Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print October 10, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.061663
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/1/68    most recent
biolreprod.107.061663v1
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 78, 68–76 (2008)
DOI: 10.1095/biolreprod.107.061663
© 2008 by the Society for the Study of Reproduction, Inc.

Pregnancy; 

Nuclear Factor-Kappa B Regulates Inducible Prostaglandin E Synthase Expression in Human Amnion Mesenchymal Cells1

William E Ackerman, IV 2 3, Taryn L.S Summerfield 3, Dale D Vandre 5, John M Robinson 5, and Douglas A Kniss 3 4

Department of Obstetrics and Gynecology, Laboratory of Perinatal Research and Division of Maternal-Fetal Medicine,3 Department of Biomedical Engineering,4 and Department of Cell Biology and Physiology,5 The Ohio State University, Columbus, Ohio 43210

ABSTRACT

The human amnion is a major intrauterine source of prostaglandin (PG) E2, a potent mediator of uterine contractions and cervical ripening. During parturition, inflammatory cytokines promote PGE2 production through increased prostaglandin-endoperoxide synthase-2 (PTGS2, also known as cyclooxygenase-2) expression. This is mediated, in part, through activation of the transcription factor nuclear factor kappa B (NFkappaB). Prostaglandin E synthase (PTGES, also known as microsomal PGE synthase-1) acts downstream of PTGS2 and is inducibly expressed in most systems. We hypothesized that NFkappaB might regulate cytokine-induced PTGES expression in amnion cells. With amnion mesenchymal cells, we found that proinflammatory cytokines coordinately upregulated PTGS2 and PTGES mRNA expression. In parallel, increased expression of the PTGS2 and PTGES proteins was observed. In comparison, the expression of two other PGE synthases (PTGES2 and PTGES3) was unmodified. PTGES induction was blocked both in the presence of pharmacological NFkappaB inhibitors and following adenovirus-mediated overexpression of a dominant-negative NFkappaB pathway protein. In cells transiently transfected with a luciferase reporter bearing a portion (–597/+33) of the human PTGES gene promoter, interleukin-1beta (IL1B) produced a moderate increase in luciferase activity; this effect was abrogated in the presence of an indirect NFkappaB inhibitor (MG-132). Finally, a kappaB-like regulatory element was identified that, when mutated, markedly attenuated IL1B-responsive PTGES promoter activity. In conclusion, our results support a role for NFkappaB in cytokine-induced PTGES expression in amnion mesenchymal cells in vitro. By coordinately regulating PTGS2 and PTGES, NFkappaB may contribute to an inducible PGE2 biosynthesis pathway during human parturition.

cytokines, gene regulation, parturition, placenta, pregnancy

INTRODUCTION

The generation of prostaglandins (PGs) by amnion and decidual tissues is fundamental to both the onset and continuance of human labor. Uterotonic eicosanoids such as PGE2 accumulate in amniotic fluid with advancing gestation [1], and the enzymatic pathways responsible for PGE2 production are active in amnion cells prior to the onset of active labor [2]. PGs generated toward the end of gestation are thought to contribute to early labor-associated events, such as myometrial activation and cervical ripening [3]. During spontaneous active labor, a surge in PG production accompanies a series of biochemical events resembling a localized, acute inflammatory response (reviewed in [4]).

PG production requires the coordination of three major enzymatic reactions. In this pathway, arachidonic acid liberated from intracellular membranes by phospholipases is converted to intermediates by isoforms of prostaglandin-endoperoxide synthase (PTGS, also known as prostaglandin G/H synthase or cyclooxygenase); these are subsequently isomerized by terminal PG synthases to form active PGs. Two well-characterized PTGS isoforms, PTGS1 and PTGS2, catalyze the committing and rate-limiting step in this cascade. Whereas PTGS1 is constitutively expressed and contributes to immediate, low-amplitude PG release, the PTGS2 isoform is required for high-amplitude, delayed PG production [5]. In the context of parturition, copious PG production is associated with increased de novo expression of PTGS2 [6, 7].

For biosynthesis of PGE2, the major PG released by human amnion, several distinct gene products bearing terminal PGE isomerase activity have been described, including prostaglandin E synthase (PTGES, also known as microsomal PGE synthase-1), PTGES2 (also known as microsomal PGE synthase-2), and PTGES3 (also known as cytosolic PGE synthase) [8, 9]. Among these, PTGES is distinguished in that 1) it is upregulated in response to proinflammatory stimuli in many systems [10–12], including intrauterine cells [13, 14]; 2) it exhibits catalytic efficiency exceeding that of other PGE isomerases [15]; and 3) it is functionally coupled with PTGS2, an assertion that is supported by both biochemical [16] and gene deletion studies [17–19]. Collectively, such observations suggest that PTGH2 and PTGES comprise an inducible pathway for inflammatory PGE2 release.

Given that both PTGES and PTGS2 are induced in response to common proinflammatory stimuli, we hypothesized that the genes encoding these proteins might share common regulatory mechanisms. In the present study, we addressed whether the transcription factor nuclear-factor kappa B (NF{kappa}B), a master transcription factor for inflammatory gene expression and a major regulator of PTGS2 transcription, might also govern the expression of PTGES in amnion cells in vitro.

MATERIALS AND METHODS

Materials

Recombinant human interleukin-1β (IL1B) and tumor necrosis factor (TNF) alpha were purchased from R&D Systems (Minneapolis, MN). The NF{kappa}B inhibitors MG-132, SN50, and SN50M were obtained from BIOMOL International (Plymouth Meeting, PA). The Dual-Luciferase Reporter Assay System and the Renilla luciferase control expression vector (pRL-SV40) were obtained from Promega (Madison, WI). A luciferase reporter plasmid (pGL2–651) containing a region of the human PTGES gene promoter (–651/–20 relative to the translation start site, –597/+33 in relation to the transcription start site) was generously donated by Ralf Morgenstern (Karolinska Institute, Stockholm, Sweden) and has been described previously [11]. The QuikChange Site-Directed Mutagenesis Kit was obtained from Stratagene (La Jolla, CA). Arachidonic acid, PGE2 ELISA kits, and antibodies against PTGES (catalog numbers 160140 and 10004350), PTGES2 (catalog number 160145), and PTGES3 (catalog number 160150) were purchased from Cayman Chemical Company (Ann Arbor, MI). TRIzol reagent, cell culture media and sera, oligonucleotide primers, reagents for RT-PCR, Prolong Gold antifade mounting reagent, and Alexa Fluor-conjugated secondary antibodies (used in immunofluorescence experiments) were purchased from Invitrogen (Carlsbad, CA). SuperSignal chemiluminescent detection reagents were obtained from Pierce Biotechnology (Rockford, MA). Antibodies against PTGS2, 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), inhibitory factor {kappa}Bβ (I{kappa}Bβ, also known as nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor beta, NFKBIB), I{kappa}B kinase {alpha} (IKK{alpha}, also known as conserved helix-loop-helix ubiquitous kinase, CHUK), and NF{kappa}B subunit p65 (also known as reticuloendotheliosis viral oncogene homolog A, RELA), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Chemicon International (Temecula, CA). Antibodies recognizing phosphorylated forms of NFKBIA (I{kappa}B{alpha}; Ser32), RELA (p65; Ser536), and CHUK/IKBKB (IKK{alpha}/IKKβ; Ser180/Ser181) were from Cell Signaling Technology (Beverly, MA). Assays-on-Demand gene expression target assay mixtures (assay numbers Hs00610420_m1, Hs00228159_m1, Hs00832847_gH, Hs00153133_m1, and Hs99999905_m1 for PTGES, PTGES2, PTGES3, PTGS2, and GAPDH, respectively) and TaqMan Universal Master Mix were obtained from Applied Biosystems (Foster City, CA). ViraDuctin reagent and adenovirus particles expressing either recombinant E. coli β-galactosidase (Ad-lacZ, catalog number ADV-002) or a dominant-negative form of the human NFKBIA (I{kappa}B{alpha} protein [Ad-DN-NFKBIA], catalog number ADV-302) were purchased from Cell Biolabs (San Diego, CA). The β-galactosidase reporter gene histochemical staining kit, collagenase A, trypsin inhibitor, and monoclonal anti-vimentin and anti-cytokeratin antibodies were purchased from Sigma (St. Louis, MO); all other reagents unless otherwise specified also were obtained from Sigma.

Isolation of Amnion Mesenchymal Cells

Fetal membranes were collected at the time of uncomplicated scheduled cesarean delivery at term in the absence of labor or membrane rupture. There was no clinical evidence of infection in any of these subjects. Informed consent was obtained by a protocol approved by the Institutional Review Board of The Ohio State University (Columbus, OH).

Human amnion mesenchymal cells were obtained from the reflected fetal membranes following blunt dissection of the choriodecidua and digestion with 1 mg/ml of collagenase A, as previously described [20, 21]. Cells were seeded onto culture dishes coated with 0.1% (w/v) type I collagen and grown in Dulbecco modified Eagle medium (4500 mg/L of D-glucose) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µg/ml of gentamicin sulfate, 0.5 µg/ml of amphotericin B, and 10% (v/v) fetal bovine serum. Cultures attained confluence within 4–7 days and were subsequently expanded and plated for experiments at the lowest possible passage number (between the first and third passages for the experiments described). In some experiments, cultures enriched for amnion epithelial cells were derived simultaneously with mesenchymal cells with a series of incubations in 0.4% (w/v) trypsin [22]; these were grown in primary culture as described previously [23].

Immunofluorescence

Immunolabeling of cultured cells was performed as described previously [23] with antibodies directed against NF{kappa}B subunit RELA (p65, sc-8008; Santa Cruz), vimentin (Sigma), or cytokeratin (Sigma). In control experiments, an equivalent concentration of isotype-specific normal mouse immunoglobulin G (IgG) or an irrelevant antibody (anti-FLAG epitope) was substituted for the primary antibody; these controls exhibited only faint, nonspecific staining in all cases. After stringent washing in PBS, the coverslips were exposed to fluorophore-conjugated secondary antibodies (Molecular Probes). Nuclei were counterstained with 5 µg/ml of 4',6-diamidino-2-phenylindole (Sigma). Cells were mounted with the ProLong Antifade kit (Molecular Probes) and visualized with conventional epifluorescence (Nikon Instruments, Melville, NY) or confocal laser scanning microscopy (Zeiss 510 META; Carl Zeiss Inc., Thornwood, NY).

Immunoblot Analysis

Cellular proteins were extracted as previously described [23]. Proteins (20 µg/lane) were resolved by SDS-PAGE and transferred to nitrocellulose. Immunoblotting was performed with antibodies directed against targets described in the text and figure legends in Tris-buffered saline (pH 8.0) containing 0.05% (v/v) Tween-20 and 5% (w/v) nonfat dry milk. Following exposure to horseradish peroxidase-conjugated secondary antibodies, chemiluminescent signals were detected with SuperSignal chemiluminescent detection reagents (Pierce). Immunoreactive proteins were visualized with the VersaDoc Imaging System and analyzed by Quantity One software (Bio-Rad, Hercules, CA).

RNA Isolation and Real-Time RT-PCR

Total RNA from cultured amnion mesenchymal cells was purified with TRIzol reagent (Invitrogen) according to the method of Chomczynski and Sacchi [24]. Complementary DNA was prepared from 2 µg of RNA with oligo-dT primers and SuperScript II RT (Invitrogen). For quantitative real-time RT-PCR, cDNA was amplified with Assays-on-Demand gene expression target assay primer/probe mixtures (Applied Biosystems) and detected with an ABI PRISM 7700 sequence detector (Applied Biosystems). Amplification mixtures contained 2.5 µl of the appropriate 20x target assay mixture, 1 µl of first-strand cDNA synthesis mixture (corresponding to 50 ng of reverse-transcribed RNA), 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. Fold change was computed relative to control by the 2–{Delta}{Delta}CT method [25]. Samples were normalized to GAPDH expression, determined in parallel reactions.

Site-Directed Mutagenesis

In vitro mutagenesis was performed with the QuikChange Site-Directed Mutagenesis Kit (Stratagene), according to the manufacturer's instructions. Briefly, the supercoiled template plasmid (pGL2–651) was replicated in the presence of complementary mutagenic primers (sequences for the plus strand: 5'-CGA AAT CCC ATG TtA cAA GaC agT TTT GCC ACA TAG TCA CAG TCA CGG; lowercase letters refer to mutated oligonucleotides of a putative {kappa}B response element, 5'-GAAAAGTCCC). Subsequently, the methylated template plasmid was digested with the restriction endonuclease Dpn I, and the variant plasmid (pGL2–651 [m{kappa}B]) was transformed into chemically competent E. coli. The desired mutation was verified by sequence analysis (Plant-Microbe Genomics Facility, The Ohio State University, Columbus, OH).

Transient Transfection of Amnion Mesenchymal Cells

Transient transfections were performed in a 24-well tissue culture plate seeded at 4 x 104 cells per well. For each cotransfection, 0.7 µg of firefly luciferase reporter plasmid (pGL2–651 or pGL2–651 [m{kappa}B]), 0.1 µg of Renilla luciferase control plasmid (pRL-SV40), and 2.5 µl of LIPOFECTAMINE 2000 reagent (Invitrogen) were diluted into OPTI-MEM I Reduced Serum Medium (Invitrogen), according to the manufacturer's instructions. By this method, transfection efficiencies for individual amnion mesenchymal cell preparations ranged from 10% to 30%, as determined with a reporter plasmid expressing a variant of Aequorea victoria green fluorescent protein (pEGFP-N1; BD Biosciences Clontech, Mountain View, CA).

Luciferase assays were performed with the Dual-Luciferase Reporter Assay System (Promega) on either a Dynex MLX microplate luminometer (Dynex Technologies, Chantilly, VA) or a Wallac 1420 VICTOR3 multilabel plate reader (PerkinElmer, Waltham, MA). Reporter activity was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity.

Adenovirus-Mediated Gene Transfer to Amnion Mesenchymal Cells

All recombinant adenovirus particles were prepared by Cell Biolabs (San Diego, CA). Permission to use these vectors was granted by the Institutional Biosafety Committee of The Ohio State University. Preconfluent amnion mesenchymal cells were infected with adenoviral vectors (Ad-lacZ and Ad-DN-Ad-DN-NFKBIA, with a multiplicity of infection (MOI) of 1–1000 plaque-forming units per cell in the absence or presence of ViraDuctin (9 µl/106 cells). Cells were incubated for 48 h in the presence of viral particles prior to washing and exchange with fresh serum-containing medium. Following an additional 24-h incubation, cells were assessed for the expression and function of the transferred genes. All experiments were conducted with cells infected at an MOI of 500, which resulted in maximal expression levels 3 days after infection.

Staining for β-Galactosidase (lacZ) Reporter Gene Activity

Following infection with Ad-lacZ, cells were fixed in 2% (v/v) formaldehyde/0.2% (v/v) glutaraldehyde/PBS for 10 min at room temperature. The activity of the β-galactosidase reporter gene was detected by application of X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) in PBS solution containing magnesium chloride, potassium ferricyanide, and potassium ferrocyanide, according to the instructions provided by the manufacturer (Sigma). Cells expressing the transgene stained an indigo blue that could be identified with brightfield microscopy.

Prostaglandin E2 ELISA

Cells were plated in 48-well tissue culture plates in serum-free culture medium supplemented with 5 µM arachidonic acid. Following 4-h incubations with test substances, the media were collected and frozen at –80°C until assays were performed. Media were analyzed for PGE2 content by competitive ELISA (Cayman Chemical). Consistent with our prior experience [23, 26, 27], the intra- and interassay coefficients of variation for this assay were <10%, and the limit of detection was 15 pg/ml in undiluted samples. When used to compare PGE2 output from amnion cell subpopulations, each replicate was assayed with a series of dilutions (undiluted, 1:5, 1:10, and 1:20) to accommodate measurements occurring at extremes of the standard curve; those values occurring within the linear range of the standard curve (when plotted logarithmically) were interpreted as being the most accurate.

Statistical Analyses

All experiments in this study were repeated a minimum of three times. ELISA, densitometric, real-time RT-PCR, and luminometric data were assessed by one-way analysis of variance, followed by the Bonferroni multiple comparisons post hoc test, by means of Prism version 4.00 software (GraphPad Software, San Diego, CA); P < 0.05 was considered statistically significant.

RESULTS

Amnion Mesenchymal Cells Produce More PGE2 than Amnion Epithelial Cells In Vitro

The amniotic membrane consists of a single layer of epithelial cells overlying a layer of mesenchymal cells. Although the amnion is generally accepted to be an intrauterine source for PGE2 [3, 28], controversy has surrounded the relative contribution of these two cell types to inducible PGE2 synthesis. Studies by Whittle et al. [20] and Gibb and Sun [29] have highlighted the importance of subepithelial amnion cells in human parturition by demonstrating that the potential for cytokine-induced PTGS2 expression and PGE2 release by mesenchymal cells greatly exceeds that for epithelial cells.

We revisited this issue by conducting experiments with primary cultures enriched for either of these two cell types. We found that, for equal numbers of plated cells and in the presence of exogenous arachidonic acid, basal PGE2 release from mesenchymal cells (1260 ± 160 pg/ml/4 h, mean ± SD) was 5-fold higher than induced PGE2 release from epithelial cells (233 ± 71 pg/ml/4 h) (Fig. 1A, P < 0.001). In the presence of IL1B, a 3-fold increase in mean PGE2 release from amnion mesenchymal cells (3820 ± 730 pg/ml/4 h, P < 0.001) was observed. For amnion epithelial cells, cytokine-induced PGE2 release was modest (1.4-fold, Fig. 1A, inset) and did not achieve the level of statistical significance in limited experiments.


Figure 01
View larger version (62K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 Characterization of amnion cell subpopulations in vitro. A) Equivalent numbers (2 x 105 cells/well) of amnion epithelial (white bars) and mesenchymal (black bars) cells were incubated with 5 µM of exogenous arachidonic in the absence or presence of IL1β (10 ng/ml) for 4 h. PGE2 released into the media was analyzed by ELISA (mean ± SEM for quadruplicate determinations, n = 2 individual experiments). Values in each column with different letters differ significantly, P < 0.001 (ANOVA). B) Representative photomicrographs demonstrating characteristic vimentin-like immunostaining in amnion mesenchymal cells. Greater than 90% of the cells in five individual preparations expressed vimentin, consistent with mesenchymal origin. A higher magnification image (inset) shows details of the intracellular staining pattern for this intermediate filament protein. Bar = 20 µm.

In primary cultures, amnion mesenchymal cells were identified with vimentin immunolabeling, whereas epithelial cells were recognized by with anti-cytokeratin antibodies [21, 29]. Although both epithelial and mesenchymal cells were found in all amnion cell cultures, the majority (>90%) of cells in five individual mesenchyme-enriched preparations exhibited strong vimentin-like immunostaining, similar to that shown in Figure 1B.

On the basis of these results, we chose amnion mesenchymal cells as a relevant model system in which to focus the remainder of our experiments.

Cytokine-Induced Activation of the Canonical NF{kappa}B Signaling Pathway in Amnion Mesenchymal Cells

The general framework for canonical (or classical) NF{kappa}B activation is well established [30, 31]. In quiescent cells, a majority of NF{kappa}B resides in the cytoplasm coupled with inhibitor (I{kappa}B) proteins. In response to a variety of extracellular signals, an I{kappa}B kinase complex (IKK) is transiently activated [30, 31], which, in turn, phosphorylates I{kappa}B proteins, evoking a mechanism that ultimately results in proteolytic degradation of this inhibitor. Once liberated from I{kappa}B, NF{kappa}B translocates to the cell nucleus, where it modulates transcription. To characterize the kinetics of NF{kappa}B pathway activation in amnion mesenchymal cells, we conducted a series of time course experiments. Following IL1B challenge, we observed that the alpha and beta subunits of I{kappa}B kinase complex IKK (CHUK and IKBKB, respectively) were rapidly but transiently phosphorylated (Fig. 2A), a posttranslational event associated with increased IKK complex activity. IKK complex activation was substantiated by the concomitant phosphorylation of NFKBIA (I{kappa}B{alpha} at serinyl residue 32, which immediately preceded its degradation [between 15 and 30 min]). By 60 min, NFKBIA (I{kappa}B{alpha}) reappeared, likely the result of rapid de novo resynthesis (as NFKBIA is highly induced by NF{kappa}B). A second IKK complex substrate, NFKBIB (I{kappa}Bβ), was degraded with delayed kinetics relative to NFKBIA (I{kappa}B{alpha}). Nuclear translocation of NF{kappa}B was confirmed with immunofluorescence (Fig. 2B, identified by changes in cellular distribution of RELA [p65] immunoreactivity [23, 27]). At the same time, signal-coupled phosphorylation of RELA (p65) at serinyl residue 536 was observed (Fig. 2A); this latter event, also mediated by the IKK complex, is associated with enhanced NF{kappa}B transcriptional activity [30]. Despite resynthesis of NFKBIA (I{kappa}B{alpha}), net RELA (p65) immunostaining remained nuclear through the 60-min time point. Given that inhibitors such as NFKBIB (I{kappa}Bβ) or NFKBIE (I{kappa}B{varepsilon}) are degraded more slowly than NFKBIA (I{kappa}B{alpha} following cytokine stimulation (reviewed in [30] and [32]), this finding may reflect that certain pools of RELA (p65)-containing NF{kappa}B:I{kappa}B protein complexes exhibit delayed nuclear translocation. Overall, these results were highly consistent with those of Yan et al. [21], who observed that IL1B-induced NF{kappa}B binding activity in amnion mesenchymal cells was maximal 1 h following stimulation.


Figure 02
View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 Temporal course of IL1B-induced NF{kappa}B activation in amnion mesenchymal cells. Amnion mesenchymal cells were stimulated with IL1B (10 ng/ml) for the indicated times and prepared either for immunoblot analysis (A) or immunofluorescence (B). A) Representative results from a series of immunoblots probed with antibodies directed against native (black lettering) and phosphorylated (blue lettering) forms of major NF{kappa}B pathway component proteins. GAPDH was used as a control for loading. B) Intracellular localization of NF{kappa}B subunit RELA (p65) was detected by immunofluorescence. The predominantly cytoplasmic distribution observed in untreated cells (0', in which double arrows denote cell nuclei with relatively little p65-like immunoreactivity) shifted to nuclear staining in the presence of IL1B (arrowheads in remaining panels). Bar = 20 µm.

Blockade of NF{kappa}B Signaling Attenuates Cytokine-Induced PTGES Expression

The NF{kappa}B signaling pathway has been shown to mediate PTGES induction in response to IL1B in pulmonary A549 cells [33]. To determine whether NF{kappa}B serves an analogous role in amnion mesenchymal cells, we examined the effect of MG-132, an indirect NF{kappa}B inhibitor, on basal- and cytokine-induced PTGES protein expression. In these experiments, amnion cells were preincubated for 30 min with MG-132 or vehicle and then incubated for 4 h with either IL1B or TNF. By immunofluorescence, we confirmed that MG-132 prevented IL1B-induced nuclear translocation of the NF{kappa}B subunit RELA (p65) (Fig. 3A).


Figure 03
View larger version (46K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 Effects of cytokines and MG-132 on the expression of prostaglandin E biosynthetic pathway enzymes. A) Immunolocalization of NF{kappa}B subunit RELA (p65) following 15 min of IL1B challenge in the absence (top panel) or presence (bottom panel) of MG-132 (30 µM); RELA (p65) nuclear localization was blocked in the presence of this inhibitor. Bar = 20 µm. B) Representative immunoblots prepared from amnion mesenchymal cell lysates 4 h following challenge with media (CTRL), IL1B (10 ng/ml), or TNF (25 ng/ml) in combination with vehicle (Veh, 0.3% v/v of ethanol) or MG-132 (30 µM). C–F) Values obtained from densitometric analysis of immunoreactive PTGS2 (C), PTGES (D), PTGES2 (E), and PTGES3 (F) following treatment with vehicle (white bars) or MG-132 (black bars), alone or in combination with cytokine. Data were normalized to GAPDH levels for each treatment group and have been expressed as fold-change in relation to control (mean ± SEM, n = 3 independent experiments). Values in each column with different letters differ significantly, P < 0.05 (ANOVA).

Following exposure to either cytokine, PTGES protein expression was increased ~2-fold (Fig. 3, B and C). In the presence of MG-132, cytokine-elicited PTGES induction was blocked. Under identical treatment conditions, cytokine challenge increased PTGS2 expression by 3- to 6-fold following TNF and IL1B treatment, respectively (Fig. 3, B and D). Unexpectedly, rather than blocking PTGS2 induction, MG-132 exposure resulted in a 5-fold increase in PTGS2 protein relative to control. Notably, this occurred even in the absence of cytokine exposure. By comparison, we observed no changes in the levels of PTGES2 or PTGES3 proteins with cytokine or MG-132 treatments (Fig. 3, B, E, and F).

We next examined the effect of MG-132 on PTGES mRNA expression by real-time RT-PCR. As in the prior series of experiments, cells were preincubated for 30 min with MG-132 or vehicle and then challenged for 4 h with cytokine (IL1B) prior to RNA extraction. As shown in Figure 4A, PTGES expression increased by an average of 5-fold (P < 0.05) in response to IL1B. MG-132 significantly decreased induced, as well as basal, PTGES mRNA levels. Under the same conditions, PTGS2 mRNA levels increased robustly (120-fold, P < 0.001) following IL1B (Fig. 4B). In contrast to its effect on PTGS2 protein expression, MG-132 attenuated PTGS2 induction by 50%. Neither PTGES2 expression (Fig. 4C) nor that of PTGES3 (Fig. 4D) was significantly altered under these treatment conditions. Similar results were obtained with a cell-permeable peptide inhibitor of NF{kappa}B nuclear import, SN50. This second compound, but not an equivalent amount of control SN50M peptide, blocked IL1B-induced PTGES mRNA expression (Fig. 4E, P < 0.001).


Figure 04
View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 Pharmacological NF{kappa}B inhibitors abrogate cytokine-induced PTGS2 and PTGES mRNA expression. A–D) Real-time RT-PCR was used to assess PTGES (A), PTGS2 (B), PTGES2 (C), and PTGES3 (D) mRNA expression in amnion mesenchymal cells treated with vehicle (0.3% v/v of ethanol, white bars) or 30 µM of MG-132 (black bars) alone or in the presence of IL1B (10 ng/ml) for 4 h. E, F) Real-time RT-PCR was used to assess PTGES (E) and PTGS2 (F) expression in amnion mesenchymal cells following treatment with IL1B (10 ng/ml) alone or with 150 µg/ml of either SN50 or SN50M. For each set of experiments, the threshold cycle (CT) at which each mRNA species was detected was normalized to that of GAPDH. The fold-change in transcript expression was calculated in relation to the control group for individual assays (mean ± SEM, n = 3 independent experiments). Values in each column with different letters differ significantly, P < 0.05 (ANOVA).

Given the potential for nonspecificity when pharmacological NF{kappa}B inhibitors are used [34], we performed additional experiments with a dominant-negative variant of human NFKBIA (I{kappa}B{alpha}). This construct, harboring a serine-to-alanine substitution at amino acid 32, is resistant to phosphorylation-induced degradation and acts by sequestering the cytoplasmic NF{kappa}B pool in a manner that is insensitive to extracellular stimuli. Given the relatively low transfection efficiency (10%–30%) of the primary cultures with lipidic reagents, it was necessary to employ adenovirus-mediated gene transfer to achieve levels of expression relevant for use of this construct. In pilot experiments with Ad-lacZ, it was determined that infection at an MOI of 500 resulted in >85% transduction efficiency in amnion mesenchymal cells; however, this was observed only in the presence of ViraDuctin (Fig. 5A), indicating that amnion mesenchymal cells do not express the coxsackievirus and adenovirus receptor (required for adenoviral infectivity in this case) in abundance [35]. Following transduction with the dominant-negative adenoviral vector (Ad-DN-NFKBIA, we verified that the recombinant protein was resistant to proteasome-mediated degradation following cytokine challenge (Fig. 5B). By real-time RT-PCR, we found that both IL1B and TNF significantly increased PTGES expression in cells infected with Ad-lacZ (P < 0.05). Consistent with the results obtained with pharmacological agents, PTGES induction by either cytokine was blocked in cells expressing dominant-negative NFKBIA (Fig. 5C). Simultaneously, Ad-DN-NFKBIA infection resulted in a 67% decrease in PTGS2 expression following IL1B stimulation relative to cells infected with Ad-lacZ. A similar trend (which did not reach a level of statistical significance) was observed following TNF challenge. Collectively, these data suggest that NF{kappa}B contributes to the coordinate regulation of PTGS2 and PTGES but does not influence the expression of PTGES2 or PTGES3.


Figure 05
View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 Adenovirus-mediated overexpression of a dominant-negative NFKBIA (I{kappa}B{alpha}) variant inhibits cytokine-induced PTGES mRNA expression. A) Representative photomicrographs demonstrating staining for β-galactosidase expression in nuclei following infection with Ad-lacZ at an MOI of 500 in the absence (left panel) or presence (right panel) of ViraDuctin. Bar = 200 µm. B) Adenovirus-expressed dominant-negative NFKBIA (I{kappa}B{alpha}) is resistant to proteasome-mediated degradation. Amnion mesenchymal cells were infected either with Ad-lacZ (control) or Ad-DN-NFKBIA at an MOI of 500 with ViraDuctin and then challenged for 15 min with IL1B (10 ng/ml) and prepared for immunoblot analysis. When probed with antibodies directed against NFKBIA (I{kappa}B{alpha}), only the endogenous protein was degraded (lane 1). C, D) Real-time RT-PCR was used to assess PTGES (C) and PTGS2 (D) mRNA expression in amnion mesenchymal cells following infection either with Ad-lacZ (control, white bars) or Ad-DN-NFKBIA (black bars) for 72 h, followed by treatment with media alone, IL1B (10 ng/ml), or TNF (25 mg/ml) for 4 h. For each set of experiments, the threshold cycle (CT) at which each mRNA species was detected was normalized to that of GAPDH. The fold-change in transcript expression was calculated in relation to the control group (mean ± SEM, n = 2 independent experiments). Values in each column with different letters differ significantly, P < 0.05 (ANOVA).

Identification of a {kappa}B-Like Response Element Within the Human PTGES Gene Promoter

Several potential transcriptional elements have been identified in the 5'-promoter region of the human PTGES gene, including GC boxes (involved in regulation by the early growth response gene-1 [EGR1] transcription factor); activator protein-1 (AP-1) response elements; CCAAT/enhancer-binding protein {alpha} and β biding sites (c/EBP{alpha} and β); and response elements for progesterone and glucocorticoid nuclear receptors [9, 36]. Despite data supporting the involvement of NF{kappa}B in PTGES transcriptional activation [33], there has been no evidence that its gene promoter contains a functional {kappa}B binding site. We therefore used the TESS (Transcription Element Search Software; http://www.cbil.upenn.edu/tess [37]) program to identify potential NF{kappa}B response elements ({kappa}BRE) within the PTGES 5'-promoter sequence. One region, located –542/–533 upstream of the PTGES transcription start site and bearing the sequence 5'-GAAAAGTCCC, was identified as having a high likelihood for NF{kappa}B binding. In the complementary direction, this sequence shares 90% identity with the canonical NF{kappa}B B-enhancer sequence, 5'-GGGRNYYYCC (where R is a purine, Y is a pyrimidine, and N is any nucleic acid) [30]. In the forward direction, this sequence more closely resembles NF{kappa}B-like sites that diverge from consensus, such as those found in the 5'-promoter regions of cytokine/chemokine genes such interleukin-2 (IL2) [38] and interleukin-8 (IL8) [39], as well as several apoptosis-related genes [40, 41].

To examine the effect of IL-1β on PTGES-promoter activity, cells were transiently transfected with a luciferase reporter plasmid containing a segment (–597/+33 in relation to the transcription start site) of the human PTGES gene promoter (pGL2–651). IL-1β elicited a 2.8-fold increase in reporter activity relative to untreated cells (P < 0.01; Fig. 6B), confirming the presence of cytokine-responsive transcriptional elements within this promoter region. We additionally observed that this cytokine-induced increase in promoter activity was blocked in the presence of MG-132 (Fig. 6B), providing further evidence that NF{kappa}B contributes to the transcription activation of this gene.


Figure 06
View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6 Functional characterization of a {kappa}B-like response element within the PTGES 5'-promoter. A) Diagram of human PTGES promoter region cloned upstream of firefly luciferase to generate the pGL2–651 luciferase reporter plasmid [11]. The position of a {kappa}B-like response element ({kappa}BRE) in relation to the transcriptional start site is shown. B) Cells were transiently transfected with pGL2–651 for 24 h and then stimulated with IL1B (10 ng/ml) in the absence or presence of MG-132 (30 µM) for 4 h. Relative luciferase activity was quantified with a luminometer. Each bar represents the mean ± SD of six replicates from a representative experiment. C) Cells were transiently transfected either with wild-type (pGL2–651) or mutant (pGL2–651 [m{kappa}B]) reporter plasmids for 24 h and then incubated in the absence (white bars) or presence of 10 ng/ml of IL1B (black bars) for 4 h (mean ± SEM, n = 4 independent experiments). Values in each column with different letters differ significantly, P < 0.05 (ANOVA).

Next, to determine whether the putative {kappa}B binding site in the PTGES promoter might correspond to the functional regulation of PTGES in response to IL-1β, site-directed mutagenesis was used to introduce five-point mutations within this sequence (5'-tAcAAGaCag, where lowercase letters refer to mutated oligonucleotides). As shown in Figure 6C, responsiveness of the PTGES promoter to cytokine stimulation was diminished when this potential regulatory element was altered.

DISCUSSION

Although the molecular mechanisms responsible for human parturition remain incompletely understood, there is abundant evidence to suggest that inflammation plays an important role in this process (reviewed in [4, 42]). Concentrations of cytokines, chemokines, and inflammatory lipids increase in amniotic fluid toward term in normal pregnancies (reviewed in [43]). In addition, during spontaneous active labor, there is significant upregulation in the expression of proinflammatory mediators within intrauterine tissues [44, 45]. Many of these labor-associated genes are regulated at the transcription level by NF{kappa}B. The contribution of NF{kappa}B to human labor has been addressed by several groups (see [46, 47] for recent reviews), and although biochemical [48] and immunohistochemical [49] studies have been conflicting, the present consensus suggests that NF{kappa}B activation both precedes and accompanies parturition.

Prior reports have established the importance of NF{kappa}B in regulating PTGS2 expression in amnion and myometrial cells [21, 48, 50]. In the present study, we provide further evidence that NF{kappa}B contributes significantly, albeit not exclusively, to cytokine-mediated PTGS2 induction. One unanticipated observation was that MG-132, rather than blocking PTGS2 induction, resulted in a 5-fold increase in the PTGS2 protein level (even in the absence of stimulation). Such results were inconsistent with the abrogating effect of MG-132 on PTGS2 mRNA induction (Fig. 5B). It is reasonable to speculate that MG-132, an inhibitor of the 26 S proteasome complex, interferes with tonically active PTGS2 protein degradation. Consistently, Mbonye et al. [51] recently demonstrated that normal PTGS2 turnover is mediated through an endoplasmic reticulum-associated degradation system that is ultimately dependent on proteasome-mediated destruction.

In addition to PTGS2, we provide evidence that NF{kappa}B contributes to cytokine-induced PTGES gene expression in amnion mesenchymal cells in vitro. In support of this, we observed that pharmacological inhibition of NF{kappa}B signaling significantly attenuated cytokine-elicited PTGES mRNA (Fig. 4, A and E) and protein (Fig. 3D) expression. Similarly, overexpression of a dominant-negative variant of human NFKBIA (I{kappa}B{alpha}) blocked the induction of PTGES mRNA expression by IL1B and TNF (Fig. 5E). Furthermore, NF{kappa}B inhibitors reduced cytokine-responsive PTGES promoter activity (Fig. 6B). Although MG-132 also significantly decreased PTGES mRNA expression in the absence of stimulation (Fig. 4A), suggesting that basal NF{kappa}B activity contributes to PTGES expression in resting cells, this effect was not replicated with a second NF{kappa}B inhibitor (SN50) or the dominant-negative construct; as such, we cannot discount that off-target activity might have contributed to this effect. Overall, these results are consistent with the observations of Catley et al. [33], who also used a dominant-negative construct to demonstrate that intact NF{kappa}B signaling was required for cytokine-induced PTGES transactivation in A549 cells.

Given that NF{kappa}B transcriptional regulation requires the presence of cognate enhancer elements, we used a bioinformatics search algorithm to discern potential {kappa}B binding sites within the human PTGES gene promoter. One such region was identified that, when altered by site-directed mutagenesis, significantly decreased cytokine-elicited activation of a segment of the PTGES promoter (Fig. 6C). Although it remains to be determined which NF{kappa}B subunits might bind to this site, these current data are the first to suggest that this region represents an important PTGES regulatory element. It is noteworthy that the PTGES {kappa}B-like sequence is similar to a {kappa}BRE described previously within the IL8 gene promoter [39]. By introducing point mutations (similar to those described herein) into the IL8 {kappa}BRE, Elliott et al. [52] found this region to be important for IL1B-stimulated gene expression in amnion epithelial cells. It is interesting that the {kappa}B response elements in these two genes, as well as those in several other cytokine and chemokine promoters [38, 40, 41], share sequence similarity that differs from the traditional {kappa}BRE consensus sequence [30]. Inasmuch as prior and current evidence supports the functionality of such "variant" {kappa}BREs, we speculate that the {kappa}B-like motif in the PTGES promoter is likely to represent a bona fide NF{kappa}B response element.

In addition to NF{kappa}B, PTGES and PTGS2 may share coordinate regulation through other means. Indeed, both PTGS2 and PTGES genes contain multiple transcription factor binding motifs, and it is likely that combinatorial control of these transcripts contributes to their optimal expression. For example, effectors of the mitogen-activated protein kinase 1 (MAPK1, p38) could contribute to the coinduction of PTGS2 [27, 53] and PTGES [54]. In PTGES, such effects might manifest directly or via the induction of an intercessor (early growth response gene-1 [EGR1], another master gene regulator whose product transactivates the PTGES promoter [55]). EGR1 may act either cooperatively [56] or antagonistically [57] with NF{kappa}B (depending on context), and limited evidence additionally suggests that the EGR1 gene itself is regulated by NF{kappa}B [58]. How such nonlinear interactions might contribute to the regulation of PTGES expression remains to be determined. In light of the potential for combinatorial regulatory control, it is somewhat surprising that inhibition of the NF{kappa}B pathway had such a pronounced effect on PTGES induction in the current study. We cannot completely discount that the pharmacological inhibitors (or perhaps even the dominant-negative construct) acted through the blockade multiple-signaling pathways, contributing to the dramatic reduction in cytokine-mediated PTGES expression that we have attributed to NF{kappa}B. Nor did we anticipate that point mutations in a single putative response element would so significantly decrease cytokine-elicited PTGES promoter activation. Nevertheless, our results provide compelling evidence that the transcriptional control of PTGES warrants additional scrutiny.

Inasmuch as prior reports have suggested that labor has little, if any, effect on the expression of PTGES [59, 60], it would appear that PTGES induction is not rate limiting for PGE2 synthesis in fetal membranes during parturition. Therefore, it is unclear to what extent upregulation, rather than mere presence, of PTGES contributes to this process. Although it is difficult to reconcile our own in vitro data, and those of others [13, 14], to the in vivo context of labor, our results suggest that coordinate regulation of PTGS2 and PTGES facilitates cytokine-elicited PGE2 production within amnion mesenchymal cells. It may be possible to infer that a similar, albeit more complicated, molecular mechanism governs the synthesis of PGE2 within the intact amniochorion and decidua.

ACKNOWLEDGMENTS

The authors thank Dr. Ralf Morgenstern (Karolinska Institutet, Stockholm, Sweden) for generously donating the human PTGES 5'-promoter luciferase reporter plasmid. The authors are indebted to Jay Leng, Ph.D., of Cell Biolabs (San Diego, CA), who provided helpful technical advice related to the adenovirus-mediated gene transfer experiments.

FOOTNOTES

1Supported by NIH grants F32 HD042910 and K08 HD049628 and The Ohio State University Perinatal Research and Development Fund. Portions of this study were presented in abstract form at the 53rd Annual Meeting of the Society for Gynecologic Investigation, March 22–25, 2006, Toronto, ON, Canada. Back

Correspondence: 2William E. Ackerman IV, Laboratory of Perinatal Research, Department of Obstetrics and Gynecology, The Ohio State University, 5th Floor Means Hall, 1654 Upham Dr., Columbus, OH 43210. FAX: 614 293 5728; e-mail: ackerman.72{at}osu.edu

Received: 20 March 2007.

First decision: 21 April 2007.

Accepted: 1 October 2007.

REFERENCES

  1. Romero R, Munoz H, Gomez R, Parra M, Polanco M, Valverde V, Hasbun J, Garrido J, Ghezzi F, Mazor M, Tolosa JE, Mitchell MD. Increase in prostaglandin bioavailability precedes the onset of human parturition Prostaglandins Leukot Essent Fatty Acids 1996 54187–191[CrossRef][Medline]
  2. Brown NL, Alvi SA, Elder MG, Bennett PR, Sullivan MH. A spontaneous induction of fetal membrane prostaglandin production precedes clinical labour J Endocrinol 1998 157R1–R6[Abstract]
  3. Challis JRG, Matthews SG, Gibb W, Lye SJ. Endocrine and paracrine regulation of birth at term and preterm Endocr Rev 2000 21514–550[Abstract/Free Full Text]
  4. Romero R, Espinoza J, Goncalves LF, Kusanovic JP, Friel LA, Nien JK. Inflammation in preterm and term labour and delivery Semin Fetal Neonatal Med 2006 11317–326[CrossRef][Medline]
  5. Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: structural, cellular, and molecular biology Annu Rev Biochem 2000 69145–182[CrossRef][Medline]
  6. Slater DM, Berger LC, Newton R, Moore GE, Bennett PR. Expression of cyclooxygenase types 1 and 2 in human fetal membranes at term Am J Obstet Gynecol 1995 17277–82[CrossRef][Medline]
  7. Mijovic JE, Zakar T, Nairn TK, Olson DM. Prostaglandin endoperoxide H synthase (PGHS) activity and PGHS-1 and –2 messenger ribonucleic acid abundance in human chorion throughout gestation and with preterm labor J Clin Endocrinol Metab 1998 831358–1367[Abstract/Free Full Text]
  8. Murakami M, Nakatani Y, Tanioka T, Kudo I. Prostaglandin E synthase Prostaglandins Other Lipid Mediat 2002 68–69383–399
  9. Murakami M and Kudo I. Recent advances in molecular biology and physiology of the prostaglandin E2-biosynthetic pathway Prog Lipid Res 2004 433–35[CrossRef][Medline]
  10. Jakobsson PJ, Thoren S, Morgenstern R, Samuelsson B. Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target Proc Natl Acad Sci U S A 1999 967220–7225[Abstract/Free Full Text]
  11. Forsberg L, Leeb L, Thoren S, Morgenstern R, Jakobsson P. Human glutathione dependent prostaglandin E synthase: gene structure and regulation FEBS Lett 2000 47178–82[CrossRef][Medline]
  12. Thoren S and Jakobsson PJ. Coordinate up- and down-regulation of glutathione-dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells. Inhibition by NS-398 and leukotriene C4 Eur J Biochem 2000 2676428–6434[Medline]
  13. Premyslova M, Li W, Alfaidy N, Bocking AD, Campbell K, Gibb W, Challis JR. Differential expression and regulation of microsomal prostaglandin E(2) synthase in human fetal membranes and placenta with infection and in cultured trophoblast cells J Clin Endocrinol Metab 2003 886040–6047[Abstract/Free Full Text]
  14. Roman AS, Schreher J, Mackenzie AP, Nathanielsz PW. Omega-3 fatty acids and decidual cell prostaglandin production in response to the inflammatory cytokine IL-1beta Am J Obstet Gynecol 2006 1951693–1699[CrossRef][Medline]
  15. Thoren S, Weinander R, Saha S, Jegerschold C, Pettersson PL, Samuelsson B, Hebert H, Hamberg M, Morgenstern R, Jakobsson PJ. Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination J Biol Chem 2003 27822199–22209[Abstract/Free Full Text]
  16. Murakami M, Naraba H, Tanioka T, Semmyo N, Nakatani Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh S, Kudo I. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2 J Biol Chem 2000 27532783–32792[Abstract/Free Full Text]
  17. Kapoor M, Kojima F, Qian M, Yang L, Crofford LJ. Shunting of prostanoid biosynthesis in microsomal prostaglandin E synthase-1 null embryo fibroblasts: regulatory effects on inducible nitric oxide synthase expression and nitrite synthesis FASEB J 2006 20E1704–E1715
  18. Uematsu S, Matsumoto M, Takeda K, Akira S. Lipopolysaccharide-dependent prostaglandin E(2) production is regulated by the glutathione-dependent prostaglandin E(2) synthase gene induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway J Immunol 2002 1685811–5816[Abstract/Free Full Text]
  19. Boulet L, Ouellet M, Bateman KP, Ethier D, Percival MD, Riendeau D, Mancini JA, Methot N. Deletion of microsomal prostaglandin E2 (PGE2) synthase-1 reduces inducible and basal PGE2 production and alters the gastric prostanoid profile J Biol Chem 2004 27923229–23237[Abstract/Free Full Text]
  20. Whittle WL, Gibb W, Challis JR. The characterization of human amnion epithelial and mesenchymal cells: the cellular expression, activity and glucocorticoid regulation of prostaglandin output Placenta 2000 21394–401[CrossRef][Medline]
  21. 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 661667–1671[Abstract/Free Full Text]
  22. 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 19117–126[Medline]
  23. Ackerman WE IV, Zhang XL, Rovin BH, Kniss DA. Modulation of cytokine-induced cyclooxygenase 2 expression by PPARG ligands through NFkappaB signal disruption in human WISH and amnion cells Biol Reprod 2005 73527–535[Abstract/Free Full Text]
  24. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 1987 162156–159[Medline]
  25. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Methods 2001 25402–408[CrossRef][Medline]
  26. Ackerman WE, Robinson JM, Kniss DA. Despite transcriptional and functional coordination, cyclooxygenase-2 and microsomal prostaglandin E synthase-1 largely reside in distinct lipid microdomains in WISH epithelial cells J Histochem Cytochem 2005 531391–1401[Abstract/Free Full Text]
  27. Ackerman WE IV, Rovin BH, Kniss DA. Epidermal growth factor and interleukin-1beta utilize divergent signaling pathways to synergistically upregulate cyclooxygenase-2 gene expression in human amnion-derived WISH cells Biol Reprod 2004 712079–2086[Abstract/Free Full Text]
  28. Olson DM, Skinner K, Challis JR. Prostaglandin output in relation to parturition by cells dispersed from human intrauterine tissues J Clin Endocrinol Metab 1983 57694–699[Abstract/Free Full Text]
  29. Gibb W and Sun M. Cellular specificity of interleukin-1beta-stimulated expression of type-2 prostaglandin H synthase in human amnion cell cultures Biol Reprod 1998 591139–1142[Abstract/Free Full Text]
  30. Chen LF and Greene WC. Shaping the nuclear action of NF-kappaB Nat Rev Mol Cell Biol 2004 5392–401[CrossRef][Medline]
  31. Perkins DJ and Kniss DA. Rapid and transient induction of cyclo-oxygenase 2 by epidermal growth factor in human amnion-derived WISH cells Biochem J 1997 321677–681[Medline]
  32. Hayden MS and Ghosh S. Signaling to NF-kappaB Genes Dev 2004 182195–2224[Abstract/Free Full Text]
  33. Catley MC, Chivers JE, Cambridge LM, Holden N, Slater DM, Staples KJ, Bergmann MW, Loser P, Barnes PJ, Newton R. IL-1beta-dependent activation of NF-kappaB mediates PGE2 release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase FEBS Lett 2003 54775–79[CrossRef][Medline]
  34. NF-kappaB/Rel Transcription Factor Family Tergaonkar V, Li Q, Verma IM. Inhibitors of NF-kappaB activity: tools for treatment of human ailments 2006Austin, TX Springer US162–178 In:
  35. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5 Science 1997 2751320–1323[Abstract/Free Full Text]
  36. Sampey AV, Monrad S, Crofford LJ. Microsomal prostaglandin E synthase-1: the inducible synthase for prostaglandin E2 Arthritis Res Ther 2005 7114–117[CrossRef][Medline]
  37. Christian Overton, Technical Report CBIL-TR-1997–1001-v0.0 TESS: Transcription Element Search Software, Jonathan Schug and G. 1997 Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania World Wide Web (URL: http://www.cbil.upenn.edu/tess). (March, 2007)
  38. Maggirwar SJ, Harhaj EW, Sun S-C. Regulation of the interleukin-2 CD28-responsive element by NF-ATp and various NF-kappaB/Rel transcription factors Mol Cell Biol 1997 172605–2614[Abstract]
  39. Okamoto S, Mukaida N, Yasumoto K, Rice N, Ishikawa Y, Horiguchi H, Murakami S, Matsushima K. The interleukin-8 AP-1 and kappa B-like sites are genetic end targets of FK506-sensitive pathway accompanied by calcium mobilization J Biol Chem 1994 2698582–8589[Abstract/Free Full Text]
  40. Chen F, Demers LM, Vallyathan V, Lu Y, Castranova V, Shi X. Involvement of 5'-flanking kappaB-like sites within bcl-x gene in silica-induced Bcl-x expression J Biol Chem 1999 27435591–35595[Abstract/Free Full Text]
  41. Hong SY, Yoon WH, Park JH, Kang SG, Ahn JH, Lee TH. Involvement of two NF-kappa B binding elements in tumor necrosis factor alpha-, CD40-, and Epstein-Barr virus latent membrane protein 1-mediated induction of the cellular inhibitor of apoptosis protein 2 gene J Biol Chem 2000 27518022–18028[Abstract/Free Full Text]
  42. Romero R, Emamian M, Quintero R, Wan M, Hobbins JC, Mitchell MD. Amniotic fluid prostaglandin levels and intra-amniotic infections Lancet 1986 11380[Medline]
  43. Bowen JM, Chamley L, Keelan JA, Mitchell MD. Cytokines of the placenta and extra-placental membranes: roles and regulation during human pregnancy and parturition Placenta 2002 23257–273[CrossRef][Medline]
  44. Keelan JA, Blumenstein M, Helliwell RJ, Sato TA, Marvin KW, Mitchell MD. Cytokines, prostaglandins and parturition—a review Placenta 2003 24(Suppl A)S33–S46[CrossRef][Medline]
  45. Haddad R, Tromp G, Kuivaniemi H, Chaiworapongsa T, Kim YM, Mazor M, Romero R. Human spontaneous labor without histologic chorioamnionitis is characterized by an acute inflammation gene expression signature Am J Obstet Gynecol 2006 195394e1–394e24
  46. Lindstrom TM and Bennett PR. The role of nuclear factor kappa B in human labour Reproduction 2005 130569–581[Abstract/Free Full Text]
  47. Lappas M and Rice GE. The role and regulation of the nuclear factor kappa B signalling pathway in human labour Placenta 2006 5–6543–556
  48. Allport VC, Pieber D, Slater DM, Newton R, White JO, Bennett PR. Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the ‘functional progesterone withdrawal.' Mol Hum Reprod 2001 7581–586[Abstract/Free Full Text]
  49. Yan X, Sun M, Gibb W. Localization of nuclear factor-kappa B (NF kappa B) and inhibitory factor-kappa B (I kappa B) in human fetal membranes and decidua at term and preterm delivery Placenta 2002 23288–293[CrossRef][Medline]
  50. Soloff MS, Cook DL Jr, Jeng YJ, Anderson GD. In situ analysis of interleukin-1-induced transcription of cox-2 and il-8 in cultured human myometrial cells Endocrinology 2004 1451248–1254[Abstract/Free Full Text]
  51. Mbonye UR, Wada M, Rieke CJ, Tang HY, DeWitt DL, Smith WL. The 19-amino acid cassette of cyclooxygenase-2 mediates entry of the protein into the endoplasmic reticulum-associated degradation system J Biol Chem 2006 28135770–35778[Abstract/Free Full Text]
  52. Elliott CL, Allport VC, Loudon JA, Wu GD, Bennett PR. Nuclear factor-kappa B is essential for up-regulation of interleukin-8 expression in human amnion and cervical epithelial cells Mol Hum Reprod 2001 7787–790[Abstract/Free Full Text]
  53. Sooranna SR, Engineer N, Loudon JA, Terzidou V, Bennett PR, Johnson MR. The mitogen-activated protein kinase dependent expression of prostaglandin H synthase-2 and interleukin-8 messenger ribonucleic acid by myometrial cells: the differential effect of stretch and interleukin-1{beta} J Clin Endocrinol Metab 2005 903517–3527[Abstract/Free Full Text]
  54. Masuko-Hongo K, Berenbaum F, Humbert L, Salvat C, Goldring MB, Thirion S. Up-regulation of microsomal prostaglandin E synthase 1 in osteoarthritic human cartilage: critical roles of the ERK-1/2 and p38 signaling pathways Arthritis Rheum 2004 502829–2838[CrossRef][Medline]
  55. Subbaramaiah K, Yoshimatsu K, Scherl E, Das KM, Glazier KD, Golijanin D, Soslow RA, Tanabe T, Naraba H, Dannenberg AJ. Microsomal prostaglandin E synthase-1 is overexpressed in inflammatory bowel disease. Evidence for involvement of the transcription factor Egr-1 J Biol Chem 2004 27912647–12658[Abstract/Free Full Text]
  56. Cogswell PC, Mayo MW, Baldwin AS Jr. Involvement of Egr-1/RelA synergy in distinguishing T cell activation from tumor necrosis factor-alpha-induced NF-kappa B1 transcription J Exp Med 1997 185491–497[Abstract/Free Full Text]
  57. Chapman NR and Perkins ND. Inhibition of the RelA(p65) NF-kappaB subunit by Egr-1 J Biol Chem 2000 2754719–4725[Abstract/Free Full Text]
  58. Kim JH, Kim WS, Kang JH, Lim HY, Ko YH, Park C. Egr-1, a new downstream molecule of Epstein-Barr virus latent membrane protein 1 FEBS Lett 2007 581623–628[CrossRef][Medline]
  59. Alfaidy N, Sun M, Challis JR, Gibb W. Expression of membrane prostaglandin E synthase in human placenta and fetal membranes and effect of labor Endocrine 2003 20219–225[CrossRef][Medline]
  60. Meadows JW, Eis AL, Brockman DE, Myatt L. Expression and localization of prostaglandin E synthase isoforms in human fetal membranes in term and preterm labor J Clin Endocrinol Metab 2003 88433–439[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
J. Frasor, A. E. Weaver, M. Pradhan, and K. Mehta
Synergistic Up-Regulation of Prostaglandin E Synthase Expression in Breast Cancer Cells by 17{beta}-Estradiol and Proinflammatory Cytokines
Endocrinology, December 1, 2008; 149(12): 6272 - 6279.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/1/68    most recent
biolreprod.107.061663v1
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