Molecular Human Reproduction, Vol. 6, No. 6, 561-565,
June 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
NF-
B and AP-1 are required for cyclo-oxygenase 2 gene expression in amnion epithelial cell line (WISH)
1 Imperial College School of Medicine, Institute of Obstetrics and Gynaecology, Queen Charlotte's and Chelsea Hospital, Goldhawk Road, London W6 OXG, and 2 National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK
Abstract
Prostaglandins are known to play an important role in human labour and are used clinically to induce labour onset. Cytokines, e.g. interleukin 1 ß (IL-1ß), are up-regulated in the amniotic fluid late in gestation and can increase prostaglandin production through the expression of cyclo-oxygenase 2 (COX-2), the prostaglandin synthetic isoform involved in human labour. We demonstrate in immortalized amnion epithelial (WISH) cells, that IL-1ß causes increased transcription of the COX-2 gene. Luciferase reporter constructs with site-directed mutagenesis of the two NF-
B sites and an AP-1 site in the COX-2 promoter showed reduced expression of luciferase in transient transfection studies. This suggests that the binding of transcription factors to these sites is essential for the regulation of COX-2 transcription in IL-1ß-treated WISH cells.
cyclo-oxygenase 2/gene expression/parturition/transcription factors
Introduction
Prostaglandins, specifically prostaglandin E2 (PGE2), play an important role in the onset of human labour. They are required for both cervical ripening and fundally dominant myometrial contractions. Prostaglandins are produced from arachidonic acid by the action of the enzyme, cyclo-oxygenase (COX). It is the inducible type-2 COX enzyme (COX-2) that is important for increased prostaglandin production in association with human labour (Slater et al., 1994
). Within the uterus levels of the prostaglandin precursor, arachidonic acid, are found to be highest in the amnion. It is also in amnion that the expression of COX-2 is found to increase exponentially with increasing gestational age to term and with a further doubling in association with labour onset (Slater et al., 1995). Interleukin-1ß (IL-1ß) is one putative stimulant for the onset of labour. Induction of IL-1ß production by bacterial products has been shown to stimulate prostaglandin synthesis from the amnion, decidua and myometrium (Mitchell et al., 1991). However, IL-1ß is not only evident in labour induced by bacterial infection. IL-1ß also increases at term in women with no clinical signs of infection (Romero et al., 1990).
The promoter of the human COX-2 gene has been cloned and sequenced and several putative transcription factor binding sites have been identified, including a cyclic AMP response element (CRE), a nuclear factor (NF)–interleukin 6 (IL6) site and two NF-B sites. Each of these has been demonstrated in various cells to regulate COX-2 transcription. CRE is required for COX-2 transcription in human chondrocytes (Miller et al., 1998) and is involved in the suppressive effect of glucocorticoids in WISH cells (Wang and Tai, 1999). NF–IL6 is important in rat aortic smooth muscle cells (Chen et al., 1998), rat granulosa cells (Sirois and Richards, 1993) and the mouse skin carcinoma cell line, JWF2 (Kim and Fischer, 1998). NF-B has been implicated in several cell types and disease situations including bronchial epithelial cells (Newton et al., 1997), immortalized human myometrial cells (Belt et al., 1999), human brain (Lukiw and Bazan, 1998), and rheumatoid synoviocytes (Crofford et al., 1997). The transcription factor NF-B functions as a homo- or heterodimer of the Rel protein family. These proteins all contain a Rel homology domain (RHD) whose function is DNA binding, dimerization and nuclear localization. Dimers of NF-B exist in the cytoplasm of cells bound by an inhibitor (IB) protein which masks the nuclear localization signal of the NF-B dimer. Phosphorylation of the inhibitor protein at specific serine residues signals ubiquitination and then degradation by the 26S proteasome and NF-B translocates to the nucleus, binds DNA and regulates the transcription of genes (Siebenlist et al., 1994; Baldwin, 1996).
We have previously shown the up-regulation of expression of COX-2 in an immortalized amnion epithelial derived cell line, WISH, by IL-1ß (Xue et al., 1996). Here we report a study to determine in WISH cells the transcription factors involved in the regulation of expression of COX-2 by IL-1ß.
Materials and methods
Cell culture
WISH cells were cultured in Dulbecco's modified Eagle's medium (Sigma Chemical Co Ltd, Poole, UK) containing 10% fetal calf serum (FCS; Sigma Chemical Co), 2 mmol/l L-glutamine (Life Technologies, Paisley, UK) 100 IU/ml penicillin (Life Technologies) and 100 µg/ml streptomycin (Life Technologies) at 37°C, 5% CO2 in air. Cells were passaged using trypsin–EDTA (Life Technologies) when 85–90% confluent and the medium changed every 2–3 days. For experimentation, cells were plated in 12-well plates at 0.75x106cells/well. At 24 h prior to treatment the cells were washed and cultured in serum-free and Phenol Red-free medium. IL-1ß (1 ng/ml; R&D systems, Abingdon, UK) was added as appropriate in fresh serum-free media and incubated for 24 h. Medium was removed and stored at –80°C for PGE2 measurement. Each treatment was carried out in triplicate and three control, no-stimulant (NS) wells were allocated per plate.
Radioimmunoassay
PGE2 (Sigma Chemical Co Ltd, Poole, UK) standards were prepared from 40 to 0.078 ng/ml in a two fold dilution series. Sample and standards were made up to a total volume of 200 µl with buffer (1:1; 0.05 mol/l Tris–HCl pH 7.4: 0.05 mol/l Tris–HCl pH7.4, 0.1% gelatine). [3H]-PGE2 (Amersham Pharmacia Biotech, Little Chalfont, UK) and rabbit anti-PGE2 immunoglobulin G (IgG) with <3.5% cross-reactivity with other prostaglandins (Sigma Chemical Co) were used according to the manufacturer's instructions. Protein assays (Bio-Rad, Hemel Hempstead, UK) were performed and data were expressed as PGE2 produced per µg total protein.
Reverse transcription–polymerase chain reaction (RT–PCR)
Total RNA was isolated using a standard protocol (Chomczynski and Sacchi, 1987). 1 µg of RNA was reverse transcribed and 1/20th of resultant cDNA was used for amplification. Polymerase chain reactions (PCR) were performed in a 25 µl volume containing 1.5 mmol/l MgCl2, 0.2 mmol/l dNTPs, 0.125 µg each primer and 1 IU Biotaq polymerase (Bioline, London, UK).
Cycling parameters were; denaturing 94°C, 30 s; annealing, 30 s; 72°C, 30 s. Annealing temperatures were 58°C (COX-2) and 58°C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Products were isolated from an agarose gel and subcloned into pGEM-Teasy (Promega, Southampton, UK). Double-stranded sequencing was performed using primers to the T7 and Sp6 sites within the vector to verify the clone.
Transfections
Tfx-50 (Promega) was prepared according to manufacturer's instructions and then left at –20°C for at least 16 h before use. 2.5x105 cells were seeded into 24-well plates and allowed to grow to 85–90% confluence. The transfection procedure was carried out according to instructions using a charge ratio of 3:1, 0.5 µg DNA/well and 1 h incubation time. Cells were cultured in serum-containing media for 24 h then washed and cultured for a further 24 h in serum and Phenol Red-free media. Treatments were performed in the final 8 h of this period. Lysates were stored at –80°C until analysis. All treatments were carried out in triplicate.
Luciferase assay buffer (Promega) was prepared according to manufacturer's instructions. All solutions were allowed to reach room temperature before analysis. Samples were centrifuged for 30 s at 13000 rpm. The light released was measured by a luminometer (Turner Design TD 20/20; Promega).
Site-directed mutagenesis
Site directed mutagenesis was performed using the Gene Editor kit (Promega) following the manufacturer's instructions. An oligonucleotide containing the relevant mutation was phosphorylated and annealed to the alkali-denatured vector. T4 DNA polymerase and T4 DNA ligase were added and incubated for 90 min at 37°C to allow synthesis of the second strand. BMH 71–18 mutS competent cells were transformed with 10 ng of vector and cultured overnight. DNA was prepared from these cultures and was used to transform JM109 cells. Sequence analysis was used to verify the presence of the mutation. The oligonucleotides used were AP1mut 5'-GATGAAATATCTGTAGAGGAG-3', NF2mut 5'-CGGGAGAGGCCATTCCCTGC-3' and NF1mut 5'-CAGGAGAGTGGCCACTACCCCC-3' (mutated sites underlined).
Western analysis
Total protein was extracted from cells after treatment. Culture medium was removed and the cells were scraped in 400 µl of T-wash solution. Samples were centrifuged for 2 min at 14 000 g, 4°C and the supernatant stored at –80°C. Protein assay (Bio-Rad) was used to assess protein concentration. 10 µg of protein was made up to a total volume of 10 µl with T-wash and an equal volume of loading buffer added to each sample. Samples were denatured by boiling for 5 min and run on an acrylamide gel and then transferred by electrophoresis to a nitrocellulose membrane. The membrane was blocked overnight at 4°C in 5% milk protein (Marvel), washed and hybridized with the primary antibody for 1 h at 4°C in a 1% milk protein solution. This process was repeated with the secondary antibody and after washing the ECL solution (ICN Pharmaceuticals, Basingstoke, UK) was added and the membrane autoradiographed. Primary antibodies used were goat polyclonal IgG anti-p65, non-cross reactive with c-Rel (p75) or Rel-B (p68), goat polyclonal IgG anti-p50, non-cross reactive with p52 (Diaska and Weiss, 1999), and rabbit polyclonal IgG anti-IB
, specific for IB (Han et al., 1999) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies were also obtained from Santa Cruz.
Results
To show the induction of prostaglandin production in WISH cells by IL-1ß, cells were treated with increasing concentrations of IL-1ß and the subsequent release of PGE2 was measured by radioimmunoassay. IL-1ß caused a dose-dependent increase in PGE2 production from WISH cells. This increase was demonstrated to be due, at least in part, to an increase in COX-2 expression. RNA was extracted from cells after IL-1ß treatment (1 ng/ml), over a 24 h period and RT–PCR for COX-2 (302 bp) and GAPDH (600 bp) performed. COX-2 mRNA concentration increased after 15 min, peaking at 1.5 h and maintained this level up to 24 h of IL-1ß treatment (Figure 1).
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The regulation of expression due to IL-1ß treatment was assessed by the use of a series of reporter constructs. 2.2 kb of the COX-2 promoter was linked to the luciferase reporter gene in the pGL3 vector (Promega). A deletion series of the promoter was created in this reporter vector and the constructs were transiently transfected into WISH cells. Constructs were either non-stimulated (NS) or treated with IL-1ß. The data is expressed as the ratio of IL-1ß treated over non-stimulated relative luciferase units for each individual construct, i.e. the fold activation of expression of each individual construct due to IL-1ß treatment. These data show that only the C2.2 reporter construct containing the full-length (2.2 kb) promoter is able to cause a significant increase in reporter expression with IL-1ß treatment when compared with the control `basic' vector (Figure 2
).
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Western analysis of total protein extracted from WISH cells, which had been either non-stimulated (NS) or treated with IL-1ß, demonstrates clearly the presence of both the p50 and p65 subunits of NF-
B. Analysis of WISH cells treated with IL-1ß over a time course of 90 min shows the degradation and subsequent reappearance of the inhibitor B (IB) protein, suggesting that with IL-1ß treatment NF-B is activated in WISH cells (Figure 3a,b). To determine whether the putative activation of NF-B was transcriptionally active in these cells a luciferase reporter construct containing 6 tandem repeats of the consensus NF-B binding site (pGL3.6B.BG.luc) was transiently transfected into WISH cells. Two control constructs were also used, these contained either no transcription factor binding sites or six copies of a mutated consensus NF-B site. Neither control construct showed any effect of IL-1ß treatment, however, expression from the NF-B dependent construct demonstrated a three-fold increase in reporter expression with IL-1ß treatment (Figure 3c).
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Site-directed mutagenesis of the two NF-
B (–222 and –470 bp) DNA binding sites and of a putative AP-1 site, identified using `Transfac' (www.hgmp.mrc.ac.uk) a computer-based search at position –1593 bp in the C2.2 promoter construct, was performed and these constructs were transiently transfected into WISH cells. Reporter expression with and without IL-1ß treatment was compared with that of the wild-type C2.2 construct. There was a significant reduction in reporter expression from the mutated AP-1 site and both NF-B sites (AP1mut, P = 0.02, NF1mut, P = 0.004 and NF2mut, P = 0.0003) in IL-1ß treated cells but no significant effect on non-stimulated reporter expression (Figure 4).
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Discussion
These data demonstrate that IL-1ß treatment caused significantly increased transcription of COX-2 in WISH cells but that this requires a minimum length of 2.2 kb of the promoter sequence. It has been suggested that, under non-stimulated conditions, there is significantly increased transcription of a 900 bp region compared with 600 bp and that this in turn gives significantly higher expression than a 300 bp region (Wang and Baldwin, 1998). We were unable to show any differences in reporter expression with these fragment sizes of the promoter either under non-stimulated or IL-1ß-treated conditions.
We suggest that either transcription factor(s) bind within the upstream region of the promoter, between –1000 and –2200 bp, to cause transcriptional regulation, or that the whole length of the promoter is required for regulation through the co-operative binding of several factors along the entire length of the promoter. The binding of more than one transcription factor within a promoter for gene transcription is not a novel finding. Indeed, COX-2 has been shown to require both the NF-IL6 and CRE transcription factor binding sites for expression after treatment with lipopolysaccharide and 4ß-phorbol-12-myrisate-13-acetate in bronchial airway epithelial cells (Inoue et al., 1995) and both the AP-1 and CRE binding proteins are required in human chondrocytes (Miller et al., 1998). The transcription factor NF-B has also been shown to be involved in the regulation of COX-2 transcription in many different cell types including bronchial epithelial cells (Newton et al., 1997), immortalized human myometrial cells (Belt et al., 1999), HUVEC and HMEC-1 (Schmedtje et al., 1997), human brain (Lukiw and Bazan, 1998), WISH cells (Wang and Tai, 1999), rheumatoid synoviocytes (Crofford et al., 1997) and U937 cells (Inoue and Tanabe, 1998).
Our data verified the presence of both the p50 and p65 subunits of NF-B in WISH cells and after treatment with IL-1ß the degradation and subsequent reappearance of the inhibitor protein, IB. Transcriptional activation of a NF-B-dependent reporter construct (NFBG) with IL-1ß demonstrates the functional activation of NF-B in WISH cells. Some workers have shown that NF-B regulates COX-2 mRNA expression in bronchial epithelial cells with a preference for the most 5', upstream site (Newton et al., 1997). We show that mutation of either NF-B DNA binding site causes significant reduction in reporter expression. However, mutation of the AP-1 site also causes significant loss of reporter expression. We suggest that it is a combination of these transcription factor binding sites that is required for significant up-regulation of COX-2 expression although we cannot rule out the possible involvement of other transcription factors that have not yet been studied.
Proteins that bind to AP-1 sites are dimers composed of the proteins Jun (v-jun, c-jun, JunB, JunD), Fos (v-Fos, c-Fos, FosB, Fra1, Fra2) or activating transcription factor (ATF2, ATF3, B-ATF) and the bZIP proteins. AP-1 activity is regulated both by the transcription of the component proteins and by their stability. Phosphorylation of c-jun by MAPK reduces ubiquitination and degradation, therefore increasing its stability (Toone and Jones, 1999). Transcriptional activity also increases after phosphorylation and has been suggested to be due to a higher affinity for the co-activator CBP. Fos is also rapidly and transiently induced by CRE, SIE or SRE sites. Xie et al. (1995) have shown that COX-2 expression due to v-src treatment requires the AP-1 proteins c-jun and possibly c-fos but that these proteins activate transcription through the CRE site at the 3' end of the promoter (Xie and Herschman, 1995). Further study is required to determine which proteins are involved in binding to the AP-1 site at –1593bp.
In summary, we have demonstrated that a minimal length of 2.2 kb is required for significant reporter expression due to IL-1ß stimulation of the COX-2 gene promoter in WISH cells. We have identified an AP-1 site in the 5' region that regulates transcription of the reporter gene and have also shown the importance of both of the identified NF-B sites. We suggest that it is the co-operative interaction of proteins that bind to the AP-1 and NF-B sites within the promoter that regulate IL-1ß stimulated COX-2 gene expression in WISH cells.
Acknowledgments
We would like to thank Wellbeing and the MRC for funding this research.
Notes
3 To whom correspondence should be addressed at: Imperial College School of Medicine, Institute of Obstetrics and Gynaecology, Queen Charlotte's and Chelsea Hospital, Goldhawk Road, London, W6 OXG, UK. E-mail v.allport{at}ic.ac.uk 
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Submitted on January 24, 2000; accepted on March 21, 2000.
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M. Lappas, M. Permezel, and G. E. Rice N-Acetyl-Cysteine Inhibits Phospholipid Metabolism, Proinflammatory Cytokine Release, Protease Activity, and Nuclear Factor-{kappa}B Deoxyribonucleic Acid-Binding Activity in Human Fetal Membranes in Vitro J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1723 - 1729. [Abstract] [Full Text] [PDF]
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Y. Lee, V. Allport, A. Sykes, T. Lindstrom, D. Slater, and P. Bennett The effects of labour and of interleukin 1 beta upon the expression of nuclear factor kappa B related proteins in human amnion Mol. Hum. Reprod., April 1, 2003; 9(4): 213 - 218. [Abstract] [Full Text] [PDF]
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Y. Ikoma, S. Nomura, T. Ito, Y. Katsumata, M. Nakata, K. Iwanaga, M. Okada, F. Kikkawa, K. Tamakoshi, T. Nagasaka, et al. Interleukin-1{beta} stimulates placental leucine aminopeptidase/oxytocinase expression in BeWo choriocarcinoma cells Mol. Hum. Reprod., February 1, 2003; 9(2): 103 - 110. [Abstract] [Full Text] [PDF]
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 T. Suganuma, K. Irie, E. Fujii, T. Yoshioka, and T. Muraki Effect of Heat Stress on Lipopolysaccharide-Induced Vascular Permeability Change in Mice J. Pharmacol. Exp. Ther., November 1, 2002; 303(2): 656 - 663. [Abstract] [Full Text] [PDF]
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 M.-E. Janelle, A. Gravel, J. Gosselin, M. J. Tremblay, and L. Flamand Activation of Monocyte Cyclooxygenase-2 Gene Expression by Human Herpesvirus 6. ROLE FOR CYCLIC AMP-RESPONSIVE ELEMENT-BINDING PROTEIN AND ACTIVATOR PROTEIN-1 J. Biol. Chem., August 16, 2002; 277(34): 30665 - 30674. [Abstract] [Full Text] [PDF]
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M. Lappas, M. Permezel, H. M. Georgiou, and G. E. Rice Nuclear Factor Kappa B Regulation of Proinflammatory Cytokines in Human Gestational Tissues In Vitro Biol Reprod, August 1, 2002; 67(2): 668 - 673. [Abstract] [Full Text] [PDF]
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R. F. Johnson, C. M. Mitchell, W. B. Giles, W. A. Walters, and T. Zakar The in Vivo Control of Prostaglandin H Synthase-2 Messenger Ribonucleic Acid Expression in the Human Amnion at Parturition J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2816 - 2823. [Abstract] [Full Text] [PDF]
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M.H.F. Sullivan, S.A. Alvi, N.L. Brown, M.G. Elder, and P.R. Bennett The effects of a cytokine suppressive anti-inflammatory drug on the output of prostaglandin E2 and interleukin-1{beta} from human fetal membranes Mol. Hum. Reprod., March 1, 2002; 8(3): 281 - 285. [Abstract] [Full Text] [PDF]
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 I. Vancurova, R. Wu, V. Miskolci, and S. Sun Increased p50/p50 NF-{kappa}B Activation in Human Papillomavirus Type 6- or Type 11-Induced Laryngeal Papilloma Tissue J. Virol., February 1, 2002; 76(3): 1533 - 1536. [Abstract] [Full Text] [PDF]
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C. Tan, A. Mui, and S. Dedhar Integrin-linked Kinase Regulates Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Expression in an NF-kappa B-dependent Manner J. Biol. Chem., January 25, 2002; 277(5): 3109 - 3116. [Abstract] [Full Text] [PDF]
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 M. S. Chin, C. N. Nagineni, L. C. Hooper, B. Detrick, and J. J. Hooks Cyclooxygenase-2 Gene Expression and Regulation in Human Retinal Pigment Epithelial Cells Invest. Ophthalmol. Vis. Sci., September 1, 2001; 42(10): 2338 - 2346. [Abstract] [Full Text] [PDF]
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C.L. Elliott, V.C. Allport, J.A.Z. Loudon, G.D. Wu, and P.R. Bennett Nuclear factor-kappa B is essential for up-regulation of interleukin-8 expression in human amnion and cervical epithelial cells Mol. Hum. Reprod., August 1, 2001; 7(8): 787 - 790. [Abstract] [Full Text] [PDF]
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