Mol. Hum. Reprod. Advance Access originally published online on January 6, 2006
Molecular Human Reproduction 2005 11(12):853-858; doi:10.1093/molehr/gah194
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The role of CCAAT/enhancer-binding protein ß in the transcriptional regulation of COX-2 in human amnion
Imperial College Parturition Research Group, Institute of Reproductive and Developmental Biology, Hammersmith Hospital Campus, London, UK
1 To whom correspondence should be addressed at: Imperial College Parturition Research Group, Institute of Reproductive and Developmental Biology, Hammersmith Hospital Campus, London, UK. E-mail: yun.lee{at}imperial.ac.uk
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Abstract |
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Human labour is associated with increased prostaglandin synthesis within the uterus by the action of the inducible type-2 cyclo-oxygenase enzyme (COX-2). A major source of prostaglandin is the fetal membranes, in particular the amnion, in which expression of COX-2 increases in late pregnancy and with labour. The COX-2 gene promoter contains several putative transcription factor binding sites including those for NF-
B, AP-1 and C/EBP and therefore has the features of a rapid response gene. We have previously shown that, in amnion, the NF-B DNA-binding sites in the COX-2 promoter are essential for gene expression and that there is an increase in NF-B activity in amnion with the onset of labour. In this study, we demonstrate that in primary human amnion cells, CCAAT/enhancer-binding protein ß (C/EBPß) DNA-binding sites are crucial for the function of the COX-2 gene promoter. Three potential C/EBPß DNA-binding sites were identified within the COX-2 promoter which were shown to bind to C/EBPß but not to C/EBP
, C/EBP
, CREB (cAMP responsive element modulator) or CREM. Luciferase reporter constructs with site-directed mutagenesis of the three C/EBPß sites in the COX-2 promoter showed reduced expression of luciferase in transient transfection studies. However, comparison of C/EBPß protein levels and their DNA-binding activity from cells obtained before and after labour showed no significant differences. This suggests that although C/EBPß plays an essential constitutive role in the expression of COX-2, C/EBPß may not be directly involved in its regulation in association with human labour. Key words: Amnion/COX-2/C/EBPß/Labour
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Introduction |
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The onset of human labour is associated with an increase in synthesis of both prostaglandins (PGs) and inflammatory cytokines such as interleukin-1 beta (IL-1ß) and interleukin-8 (IL-8) within the uterus. PGs, specifically PG estradiol (E2) are required for both cervical ripening and fundally dominant myometrial contractions. Cyclooxygenase (COX), also known as PG endoperoxidase H synthase (PGHS), is the key enzyme in the biosynthetic pathway of PGs from arachidonic acid. The enzyme is bifunctional and has both COX activity and hydroperoxidase activity. There are two major isozymes called COX-1 and COX-2 which are found in mammalian tissues, although a third isoform, COX-3, which is a variant of COX-1, has been identified recently (Chandrasekharan et al., 2002
). COX-1 is generally considered to be a constitutive enzyme, whereas COX-2 is rapidly and transiently induced by various cytokines, hormones and tumour promoters (Herschman, 1996; Smith et al., 1996). For increased PG production in association with human labour it is the inducible type, COX-2 that is important (Sadovsky et al., 2000; Sawdy et al., 2000). A major source of uterine PGs in human is the amnion. It is within the amnion epithelial layer that the expression of COX-2 is found to increase exponentially with increasing gestational age to term and with a further doubling with the onset of labour (Slater et al., 1995). IL-1ß is one of the putative stimulants for labour onset. It has been shown that the PG synthesis from the amnion, decidua and myometrium is stimulated by IL-1ß production, which may be induced by bacterial infection (Mitchell et al., 1991) but which also increases in women at term with no clinical signs of infection (Romero et al., 1990).
The promoter of the human COX-2 gene contains several putative transcription factor binding sites including a cyclic AMP response element (CRE), and binding sites for NF-IL6 [CCAAT/enhancer-binding protein ß (C/EBPß)], AP-1, AP-2, SP-1 and NF-B (Kosaka et al., 1994). Among these factors CRE, C/EBPß and NF-B were shown to act as positive regulators for COX-2 transcription in various cell types (Sirois and Richards, 1993; Xie et al., 1994; Newton et al., 1997; Potter et al., 2000). C/EBPß is a member of CCAAT/enhancer binding protein, a family of transcription factors containing a highly conserved, basic region/leucine zipper domain at the C-terminus that is involved in dimerization and DNA binding. At least five more members of the family have been isolated and characterized to date (C/EBP–C/EBP
) with further diversity produced by the generation of different sized polypeptides, predominantly by differential use of translation initiation sites, and extensive protein–protein interactions both within the family and with other transcription factors. C/EBPß was originally identified because of its inducibility by IL-6 or by IL-1 in human hepatoma cells and in glioblastoma cell line, respectively (Akira et al., 1990; Poli et al., 1990) and is therefore also referred as NF-IL6. It has been subsequently determined by many independent studies that both C/EBPß and C/EBP are strongly up-regulated at the transcriptional level by inflammatory stimuli including bacterial lipopolysaccharide and by cytokines such as IL-6, IL-1 and tumour necrosis factor (TNF) (Akira et al., 1990). C/EBPß is involved in the regulation of a variety of genes including those coding for acute phase proteins. C/EBPß mRNA can cause three isoforms. These are the liver-enriched activatory protein with a molecular weight of 38 kDa (LAP*), 35 kDa (LAP) and the inhibitory protein of 20 KDa (LIP), with the LAP and the LIP forms being the major polypeptides produced in cells. LIP lacks the trans activation domains that are present in LAP, therefore can act as a potent repressor of LAP-induced transcriptional activation (Descombes and Schibler, 1991).
Potter et al. (2000) have previously reported studies of the role of the COX-2 C/EBP DNA-binding sites in an immortalized amnion-derived cell line, but these studies used only a 203 bp length reporter construct. We have previously shown that the full function of the COX-2 promoter requires over 2 kb of promoter sequence and that regulation of the promoter is different between primary amnion cells and immortalized amnion-derived cell lines (Allport et al., 2000, 2001) Therefore, in this study we have investigated the possible role of C/EBPß in the regulation of COX-2 gene expression in the context of the full promoter sequence in human primary amnion cells.
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Materials and methods |
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Cell preparation
Fetal membranes were obtained from elective caesarean section (L–) before labour at term or after spontaneous vaginal delivery at term (L+) where term was defined as 37–42 completed weeks of pregnancy. Institutional ethics committee approval was granted for the study, and patients gave informed consent. Amnion cells were prepared from tissue as previously described (Bennett et al., 1987
). In brief, amnion was separated from the chorion and washed in phosphate-buffered saline (PBS). The membrane was cut into strips and incubated in 0.5 mmol/l EDTA (BDH Laboratory Supplies Ltd), for 15 min at room temperature. The strips were rinsed twice in PBS followed by incubation in Dispase (Life Technologies, Paisely, UK) 2.5 g/l for 40 mins at 37°C. Amnion epithelial cells were isolated by vigorous shaking for 3 min and strips removed. Individual cells were collected by centrifugation at 400g for 10 min and grown 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) at 5% CO2.
Nuclear protein extracts
Monolayer amnion cells were lysed in buffer containing 10 mM HEPES, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 mM DTT (1-4, Dithrothreitol), 1% (v/v) NP-40 and complete protease inhibitor diluted according to manufacturer’s instruction (Roche). Cytosolic protein extracts were obtained by the centrifugation of the cell lysate for 30 s at 12 000 g at 4°C. The nuclear extracts are then attained by resuspending the pellets in buffer containing 10 mM HEPES, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 m MDTT, 400 mM NaCl and 1% (v/v) NP-40 with complete protease inhibitor as above. Samples were shaken vigorously for 15 min on ice followed by centrifugation at 12 000 g for 5 mins at 4°C. Supernatant which contains the nuclear extracts are then transferred and stored at –80°C.
Electromobility shift assay
Oligonucleotides used were synthesized commercially (Thermo Hybaid, Ulm, Germany) as single strands, and the corresponding complementary strands were annealed in a buffer containing 10 mM Tris-Cl, pH7.5 and 100 mM NaCl and 1 mM EDTA. Double-stranded oligonucleotides were labelled with [
-32P] ATP with T4 polynucleotide kinase. 5-µg nuclear protein were used in binding reaction as described previously (Dignam et al., 1983). Specificity was determined by competition with 100-fold excess of respective non-labelled oligonucleotide. Supershift analysis was performed by addition of C/EBPß antibody (SC 7962X, Santa Cruz, CA, USA), C/EBP antibody (SC9314X Santa Cruz, CA, USA), C/EBP antibody (SC633X Santa Cruz, CA, USA) or CREB/CREM antibody (SC633X Santa Cruz, CA, USA) on ice 60 min before addition of labelled probe. Electrophoresis was carried out on a 4.5% non-denaturing acrylamide gel in 0.25x Tris–borate–EDTA (TBE) for 2 h at 200 V. The gel was dried under vacuum for 1 h at 80°C and protein–DNA complexes were visualized by enhanced chemiluminescence (ECL).
Transient transfections
Amnion cells from were grown in 24-well plates to 80–85% confluence, and the transfections were achieved using the liposome-mediated Transfast transfection reagent (Promega, London, UK), prepared according to the manufacturer’s instruction. Transfections used a charge ratio of 3:1, 1.0 µg of DNA per well in triplicates with 1 h incubation period. CMV-Renilla vector (Promega, London, UK) (1/10 of reporter) was used as control for transfection efficency. Cells were cultured for total of 48 h followed by analysis with a dual firefly/renilla luciferase assay (Luclite, Packard Biosciences, Groningen, NL, USA and Coelenterazine CN Biosciences, CA, USA).
Site directed mutagenesis
The COX-2 promoter construct, PGL3.C2.2.luc, containing the promoter fragment (–2375/+43) in PGL3 basic vector was used to create 3 individual mutations in C/EBPß DNA-binding sites (Table I). QuickChange Site-Directed Mutagenesis Kit (Stratagene) was used following the manufacturer’s instructions. The mutagenic oligonucleotide primers were designed individually, and the introduced mutations were verified with EMSA (Electromobility shift assay) to ensure that the oligonucleotides did not contain any specific DNA-binding sequence.
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Western analysis
Protein samples (30 µg) were denatured by boiling for 5 min and run on a 10% SDS-polyacrylamide gel for 60 min at 140 V, followed by a transfer to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, New Jersey, USA). The membrane was blocked in 5% milk protein solution (Marvel Lincs, UK) overnight, washed and hybridized with the primary antibody for 1 h at room temperature in a fresh blocking buffer (1x PBS, 1% milk protein and 0.1% Tween-20) containing mouse anti-human C/EBPß (SC7962 Santa Cruz Biotechnology, Santa Cruz, CA, USA). This process was repeated with the secondary antibody also obtained from Santa Cruz. For ECL detection of horse-radish peroxidase, ECL Plus from Amersham Biosciences was used. Exposure for detection was at 25°C for 1 min. To confirm equal loading of each well, the blot membrane was treated with a stripping buffer (2% SDS, 62.5 mM Tris–HCl, pH 6.7 and 100 mM 2-mercaptoethanol) for 30 min at 50°C, washed in PBS-T, and then preblocked and reprobed with an antibody to human ß-actin. To confirm good nuclear/cytosolic separation a single representative sample was also subjected to western analysis for Lamin B1 (Calbiochem NA12) whose expression is limited to the nucleus.
Statistical analysis
Multiple comparisons were analysed using a one-way analysis of variance (ANOVA), and the Bonferroni/Dunn test was used to determine variation within each group. Comparison of IL-1ß with non-stimulated (NS) effects were analysed using t-test. Differences with probability value of P 
0.05 were considered to be statistically significant.
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Results |
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Using ‘TFSEARCH’ (www.cbrc.jp/htbin/nph-tfsearch), six putative C/EBPß DNA-binding sites were identified in the region of the COX-2 promoter 0 to –650. Preliminary EMSA studies (data not shown) demonstrated that three of these sites did not bind amnion cell nuclear proteins (-974 to –963, –1600 to –1587 and –1683 to –1871). The remaining three, which did bind nuclear protein, were therefore selected for further study (Figure 1). These were at –63 to –50, 5'-ACAGTCATTTCGTCACATGGGC-3' (C/EBPß1); –135 to –122, 5'-ACCGGGCTTACGCAATTTTTTT-3' (C/EBPß2) and –543 to –530: 5'-GTCAGCCTTTCTTAACCTTACT-3' (C/EBPß3).
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Preliminary transfection experiments using a B-gal reporter vector showed transfection efficiency to be 60–80%. In subsequent experiments, variations in transfection efficiency were controlled for by co-transfection of a CMV-Renilla vector, analysis with a dual firefly/renilla luciferase assay. Firefly luciferase reporter construct activity was expressed as a ratio to that of CMV-Renilla. To determine the COX-2 gene transcriptional activity of these C/EBPß sites in association with labour, amnion cells were transiently transfected with site directed mutants of each of the three C/EBPß DNA-binding sites (Figure 2). Reporter expression was compared with that of the wild type C2.2 construct which contains 2.2 Kb of the COX-2 promoter linked to the luciferase reporter gene in the pGL3 vector (Promega). C/EBPß1 mutant (–63 to –50) showed greater than 50% reduction in reporter expression in both pre- and post-labour amnions. C/EBPß2 mutant (–135 to –122) showed a smaller reduction of 20%, but the most significant reduction in reporter expression was seen in C/EBPß3 mutant (–543 to –530) where there was greater than 90% decrease in COX-2 promoter activity (Figure 2A and B).
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EMSA analysis was carried out to confirm that the three putative C/EBPß binding sites identified earlier did bind C/EBPß and to determine the amount of protein available for DNA binding. EMSA demonstrated that there was binding of C/EBPß to all three putative sites verified by supershift with the antibody to C/EBPß (Figure 3A, B and C). In nuclei from both pre-labour and post-labour cells, protein binding is increased by IL-1ß stimulation which can be seen more clearly in the supershifted band by C/EBPß antibody (Figure 3). However, there appeared no significant differences in general C/EBPß binding between nuclear extracts obtained from NS amnion cells before or after labour.
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Because the C/EBPß supershift antibody used in the EMSA studies is weakly cross-reactive with C/EBP and C/EBP, supershift studies were repeated with antibodies to C/EBP and C/EBP. Furthermore, because the C/EBPß1 site has also been shown to act as a CRE, supershift studies were also performed, for each of the three sites, using an antibody which recognizes both CREB and CREM. No supershift was seen when using antibodies for C/EBP, C/EBP or CREB/CREM at any of the three sites.
Western analysis of nuclear and cytoplasmic protein extracted from NS and IL-1ß treated cells derived from both pre- and post-labour amnions showed the presence of C/EBPß LAP and LIP. C/EBPß was only present in the nucleus (Figure 4A). There were no differences in expression of C/EBPß between pre-labour and post-labour amnion cells. In both pre-labour and post-labour amnion cells IL-1ß stimulation increased the expression of C/EBPß (Figure 4B, D and E). Neither labour nor incubation with IL-1ß changed the ratio of C/EBPß LAP to LIP (Figure 4F).
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Discussion |
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Several investigators have shown that the increased synthesis of PG E2 seen in amnion in association with labour is due to increased expression of COX-2 (Teixeira et al., 1994
; Hirst et al., 1995; Slater et al., 1995; Sadovsky et al., 2000; Sawdy et al., 2000). Johnson et al. 2002 have shown that the COX-2 mRNA is stable in term human amnion and that its expression is regulated principally at the level of transcription. We have previously shown that COX-2 promoter activity in term human amnion is dependent upon binding of NF-B (Allport et al., 2001). We identified two NF-B sites in the COX-2 promoter. Mutation of the upstream site, (–448 to –439) which we have designated NF2 leads to almost complete inhibition of COX-2 promoter activity, whereas mutation of the downstream site (–223 to –214), designated NF1, reduces COX-2 promoter activity by 50%. We found a significant increase in NF-B activity in post-labour when compared to pre-labour amnion cells. This suggests that activation of NF-B is important in regulating the increased COX-2 expression seen at the time of labour.
Working with an immortalized amnion cell line, AV-3, Potter et al. (2000) suggested that the DNA region (–59 to –52), which they and others have designated as a CRE but which we have designated C/EBPß1, was critical to both basal and IL-1ß stimulated COX-2 promoter activity, that mutation of C/EBPß2 reduced promoter activity by 70%, whereas the NF-B DNA-binding sites were of no importance. However, these studies used promoter constructs containing only 203 bp of promoter region. We have previously reported (Allport et al., 2000) that, in the immortalized amnion cell line WISH (Wistar Institute Susan Hayflick), shortening of the COX-2 promoter from 2375 bp to 917 bp leads to a 50% reduction in activity. Although further shortening of the promoter to remove the two NF-B binding sites does not further effect reporter activity, mutation of the NF-B sites, in the context of the full 2375 bp does reduce promoter activity. This suggests us that studies of the ‘COX-2 promoter’ which use very short regions of promoter sequence may not be representative of genuine COX-2 promoter function. Therefore, in our current study, all of the C/EBPß binding sequence mutations have been performed in the context of 2375 bp of COX-2 promoter sequence. We also found that the importance of the two NF-B binding sites is different in WISH when compared to primary amnion epithelial cells.
Our present data show that C/EBPß appears important for COX-2 promoter activity in term human amnion but that there is no increase in C/EBPß expression, nuclear localization or DNA binding associated with the onset of labour. C/EBPß, therefore, appears critical for COX-2 promoter function but not to be a factor which regulates the increase in COX-2 expression seen in amnion at the time of labour.
We found that the COX-2 promoter requires the C/EBPß3 DNA sequence for its expression in human amnion at term. The C/EBPß1 site also appears important for full expression but the C/EBPb2 DNA sequence does not appear of critical importance for COX-2 promoter activity in primary amnion. Both our study and that of Potter et al. (2000) shows that C/EBP binds to the DNA region (–59 to –52, CRE or C/EBPß1). We have found that the C/EBPß2 region is of little importance in primary amnion whereas Potter et al. found it to be apparently of importance in AV-1 cells, but this was in the context of only 203 bp of COX-2 promoter and in an immortalized cell line.
C/EBPß mRNA can cause three isoforms. The activatory proteins (LAP*, LAP) have molecular weights of 38 kDa (LAP*) and 35 kDa (LAP), and the inhibitory protein LIP has a molecular weight 20 KDa. (Descombes and Schibler, 1991). Our data show that the 35 kDa LAP and 20 kDa LIP are expressed in human amnion epithelial cells and that expression is principally confined to the nucleus. Although there was no change in expression or nuclear localization associated with labour, IL-1ß stimulated an increase in nuclear concentrations and DNA binding in both pre- and post-labour amnion. However, the ratio of LIP : LAP remained unchanged. Whether the expression of the LIP isoform of C/EBPß plays any role in regulation of C/EBPß function in the amnion is unknown but our data would suggest that LIP does not play a role in either labour-associated or IL-1–stimulated increases in COX-2 expression.
This, and our previous studies (Allport et al., 2001), have shown that both C/EBPß and NF-B activity is stimulated by IL-1ß. Labour is associated with increased concentrations of IL-1ß within the uterus. We previously found a significant increase in NF-B activity in post-labour when compared to pre-labour amnion cells, but this was despite high levels of inhibitory IB protein which would be degraded by IL-1ß. Increased NF-B activity associated with labour is therefore probably independent of IL-1ß stimulation. We have not found any labour-associated differences in C/EBPß, activity which further supports the concept that, in normal term labour, IL-1ß does not play an important role in stimulation of COX-2 gene expression in the amnion. It remains possible that COX-2 may be up-regulated through IL-1ß stimulation of both C/EBPß and NF-B activity in the context of infection or inflammation.
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Submitted on April 15, 2005; accepted on May 24, 2005.
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