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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 5 2365-2372
Copyright © 2004 by The Endocrine Society

Regulation of Phospholipase Isozymes by Nuclear Factor-{kappa}B in Human Gestational Tissues in Vitro

Martha Lappas, Michael Permezel, Harry M. Georgiou and Gregory E. Rice

Department of Obstetrics and Gynaecology, The University of Melbourne and Mercy Perinatal Research Centre, Mercy Hospital for Women, East Melbourne, Victoria, Australia 3002

Address all correspondence and requests for reprints to: Martha Lappas, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, 126 Clarendon Street, East Melbourne, 3002 Victoria, Australia. E-mail: mlappas{at}unimelb.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phospholipid-derived mediators are implicated in the initiation and progression of human labor and delivery, particularly in relation to infection-induced preterm labor. We previously demonstrated that, in human intrauterine tissues, lipopolysaccharide (LPS)-stimulated nuclear factor-{kappa}B (NF-{kappa}B) DNA binding activity, and subsequent cytokine release can be suppressed by sulfasalazine (SASP) concentrations greater than 5 mM. The aim of this study was to elucidate the effect the SASP on secretory type II phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2), cyclooxygenase (COX) isozymes, and subsequent prostaglandin F2{alpha} (PGF2{alpha}) production in human gestational tissues. Human placenta, amnion, and choriodecidua (n = 4–9 separate placentas) were incubated in the presence of SASP (0.1, 1, 5, and/or 10 mM) under either basal or LPS (10 µg/ml) conditions. After 6 h incubation, the tissues were collected and assayed for type II PLA2 by ELISA and cPLA2, COX-1, and COX-2 content by Western blotting. The incubation medium was collected and assayed for type II PLA2 and 13,14-dihydro-15-keto PGF2{alpha} release by ELISA and PGF2{alpha} by RIA. Treatment of placenta, amnion, and choriodecidua with SASP concentrations greater than 5 mM significantly inhibited basal and/or LPS-stimulated type II PLA2 content and release, 13,14-dihydro-15-keto PGF2{alpha} release, and cPLA2 protein content (ANOVA, P < 0.05); however, no effect of SASP was observed on basal or LPS-stimulated COX-1 or COX-2 protein. Although no effect of SASP was observed on basal and LPS-stimulated PGF2{alpha} release from placenta and amnion, it significantly increased both basal and LPS-stimulated PGF2{alpha} release from choriodecidua. In addition, SASP concentrations of 5 mM or greater significantly suppressed NF-{kappa}B DNA binding activity. These data are consistent with the hypothesis that NF-{kappa}B regulates the expression and release of phospholipase isozymes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A SUCCESSFUL OUTCOME to labor and delivery is dependent on common proximal events that result in an increase in the bioactivity and/or availability of phospholipid-derived mediators (e.g. prostaglandins) and cytokines (1, 2). The mechanisms by which these processes are coordinated and coupled with fetal maturation (i.e. normal spontaneous delivery at term) or uncoupled from fetal development (i.e. preterm birth) remain to be fully elucidated. Previously the involvement of upstream, multifunction regulators that may coordinate proximal labor-associated events has been proposed, including nuclear factor-{kappa}B (NF-{kappa}B) signaling pathway. We recently demonstrated that NF-{kappa}B regulates the release of proinflammatory cytokines TNF{alpha}, IL-6, and IL-8 from human intrauterine tissues (3, 4). Therefore, the aim of this study was to assess the involvement of NF-{kappa}B in regulating the expression of components of the phospholipid metabolizing pathway in human gestational tissues, under both basal and lipopolysaccharide (LPS) stimulated conditions.

Prostaglandins are formed via the action of multiple enzyme pathways, involving phospholipase A2 (PLA2) and cyclooxygenase (COX) isozymes. PLA2 isozymes catalyze the release of free arachidonic acid from the sn-2 position of membrane phospholipids (reviewed in Ref.1). At least two distinctly different calcium-dependent PLA2 enzymes have been shown to contribute to the synthesis of prostaglandins. One is a 14-kDa secretory type II PLA2 that has been localized to a number of cell types, and the other is a 85-kDa cytosolic PLA2 (cPLA2). The presence of type II PLA2 mRNA, immunoreactive protein, and enzymatic activity in placenta, amnion, and choriodecidua has been established (reviewed in Ref.1). Consistent with its extracellular site of action, type II PLA2 is secreted by human gestational tissues in vitro. In human gestational tissues, cPLA2 has been detected in placenta, amnion, and choriodecidua, in which it is expressed in greater abundance in the fetal membranes than in the placenta. COX-1 and COX-2 mRNA transcripts and protein have been identified in fetal membranes, placenta, and decidua at term (5, 6, 7, 8). Studies have confirmed that there is a marked increase in COX-2 activity in both amnion and chorion during term spontaneous labor onset when compared with term cesarean not in labor (5, 7). However, the importance of COX-1 in parturition has been demonstrated, in which transgenic mice harboring a null mutation for COX-1 showed delays in normal parturition, compared with wild-type littermates (9).

Despite its importance in parturition, the regulation of phospholipid metabolism in pregnancy remains to be elucidated. The promoter region for type II PLA2, cPLA2, and COX-2 gene contains several putative binding sites for transcription factors, including NF-{kappa}B (10, 11, 12). NF-{kappa}B is a transcription factor that upon activation (e.g. with LPS) leads to the coordinated expression of many inflammatory gene products (13). NF-{kappa}B subunits p50 and RelA have been demonstrated in amnion-derived WISH cells (14, 15), human myometrial cells (16), and human cytotrophoblasts (17, 18). King et al. (19) demonstrated the differential expression of mRNA transcripts encoding for NF-{kappa}B pathway intermediates throughout the peripartum period. Furthermore, the NF-{kappa}B signaling pathway also regulates proinflammatory cytokine gene expression and release by human gestational tissues (3, 4, 15).

We previously demonstrated that NF-{kappa}B DNA binding activity in human gestational tissues is suppressed by the antiinflammatory agent sulfasalazine (SASP) (3) and demonstrated that the NF-{kappa}B inhibitor, N-acetyl-cysteine, through its ability to act as an free radical scavenger, inhibits LPS-stimulated type II PLA2 and cPLA2 in human fetal membranes (4). Therefore, the aim of this study was to investigate whether SASP regulates the expression and/or release of both basal and LPS-stimulated type II PLA2, cPLA2, COX isozymes, and subsequent prostaglandin F2{alpha} (PGF2{alpha}) and 13,14-dihydro-15-keto PGF2{alpha} release, from human gestational tissues, in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

All chemicals were purchased from BDH Chemicals Australia (Melbourne, Australia) unless otherwise stated. RPMI 1640 (phenol red free) was obtained from Life Technologies, Inc. Laboratories (Grand Island, NY). BSA (RIA grade), EDTA, LPS (from Escherichia coli 026:B6), and SASP were supplied by Sigma (St. Louis, MO). Starscint scintillation fluid was purchased from Packard (Meriden, CT). Human {gamma}-globulin was provided by the Commonwealth Serum Laboratories (Parkville, Australia). Goat polyclonal antiserum raised against PGF2{alpha} was generously provided by Dr. Meg Ralph (Department of Physiology, Monash University, Clayton, Australia). Alkaline phosphatase (calf intestine grade 1) was obtained from Roche Molecular Biochemicals Australia (Melbourne, Australia). Type II human PLA2 monoclonal antibodies (3G3 and 2A9) were prepared by Bioquest (Sydney, Australia) and recombinantly expressed human type II PLA2 standard was isolated from Chinese hamster ovary cell line stably transfected with human type II PLA2 under a metallothionine promoter. Rabbit polyclonal antibodies raised against human cPLA2, COX-1, COX-2, alkaline phosphataseconjugated goat antirabbit IgG, and 5-bromo-4-chloro-3-indoyl-phosphate/4-nitro blue tetrazolium chloride (BCIP/NBT) color substrate were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Acrylamide, ammonium persulfate, bisacrylamide, bromophenol blue, Coomassie Brilliant Blue, rainbow-colored protein molecular-weight markers, TEMED, and 3H-PGF2{alpha} were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Pefabloc SC (AEBSF) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). The transcription factor consensus oligonucleotides for NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3') and activator protein-1 (AP-1) (5'-TTCCGGCTGACTCATCAAGCG-3'), HeLa scribe nuclear extract, gel shift binding buffer, and polynucleotide kinase for labeling of 5'OH blunt-ended probes were purchased from Promega (Madison, WI). NF-{kappa}B p65 ELISA was purchased from BD Biosciences Clontech (Palo Alto, CA).

Tissue collection and preparation

Human placentae and attached fetal membranes were obtained (with Institutional Research and Ethics Committee approval) from women who delivered healthy, singleton infants at term (>=37 weeks gestation) undergoing elective cesarean section (indications for cesarean section were breech presentation and/or previous cesarean section). A human explant system was used to establish the effect of SASP on phospholipid metabolism from human gestational tissues (as detailed in Refs.3 ,4). The explants were incubated, in duplicate, in 2 ml RPMI 1640 containing penicillin G (100 U/ml) and streptomycin (100 µg/ml), in the presence of 0 (control), 0.1, 1, 5, or 10 mM SASP (n = 9), either under basal or 10 µg/ml LPS stimulation. All concentrations used in this study were based on results obtained from our previous studies (3).

Experimental assays

Separate tissue samples (n = 9) were incubated for 6 h, and both the tissue and incubation medium was collected and assayed for type II PLA2 as previously described (20, 21). The concentration of type II PLA2 was quantified by a noncompetitive sandwich ELISA using two monoclonal antibodies raised against recombinant human type II PLA2 as described previously (21), and data were corrected for protein and expressed as nanograms per milligram protein. The concentration of PGF2{alpha} in the incubation medium was determined by a RIA as previously described (21), and data were corrected for total protein and expressed as picomole per milligram protein. The release of 13,14-dihydro-15-keto PGF2{alpha} into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer’s specifications (Cayman Chemical Co., Ann Arbor, MI), data were corrected for total protein and expressed as picogram per milligram protein. SASP did not interfere with the type II PLA2 ELISA and PGF2{alpha} RIA. The protein content of tissue homogenates was determined using a BCA protein assay (Pierce, Rockford, IL), using BSA as a reference standard (described in Ref.3).

Nuclear protein extraction and assessment of NF-{kappa}B DNA binding activity

After the 6 h incubation, placental tissues were collected and nuclear protein was extracted as previously described (3, 4). Nuclear protein (12 µg for placenta and 20 µg for fetal membranes) was then subjected to EMSA using a double-stranded NF-{kappa}B oligonucleotide that was end labeled using T4 polynucleotide kinase and [{gamma}-32P] ATP (detailed in Ref.3). After electrophoresis, the gel was dried and exposed to X-OMAT AR film (Kodak, Rochester, NY) overnight at –80 C. NF-{kappa}B DNA binding in nuclear protein extracts were also assessed using a commercially available NF-{kappa}B p65 ELISA according to manufacturer’s instructions (BD Biosciences Clontech) in which TNF{alpha}-stimulated HeLa nuclear protein extract was used as a positive control for NF-{kappa}B activation, and specificity of NF-{kappa}B binding was assessed using wild-type and mutated consensus oligonucleotides. A Benchmark microplate reader (Bio-Rad Laboratories, Hercules, CA) was used to read the sample absorbance, with data are expressed as absorbance at 655 nm.

Western blotting

Assessment of cPLA2, COX-1, and COX-2 protein expression in tissues was analyzed by Western blotting. Forty micrograms of tissue protein extracts were separated on a 10% polyacrylamide gel and transferred to nitrocellulose as previously described (4). The blots were blocked in PBS containing 0.1% Tween 20 and 5% nonfat dry milk and then incubated overnight at 4 C with a 1:200 dilution of the rabbit polyclonal primary antibody. The blots were then washed three times for 10 min with PBS-Tween 20 and incubated for 1 h with a 1:1000 dilution of either a goat antirabbit IgG alkaline phosphatase (for experiments performed in the presence of LPS) or a goat antirabbit IgG horseradish peroxidase (for basal experiments) in PBS-Tween 20. After washing three times with PBS-Tween 20 and two times in alkaline phosphatase buffer, the blots were developed using the nitro blue tetrazolium chloride/5-bromo-chloro-3 indolyl phosphate or enhanced chemiluminescence detection system, respectively. Protein expression of cPLA2, COX-1, and COX-2 was identified by comigration with a positive control (3T3/NIH cell whole lysate) and by comparison with the mobility of protein standard.

Statistical analysis

Statistical analyses were performed using a commercially available statistical software package (Statgraphics, STSC, Rockville, MD). Homogeneity of data was assessed by Bartlett’s test (22), and when significant, data were logarithmically transformed before further analysis. Data were subjected to a one-way ANOVA. Statistical difference was indicated by P < 0.05. Data are expressed as mean ± SEM of three to nine different tissues.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Validation of explant cultures and viability

To validate the integrity of explants in the presence of SASP, cell viability was investigated using lactate dehydrogenase (LDH) release from explants. LDH release was investigated over the 6-h time course of placental, amnion, and choriodecidual explants (n = 3). Explants were incubated either in control (basal or LPS-stimulated) media or media containing SASP (see Table 1Go). Data are presented as a percentage of total tissue LDH, which was calculated for tissues collected at time zero and homogenized to release cytosolic LDH. Compared with the control, treatment with SASP, at all concentrations tested, did not significantly affect basal or LPS-stimulated LDH release from placenta, amnion, and choriodecidua, indicating that the concentrations used were not toxic to the tissue explants.


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  		TABLE 1. Effect of SASP on basal and LPS-stimulated LDH release from human gestational tissues (n = 3)

 
NF-{kappa}B EMSA validation studies

The specificity of NF-{kappa}B DNA binding was confirmed in competition experiments. Incubation with 100-fold excess of an unrelated oligonucleotide spanning AP-1 binding site did not antagonize NF-{kappa}B binding (Fig. 1Go, lane 4), whereas competition with 100-fold excess unlabeled NF-{kappa}B oligonucleotide inhibited binding activity (Fig. 1Go, lane 3). Negative and positive (Fig. 1Go, lanes 1 and 2, respectively) controls were run in parallel. To characterize the NF-{kappa}B subunits, antibodies to the NF-{kappa}B heterodimers p50 and p65 were added to nuclear protein extracts. When compared with control (Fig. 1Go, lane 5), antibody binding of p50 and p65 resulted in a higher shift, or supershift, on EMSAs with a reciprocal decrease in the intensity of the NF-{kappa}B band (Fig. 1Go, lane 6 and 7, respectively). Furthermore, the addition of both p50 and p65 antibodies eliminated the NF-{kappa}B band (Fig. 1Go, lane 8).



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  		FIG. 1. EMSA demonstrating the specificity and composition of NF-{kappa}B in human placenta. Supershift assays where performed to characterize the NF-{kappa}B subunits in human placenta. NF-kB binding reactions were performed in the absence and presence of antibodies to p50, p65, and both. When compared with control (lane 5), the addition of anti-NF-{kappa}B p50 (lane 6) or anti-NF-{kappa}B p65 (lane 7) resulted in reduced intensity of the NF-{kappa}B band and caused the appearance of a slow migrating band (shift). The addition of p50 and p65 antibodies resulted in a complete loss of the NF-{kappa}B band (lane 8). The specificity of binding activity was also confirmed by competition experiments, in which the addition of unlabeled NF-{kappa}B oligonucleotide resulted in the loss of NF-{kappa}B binding activity (lane 3) but not with the nonspecific competitor, AP-1 oligonucleotide (lane 4). Similar results were obtained with amnion and choriodecidua.

 
Effect of SASP on basal NF-{kappa}B DNA binding activity as determined by EMSA

We previously demonstrated that SASP concentrations greater than 5 mM suppress LPS-stimulated NF-{kappa}B DNA binding activity in nuclear extracts from human placenta, amnion, and choriodecidua (3). In this study, we also demonstrated that treatment of tissue explants with SASP concentrations equal to or greater than 5 mM caused a significant suppression of basal NF-{kappa}B DNA binding activity in nuclear extracts prepared from human placenta, amnion, and choriodecidua (Fig. 2AGo, n = 3).



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  		FIG. 2. A, A representative gel shift assay demonstrating the effect of SASP on basal NF-{kappa}B DNA binding activity in human placenta, amnion, and choriodecidua. Basal NF-{kappa}B DNA binding activity was significantly inhibited by concentrations of SASP 5 mM. Results are representative of four gel shift assays of four independent experiments placentas. B, Effect of SASP on basal NF-{kappa}B DNA binding activity in human amnion choriodecidua as determined by NF-{kappa}B p65 ELISA. Data represent the mean ± SEM of three separate tissue explants. Significant differences, compared with control, are represented by * (P < 0.05, ANOVA).

 
Effect of SASP on basal NF-{kappa}B p65 DNA binding activity as determined by ELISA

The binding ability of NF-{kappa}B p65 to DNA consensus sequences was also measured using a commercially available kit. An NF-{kappa}B wild-type consensus oligonucleotide was used to monitor the specificity of the assay. The wild-type oligonucleotide, by competing for NF-{kappa}B binding to the probe immobilized on the plate, acted as an effective competitor for NF-{kappa}B p65 binding (data not shown). Specificity of binding was also demonstrated using wells coated with mutated consensus oligonucleotide. In these experiments, no binding was detected in the presence of the positive control (data not shown). Incubation of amnion and choriodecidua with SASP concentration greater than 5 mM significantly inhibited NF-{kappa}B p65 DNA binding activity (Fig. 2BGo, n = 3), thus confirming the EMSA results.

Effect of SASP on type II PLA2 content and release

SASP at concentrations 5 mM or greater significantly reduced both basal and LPS-induced type II PLA2 tissue content in placenta (Fig. 3AGo, n = 9), amnion (Fig. 3BGo, n = 4), and choriodecidua (Fig. 3CGo, n = 4). Compared with control, 5 mM SASP caused a significant decrease in both basal and LPS-stimulated type II PLA2 release from placenta (Fig. 4Go, n = 9). The release of type II PLA2 from amnion and choriodecidua is not detectable using this assay.



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  		FIG. 3. Effect of SASP on basal and LPS-stimulated type II PLA2 tissue content in human placenta (A), amnion (B), and choriodecidua (C). Data represent the mean ± SEM of five to nine separate tissue explants. Significant differences, compared with control, are represented by * (P < 0.05, ANOVA).

 


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  		FIG. 4. Effect of SASP on basal and LPS-stimulated type II PLA2 release from human placental explants. Data represent the mean ± SEM of nine separate tissue explants. Significant differences, compared with control, are represented by * (P < 0.05, ANOVA).

 
Effect of SASP on tissue cPLA2 protein

The mature cPLA2 protein has a calculated molecular weight of 85 kDa, although it migrates as 85–110 kDa on SDS-PAGE (23). In this study, the identification of cPLA2 protein was based on the use of a positive control (3T3/NIH whole-cell lysate). A band corresponding to approximately 105 kDa was identified as cPLA2 and was present in all samples of placenta, amnion, and choriodecidua. SASP concentrations greater than 5 mM significantly reduced basal and/or LPS-stimulated cPLA2 protein in placenta (Fig. 5AGo), amnion (Fig. 5BGo), and choriodecidua (Fig. 5CGo).



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  		FIG. 5. A representative Western blot is shown demonstrating the effect of SASP on basal and LPS-stimulated cPLA2 protein in human placenta (A), amnion (B), and choriodecidua (C). When compared with control, SASP at concentrations greater than 5 mM decreased cPLA2 protein from all three tissues. Results are representative of four Western blots of four independent experiments.

 
Effect of SASP on tissue COX-1 and COX-2 protein

In this study, the effect of SASP on COX-1 and COX-2 abundance in gestational tissue samples was quantified by Western blotting using specific rabbit anti-COX-1 or -COX-2 polyclonal antibodies. For COX-1, a band was detected at approximately 72 kDa and, on the basis of its molecular weight and its comigration with a positive control (3T3/NIH whole-cell lysate), was identified as COX-1. A doublet band was detected at approximately 66–72 kDa and, on the basis of its molecular weight and its comigration with a reference standard, was identified as COX-2. Similarly, previous studies by Sawdy et al. (8) used these rabbit anti-COX-1 or -COX-2 polyclonal antibodies to identify COX protein from human gestational tissue samples. In this study, COX-1 and COX-2 protein was detected in only a few of the basal samples and all LPS-stimulated samples; thus, analysis was performed only on LPS-stimulated tissues. SASP, at all concentrations tested, did not have an effect on LPS-stimulated COX-1 or COX-2 protein in placenta (Fig. 6AGo), amnion (Fig. 6BGo), and choriodecidua (Fig. 6CGo).



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  		FIG. 6. A representative Western blot demonstrating the effect of SASP on LPS-stimulated COX-1 and COX-2 protein in human placenta (A), amnion (B), and choriodecidua (C). SASP at all concentrations tested did not alter COX-1 or COX-2 protein from placenta, amnion, and choriodecidua. Results are representative of four Western blots of four independent experiments.

 
Effect of SASP on PGF2{alpha} release

SASP, at all concentrations tested had no significant effect on basal or LPS-stimulated PGF2{alpha} release from placenta (Fig. 7AGo, n = 6). Although 5 and 10 mM SASP caused an increase in PGF2{alpha} release from amnion (Fig. 7BGo, n = 6), this did not reach significance. In choriodecidua, SASP at all concentrations tested increased the basal release of PGF2{alpha}; however, only 10 mM SASP significantly increased LPS-stimulated PGF2{alpha} release (Fig. 7CGo, n = 6).



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  		FIG. 7. Effect of SASP on basal and LPS-stimulated PGF2{alpha} release by human placenta (A), amnion (B), and choriodecidua (C). Data represent the mean ± SEM of five to nine separate tissue explants. Significant differences, compared with control, are represented by * (P < 0.05, ANOVA).

 
Effect of SASP on 13,14-dihydro-15-keto PGF2{alpha} release

In amnion, 10 mM SASP significantly decreased basal 13,14-dihydro-15-keto PGF2{alpha} release (Fig. 8Go, n = 4), but this did not reach significance. In choriodecidua, SASP at all concentrations tested decreased the basal release of PGF2{alpha}; however, only 5 and 10 mM SASP significantly decreased 13,14-dihydro-15-keto PGF2{alpha} release (Fig. 8Go, n = 4).



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  		FIG. 8. Effect of SASP on basal 13,14-dihydro-15-keto PGF2{alpha} release by human amnion and choriodecidua. Data represent the mean ± SEM of four separate tissue explants. Significant differences, compared with control, are represented by * (P < 0.05, ANOVA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Prostaglandins play a central role in the initiation of human labor and delivery, mediating uterine contractions and cervical ripening (reviewed in Refs. 1 and 24). Changes in the concentrations of prostaglandins correlate with the physiological changes that occur during labor. The formation of these prostaglandins by human gestational tissues is dependent on the activity of a number of enzymes, including PLA2 and COX isozymes. In human gestational tissues, sPLA2 and cPLA2 isozymes are postulated to play significant roles in the parturition process (reviewed in Ref.1). They catalyze the first step in the synthesis of prostaglandins, i.e., the release of arachidonic acid from membrane phospholipids. The promoter regions for both type II PLA2 and cPLA2 gene contains several oligonucleotide elements homologous with consensus sequences for binding of transcription factors, including NF-{kappa}B (25). COX isozymes catalyze the conversion of free arachidonic acid to PGH2. Whereas COX-1 is constitutively expressed and contains no putative NF-{kappa}B binding site, COX-2 can be induced by LPS and proinflammatory cytokines and contains at least two NF-{kappa}B binding sites (reviewed in Ref.26).

SASP, a potent antiinflammatory agent, was synthesized in the 1930s to combine an antibiotic, sulfapyridine, linked by an azo bond to a salicylate moiety, 5-aminosalicylic acid. It has been used successfully in the treatment of ulcerative colitis, and rheumatoid arthritis for nearly 50 yr. Recently it has been demonstrated that these antiinflammatory effects of SASP may be attributed to its ability to suppress NF-{kappa}B activation (3, 4, 27, 28). We previously demonstrated that SASP suppresses LPS-induced NF-{kappa}B DNA binding activity in human gestational tissues (3). The data presented in this study demonstrate that SASP at concentrations greater than 5 mM significantly inhibited both basal and LPS-stimulated type II PLA2 content and release and cPLA2 protein in placenta, amnion, and choriodecidua. Although the exact mechanism by which SASP and other salicylates inhibit NF-{kappa}B activation is not understood, it is believed that inhibition is the result of preventing nuclear translocation of RelA due to the inhibition of inhibitory {kappa}B{alpha} (I{kappa}B{alpha}) phosphorylation and subsequent degradation (27, 28).

The transcriptional control of PLA2 isozymes and protease activity by NF-{kappa}B is supported by other studies. In rat mesangial cells, aspirin inhibits IL-1ß-induced PLA2 activity and expression with concurrent reduction in NF-{kappa}B activity (29), whereas cyclosporin A induces type II PLA2 expression and activity via up-regulation of NF-{kappa}B DNA binding activity (30). In human rheumatoid synovial fibroblasts, the use of oligonucleotide decoys and antisense has demonstrated the involvement of NF-{kappa}B in the regulation of cPLA2 (31).

In this study, although the inhibition of NF-{kappa}B DNA binding activity by SASP resulted in decreased synthesis of prostaglandin-forming enzymes cPLA2 and type II PLA2, no decrease in COX isozymes and PGF2{alpha} release was observed. In fact, in the amnion and choriodecidua, PGF2{alpha} secretion increased with increasing SASP concentrations. The influence of SASP on prostaglandin production remains equivocal, with both increases and decreases in prostaglandin production reported (32, 33, 34, 35). Punchard et al. (36) reported that the outcome is dependent on the incubation conditions, i.e. the concentration of the drug, the prostaglandin measured, and the stimulant used all playing a role.

The finding that SASP did not inhibit COX isozymes suggests that the NF-{kappa}B signaling pathway does not regulate COX in human placenta, amnion, and choriodecidua. However, previous studies implicated NF-{kappa}B in regulating COX-2 expression in the amnion-derived WISH cell line (14) and human myometrial cells (16). Site-directed mutagenesis of the two NF-{kappa}B sites in both amnion-derived WISH cell line and primary amnion cells demonstrated that these sites were essential for the increase in COX-2 transcription by IL-1ß, although they did not affect basal nonstimulated reporter expression (14). Belt et al. (16) reported that when I{kappa}B-{alpha} degradation was blocked, TNF{alpha} stimulated NF-{kappa}B translocation, and COX-2 mRNA and prostaglandin synthesis were inhibited. In contrast, in amnion-derived AV3 cells, there was no requirement for NF-{kappa}B activity in basal and IL-1ß- and TNF{alpha}-induced COX-2 transcription (37, 38). In these cells, the region that included the NF-{kappa}B site had little influence on COX-2 expression; however, mutation in the region containing the nuclear factor-IL-6 and cAMP response element sites reduced basal and IL-1ß- and TNF{alpha}-stimulated COX-2 production. These conflicting data suggest that COX-2 regulation differs markedly from cell line to cell line.

Several possible mechanisms exist to account for the lack of effect of SASP on placenta and the increased accumulation of PGF2{alpha} in incubation medium released from the fetal membranes in this study. First, SASP is a potent inhibitor of prostaglandin dehydrogenase (PGDH), the enzyme responsible for the initial metabolic inactivation of prostaglandins (39, 40). Because PGDH has previously been localized to syncytiotrophoblasts in placenta and trophoblast cells on chorion (41), the addition of SASP to these tissues may therefore act to prevent the breakdown of prostaglandins. The finding that SASP increased PGF2{alpha} but decreased 13,14dihydro-15-keto PGF2{alpha} release from amnion and choriodecidua confirms that SASP may be acting as an inhibitor of PGDH. Second, the formation of prostaglandins by COX isozymes gives rise to the intracellular reactive oxygen species superoxide, which is capable of causing COX inactivation. SASP is a scavenger of free radicals that has been shown to inhibit reactive oxygen species production in a number of test systems (42). SASP, acting as an antioxidant, may inhibit free radical-mediated inactivation of cyclooxygenase, extending the life of the enzyme. This proposal is consistent with the finding that SASP did not alter COX-2 mRNA expression in osteoblasts, although PGE2 production from these cells was increased (35).

The importance of NF-{kappa}B in prostaglandin formation, however, has been demonstrated in human myometrial cells and amnion-like WISH cells (14, 16, 18). After challenge with TNF{alpha}, placental trophoblasts showed activated NF-{kappa}B and increased COX-2 gene and protein expression with concurrent increases in the production of PGE2 and PGF2{alpha} (18). Belt et al. (16) reported that when I{kappa}B-{alpha} degradation was blocked in immortalized human myometrial cells, NF-{kappa}B translocation, COX-2 mRNA, and prostaglandin synthesis were inhibited.

In this study, we demonstrate that NF-{kappa}B is involved in the regulation of PLA2 isozymes from human gestational tissues. Consequently, the development of specific inhibitors of NF-{kappa}B will be both beneficial in further dissecting the role of NF-{kappa}B in the initiation of human labor and could potentially be clinically useful in the management and/or treatment of preterm labor.


   Acknowledgments
 
The authors gratefully acknowledge the assistance of the Clinical Research Midwives Angie Denning, Val Bryant, Ellen Smith, and Melissa Ryan and the Obstetrics and Midwifery staff of the Mercy Hospital for Women for their cooperation. This manuscript is dedicated to the memory of our dear friend and valued colleague Lyn Tuttle for her unfailing and inspirational dedication to both midwifery and research.


   Footnotes
 
This work was supported by the Medical Research Foundation for Women and Babies and National Health and Medical Research Council Grant 114106. M.L. received a Postdoctoral Research Fellowship from the Elizabeth and Vernon Puzey Foundation.

Abbreviations: AP-1, Activator protein-1; COX, cyclooxygenase; cPLA2, cytosolic PLA2; I{kappa}B{alpha}, inhibitory {kappa}B{alpha}; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NF-{kappa}B, nuclear factor-{kappa}B; PGDH, prostaglandin dehydrogenase; PGF2{alpha}, prostaglandin F2{alpha}; PLA2, phospholipase A2; SASP, sulfasalazine.

Received August 7, 2003.

Accepted February 3, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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