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Endocrinology Vol. 148, No. 4 1850-1857
Copyright © 2007 by The Endocrine Society

The Effect of Mechanical Stretch on Cyclooxygenase Type 2 Expression and Activator Protein-1 and Nuclear Factor-{kappa}B Activity in Human Amnion Cells

Aarthi R. Mohan, Suren R. Sooranna, Tamsin M. Lindstrom, Mark R. Johnson and Phillip R. Bennett

Imperial College London (A.R.M., T.M.L., P.R.B.), Parturition Research Group, Institute of Reproductive and Developmental Biology, Hammersmith Hospital Campus, London W12 ONN, United Kingdom; and Department of Obstetrics and Gynaecology (S.R.S., M.R.J.), Imperial College, Chelsea Westminster Hospital, London SW10 9NH, United Kingdom

Address all correspondence and requests for reprints to: Dr. A. Mohan, Parturition Research Group, Institute of Reproductive and Developmental Biology, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, United Kingdom. E-mail: a.mohan{at}imperial.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stretch of the uterus plays a role in parturition. Uterine stretch also leads to stretch of the fetal membranes, including the amnion, an important source of prostaglandin E2 (PGE2). We tested the hypothesis that stretch of the amnion leads to increased cyclooxygenase (COX)-2 expression and PGE2 synthesis and investigated the mechanisms involved. We obtained amnion from women undergoing term elective cesarean section and isolated amnion epithelial cells. These cells were subjected to 11% static stretch. Stretch increased COX-2 expression and PGE2 production. EMSA studies showed that stretch increased both activator protein-1 (AP-1) and nuclear factor-{kappa}B (NF-{kappa}B) DNA binding at 1 and 6 h. In contrast, IL-1ß increased both AP-1 and NF-{kappa}B DNA binding at 1 h only. Chromatin immunoprecipitation studies confirmed that stretch increased binding of NF-{kappa}B to the COX-2 promoter in vivo. Stretch had no effect on inhibitory-{kappa}B{alpha} (I{kappa}B{alpha}) levels at the early time points but caused a decrease at 4 h. IL-1ß stimulation decreased I{kappa}B{alpha} levels after 30 min. MG132, a proteasome inhibitor, inhibited only the second stretch-induced increase in NF-{kappa}B binding. This suggests that stretch initially activates NF-{kappa}B via a nonclassical pathway, which does not involve the inhibitory-{kappa} kinase-induced degradation of I{kappa}B{alpha}. The second peak of NF-{kappa}B activation may be mediated by the classical mechanism. Stretch of the amnion may contribute to increased expression of COX-2- and other AP-1- and NF-{kappa}B-regulated genes with the onset of labor in the human.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ONSET OF human parturition is preceded by changes in the fetal membranes (1). There is degradation of the extracellular matrix (2) and physical separation of the amnion from the choriodecidua before membrane rupture (3). Many of the biochemical events involved in parturition resemble an inflammatory reaction (4). Human labor is associated with increased synthesis of prostaglandins (5) and inflammatory cytokines such as IL-1ß within the uterus (6). Prostaglandins, specifically prostaglandin E2, act to cause both cervical ripening and uterine contractions. IL-1ß increases both cytokine and prostaglandin synthesis (7) and probably acts to augment the labor process.

There is mounting evidence that stretch of the myometrium plays a physiological role in the onset of labor at term, and that overstretch of the uterus may contribute to the risk of preterm delivery (8). Studies in the sheep, rat, and wallaby have shown that stretch increases myometrial expression of cyclooxygenase (COX)-2 (9), the oxytocin receptor (9, 10), and connexin-43 (Cx-43) (11). Myometrial expression of the activator protein-1 (AP-1) family members Fos and Jun increase in the rat myometrium before the onset of labor secondary to mechanical stretch (12). AP-1 is a proinflammatory transcription factor that contributes to the initiation of the inflammatory response in disease states (13). AP-1 binding sites are found in the promoter regions of many proinflammatory genes, including cytokines, adhesion molecules, and cell proliferation growth factors. The AP-1 family of transcription factors function as either homodimers of the Jun family members or heterodimers of Fos and Jun family proteins. These different dimers have different DNA-binding and transcriptional activation characteristics. The oxytocin receptor, COX-2, and Cx-43 genes contain AP-1 binding sites in their promoters and temporal correlation has been demonstrated between the expression of c-fos and Cx-43 in rat myometrium during labor. Overexpression of c-Fos and c-Jun in myometrial cells activates the Cx-43 promoter through the AP-1 site (14). Stretch of human myocytes in vitro enhances the expression of OTR, COX-2, and IL-8 (15, 16, 17), which is also associated with activation of AP-1 (17).

Stretch of the uterus leads to not only myometrial stretch but also stretch of the fetal membranes, which adhere to the inner surface of the uterus. The fetal membranes enlarge rapidly by hyperplasia in the first half of pregnancy, but by the end of the second trimester, their growth has significantly slowed, as shown by a decline in mitotic rate (18). The fetal membranes do not line the uterine cavity passively; they are in a state of continuous stretch and tension during pregnancy. At term, the fetal membranes, in utero, are stretched to almost double their size after delivery, and more stretching will occur during uterine contractions (18, 19, 20). Human amnion is a major source of prostaglandin E2 whose synthesis increases in association with labor. Amnion contains high concentrations of the prostaglandin precursor, arachidonic acid. COX is the central enzyme in the synthesis of prostaglandins from arachidonic acid. Amnion expresses both constitutive COX-1 and inducible COX-2. The current evidence supports a major role for COX-2 in the onset of human parturition (21, 22). COX-2 mRNA and protein levels increase in amnion at term before and during labor (23, 24, 25). In term amnion, COX-2 mRNA is transcriptionally regulated and constitutively stable (26).

Expression of COX-2 in amnion has been shown to be dependent on activation of the nuclear factor-{kappa}B (NF-{kappa}B) transcription factor system (27, 28). NF-{kappa}B is activated in the amnion at the time of the onset of labor (28, 29). NF-{kappa}B functions as a homo- or heterodimer of the Rel family of proteins, of which there are five presently identified: NF-{kappa}B1 (p50 and its precursor p105), NF-{kappa}B2 (p52 and its precursor p100), p65 (RelA), cRel, and RelB. The most common combination is a heterodimer of the NF-{kappa}B proteins p50 and p65, and these appear to be the principal dimers involved in DNA binding in amnion (29). The transcriptional activity of the NF-{kappa}B Rel proteins is regulated by their association with members of the inhibitory molecule family, I{kappa}B. I{kappa}B{alpha} binding masks the nuclear localization signal of Rel proteins and therefore sequesters NF-{kappa}B in the cytosol. I{kappa}B{alpha} also prevents NF-{kappa}B from binding to DNA by masking its DNA-binding domain and leads to reexportation of NF-{kappa}B into the cytoplasm. Exposure of cells to certain stimuli, such as the cytokines, IL-1ß, and TNF or bacterial endotoxin, leads to phosphorylation of the I-{kappa} kinase enzymes and then to phosphorylation of I{kappa}B{alpha} with subsequent ubiquitination, followed by degradation via the 26S proteasome. This allows active NF-{kappa}B to translocate to the nucleus, in which it binds to its consensus sequences within the promoter regions of genes, thus activating transcription. The I{kappa}B{alpha} gene contains an NF-{kappa}B binding site within its promoter. Therefore, activation of NF-{kappa}B leads initially to degradation of I{kappa}B{alpha}, followed by resynthesis. Newly synthesized I{kappa}B{alpha} then causes DNA dislocation and export into the cytoplasm of NF-{kappa}B and limits the duration of the response (30).

The mechanism by which NF-{kappa}B is activated in amnion cells at the time of labor is currently unknown. It is possible that stretch of the fetal membranes may cause or contribute to activation of NF-{kappa}B in amnion at term. We therefore explored the hypothesis that stretch of the amnion, as with the myometrium, would increase expression of COX-2 and therefore synthesis of prostaglandins and investigated the effect of stretch on activation of AP-1 and NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue specimens
Intact fetal membranes were obtained from patients at elective cesarean section before labor at term (38–40 wk). Indications for term elective cesarean section were breech presentation, previous cesarean section, or maternal request. Institutional ethics committee approval was granted for the study and all patients gave informed consent.

Cell culture
Amnion was separated from the chorion-decidua and washed three times in PBS. The membrane was cut into strips and incubated in 0.5 mmol/liter EDTA (BDH, Poole, UK), for 15 min at 20 C. The strips were washed twice in PBS and then incubated in dispase (Life Technologies, Paisley, UK) 2.5 g/liter for 35 min at 37 C. Amnion epithelial cells were separated by vigorous shaking in DMEM, 10% fetal calf serum (Sigma, Poole, UK), for 3 min, the remaining strips were discarded, and the cell suspension pelleted at 175 g for 10 min. Cell pellets were resuspended in DMEM supplemented with 10% fetal calf serum 2 mmol/liter, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). This technique creates a cell culture containing 95–99% epithelial cells with only very few fibroblasts. The cells were cultured in 6-well flexible-bottomed culture plates precoated with collagen type I (Dunn Labortechnik, Asbach, Germany) in 2.5 ml DMEM medium. When cells were 95% confluent (d 3–4), medium was removed and replaced with 2.5 ml of fresh serum-free medium.

Quantitative RT-PCR
Total RNA was extracted and purified from amnion epithelial cells grown in 6-well flexible-bottomed culture plates using an RNeasy minikit from QIAGEN (Crawley, West Sussex, UK). After quantification 1.0 µg was reverse transcribed with oligo dT random primers using Moloney leukemia virus reverse transcriptase (Applied Biosystems, Warrington, Cheshire, UK). Paired oligonucleotide primers for amplification of human prostanoid receptors were designed using Primer Designer (Scientific and Educational Software, Durham, NC) against the sequence downloaded from GenBank. The primer sets used (Table 1Go) produced amplicons of the expected size and flanked intron/exon junctions. Assays were validated for all primer sets by confirming that single amplicons of appropriate size and sequence were generated. Quantitative PCR was performed in the presence of SYBR Green (QIAGEN), and amplicon yield was monitored during cycling in a RotorGene sequence detector (Corbett Research, Mortlake, Sydney, Australia) that continually measures fluorescence caused by the binding of the dye to double-stranded DNA. Pre-PCR cycle was 10 min at 95 C followed by up to 45 cycles of 95 C for 20 sec, 58–60 C for 20 sec, and 72 C for 20 sec followed by an extension at 72 C for 15 sec. The final procedure involves a melt over the temperature range of 72–99 C rising by 1 C steps with a wait for 15 sec on the first step followed by a wait of 5 sec for each subsequent step. The cycle at which the fluorescence reached a preset threshold (cycle threshold) was used for quantitative analyses. The cycle threshold in each assay was set at a level where the exponential increase in amplicon abundance was approximately parallel between all samples. All mRNA abundance data were expressed relative to the amount of the constitutively expressed glyceraldehyde-3-phosphatedehydrogenase (GAPDH). The r2 value for each curve and efficiency of each primer pair is summarized in Table 1Go. Conventional PCR was performed using Ampli-Taq Gold DNA polymerase (Applied Biosystems). Pre-PCR cycle was 10 min at 95 C followed by 35 cycles of 95 C for 1 min, 56–60 C for 1 min, and 72 C for 1 min followed by final extension 72 C for 10 min.


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  		TABLE 1. The primer pairs, GenBank accession numbers, and nucleotide sequence numbers for the primer pairs used

 
ELISA
The concentration of prostaglandin E2, IL-1ß, IL-8, and TNF{alpha} in medium collected from unstretched and stretched cells was measured using ELISA kits purchased from GE Healthcare (Milwaukee, WI), R & D Systems Ltd. (Minneapolis, MN), and BD Biosciences (Franklin Lakes NJ), respectively, according to manufacturers’ instructions.

Protein extraction
Monolayer amnion cells were scraped and lysed using a buffer containing 10 mM HEPES, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM dithiothreitol (DTT), 1% Nonidet P-40, and Complete protease inhibitor tablet (diluted according to manufacturer’s instruction; Roche, Stockholm, Sweden). Cytosolic protein extracts were obtained in the supernatant after centrifugation of the cell lysate for 30 sec at 12,000 g at 4 C. The pellet was resuspended in buffer containing 10 mM HEPES, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM DTT, 400 mM NaCl, 1% (vol/vol) Nonidet P-40, and Roche Complete protease inhibitor tablet. Samples were then shaken vigorously for 15 min on ice. Nuclear protein extracts were obtained in the supernatant after centrifugation for 5 min at 12,000 g at 4 C. All extracts were processed for protein quantitation by the Lowry method using protein assay reagents (Bio-Rad Laboratories, Hercules, CA) according to manufacturer’s instructions.

Western immunoblotting
Ten micrograms of protein were made up to a total volume of 10 µl with T-wash [0.1% Tween in PBS (PBS-T)] and added to an equal volume of Laemmli sample buffer containing ß-mercaptoethanol (5%) and denatured by boiling for 5 min. Proteins were then separated by SDS-PAGE (12–14% gels) and transferred onto polyvinyl difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were blocked overnight in 5% nonfat milk prepared in PBS-T buffer, at 4 C. The blots were incubated with the primary antibody in 1% nonfat milk in PBS-T buffer for 1 h and washed three times (10 min each) in PBS-T with vigorous shaking. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody (diluted 1:2000 in 1% nonfat milk in PBS-T buffer) for 1 h and washed three times (10 min each) in PBS-T. Signal detection was achieved using enhanced chemiluminescence (ECL Plus system) (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. To reprobe membranes, blots were incubated for 30 min in 50 C stripping buffer [2% sodium dodecyl sulfate, 62.5 mM Tris-HCl (pH 6.7), 100 mM 2-mercaptoethanol], washed 2 times in PBS-T, placed in blotto overnight, and then probed with a new antibody as above.

EMSA
Sense and antisense strands (175 nmol/ml each) were incubated in annealing buffer [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA] for 10 min at 65 C and allowed to cool at room temperature for 2 h. then 3.5 pmol double-stranded oligonucleotides were end labeled with 0.37 MBq 32P({gamma}ATP) by incubating for 30 min at 37 C with T4 polynucleotide kinase. Labeled oligonucleotides were recovered by centrifugation at 3000 rpm for 2 min through MicroSpin G-25 Sephadex columns (Amersham Biosciences, Piscataway, NJ). Four micrograms nuclear protein extracts were incubated on ice for 1 h with nonradiolabeled (cold) specific competitive and nonspecific competitive oligonucleotide (Oct-1) (1.75 pmol) in binding buffer with poly-dIdC to minimize nonspecific binding (4% glycerol, 1 mM MgCl2, 0.4 mM EDTA, 10 mM Tris-HCl, 50 mM NaCl, 0.4 mM DTT) and then incubated for 45 min on ice with a 0.035 pmol32P ({gamma}ATP)-end labeled oligonucleotide containing NF-{kappa}B or AP-1 consensus sequence (Promega Life Science, Madison, WI): NF-{kappa}B, 5'-GT TGA GGG GAC TTT CCC AGG C-3' and AP-1 (c-jun) 5'-CGC TTG ATG AGT CAG CCG GAA-3'. Oct-1 consensus sequence, 5'-TGT CGA ATG CAA ATC ACT AGA A-3' was used as a control for an NF-{kappa}B-specific effect. Nonradiolabeled oligonucleotides were at 200-fold excess to the 32P-labeled probes for specific and nonspecific competition for DNA binding. The resulting protein/DNA complexes were separated in a 4% acrylamide gel run at 250 V for 1 h. The gel was dried under vacuum for 1 h at 80 C and exposed to x-ray film. For supershift analysis, samples were preincubated with 2 µg of antibody for phospho-c-jun and p50 and p65 (Santa Cruz Biotechnology, Santa Cruz, CA) for 60 min before the addition of oligonucleotides, which confirmed specificity (Figs. 2BGo and 3CGo).


Figure 2
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  		FIG. 2. Amnion epithelial cells were either stretched (A) or stimulated with IL-1ß (C) (1 ng/ml) for up to 6 h. AP-1 DNA binding was measured by EMSA in nuclear protein extracts from cells, 4 µg protein loaded. Values are presented as ratios of nonstretched cells, mean + SEM, n = 4 (*, P < 0.05). Nuclear protein extract was also incubated with a cold oligonucleotide containing a consensus AP-1 DNA binding sequence as well as with an oligonucleotide containing a consensus Oct-1 DNA binding sequence as controls, and supershift by anti-phospho-c-jun antibodies, were also used to confirm specificity.

 

Figure 3
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  		FIG. 3. Amnion epithelial cells were either stretched (A) or stimulated with IL-1ß (C) (1 ng/ml) for up to 6 h. NF-{kappa}B DNA binding was measured by EMSA in nuclear protein extracts from cells, 4 µg protein loaded. Values are presented as ratios of nonstretched cells, mean + SEM, n = 4. Nuclear protein extract was also incubated with a cold oligonucleotide containing a consensus NF-{kappa}B DNA binding sequence as well as with an oligonucleotide containing a consensus Oct-1 DNA binding sequence as controls. Supershift by anti-phospho-c-jun antibodies was also used to confirm specificity. Western blot analysis of cytosolic protein extracts, 40 µg protein loaded, from amnion epithelial cells was conducted. Cells were either subjected to 11% stretch for up to 6 h (B) or incubated with IL-1ß (1 ng/ml) (E) for up to 6 h, after which cytosolic protein was extracted and expression of I{kappa}B{alpha} was assessed by Western analysis (n = 4, representative blot shown). Membranes were probed with antibodies against I{kappa}B{alpha} and ß-actin. ß-Actin was used to confirm equal protein loading. EMSA analysis of nuclear protein extracts from nonstretched cells using consensus NF-{kappa}B probe. MG132 (40 µM) was added 1 h before stimulation by either stretch (11%) for up to 6 h (F) or IL-1ß (1 ng/ml) for 15 min (G) (n = 4, representative blots shown).

 
Chromatin immunoprecipitation (ChIP) assay
DNA-protein complexes were cross-linked in situ with 1% formaldehyde. The cells were then lysed and chromatin was sheared into 200- to 1000-bp fragments by sonication. Antibodies recognizing the C-terminal of p65 or acetyl-histone H4 were used for immunoprecipitation, and the chromatin fragments containing the cross-linked protein were purified by immunoadsorption and elution from protein A/G beads. The cross-links were reversed and the DNA was purified using a QIAquick nucleotide removal kit (QIAGEN Inc., Valencia, CA). The DNA region cross-linked to the protein was determined by PCR analysis and primers were designed to amplify three putative NF-{kappa}B DNA binding sites: NF-{kappa}B-1, –214/223; NF-{kappa}B-2, –439/448; NF-{kappa}B-3 –1768/88. For primers sequences, see Table 1Go.

Statistical analysis
Differences between COX-2 to GAPDH mRNA, IL-1ß to GAPDH mRNA, and IL-8 to GAPDH mRNA ratios from unstretched and stretched cells were assessed by Wilkoxin signed ranks test (nonparametric test for related samples) using SPSS (version 10.0; Chicago, IL) Kolmogorov-Smirnov and Shapiro-Wilk statistics were used to determine normality of each sample pair. Differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stretch of human amnion cells increases expression of COX-2 and synthesis of prostaglandin E2
Expression of COX-2 mRNA was increased by stretch and this was significant at all time points (P < 0.05, Fig. 1AGo). Synthesis of COX-2, measured by Western analysis, increased at 60 min and remained elevated for 6 h (P < 0.05, Fig. 1BGo). Prostaglandin E2 concentrations in the culture supernatant, measured by ELISA, increased 8-fold after 2 h and 10-fold after 6 h (P < 0.05, Fig. 1CGo).


Figure 1
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  		FIG. 1. A, Expression of COX-2 mRNA measured by real-time RT-PCR in amnion cells after stretch (n = 8). Results are corrected for GAPDH expression and given as a ratio to nonstretched samples (*, P < 0.05). B, Western blot analysis of cytosolic protein extracts, 40 µg protein loaded, from unstimulated or stretched cells were subjected to 11% stretch for up to 6 h (n = 4). Membranes were probed with antibodies against COX-2, and results are shown as a ratio to nonstretched cells (*, P < 0.05). C, Prostaglandin E2 concentrations were measured in the amnion culture supernatant by ELISA, after stretching for up to 6 h. Results are mean + SEM (n = 4) (*, P < 0.05).

 
Both IL-1ß stimulation and stretch of human amnion cells increase AP-1 DNA binding
AP-1 DNA binding was seen in cells before incubation with IL-1ß or to stretch. Stretch of amnion cells was associated with an early increase (15 min) in AP-1 DNA binding; this decreased slightly after 4 h after which there was a second increase in DNA binding at 6 h (Fig. 2AGo). Supershift using antibodies to phospho-c-jun confirmed the presence of AP-1 (Fig. 2BGo). Stimulation with IL-1ß resulted in a time-dependent increase in AP-1 DNA binding, peaking at 2 h and declining thereafter (Fig. 2CGo).

Both IL-1ß stimulation and stretch of human amnion cells increase NF-{kappa}B DNA binding
NF-{kappa}B DNA binding was seen in cells before incubation with IL-1ß or to stretch. Stretch of amnion cells was associated with an early increase in NF-{kappa}B DNA binding (15 min), which peaked at 1 h, decreased slightly at 4 h, and increased again at 6 h (Fig. 3AGo). Supershift using antibodies to p65 and p50 confirmed the presence of NF-{kappa}B (Fig. 3CGo). Stimulation with IL-1ß resulted in a time-dependent increase in NF-{kappa}B DNA binding, with an early increase at 15 min, reaching a maximum at 1 h and then decreasing but persisting to 6 h (Fig. 3DGo).

Early activation of NF-{kappa}B by stretch is independent of I{kappa}B{alpha} degradation
IL-1ß caused degradation of I{kappa}B{alpha} by 30 min followed by resynthesis leading to restoration of I{kappa}B{alpha} expression by 2 h (Fig. 3EGo). Stretch had no effect on I{kappa}B{alpha} expression at earlier time points between 15 min and 2 h (Fig. 3BGo). There was a decrease in expression seen at 4 h, which had returned to prestretch levels by 6 h. This suggested that the two peaks of NF-{kappa}B DNA binding that were induced by stretch may be through different mechanisms. The first peak, at 1 h, appears to be independent of I{kappa}B{alpha} degradation; however, the decrease in I{kappa}B{alpha} concentrations at 4 h suggests that the later peak of NF-{kappa}B DNA binding may be at least in part dependent on I{kappa}B{alpha} degradation. To further confirm the role of degradation of I{kappa}B{alpha}, experiments were repeated in the presence and absence of MG132 (40 µM), which inhibits the 26S proteasome and therefore prevent I{kappa}B{alpha} degradation. MG132 completely inhibited the increase in DNA binding at 15 min stimulated by IL-1ß, had no effect on the stretch induced early increase in DNA binding at 2 h, but did inhibit the second peak of DNA binding at 6 h (Fig. 3FGo).

Stretch causes binding of NF-{kappa}B to the COX-2 promoter in vivo
In both stretched and unstretched cells, the H4 antibody precipitated DNA fragments detected by PCR primers for each of NF-{kappa}B-1, NF-{kappa}B-2, and NF-{kappa}B-3 sites. The p65 antibody precipitated DNA fragments detected by PCR primers for NF-{kappa}B-2 in both unstretched and stretched cells but only in stretched cells for NF-{kappa}B-1 and NF-{kappa}B-3 (Fig. 4Go).


Figure 4
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  		FIG. 4. ChIP analysis of three NF-{kappa}B binding sites in the COX-2 promoter after 6 h of stretch (11%), compared with nonstretched samples. Samples without an antibody (Ab) were used as negative controls, and samples with antibodies to acetylated histone H4 were used as positive controls.

 
Stretch increases IL-1ß mRNA at 4 and 6 h only but has no effect on IL-8 mRNA
IL-1ß mRNA levels were measured after increasing times of stretch, varying from 15 min to a 6-h stretch. There was a marked increase at the 4- and 6-h stretch time points but not at the earlier times (P < 0.05, Fig. 5AGo). However, there was no increase in IL-8 expression at any of the stretch time points (Fig. 5BGo). We attempted to perform Western analysis for both IL-1ß and IL-8 but were unable to obtain adequate blots. Very low levels of IL-1ß, TNF{alpha}, and IL-8 were detected by ELISA in the supernatant, which did not increase with stretch.


Figure 5
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  		FIG. 5. Expression of IL-1ß mRNA (A) and IL-8 mRNA (B) measured by real-time RT-PCR in amnion cells after stretch. Results are corrected for GAPDH expression and given as a ratio to nonstretched samples (n = 8, *, P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data show that stretch of human amnion epithelial cells causes an increase in the expression of COX-2 and prostaglandin E2 synthesis and that this is associated with activation of both AP-1 and NF-{kappa}B transcription factor systems. We have previously shown that NF-{kappa}B and AP-1 are essential for IL-1ß-stimulated COX-2 mRNA expression in amnion (28). Stretch activates both NF-{kappa}B and AP-1, leading to an increase in COX-2 mRNA expression, COX-2 protein synthesis, and prostaglandin E2 release.

We have shown that activation of NF-{kappa}B by IL-1ß in amnion is by the classical mechanism, dependent on degradation of I{kappa}B{alpha} and sensitive to MG132. Early activation of NF-{kappa}B by stretch was not, however, associated with degradation of I{kappa}B{alpha} and was insensitive to MG132. The second peak of increased NF-{kappa}B DNA binding induced by stretch was associated with degradation of I{kappa}B{alpha} and was inhibited by MG132. This suggests that the second peak of DNA binding may be stimulated by the synthesis of cytokines such as IL-1ß and/or TNF{alpha}. When we measured mRNA levels of IL-1ß with increasing times of stretch, we found an increase in IL-1ß mRNA at 4 and 6 h of stretch, although in contrast to the fall in I{kappa}B{alpha}, which was consistent in all samples, changes in IL-1ß expression was variable. It is possible that the first peak of NF-{kappa}B activation by stretch causes an increase in IL-1ß, which induces a second peak in NF-{kappa}B activation at the 6-h stretch. However, we found no increase in the supernatant levels of either IL-1ß or TNF{alpha} in response to stretch. This may be because the levels of IL-1ß and TNF{alpha} in the supernatant are too small to detect using the ELISA. IL-8 has also been reported to activate NF-{kappa}B through the classical pathway and others have reported that stretch of other cell types induces IL-8 release (31). However, we did not find any increase in IL-8 at the mRNA or protein level, suggesting that it is not responsible for the second peak in NF-{kappa}B activation. Nemeth et al. (32) have shown that stretch of intact fetal membranes leads to increased IL-8 synthesis, suggesting that it is cells other than amnion epithelial cells which respond in this way.

The primary effect of stretch on NF-{kappa}B activation in amnion cells is therefore nonclassical. It is unlikely that stretch activates NF-{kappa}B by the alternate pathway through which p100 is metabolized to p52, which then dimerizes with RelB because we did not show supershift on EMSA studies with antibodies to p52 or RelB. Atypical activation of NF-{kappa}B may involve the direct phosphorylation of the NF-{kappa}B subunits. Recently the previously simple paradigm of NF-{kappa}B activation via I{kappa}B{alpha} degradation has dramatically changed. NF-{kappa}B is a series of potentially multisite phosphorylated, acetylated, and ubiquitinated proteins. Changes in the phosphorylation status may be induced by various kinases and may change the dynamics of NF-{kappa}B DNA binding. Stretch has been shown to activate NF-{kappa}B in several cell types including fibroblasts, osteoblasts, and pulmonary artery smooth muscle cells (33, 34, 35). In a mouse model of asthma, mechanical stretch of lung parenchyma activates NF-{kappa}B and AP-1, at least in part, through the activation of MAPK signaling pathways (36), and we have shown MAPK activation by stretch in myometrial cells (37).

A similar biphasic effect of stretch on AP-1 DNA binding was seen with an early increase in DNA binding seen at 15 min and a second increase at 6 h of stretch. A similar biphasic response in AP-1 binding was seen in bovine aortic endothelial cells exposed to unidirectional shear stress in laminar flow (38). Interestingly, AP-1 and NF-{kappa}B binding was induced by cyclical stretch of human umbilical vein endothelial cells (39). The authors reported a biphasic response for cAMP response element but not either AP-1 or NF-{kappa}B; however, review of the published figures suggests that there may be a biphasic response for both AP-1 and/or NF-{kappa}B, consistent with our data. In our study, the character of AP-1 induction was very similar to NF-{kappa}B. We have previously shown that stretch activates AP-1 in myometrial cells, but we used only two time points, so we cannot comment on the nature of the response (16).

ChIP studies showed NF-{kappa}B binding to the NF-{kappa}B-2 site in the COX-2 promoter both with and without stretch. Stretch stimulated binding to the NF-{kappa}B-1 and NF-{kappa}B-3 sites. Unstimulated binding of NF-{kappa}B to the NF-{kappa}B-2 site is probably the cause of basal NF-{kappa}B activity and COX-2 expression that we find in amnion cells without stimulation. Soloff et al. (40) found no binding of NF-{kappa}B to the COX-2 promoter in unstimulated myometrial cells. A fundamental difference between amnion and myometrium is the basal unstimulated NF-{kappa}B activity in amnion cells at term.

The maximum increase in prostaglandin E2 concentrations occurred between 1 and 2 h. The lack of any further significant increase may have been due to either degradation or metabolism of prostaglandin E2 or exhaustion of arachidonic acid precursor. The culture medium in these studies did not contain arachidonic acid however, in vivo, the amnion epithelium may replenish substrate supplies upon taking arachidonic acid from the amniotic fluid. Kanayama and Fukamizu (41) described a 9-fold increase in prostaglandin E2 levels in the media from primary cultures of amnion epithelial cells after 1 h of stretch. These experiments were performed in cells grown in different conditions with a different mechanism of stretch application and so cannot be directly compared with our data. However, increased prostaglandin E2 synthesis at 1 h of stretch would be unexpectedly rapid, given the dynamics of COX-2 mRNA expression and protein synthesis.

In vivo, the increase in prostaglandin production by the fetal membranes appears to occur first in the region of the uterus overlying the cervix. In 1997 Van Meir et al. (42) found that there is regional loss of prostaglandin dehydrogenase, which acts to inactivate prostaglandin E2, in the fetal membranes in the lower uterine segment, which would facilitate local generation of bioactive prostaglandin E2 at this site, leading to cervical ripening. The principal site of prostaglandin dehydrogenase expression is in the chorion. Our own data relate only to amnion epithelial cells, but the fetal membranes consist of a range of cell types, each of which may respond to mechanical stimuli. This is an area that requires further exploration.

During the later stages of pregnancy, the fetal head engages in the pelvis, in which it acts to stretch the lower segment of the uterus and therefore to stretch the attached fetal membranes. Malpresentation (i.e. a transverse lie) or failure of engagement of the head at term is associated clinically with poor cervical ripening and an increase in the risk of dysfunctional labor. We hypothesize that pressure from the fetal head late in the third trimester of pregnancy causes stretch-induced activation of NF-{kappa}B and AP-1 in the amnion. There is a consequent increase in prostaglandin E2 synthesis via COX-2. There may also be increased IL-1ß synthesis, which has been shown to increase in the amniotic fluid of women with labor (6), which acts locally to inhibit chorion 15-hydroxyprostaglandin dehydrogenase (43) with the overall effect of increasing local prostaglandin E2 concentrations leading to cervical ripening.

We have previously shown that labor is associated with increased NF-{kappa}B activity in human amnion and that this occurs despite high levels of I{kappa}B{alpha} (29). Because stretch of amnion cells also causes activation of NF-{kappa}B without I{kappa}B{alpha} degradation, we hypothesize that stretch may contribute to increased NF-{kappa}B activity in amnion in vivo. In preterm labor, NF-{kappa}B and AP-1 could be activated by cytokines, stretch, or bacterial endotoxins via Toll-like receptors or a combination of all three. It is possible that it is stretch that causes the physiological activation of NF-{kappa}B and AP-1 within the uterus at term. Understanding how stretch activates labor associated genes may allow novel pharmacological targets for the prevention of preterm labor to be identified.


   Footnotes
 
This work was supported by Wellbeing for Women and Tommy’s Campaign.

Disclosure Summary: All authors have nothing to disclose.

First Published Online January 11, 2007

Abbreviations: AP-1, Activator protein-1; ChIP, chromatin immunoprecipitation; COX, cyclooxygenase; Cx-43, connexin-43; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; I{kappa}B, inhibitory molecule family; NF-{kappa}B, nuclear factor-{kappa}B; Oct-1, competitive oligonucleotide; PBS-T, Tween in PBS.

Received September 19, 2006.

Accepted for publication December 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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