Molecular Human Reproduction, Vol. 6, No. 9, 771-778,
September 2000
© 2000 European Society of Human Reproduction and Embryology
Endocrinology |
NF-IL6 and CRE elements principally account for both basal and interleukin-1ß-induced transcriptional activity of the proximal 528 bp of the PGHS-2 promoter in amnion-derived AV3 cells: evidence for involvement of C/EBPß
1 Department of Pharmacology and Clinical Pharmacology, University of Auckland, Faculty of Medical and Health Sciences, Auckland, New Zealand, and 2 Department of Pathology, Yale University, School of Medicine, New Haven, CT 06520–8023, USA
Abstract
Prostaglandin H synthase (PGHS)-2 promoter fragments (–528 to +9 bp and 5' unidirectional deletions thereof) were cloned upstream of the chloramphenicol acetyl-transferase (CAT) reporter gene. These were transfected into amnion-derived AV3 cells. The region, –528 to –203, which includes NF-
B sites, had little influence on CAT expression. The region, –203 and –52, however, was responsible for most of the basal promoter activity and also conferred responsiveness to interleukin (IL)-1ß (>3-times basal). Point mutations of NF-IL6 and cAMP response element (CRE) in this region reduced both basal and IL-1ß-stimulated production of CAT; dual mutation eliminated IL-1ß responsiveness. Factors in nuclear extracts from control or IL-1ß-stimulated AV3 cells specifically complexed the NF-IL6 and CRE sequences. However, the NF-IL6 and CRE oligonucleotides cross-competed, suggesting a common factor. C/EBPß was identified by supershift assay as interacting with both sequences. To a lesser extent C/EBP
and 
also interacted with the NF-IL6 site. However, CRE binding protein (CREB), was absent from the complex with the CRE. In conclusion, NF-IL6 and CRE elements principally account in AV3 amnion cells for basal and IL-1ß-inducible transcriptional activity of the proximal 528 bp of the PGHS-2 promoter, while NF-B elements play no substantial role. C/EBPs, particularly C/EBPß, are implicated in control of PGHS-2 transcription through the NF-IL6 and CRE sites.
amnion/cytokines/interleukin 1ß/prostaglandins/PGHS-2
Introduction
A significant proportion of preterm labour has been associated with intrauterine infection (Wahbeh et al., 1984
; Romero et al., 1989a). In the presence of infection, values of pro-inflammatory cytokines, e.g. interleukin (IL)-1ß and tumour necrosis factor- (TNF-) are elevated in the amniotic fluid (Romero et al., 1989b, 1992). These cytokines, produced locally in gestational tissues in response to bacterial infection, are capable of activating fetal membrane and decidual prostaglandin (PG) production (Mitchell et al., 1991), in part, by increasing prostaglandin H synthase-2 (PGHS-2) mRNA expression and activity (Teixeira et al., 1994; Hansen et al., 1998). Furthermore, IL-1ß is capable of inducing the expression of other cytokines, e.g. IL-8 (Dudley et al., 1993), TNF- (Ikejima et al., 1990) and IL-6 (Shalaby et al., 1989), or enhancing its own production (Dinarello, 1987; Gunn et al., 1996). These observations suggest that IL-1ß production of gestational tissues during bacterial invasion in pregnancy may play an important role in the events leading to preterm labour.
PGE2 is thought to be a key mediator of the onset of labour (Keirse and Gravenhorst, 1979; Dudley and Trautman, 1994). Two isoforms of the PGHS enzyme which catalyses the committed step in the prostaglandin biosynthetic pathway have been described (Funk et al., 1991; Kujubu et al., 1991). The two isoforms, referred to as PGHS-1 and PGHS-2, are encoded by genes on different chromosomes and share a 60% amino acid identity. PGHS-1 is constitutively expressed in most cell types, whereas PGHS-2 is rapidly and transiently expressed in response to pro-inflammatory cytokines, growth factors and tumour promoters (Smith and DeWitt, 1995).
The transient nature of PGHS-2 mRNA expression in response to mitogens shares similarities with the expression patterns of immediate early genes (Herschman, 1991). Despite some knowledge concerning its structural and sequence information, it is unclear how expression of the PGHS-2 gene is regulated by external stimuli in terms of signalling pathways. Signals that ultimately trigger the accumulation of PGHS-2 mRNA have been shown to be mediated through various signal transduction pathways, depending on the cell type and stimulus used (Blanco et al., 1995; Hamasaki and Eling, 1995). Sequence analysis of the 5'-flanking region of the PGHS-2 gene has shown the presence of several potential transcription regulatory sequences, including a TATA box, an NF-IL6 site, a cAMP response element (CRE) motif and a CCAAT/enhancer binding protein (C/EBP) motif (Appleby et al., 1994; Kosaka et al., 1994). Additional cis-acting DNA elements, e.g. NF-B elements, which are found in the rat (Sirois and Richards, 1993), mouse and human PGHS-2 promoters (Fletcher et al., 1992), may also facilitate transcription from other effector pathways that regulate PGHS-2 transcription. In the present report we have sought to determine the promoter elements in the proximal 528 bp of the PGHS-2 promoter which play a role in basal and IL-1ß-mediated PGHS-2 gene transcription in amnion-derived AV3 cells.
Materials and methods
Reagents
Hams F12/Dulbecco's modified Eagle's medium (DMEM) was purchased from Irvine Scientific, CA, USA. Trypsin 1:250 powder was obtained from Difco (Detroit, MI, USA). Fetal calf serum (FCS), penicillin G sodium, streptomycin sulphate, Concert ion-exchange DNA purification columns, gel shift primers, poly dIdC and the 0.24–0.95 Kb RNA ladder were obtained from Life Technologies, Auckland, New Zealand. Human recombinant IL-1ß was obtained from Immunex, Seattle, Washington, USA. Restriction enzymes, Fugene 6 transfection reagent, CAT and ß-galactosidase (ß-gal) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Boehringer Mannheim New Zealand Ltd (Auckland, New Zealand). GeneScreen Plus membrane was obtained from Life Technologies. [-32P]-dCTP, [
-32P]-ATP and HyperfilmMP were obtained from Amersham Pharmacia (Auckland, New Zealand). Polynucleotide kinase (PNK), PNK buffer and T4 DNA ligase were purchased from Epicentre Technologies (Madison, WI, USA). Anti-C/EBP, C/EBPß and C/EBP antibodies were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Anti-CREB antibody was obtained from Upstate Biotechnology (Lake Placid, NY, USA). The random prime labelling kit was purchased from Amersham Pharmacia. The DNA purification columns were purchased from Qiagen (Hilden, Germany) and the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA, USA). Other biochemicals were purchased from Sigma Chemical Co (St Louis, MO, USA) or Riedel de Haen (Seelze, Germany).
Cell culture
Amnion-derived AV3 cells (Rivadeneira and Robbins, 1958) were maintained in Ham's-F12/DMEM plus 10% heat-inactivated FCS, 6 g/l penicillin, 10 g/l streptomycin and 40 g/l gentamycin, at 37°C in a humidified 95% air/5% CO2 atmosphere. The medium was changed every 2 days.
Isolation of total RNA
Cells were treated in triplicate, confluent, 6 cm plates with vehicle or IL-1ß (1 ng/ml). At the times indicated, medium was removed and total RNA was directly isolated by acid guanidinium thiocyanate–phenol–chloroform extraction (Chomczynski and Sacchi, 1987). RNA pellets were washed with 80% ice-cold ethanol, resuspended in deionized formamide at 55°C for 15 min and stored at –70°C. RNA concentration was determined by measuring the absorbance at 260 nm.
RNA gel electrophoresis and Northern Blot analysis
Total RNA (15 µg per sample) was fractionated on a 1% agarose gel and transferred to GeneScreen Plus membrane overnight by downward capillary transfer, as described previously (Hansen et al., 1998). The cDNA probe, a 1.8 kb EcoRI/ApaI fragment of the PGHS-2 cDNA (Hla and Neilson, 1992), was labelled with [-32P]-dCTP (100 mCi/ml) using the random prime labelling kit to a specific activity >109 cpm/µg. Hybridization and washing were carried out essentially as described previously (Hansen et al., 1998). The sodium dodecyl sulphate (SDS) concentration was reduced to 0.2% in the first two wash solutions and 2% in the final. The final wash was 50 mmol/l NaH2PO4, pH 7.2, 1 mmol/l EDTA, 2% SDS for a total of 30 min at 53°C. The hybridization signal was quantified using a Packard InstantImager (Parkland Instruments Co., Mariden, CT, USA).
Cell transfections
Preparation of CAT reporter constructs
To produce deletion constructs by long template polymerase chain reaction (PCR), a –891/+9 fragment of the PGHS-2 promoter contained within the plasmid hcox-2luc (Tazawa et al., 1994) was subcloned into pBLCAT3 (Luckow and Schutz, 1987, Genbank accession no. X64409). hcox-2luc and pBLCAT3 were linearized with HindIII and XhoI respectively. The linearized plasmids were end-filled with Kenow fragment and then digested with BamHI. Finally, the BamHI/HindIII fragment of hcox-2luc was subcloned into the BamHI/XhoI-digested pBLCAT3 to produce p891CAT.
Unidirectional deletions from the 5' end of the promoter fragment were accomplished by PCR. The upstream primers (Table I) defined the 5' ends of p321CAT, p203CAT and p52CAT as noted. A common downstream primer (5'-GGA TCC TCT AGA TCG AC-3') which annealed only to pBLCAT3 vector sequence at the upstream pBLCAT3/PGHS-2 promoter sequence junction, was utilized in subsequent PCR reactions to amplify both vector and the retained portion of the promoter. The resulting PCR products were purified by low-melting point agarose electrophoresis, phosphorylated by polynucleotide kinase, and re-annealed with T4 DNA ligase.
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PGHS-2 promoter constructs containing mutations within the NF-IL6 and/or CRE elements were generated by the QuikChange Site-Directed Mutagenesis Kit according to the manufacturers instructions. Primers for mutations of the CRE and NF-IL6 elements are shown in Table II
.
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All constructs and plasmid junctions were verified by restriction digest and automated dye terminator Sanger dideoxy sequencing (Auckland DNA Sequencing Facility, School of Biological Sciences, University of Auckland). DNA purification columns were used to purify all plasmids for transfection.
Transfection of deletion constructs into AV3 cells
AV3 cells were plated in 6 cm dishes (106 cells/dish) the day before transfection. On the day of transfection the medium was exchanged at least 1 h before transfection. A transfection reagent mix was prepared by diluting Fugene 6 with serum-free medium (3 µl/100 µl). For each plate to be transfected, 1 µg of one of the above described CAT reporter constructs, 0.75 µg of pGEM7Zf(–) and 0.25 µg pdx11 (CMV-ß-galactosidase) were added to an appropriately labelled tube. Transfection reagent mix (100 µl per plate) was added dropwise to each tube containing the CAT constructs and tubes were incubated for 15 min at room temperature. Each construct (100 µl) was then added dropwise to appropriately labelled plates and incubated for 24 h at 37°C. Cells were then treated with IL-1ß (1 ng/ml) or vehicle for 24 h, lysed and amounts of CAT and ß-gal enzymes were measured by ELISA. The results are expressed as a ratio of picogram CAT/pg ß-gal and are indicative of the transcriptional activity of the indicated regions in the human PGHS-2 promoter in AV3 cells.
To evaluate responsiveness to IL-1ß mediated by cryptic elements in the vector backbone, the construct pGLCAT4 (Nyborg et al., 1990), which contains the minimal promoter (–32 to +51) from the herpes symplex thymidine kinase (TK) gene, was used in place of the PGHS-2 promoter-CAT constructs. For estimation of the transfection efficiency, pGFP-C1 (Clontech Laboratories Inc, Palo Alto, CA, USA) was substituted for the CAT construct and pGEM7Zf(–). Green fluorescent protein expressing cells resulting from transfection with this construct were counted using a Leica DM IRB microscope and fluorescein filter set (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) and compared with the number of cells detected by white light microscopy. An efficiency of 7% was obtained by this method.
Electrophoretic mobility shift assay (EMSA)
Nuclear protein isolation
Confluent cultures in 10 cm plates were treated for 1 h with 1 ng/ml IL-1ß or carrier at 37°C. After incubation the monolayers were washed twice with ice-cold phosphate-buffered saline (PBS), pH 7.2. The cells were removed by scraping and recovered by centrifugation in pre-chilled tubes at 4°C for 5 min at 650 g. Supernatants were discarded and the cells were resuspended in 5 ml ice-cold lysis buffer (10 mmol/l Tris–Cl pH 7.4, 10 mmol/l NaCl, 3 MgCl2, 0.5% NP40). After 5 min on ice the nuclei were recovered by centrifugation at 4°C for 5 min at 650 g. The resuspension of the nuclei, incubation on ice, and centrifugation were repeated. The volume of the nuclear pellet was estimated. The pellets were resuspended gently in half of the packed nuclear volume (pnv) of low salt buffer [20 mmol/l HEPES, pH 7.9 at 4°C, 25% glycerol, 1.5 mmol/l MgCl2, 0.2 mmol/l EDTA, 20 mmol/l KCl, 0.2 mmol/l phenyl methyl sulphonyl fluoride (PMSF), 0.5 mmol/l dithiothreitol (DTT)] and transferred to microfuge tubes on ice. High salt buffer (20 mmol/l HEPES, pH 7.9 at 4°C, 25% glycerol, 1.5 mmol/l MgCl2, 0.2 mmol/l EDTA, 1600 mmol/l KCl, 0.2 mmol/l PMSF, 0.5 mmol/l DTT), half pnv, was added dropwise to the pellet. Following incubation on ice for 30 min, insolubles were removed from the nuclear extracts by centrifugation at 23 100 g for 30 min at 4°C. The concentration of nuclear protein in the extracts was determined by the bicinchoninic acid (BCA) method calibrated against bovine serum albumin (Redinbaugh and Turley, 1986).
Double-stranded oligonucleotide probe production
The sense or anti-sense oligonucleotide (5 pmol in each case) was added to separate tubes containing 10 IU polynucleotide kinase (PNK) and 15 µCi of [32P]-ATP and incubated for 30 min at room temperature. The reaction was extracted with phenol/chloroform and the complementary nucleotides were combined and purified using Sephadex G-50. Reactions were boiled for 5 min, then placed in a water bath pre-heated to 80°C which was then allowed to cool overnight.
Formation of complexes and electrophoresis
The EMSA reaction containing 5 µg of nuclear extract, 2 µl 10x reaction buffer (38 mmol/l HEPES pH 7.9, 1.85 mmol/l MgCl2, 0.18 mmol/l EDTA, 27.5% glycerol, 0.95 mmol/l DTT) and 2 µl of poly dIdC was incubated for 20 min at room temperature. To each tube 50 fmol of the appropriate double-stranded oligonucleotide probe (NF-IL6: 5'-CGG CTT ACG CAA TTT TT-3'; 5'-AAA AAT TGC GTA AGC CG-3'; CRE: 5'-ACA GTC ATT TCG TCA CAT GGG CTT G-3'; 5'-CAA GCC CAT GTG ACG AAA TGA CTG T-3') was added with the indicated molar excess of unlabelled double-stranded oligonucleotide (C/EBP consensus: 5'-TGC AGA TTG CGC AAT CTG CA-3'; 5'-TGC AGA TTG CGC AAT CTG CA-3'; NF-IL6, CRE, the mutated sequence within the CRE site, the mutated sequence within the NF-IL6 site or E-selectin NF-B). Tubes were incubated for 20 min at room temperature. For the supershift experiments, 1 mg/ml of anti-C/EBP, C/EBPß, C/EBP or CREB antibody was added to tubes and incubated for a further 20 min. Complexes were resolved on 6% polyacrylamide gels at 200 V for 2 h. Gels were vacuum-dried at 80°C for 1 h, then exposed to HyperfilmMP for 1–2 weeks with an intensifying screen at –70°C, depending on signal intensity of the bands.
Statistical analysis and data expression
Measurements of CAT and ß-gal production are expressed as a ratio of pg CAT/pg ß-gal. Results are displayed as the pooled data from three experiments performed in triplicate. Statistical significance was determined by two-way analysis of variation (ANOVA) followed by least squares analysis or by paired t-test as appropriate. CAT and ß-gal activity in cells transfected with the TK minimal promoter construct were expressed as ratios to the mean value for untreated control cells in each experiment and the significance of effects of treatment assessed by t-test. P < 0.05 was considered to be statistically significant. Errors are expressed as SEM.
Results
Northern analysis
To investigate the changes in mRNA expression in response to IL-1ß, AV3 cells were treated with fresh medium containing 1 ng/ml IL-1ß or vehicle and total RNA was isolated at the times indicated from 0–16 h. Northern blot analysis revealed a rapid IL-1ß-induced increase of PGHS-2 mRNA levels in AV3 cells within 30 min after cytokine addition, reaching maximal levels by 2 h before declining gradually thereafter (Figure 1, lanes 8–14). In control cells, with the exception of slight expression at the 1 h time point, expression of PGHS-2 mRNA was not detectable (Figure 1, lanes 1–7).
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Transcriptional regulation of PGHS-2 expression in AV3 cells
Deletion analysis of the PGHS-2 promoter in AV3 cells
Expression of CAT by the the p528CAT construct following treatment with 1 ng/ml IL-1ß for 24 h was increased three-fold (P < 0.05) compared with basal expression (Figure 2
). Deletion of the sequences located between –321 and –528 (p321CAT) which contain a NF-B response element (–447/–438) did not alter expression. Further deletion of the sequences between –203 and –321 (p203CAT) which contain a second NF-B response element (–222/–213) maintained a three-fold induction of CAT activity by IL-1ß. However, deletion of the sequences between –203 and –52 (p52CAT), a region known to contain a NF-IL6 response element (–132/ –124) and a CRE site (–59/–52), had a significant effect on both basal and IL-1ß-stimulated CAT production. Basal expression was decreased to one third of the basal expression of the p528CAT construct. The ability of IL-1ß to stimulate CAT expression was substantially reduced and was no longer significant.
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= control; 
= IL-1ß-treated). Results are the pooled data from three separate experiments. Statistical significance (P < 0.05) was determined by two-way analysis of variance (ANOVA) followed by least squares analysis.The expression of ß-galactosidase (ß-gal) was not significantly influenced by treatment with IL-1ß (1.2 ± 0.1 times control versus control: 1.0 ± 0.05, P = 0.16, n = 18 for each group). Basal expression from the heterologous minimal TK promoter was approximately half that from the p528CAT construct. This was stimulated 2.6 ± 0.4 times by IL-1ß (versus control 1.0 ± 0.1, P = 0.0003, n = 18 and n = 16 for the stimulated and unstimulated cultures respectively).
Site-directed mutagenesis of putative IL-1ß regulatory elements within the PGHS-2 promoter
Mutation of the NF-IL6 response element in p203CAT to produce a construct referred to as p203NFM resulted in a 80% decrease in basal CAT production and a 70–75% decrease in IL-1ß-stimulated CAT production compared to p203CAT (Figure 3). Mutation of this element alone did not entirely eliminate CAT expression. Mutation of the CRE site (–203CREM) resulted in a 90% decrease in basal CAT production and an 80% reduction in IL-1ß-stimulated CAT expression compared to p203CAT, although, once again, did not completely eliminate CAT expression. Double mutation of both the NF-IL6 and the CRE sites (–203NFCREM) reduced basal CAT transcription by 90% and reduced IL-1ß-stimulated expression by 95%. The effect of IL-1ß on transcriptional activity was also essentially negated by the double mutation.
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Identification of nuclear factors which regulate PGHS-2 transcription by IL-1ß
We used EMSA to determine the nuclear factors that bound the NF-IL6 and CRE elements in AV3 cells treated with 1 ng/ml IL-1ß or vehicle alone. The PGHS-2-derived [32P]-labelled NF-IL6 oligonucleotide incubated with nuclear extracts from control (Figure 4A
, lane 2) or IL-1ß-stimulated cells (Figure 4A, lane 3) produced a high molecular weight DNA–protein complex. A high molecular weight complex was not observed in the lane containing probe only (Figure 4A, lane 1). Incubation with 50-fold molar excess of unlabelled NF-IL6 (N) or a consensus C/EBP oligonucleotide (CE) (Figure 4A, lanes 4 and 5) diminished the intensity of the complex compared with that seen without added competitor. The presence of 50-fold molar excess of the mutated NF-IL6 oligonucleotide (NFM) (Figure 4A, lane 6) or the mutated CRE oligonucleotide (CREM) (Figure 4A, lane 8) failed to decrease the amount of complex observed. An additional low mobility band was observed at the top of lane 6, but is artefactual, as it was not observed upon repetition. Use of 5–, 10–, 25–, 50–, 100–, or 500– fold molar excess of the NFM oligonucleotide demonstrated no reduction in intensity of the labelled complex at less than the 100-fold excess and modest reductions thereafter (data not shown). Incubation with the E selectin (ES) oligonucleotide, which binds specifically to NF-B, also did not diminish the intensity of the complex (Figure 4A, lane 9). The presence of a 50-fold molar excess of the CRE59 (C) oligonucleotide, however, completely abolished the intensity of the complex (Figure 4A, lane 7).
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This experiment was repeated using a [32P]-labelled CRE probe (Figure 4B
). The control and IL-1ß-treated extracts both produced a high molecular weight complex resulting in a mobility shift (Figure 4B, lanes 2 and 3). A high molecular weight complex was not observed in the lane containing probe only (Figure 4B, lane 1). The intensity of the DNA–protein complex was reduced with the inclusion of 50-fold molar excess of the NF-IL6 (N), the C/EBP consensus oligonucleotide (CE) or the CRE oligonucleotide (C) (Figure 4B, lanes 4, 5 and 7). Again the mutant oligonucleotides (Figure 4B, lanes 6 and 8) and the heterologous, E selectin, oligonucleotide (Figure 4B, lane 9) failed to compete with the probe for binding to the nuclear proteins in the complex. As in Figure 4A the additional low mobility band observed at the top of Figure 4B, lane 6 is artefactual. Use of a 500-fold molar excess of the CREM oligonucleotide was required to obtain a reduction in intensity of the labelled complex and even at this excess the reduction was only slight (data not shown).
Identification of the proteins which bind NF-IL6 and CRE
Supershift EMSA was used to identify the nuclear proteins that interact with the PGHS-2-derived NF-IL6 oligonucleotide (Figure 5) in the presence or absence of IL-1ß. The reactions contained a [32P]-labelled NF-IL6 probe and polyclonal antibodies to C/EBP, C/EBPß or C/EBP (Figure 5A). Lane 1 contained probe without nuclear extract. A specific protein–DNA complex was formed with both control and IL-1ß-stimulated nuclear extracts (Figure 5A, lanes 2 and 6). The reactions containing isoform specific anti-C/EBP antibodies (Figure 5A, lanes 3–5 and 7–9) produced supershifted complexes in both the control and IL-1ß stimulated extracts as indicated by the presence of a second lower mobility band above the main complex. The greatest amount of the supershifted complex was observed with anti-C/EBPß (Figure 5A, lanes 4 and 8), followed by anti-C/EBP (Figure 5A, lanes 5 and 9) while anti-C/EBP appeared also to have produced a faint supershift (Figure 5A, lanes 3 and 7).
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Similar experiments were performed with the CRE oligonucleotide as the probe (Figure 5B
). In addition to the anti-C/EBP antibodies, an anti-CREB antibody was used in these experiments to probe for involvement of this protein in the complexing of the PGHS-2 CRE site. In contrast to the results when the NF-IL6 oligonucleotide was used as the probe, the complex formed on the CRE probe was supershifted only by anti-C/EBPß (Figure 5B, lanes 4 and 9). Notably anti-CREB did not produce such a shift (Figure 5B, lanes 6 and 11). Discussion
This study examined the mechanism by which IL-1ß induces the transcription of the PGHS-2 gene in amnion-derived AV3 cells. It has been shown previously that PGHS-2 mRNA expression can be induced by a variety of stimuli including agents which act through G protein-mediated mechanisms, the protein kinase C pathway mediated by phorbol esters and the tyrosine kinase-mediated pathways activated by growth factor receptors (Herschman, 1996). However, the signal transduction mechanisms, the transcription factors, and the regulatory regions of the PGHS-2 gene necessary for PGHS-2 enzyme induction are not well understood. A number of PGHS-2 promoter elements have previously been implicated in the transcriptional regulation of the PGHS-2 gene, including the NF-IL6 element, the CRE motif, a TATA box and the NF-B sites (Appleby et al., 1994). With respect to IL-1ß-induced up-regulation of PGHS-2 mRNA expression, it has been reported that the NF-B promoter element is involved in the transcriptional activation of the PGHS-2 gene (Newton et al., 1997; Wang and Tai, 1998).
It has been suggested that the NF-IL6 site was involved in the transcriptional regulation of the PGHS-2 gene by gonadotrophin in rat granulosa cells (Sirois and Richards, 1993). Recently, Inoue et al. (1995) showed that induction of PGHS-2 expression in vascular epithelial cells by phorbol ester or LPS involved the CRE and NF-IL6 sites, and that C/EBP functioned as a trans-acting factor, perhaps in association with CREB. Other studies have provided evidence for the involvement of the CRE regulatory element in an oncogene-induced up-regulation of the murine PGHS-2 gene (Xie and Herschman, 1995).
Our CAT reporter studies indicate that a region of the human PGHS-2 promoter responsible for significant basal transcriptional activity and IL-1ß-responsiveness in amnion-derived AV3 cells lies between bases –203 and –52 upstream from the transcription start site. This region contains CRE and NF-IL6 elements. However, our results appear to exclude a role for NF-B elements between –528 and –203 in these processes.
Both the NF-IL6 and CRE response elements seem to contribute independently to CAT expression in both stimulated and unstimulated cells. Mutation of either site reduces CAT expression, substantially. Furthermore, point mutation of both the NF-IL6 and CRE elements abolished the responsiveness of the promoter to IL-1ß. This is important to note, as cryptic sites in the backbone of the vector used in making the promoter constructs confer IL-1ß responsiveness on the minimal TK promoter–reporter construct. Abolition of IL-1ß responsiveness in the PGHS-2 promoter fragment by these mutations of the promotor sequence suggests that the effects of the cryptic sites are negligible in the context of the PGHS-2 promoter constructs, at least in the case of the –203 plasmid. Therefore, both the CRE and NF-IL6 response elements seem to be involved in the transcriptional regulation of the PGHS-2 gene in AV3 cells.
To further define the mechanism of IL-1ß-induced PGHS-2 mRNA transcription, EMSA assay was utilized. Nuclear extracts from control or IL-1ß-stimulated AV3 cells contain a factor or factors which specifically bind the PGHS-2 NF-IL6 and CRE site-containing oligonucleotides. Interestingly, however, the two oligonucleotides cross-compete for binding of the factor or factors involved. These results again suggest that both the CRE and NF-IL6 promoter elements are involved in control of transcriptional activity of the PGHS-2 gene in AV3 cells. They further suggest involvement of a common factor at the two sites. The inability of the mutated sequence within the CRE oligonucleotide (CREM) or the mutated sequence within the NF-IL6 oligonucleotide (NFM) to displace the EMSA probe oligonucleotides from the DNA–protein complex indicates that the two elements are specifically required for the binding of the factors in the nuclear extracts.
In order to evaluate which proteins may be involved in the complexes formed in the EMSA assay, antibodies to factors known to bind the response elements identified were added to the assay. C/EBP was originally described as a rat liver-specific DNA-binding protein with a leucine zipper motif (Johnson et al., 1987). Several isoforms of this trans-acting factor, C/EBP, C/EBPß and C/EBP bind to the NF-IL6 site (Akira et al., 1990; Pall et al., 1997). Therefore, these C/EBP isoforms as well as the CRE binding protein, CREB, were evaluated. Our results indicate that C/EBPß is involved in the complexes formed with both the NF-IL6 and CRE response elements and that CREB does not participate in the complex between the CRE site and nuclear factors from the AV3 cells. Interestingly, C/EBP isoform specificity at the NF-IL6 site is more relaxed than at the CRE site with evidence that C/EBP and possibly C/EBP also bind the NF-IL6 site. The effectiveness of the CRE oligonucleotide to compete with the NF-IL6 oligonucleotide probe (Figure 4A, lane 5) for binding of the factors in the nuclear extracts suggests that C/EBPß is the major C/EBP binding the NF-IL6 site.
IL-1ß did not induce significant changes in either the CRE or NF-IL6 binding capacity in AV3 cell nuclear extracts. Rapid up-regulation, such as that observed for PGHS-2, tends to involve postranslational mechanisms for activation of the factors responsible. C/EBP function is regulated extensively by phosphorylation (Takiguchi, 1998). Of particular note is that binding of C/EBPß to a promoter has been reported to be unaffected by cAMP, while transcriptional activation by the factor required cAMP-induced phosphorylation (Tae et al., 1995). Thus the lack of significant change in binding cannot be taken to contradict the (modest) up-regulation observed in the promoter-reporter study. The large contrast between the induction of PGHS-2 expression by IL-1ß observed in the Northern analyses and the analyses of the cloned promoter fragments, nevertheless, suggests that regions of the promoter outside of the –528 to +9 region may be important in mediating the up-regulation by IL-1ß.
C/EBPß is thought to be involved in the mediation of extracellular signalling and its expression is up-regulated by various agents such as cytokines, calcium, lipopolysaccharide and phorbol ester, or by glutamate receptor activation (Yano et al., 1996). Interestingly, NF-B sites are often involved in mediation of pro-inflammatory cytokine induction (Tazawa et al., 1994; Collins et al., 1995). Furthermore, involvement of both NF-B sites in the –591 to –203 region of the promoter in induction of PGHS-2 by IL-1ß has been reported in the amnion-derived WISH cell line (Wang and Tai, 1998). Yet AV3 cells clearly do not utilize these NF-B sites in our experiments. This might result from differing site specificities of the subsets of the NF-B/Rel family members expressed in the cells or activated during IL-1ß-induced signalling (Collins et al., 1995; Perkins, 1997). These matters, as well as the question of whether the utilization of NF-B by WISH cells or AV3 cells is more reflective of the cells of the amnion, will require further experimentation to resolve. Our evidence suggests that evaluation of additional regulatory sequences outside the proximal promoter region examined here may be required for full understanding of regulation of PGHS-2 gene transcription by IL-1ß. However, the data strongly supports a role for C/EBPs, particularly C/EBPß, binding at the –132 NF-IL6 and –59 CRE sites in regulation of transcription of PGHS-2 in AV3 cells.
Acknowledgments
This research was supported by grants from The Royal Society of New Zealand Marsden Fund, The Health Research Council of New Zealand, and The Lottery Grants Board of New Zealand. The authors would like to thank Dr Larry Chamley (National Womens Hospital, Auckland, New Zealand) for providing the AV3 cells. We would also like to thank Allan Drew for technical assistance, and Dr Marion Blumenstein and Dr Jeffrey Keelan for critical review of the manuscript.
Notes
3 To whom correspondence should be addressed at: Department of Pharmacology and Clinical Pharmacology, University of Auckland, Faculty of Medical and Health Sciences, 85 Park Road, Grafton, Auckland, New Zealand. E-mail: k.marvin{at}auckland.ac.nz 
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Submitted on November 2, 1999; accepted on June 8, 2000.
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