Molecular Human Reproduction, Vol. 9, No. 4, 213-218,
April 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
The effects of labour and of interleukin 1 beta upon the expression of nuclear factor kappa B related proteins in human amnion
Submitted on October 16, 2002; . accepted on December 16, 2002
1 Imperial College Parturition Research Group, Institute of Reproductive and Developmental Biology, Hammersmith Hospital Campus, DuCane Road, London W12 0NN and 2 Biomedical Research Institute, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
3 To whom correspondence should be addressed. e-mail: pbennett{at}ic.ac.uk
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ABSTRACT |
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Human labour is associated with persistently increased nuclear factor kappa B (NF-kB) activity in amnion. In this study we have shown that this involves only the p65 and p50 NF-kB subunits and is associated with an increase in the expression of p65 (P < 0.05), and of the NF-kB binding proteins IkBa, IkBb-1 and IkBb-2 (P < 0.05). Interleukin-1b stimulation leads to rapid degradation and resynthesis of IkBa within 2 h, and a decrease in IkBb-1 without a return to full expression by 2 h, but has little effect upon IkBb-2. IkBb-2 was found in both the cytosolic and nuclear protein fractions. These findings demonstrate that persistently increased NF-kB activity in amnion occurs despite increased expression of the inhibitory IkBa protein and is not mediated by persistant I-kappa kinase activity or inhibition of IkBa synthesis. The increased expression and nuclear localization of IkBb-2 suggests that its function may be to protect NF-kB from inactivation by IkBa and to maintain NF-kB-mediated gene transcription.
Key words: amnion/interleukin /labour/NF-kB/parturition
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Introduction |
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The onset of labour is associated with cervical remodelling, identified clinically as softening and effacement which preceeds fundally dominant myometrial contractility leading to progressive dilatation of the cervix and eventual delivery. There is an increase in synthesis of both prostaglandins and inflammatory cytokines such as interleukin-1b (IL-1b) and interleukin-8 (IL-8) within the uterus. Prostaglandins, specifically prostaglandin E2 (PGE2), act to cause both cervical ripening and uterine contractions. IL-8 is a chemokine that is a potent attractor and activator of neutrophils (Baggiolini et al., 1989) which release metalloproteinases leading to both fetal membrane and cervical remodelling (Osmers et al., 1992). PGE2 and IL-8 act synergistically to remodel the cervix (Denison et al., 1999). IL-1b concentrations also increases within the uterus with the onset of labour (Romero et al., 1990). The role of IL-1b appears to be to act as an augmentor of the labour process. IL-1b increases both IL-8 and prostaglandin synthesis (Brown et al., 1998).
The major uterine source of PGE2 is the amnion. Amnion contains the highest concentrations of the prostaglandin precursor, arachidonic acid. Although amnion contains both types 1 and 2 cyclo-oxygenase, prostaglandin synthesis in amnion is entirely via the inducible cyclo-oxygenase type 2 enzyme (COX-2) (Sadovsky et al., 2000; Sawdy et al., 2000). Expression of COX-2 increases with increasing gestational age to term and shows a further increase in association with labour onset (Slater et al., 1995). IL-1b and IL-8 are produced both in the chorion-decidua and within the amnion. Expression of both IL-1b and IL-8 increases in amnion in the third trimester in parallel with that of COX-2 (Elliott et al., 2001a).
The transcription factor nuclear factor kappa B (NF-kB) has been shown, in other cell types, to regulate IL-1b, IL-8 and COX-2 expression (Dokter et al., 1995; Newton et al., 1997; Wu et al., 1997). We have shown that, in amnion, the NF-kB DNA binding sites in the COX-2 and IL-8 promoters are essential for expression of those genes (Allport et al., 2000; Elliott et al., 2001b). We have also found that there is an increase in NF-kB activity in amnion with the onset of labour which persists in cell culture (Allport et al., 2001).
In many species the onset of labour is preceeded by the withdrawal of progesterone. Progesterone acts to inhibit myometrial contractility and cervical ripening. However, in humans there is no detectable progesterone withdrawal, although there is up-regulation of a range of pro-labour genes which are normally repressed by the presence of progesterone (Chwalisz et al., 1994; Challis et al., 2000). It has been suggested that these events may be mediated by changes in the function or expression of the progesterone receptor (PR) rather than by withdrawal of progesterone itself (Mitchell and Wong, 1993; Chaim and Mazor, 1998; Haluska et al., 2002). This may be termed ‘functional progesterone withdrawal’. Haluska et al. have shown that, in rhesus monkey, PR expression in amnion disappears at the time of labour. This may be an important mechanism of ‘functional progesterone withdrawal’. We have shown that the mutually negative interaction between NF-kB and PR described by Kalkoven et al. (1996) functions in amnion cells so that, as NF-kB activity increases PR transcriptional activity is inhibited (Allport et al., 2001). Activation of NF-kB in the amnion at the time of labour or by inflammatory cytokines in association with preterm labour may therefore act both to increase IL-8 and PGE2 synthesis and to contribute to ‘functional progesterone withdrawal’.
NF-kB functions as homo- or heterodimers of the Rel family of proteins, which includes p50, p65, c-Rel, p52 and RelB. The most common combination of subunits is a heterodimer of the p50 and p65 proteins. The transcriptional activity of the NF-kB Rel proteins is regulated by their association with members of the inhibitory molecule family, IkB. The IkB proteins share conserved ankyrin repeat motifs which allow their association with the Rel homology region of the NF-kB proteins. IkBa binding masks the nuclear localization signal of Rel proteins and therefore sequesters NF-kB in the cytosol. IkBa also prevents NF-kB from binding to DNA by masking its DNA-binding domain and leads to re-exportation of nuclear NF-kB into the cytoplasm. Exposure of cells to certain stimuli, such as the cytokines, IL-1b and tumour necrosis factor or bacterial endotoxin, leads to phosphorylation of the I-kappa kinase enzymes (IKK) and then to phosphorylation of IkBa with subsequent ubiquitination, followed by degradation via the 26 S proteasome. This allows active NF-kB to translocate to the nucleus, where it binds to its consensus sequences within the promoter regions of genes, thus activating transcription. The IkBa gene contains an NF-kB binding site within its promoter. Therefore, activation of NF-kB leads initially to degradation of IkBa, followed by resynthesis. Newly synthesized IkBa then causes displacement of NF-kB from DNA and export into the cytoplasm, limiting the duration of the response to stimuli (Baldwin, 1996).
We have shown that, in the amnion-derived WISH cell line, IL-1b stimulation leads to degradation of IkBa within 30 min, with resynthesis within 90 min. This leads to an increase in COX-2 expression which peaks at 90 min (Allport et al., 2000). The increased NF-kB activity which we have seen in primary amnion epithelial cells associated with labour persists in cell culture for at least 7 days (Allport et al., 2001). Persistent activation of NF-kB has been demonstrated in various other cell types to be associated either with persistent activation of IKK enzymes, increased degradation of IkBa or with protection of NF-kB from IkBa inhibition by an alternative IkB protein IkBb (Suyang et al., 1996; Hirano et al., 1998; Carter et al., 2001). Two isoforms of IkBb have been described, designated IkBb-1 and IkBb-2. To determine the mechanism of persistent NF-kB activation in amnion we have performed a series of experiments to examine the effects of labour, and of IL-1b upon the NF-kB system in amnion epithelial cells.
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Materials and methods |
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Tissue collection
Intact fetal membranes were obtained from patients at elective Caesarean section prior to labour either preterm (32–35 weeks, n = 5) or at term (38–41 weeks, n = 5), or following spontaneous labour onset and vaginal delivery at term (38–41 weeks, n = 5). Indications for preterm delivery were maternal hypertension, fetal growth restriction or both. Indications for term elective Caesarean section were breech presentation or previous Caesarean section. Institutional ethics committee approval was granted for the study and all patients gave informed consent. Amnion from the portion of membrane between the placental attachment and the rupture site was separated from chorion-decidua and protein extracted for Western analysis. For cell culture studies the entire amnion except that part overlying the placenta was used.
Western analysis
Total protein was extracted from tissue or cells using a standard high salt technique. Ten micrograms of protein was made up to a total volume of 10 µl with T-wash [0.1% Tween in phosphate-buffered saline (PBS)] and an equal volume of loading buffer added to each sample. Samples were denatured by boiling for 5 min, run on a 10% acrylamide gel and then transferred by electrophoresis to a nitrocellulose membrane. The membrane was blocked overnight at 4°C in 5% marvel (dried milk powder), washed and hybridized with the primary antibody for 1 h at 4°C in a 1% marvel solution. This process was repeated with the secondary horse radish peroxidase antibody and after washing, the ECL solution (ICN) was added and the membrane autoradiographed. Antibodies to p50, p65, IkBa and IkBb were obtained from Santa Cruz Inc. (USA). Expression levels were measured on Western blots by digital densitometry.
Cell culture preparation
Amnion cells were prepared from tissue as previously described (Bennett et al., 1987). In brief, the whole amnion, except for that part overlying the placenta, was separated from the chorion and washed in PBS. The membrane was cut into strips and incubated in 0.5 mmol/l EDTA (BDH), for 15 min, 20°C. The strips were washed in PBS and then incubated in Dispase (Life Technologies, Paisley UK) 2.5 g/l for 40 min at 37°C. Amnion epithelial cells were separated by vigorous shaking for 3 min and strips removed. Cell pellets were resuspended in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Sigma, Poole, UK), 2 mmol/l L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies). In previous studies we have found that this technique creates a cell culture containing 95–99% epithelial cells with only very few fibroblasts. IL-1b (R and D Systems, UK) was added to a final concentration of 1 ng/ml and incubated for 15, 30, 60 or 120 min.
Nuclear protein extracts
Extracts were prepared according to Dignam et al. (1983). In brief, amnion cells were washed in PBS before resuspending in Buffer A on ice for 10 min then centrifuged for 10 min at 14 000 g at 4°C. The pellet was resuspended in Buffer C and incubated on ice for 1 h, with agitation before centrifugation, 10 min at 14 000 g at 4°C. Nuclear proteins were resuspended in 4 volumes of Buffer D and stored at –80°C. Nuclear protein (50 µg) from a Burkitts lymphoma cell line (Nirmalwa, Santa Cruz) was used as a positive control.
Electrophoretic mobility shift assays (EMSA)
Nuclear protein (10–20 µg) was used in binding reactions as described previously (Dignam et al., 1983) with the consensus radiolabelled NF-kB 22mer oligonucleotide (Promega, Southampton, UK). Specificity was determined by competition with 100-fold excess non-radiolabelled probe. Supershift analysis was performed by the addition of 3 µg of antisera to p65, p50, p52, RelB or cRel, according to the manufacturer’s instructions (Santa Cruz), on ice, 90 min prior to labelled probe. Electrophoresis was performed on a 6% non-denaturing acrylamide gel in 0.25x tetrabromoethane. Gels were dried and protein–DNA complexes visualized by autoradiography.
Statistical analysis
Results were analysed using analysis of variance and Fishers protected least significant difference (PLSD) test.
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Results |
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We used EMSA analysis, using a consensus NF-kB oligonucleotide, to determine the amount of nuclear NF-kB available for DNA binding and to identify which NF-kB Rel proteins were present in the term amnion cell nucleus. EMSA studies showed the presence of NF-kB in the nuclear fraction in both pre- and post-labour cells. DNA binding of NF-kB was significantly greater in the post-labour than the pre-labour cells (Figure 1). We identified three specific bands on EMSA analysis (Figure 2). One of these was supershifted only by antibody to p65, one by antibodies to both p50 and p65 and one only by antibody to p50. Antibodies to each of the other NF-kB subunits (p52, cRel, RelB) did not produce supershifts. We therefore concluded that the only NF-kB transcription factor subunits active in amnion are p65 and p50.
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Using Western analysis of whole cell protein we found that expression of p50 showed a decreasing trend when tissue collected following elective Caesarean section before term (32–35 weeks, designated preterm tissue) was compared to term pre-labour and term post-labour tissue but this did not reach statistical significance (Figure 3). Expression of p65 increased with gestational age and with labour (Figure 3). Expression of the NF-kB inhibitory protein IkBa increased dramatically when preterm cells were compared to term cells but there was no difference in expression between term pre-labour and term post-labour cells (Figure 4). IkBb-2 was the predominant IkBb isoform expressed in amnion. There was an increase in expression of IkB-2 with gestational age and with labour (Figure 5). IkBb-1 expression was very low in preterm cells, again there was an increase in expression of IkB-1 with gestational age and with labour.
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We have previously shown that IL-1b at a concentration of 1 ng/ml increases COX-2 expression through activation of NF-kB in WISH cells (Allport et al., 2000). This concentration was therefore used in the present experiments. IL-1b treatment of both pre-labour and post-labour (data not shown) amnion cells in culture caused disappearance of IkBa protein, measured by Western analysis of total cell protein, at 15 and 30 min, with reappearance at 1 h and return to full levels of expression by 2 h (Figure 6). In both pre-labour and post-labour (data not shown) amnion cells IL-1b treatment appeared to cause a decrease in IkBb-1 expression without a return to full expression by 2 h (Figure 7). IL-1b increased NF-kB DNA binding as measured by EMSA analysis (Figure 2). To examine cellular localization of IkBa, IkBb-1 and IkBb-2, Western analysis was performed using nuclear and cyoplasmic fractions from freshly isolated amnion cells. IkBa and IkBb-2 were found predominantly in the cytoplasm with lesser amounts in the nucleus (Figures 8 and 9). Nuclear concentration of IkBb-2 and IkBa appeared to be greater in post-labour than in pre-labour cells. IkbB-1 was found only in the cytoplasm (Figure 9).
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Discussion |
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We have previously reported that labour is associated with an apparently persistent activation of NF-kB in the amnion. This mediates an increase in COX-2 expression which in turn leads to increased prostaglandin synthesis (Allport et al., 2000, 2001). There is also an increase in expression of IL-8 (Elliott et al., 2000, 2001a). Together these ‘inflammatory mediators’ act in the early biochemical events of labour to cause cervical ripening, and membrane remodelling. An increase in NF-kB activity may also contribute to ‘functional progesterone withdrawal’. The concept that increased NF-kB activity in amnion is persistent suggests that this may represent a ‘watershed’ or ‘point of no return’ event in the biochemistry of human labour beyond which there will be escalation in the production of cytokines and prostaglandins leading to clinical labour and delivery. The process will only be stopped by delivery of the placenta with its attached membranes.
Our finding that IkBa expression increases dramatically at term shows that persistent activation of NF-kB in the amnion is not due to increased degradation of IkBa. In amnion, IkBa appears to have an expression pattern similar to that seen with other NF-kB dependent genes such as IL-8, whose expression also increases with gestational age but not further with the onset of labour (Elliott et al., 2001a). In term amnion therefore IkBa is responding to, but not regulating NF-kB activity. Expression of COX-2 in amnion increases at term and, unlike that of IkBa or IL-8, increases again with the onset of labour (Slater et al., 1995; Elliott et al., 2001a). We have previously shown that although IL-1b can stimulate further nuclear translocation of NF-kB in post-labour amnion cells, it does not increase NF-kB transcriptional activity (Allport et al., 2001). This suggests that, whilst IkBa and IL-8 probably share very similar regulatory mechanisms with increased transcription via NF-kB being central, the regulation of the COX-2 is more complex.
Our finding of an increase in the expression of IkBb-2 and IkBb-1 in amnion with gestational age and labour suggests a role for these proteins in increasing NF-kB activity with labour. Suyang et al. (1996) found that, in B lymphocytes, stimulation with IL-1b or lipopolysaccharide leads initially to degradation of IkBa and IkBb and then to resynthesis of unphosphorylated IkBb which acts to protect NF-kB subunits from binding to IkBa. Newly synthesized IkBb did not block either the nuclear translocation or the transciptional activity of NF-kB and could be found both in the cytosol and in the nucleus. (Suyang et al., 1996). Similarly, DeLuca et al. (1999) reported that in HIV-1 infected myeloid cells persistent NF-kB activation was associated both with protection of NF-kB from IkBa by IkBb and with constitutive activation of the IKK complex. Because we can cause degradation and resynthesis of IkBa by stimulation with IL-1b, which is associated in both pre- and post-labour cells with further nuclear translocation of NF-kB, such constitutive activation of the IKK complex is not part of the mechanism of persistent activation of NF-kB in the amnion.
Hirano et al. (1998) have shown that IkBb exists in two splicing isoforms, designated IkBb-1 and IkBb-2. Both isoforms have highest affinity for p65 homodimers and also bind strongly to heterodimers of p65/p50, whereas IkBa interacts predominantly with heterodimers containing p50. IkBb-1 is degraded following stimulation by IL-1b whereas IkBb-2 is resistant to degradation. Hirano et al. (1998) found that in B cells IkBb-1 could be found in both nucleus and cytoplasm whilst IkBb-2 was confined to the cytoplasm. They suggested that the role of IkBb-2 in B cells is to act as a dominant negative to limit responsiveness to inducing agents. In contrast, we have found that, in amnion, it is IkBb-2 which is found both within the nucleus and the cytoplasm whilst IkBb-1 is confined to the cytoplasm. The decrease in the expression of p50 and increase in expression of p65 with gestational age and labour suggests that there would be, in amnion, an increase in p65/p65 homodimers and p65/p50 heterodimers at the expense of p65/p50 heterodimers and p50/p50 homodimers. These would bind with greater affinity to IkBb than to IkBa and would increase NF-kB transcriptional activity since p50/p50 homodimers are poor mediators of transcription. In amnion, newly synthesized IkBb-2 may bind to p65-containing dimers within the nucleus and the cytoplasm, both preventing inactivation by IkBa and allowing both nuclear trafficking and NF-kB transcriptional activity.
Expression of NF-kB-regulated genes such as COX-2, IL-1b and IL-8 increases in the myometrium and decidua with advancing gestational age and labour (Slater et al., 1995; 1999; Elliott et al., 2000). Studies of the regulation of NF-kB in these tissues are currently underway in our laboratory and by others. We would predict, however, that the labour-associated persistent activation of NF-kB which we have found in the amnion will not be found in maternal tissues within the uterus since these are not expelled at the end of labour. Several pharmaceutical companies now have IKK inhibitors in development for the management of inflammation. Other anti-inflammtory drugs have been found to have a role in prevention of preterm labour. Whilst in other tissues within the uterus stimulation of NF-kB may be through the classical IKK-related pathway, any pharmacological strategy for the inhibition of NF-kB in the prevention of preterm delivery will need to take account of the concept that inhibition of IKK will probably not inhibit expression of NF-kB responsive labour-associated genes in the amnion. Lappas et al. (2002) have shown that agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma) will inhibit lipopolysaccharide-induced NF-kB activation in human amnion. PPARgamma has been shown to inhibit NF-kB through mechanisms which are independent of IKK activity and nuclear translocation of NF-kB. Pharmacological agonists of PPARgamma may therefore represent an attractive direction of research.
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Acknowledgements |
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We acknowledge the assistance of Dr Robert Newton in undertaking the EMSA studies described in this work. We are grateful to Wellbeing who funded this study.
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