97:4844-4849[Abstract/Free Full Text]
  29. Chawla A, Barak Y, Nagy L, Liao D, Tontonoz P, Evans RM. PPAR-gamma dependent and independent effects on macrophage gene expression in lipid metabolism and inflammation. Nat Med 2001 7:48-52[CrossRef][Medline]
  30. Fukushima M. Biological activities and mechanisms of action of PGJ2 and related compounds: an update. Prostaglandins Leukot Essent Fatty Acids 1992 47:1-12[CrossRef][Medline]
  31. Atsmon J, Sweetman BJ, Baertschi SW, Harris TM, Roberts LJ. Formation of thiol conjugates of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2. Biochemistry 1990 29:3760-3765[CrossRef][Medline]
  32. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 1995 83:803-812[CrossRef][Medline]
  33. Castrillo A, Diaz-Guerra MJ, Hortelano S, Martin-Sanz P, Bosca L. Inhibition of IkappaB kinase and IkappaB phosphorylation by 15-deoxy-Delta(12,14)-prostaglandin J(2) in activated murine macrophages. Mol Cell Biol 2000 20:1692-1698[Abstract/Free Full Text]
  34. Mullally JE, Moos PJ, Edes K, Fitzpatrick FA. Cyclopentenone prostaglandins of the J series inhibit the ubiquitin isopeptidase activity of the proteasome pathway. J Biol Chem 2001 276:30366-30373[Abstract/Free Full Text]
  35. Verbitski SM, Mullally JE, Fitzpatrick FA, Ireland CM. Punaglandins, chlorinated prostaglandins, function as potent Michael receptors to inhibit ubiquitin isopeptidase activity. J Med Chem 2004 47:2062-2070[CrossRef][Medline]
  36. Tazawa R, Xu XM, Wu KK, Wang LH. Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene. Biochem Biophys Res Commun 1994 203:190-199[CrossRef][Medline]
  37. Hayflick L. The establishment of a line (WISH) of human amnion cells in continuous cultivation. Exp Cell Res 1961 23:14-20[CrossRef][Medline]
  38. Kniss DA, Zimmerman PD, Fertel RH, Iams JD. Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells. Prostaglandins 1992 44:237-244[CrossRef][Medline]
  39. Bry K. Epidermal growth factor and transforming growth factor-alpha enhance the interleukin-1- and tumor necrosis factor-stimulated prostaglandin E2 production and the interleukin-1 specific binding on amnion cells. Prostaglandins Leukot Essent Fatty Acids 1993 49:923-928[CrossRef][Medline]
  40. Hansen WR, Drew A, Helsby N, Keelan JA, Sato TA, Mitchell MD. Regulation of cytosolic phospholipase A2 expression by cytokines in human amnion cells. Placenta 1999 20:303-308[CrossRef][Medline]
  41. Kniss DA, Xie Y, Li Y, Kumar S, Linton EA, Cohen P, Fan-Havard P, Redman CW, Sargent IL. ED(27) trophoblast-like cells isolated from first-trimester chorionic villi are genetically identical to HeLa cells yet exhibit a distinct phenotype. Placenta 2002 23:32-43[Medline]
  42. Delerive P, Fruchart JC, Staels B. Peroxisome proliferator-activated receptors in inflammation control. J Endocrinol 2001 169:453-459[Abstract]
  43. Lindstrom TM, Bennett PR. 15-deoxy-delta(12,14)-prostaglandin J2 inhibits IL-1beta-induced NF-kappaB in human amnion and myometrial cells: mechanisms and implications. J Clin Endocrinol Metab 2005 90:3534-3543[Abstract/Free Full Text]
  44. Perez-Sala D, Cernuda-Morollon E, Canada FJ. Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-delta(12,14)-prostaglandin J2. J Biol Chem 2003 278:51251-51260[Abstract/Free Full Text]
  45. Bishop-Bailey D, Hla T. Endothelial cell apoptosis induced by the peroxisome proliferator-activated receptor (PPAR) ligand 15-deoxy-delta 12,14-prostaglandin J2. J Biol Chem 1999 274:17042-17048[Abstract/Free Full Text]
  46. Kawahito Y, Kondo M, Tsubouchi Y, Hashiramoto A, Bishop-Bailey D, Inoue K, Kohno M, Yamada R, Hla T, Sano H. 15-deoxy-{Delta}12,14-PGJ2 induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats. J Clin Invest 2000 106:189-197[Medline]
  47. Meade EA, McIntyre TM, Zimmerman GA, Prescott SM. Peroxisome proliferators enhance cyclooxygenase-2 expression in epithelial cells. J Biol Chem 1999 274:8328-8334[Abstract/Free Full Text]
  48. Subbaramaiah K, Lin DT, Hart JC, Dannenberg AJ. Peroxisome proliferator-activated receptor gamma ligands suppress the transcriptional activation of cyclooxygenase-2. Evidence for involvement of activator protein-1 and CREB-binding protein/p300. J Biol Chem 2001 276:12440-12448[Abstract/Free Full Text]
  49. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem 1995 270:12953-12956[Abstract/Free Full Text]
  50. Mitchell MD, Kraemer DL, Strickland DM. The human placenta: a major source of prostaglandin D2. Prostaglandins Leukot Med 1982 8:383-387[CrossRef][Medline]
  51. Berryman GK, Strickland DM, Hankins GD, Mitchell MD. Amniotic fluid prostaglandin D2 in spontaneous and augmented labor. Life Sci 1987 41:1611-1614[CrossRef][Medline]
  52. Marvin KW, Eykholt RL, Keelan JA, Sato TA, Mitchell MD. The 15-deoxy-delta(12,14)-prostaglandin J(2)receptor, peroxisome proliferator activated receptor-gamma (PPARgamma) is expressed in human gestational tissues and is functionally active in JEG3 choriocarcinoma cells. Placenta 2000 21:436-440[CrossRef][Medline]
  53. Glass CK. Potential roles of the peroxisome proliferator-activated receptor-{gamma} in macrophage biology and atherosclerosis. J Endocrinol 2001 169:461-464[CrossRef][Medline]
  54. Tzameli I, Fang H, Oilero M, Shi H, Hamm JK, Kievitt P, Hollenberg AN, Flier JS. Regulated production of a peroxisome proliferator-activated receptor-g ligand during an early period of adipocyte differentiation in 3T3-L1 adipocytes. J Biol Chem 2004 279:36093-36102[Abstract/Free Full Text]
  55. Waite LL, Louie RE, Taylor RN. Circulating activators of PPARs are reduced in preeclamptic pregnancy. J Clin Endocrinol Metab 2004 90:620-626[CrossRef][Medline]



This article has been cited by other articles:


Home page
 ReproductionHome page
S. J Holdsworth-Carson, M. Permezel, G. E Rice, and M. Lappas
Preterm and infection-driven preterm labor: the role of peroxisome proliferator-activated receptors and retinoid X receptor
Reproduction, June 1, 2009; 137(6): 1007 - 1015.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
G. Pirianov, S. N. Waddington, T. M. Lindstrom, V. Terzidou, H. Mehmet, and P. R. Bennett
The Cyclopentenone 15-Deoxy-{Delta}12,14-Prostaglandin J2 Delays Lipopolysaccharide-Induced Preterm Delivery and Reduces Mortality in the Newborn Mouse
Endocrinology, February 1, 2009; 150(2): 699 - 706.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
E. Capobianco, V. White, R. Higa, N. Martinez, and A. Jawerbaum
Effects of natural ligands of PPAR{gamma} on lipid metabolism in placental tissues from healthy and diabetic rats
Mol. Hum. Reprod., August 1, 2008; 14(8): 491 - 499.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
C.M. Mitchell, R.F. Johnson, W.B. Giles, and T. Zakar
Prostaglandin H synthase-2 gene regulation in the amnion at labour: histone acetylation and nuclear factor kappa B binding to the promoter in vivo
Mol. Hum. Reprod., January 1, 2008; 14(1): 53 - 59.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
W. E Ackerman IV, T. L.S Summerfield, D. D Vandre, J. M Robinson, and D. A Kniss
Nuclear Factor-Kappa B Regulates Inducible Prostaglandin E Synthase Expression in Human Amnion Mesenchymal Cells
Biol Reprod, January 1, 2008; 78(1): 68 - 76.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
P Froment, F Gizard, D Defever, B Staels, J Dupont, and P Monget
Peroxisome proliferator-activated receptors in reproductive tissues: from gametogenesis to parturition.
J. Endocrinol., May 1, 2006; 189(2): 199 - 209.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/3/527    most recent
biolreprod.104.039032v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ackerman, W. E.
Right arrow Articles by Kniss, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ackerman, W. E., IV
Right arrow Articles by Kniss, D. A.
Agricola
Right arrow Articles by Ackerman, W. E.
Right arrow Articles by Kniss, D. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS