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The Journal of Immunology, 2006, 177: 8111-8122.
Copyright © 2006 by The American Association of Immunologists, Inc.

Cyclooxygenase-2 Gene Transcription in a Macrophage Model of Inflammation1

Yeon-Joo Kang2,*,{ddagger}, Byron A. Wingerd2,*, Toshi Arakawa{dagger} and William L. Smith3,{dagger},{ddagger}

* Cell and Molecular Biology Program and {dagger} Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824; and {ddagger} Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infections involving LPS-bearing, Gram-negative bacteria can lead to acute inflammation and septic shock. Cyclooxygenase-2 (COX-2), the target of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors, is importantly involved in these responses. We examined the dynamics of COX-2 gene expression in RAW264.7 murine macrophages treated with LPS as a model for COX-2 gene expression during inflammation. We established, using Northern blotting, nuclear run-on assays, and RT-PCR, that COX-2 transcriptional activation continues for at least 12 h after LPS treatment and involves at least three phases. Previous studies with murine macrophages identified an NF-{kappa}B site, a C/EBP site, and a cAMP response element-1 (CRE-1) as cis-acting elements in the COX-2 promoter. We identified three additional functional elements including a second CRE (CRE-2), an AP-1 site, and an E-box that overlaps CRE-1. The E-box mediates transcriptional repression whereas the other cis-elements are activating. Using electrophoretic mobility supershift and chromatin immunoprecipitation assays, we cataloged binding to each functional cis element and found them occupied to varying extents and by different transcription factors during the 12 h following LPS treatment. This suggests that the cis elements and their cognate transcription factors participate in a sequential, coordinated regulation of COX-2 gene expression during an inflammatory response. In support of this concept, we found, using inhibitors of Jun kinase and NF-{kappa}B p50 nuclear localization, that COX-2 gene transcription was completely dependent on phospho-c-Jun plus p50 at 6 h after LPS treatment but was only partially dependent on the combination of these factors at later treatment times.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Prostaglandin endoperoxide H synthases, commonly known as cyclooxygenases (COX),4 catalyze the committed step in the conversion of arachidonic acid to PGs (1, 2). There are two COX isoforms: the predominantly constitutive isoform, COX-1, and an inducible isoform, COX-2. Although both enzymes catalyze the same reaction with similar kinetics in vitro, in vivo studies with isoform-specific inhibitors and COX-1 and COX-2 knockout mice indicate that there are some physiological processes that require one specific enzyme and others where the isoforms can complement one another (3, 4).

Bacterial LPS is a potent inducer of COX-2 expression in macrophage cells. This induction is a result of LPS activation of the TLR-4, which, in turn, initiates signaling through MyD88/IL-1R-associated kinase/TNFR-associated factor 6/evolutionary conserved signaling intermediate in Toll resulting in the activation of ERK, JNK, p38, protein kinase C, and NF-{kappa}B-inducing kinase (5, 6, 7, 8), and these kinases likely exert their actions by phosphorylating either transcription factors or other downstream effectors to cause the transcriptional machinery to begin transcribing the COX-2 gene. LPS-induced COX-2 transcription is regulated through multiple redundant mechanisms involving several central response elements present in the COX-2 promoter (6, 8). The CRE-1 at –57/–52 in the murine COX-2 (mCOX-2) promoter is necessary for mediating the effects of a wide variety of stimuli, while a C/EBP site and an NF-{kappa}B response element appear to function in more specialized signaling events.

The promoters of the human, murine, rat, equine, and bovine COX-2 genes have a number of common regulatory elements. In the mouse promoter, there are both a cAMP response element 2 (CRE-2) and an NF-{kappa}B site located between 392 and 433 bp upstream of the transcription start site, a C/EBP site at –136/–128, an AP-1 site at –67/–62 and an overlapping CRE-1/E-box element at –56/–47 (Fig. 1). The NF-{kappa}B site is necessary for inducible COX-2 promoter activity in TNF-{alpha}-stimulated MC3T3-E1 cells (9). More recently, Hwang and colleagues (8, 10) have demonstrated that blocking NF-{kappa}B activation at several levels results in a large decrease in COX-2 promoter activity in RAW264.7 cells. The CRE-2 site has previously been tested in fluid sheer stress-stimulated murine osteoblastic MC3T3-E1 cells, but a mutation in this site had no effect on the transcriptional activation of a COX-2 promoter reporter gene in this system (11). However, the CRE-2 site has been demonstrated to play an important role in IL-1beta-induced COX-2 transcription in the human endometrium (12). MC3T3-E1 osteoblasts cells treated with PMA activate COX-2 transcription in a process mediated by the AP-1 site (13). The overlapping CRE-1 and E-box element of the human promoter have been studied in transfected LPS-stimulated RAW264.7 macrophages (14) and shown to be involved in stimulation of COX-2 transcription. The E-box element has also been shown to act as a positive regulatory element for COX-2 expression in bovine granulosa cells (15) and in PMA-treated human gastric epithelial cells (16). Although previous studies have suggested that the CRE-1, C/EBP, and NF-{kappa}B sites are involved in regulating COX-2 formation in RAW264.7 cells, no other cis-acting elements have been shown to be functional.


Figure 1
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  		FIGURE 1. Schematic representation of the major conserved response elements in the murine COX-2 promoter. cis-acting elements found within the COX-2 promoter are noted in the shaded boxes and their location relative to the COX-2 transcriptional start site is noted above or below each element. The consensus sequences of each cis-acting element are indicated below the shaded boxes.

 
Unlike fibroblasts where COX-2 is only transiently expressed (17), monocytes/macrophages express COX-2 for a prolonged time (18). Although the transcriptional regulation of COX-2 has been extensively studied, little is known about the dynamics of the essential cis-acting elements of the COX-2 promoter during transcriptional activation and the mechanism of sustained activation. In this study, we demonstrate the involvement of the CRE-1, CRE-2, NF-{kappa}B, and AP-1 sites in positive COX-2 transcriptional regulation and provide the first evidence of negative regulation of COX-2 gene transcription through the E-box element in LPS-treated RAW264.7 macrophages. We have also identified nuclear proteins associated with these response elements over a 12-h time period following LPS treatment and established that c-Jun phosphorylation and p50 nuclear translocation are uniquely required in the middle phase of COX-2 transcription. Cumulatively, our data demonstrate that the COX-2 gene is transcriptionally regulated in LPS-treated RAW264.7 macrophages in a coordinated manner by unique pairings of transcription factors and promoter elements to sustain COX-2 transcriptional activation for 12 h after initiating LPS treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and Abs

LPS from Salmonella minnesota was purchased from Sigma-Aldrich. Rabbit anti-p50 and anti-p65 Abs were provided by N. Rice (National Cancer Institute, Frederick, MD). CREB-1, ATF, upstream stimulating factor-1 (USF-1), and USF-2 Abs were purchased from Santa Cruz Biotechnology, Ab to phospho-CREB was purchased from Upstate Biotechnology, and Abs to CREB and CREB-binding protein (CBP) were purchased from Cell Signaling. Complete protease inhibitor mixture and Pefabloc were purchased from Roche Molecular Biochemicals. Poly(dI-dC)-poly(dI-dC) (average length 8517 bp) was purchased from Pharmacia Biotech. All other chemicals and reagents were purchased from J. T. Baker. Plasmid DNA was isolated with Qiagen Endo-Free Maxi-prep columns. Nuclei for EMSAs were isolated with NE-PER nuclear and cytoplasmic extraction reagents from Pierce. JNK inhibitor I (L-form) and the p50 inhibitor NF-{kappa}B SN50 were purchased from Calbiochem.

Plasmids

The various regions of the murine COX-2 promoter were cloned into pGL3basic (Promega). The –966/+23 construct was cloned from a KpnI and an HindIII fragment (19). The constructs containing –459/+23 and –414/+23 were cloned by PCR amplification with 3'-XhoI- and 5'- HindIII-tailed primers. Constructs containing –350/+23, –170/+23, and –98/+23 were cloned into the SmaI/HindIII sites of pGL3. Mutagenesis was performed using the Stratagene Quick Change protocol with pfu Turbo DNA polymerase (Stratagene). The sequences of the mutant and promoter constructs were verified; the mutations are summarized in Table I. pRC-p50 expression plasmid was provided by Prof. R. Schwartz (Michigan State University, East Lansing, MI) and the mCOX-2 plasmid, pSVLN-muCOX-2, was provided by Prof. D. L. DeWitt (Michigan State University, East Lansing, MI). pRSV-ACREB, pRSV-I{kappa}B S/R, and pET23b-p300 expression plasmids were provided by Prof. R. Kwok (University of Michigan, Ann Arbor, MI). The pcDNA3-USF-1/-2 expression vectors were generated by cloning HindIII-XbaI-flanked PCR products derived from USF-1/USF-2 cDNA into the HindIII-XbaI-digested pcDNA3 vector (Invitrogen Life Technologies).


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  		Table I. Oligonucleotides used for EMSAs and preparation of COX-2 promoter mutationsa

 
Cell culture and transfections

RAW264.7 cells (American Type Culture Collection) were cultured in DMEM, supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 mg/ml), and gentamicin (100 mg/ml; Invitrogen Life Technologies) and were maintained at 37°C in 5% CO2. RAW264.7 cells were cultured in 6-well plates at a density of 5 x 105 cells/ml 1 day before transfection. A luciferase reporter plasmid (2.5 µg) and a pCMV-beta-galactosidase plasmid (0.5 µg; Pharmacia) were transfected into RAW264.7 cells for 45 min using DEAE-dextran (400 µg/ml) and 100 mM Tris-HCl (pH 7.3) in 600 ml of DMEM. The transfection reaction mixture was removed and the cells were cultured in DMEM with 10% FBS for 12–24 h before LPS (200 ng/ml) stimulation for 12 h. For transfections using Superfect (Qiagen), 5 x 105 cells/well were plated 24 h before transfection. COX-2 (1 µg) plasmid and pSV-beta-galactosidase (0.5 µg) plasmids (Promega) were transfected into RAW264.7 cells according to the manufacturer’s instructions.

Chemiluminescent luciferase activity assays were performed using reagents from Promega and a Molecular Dynamics luminometer. beta-Galactosidase activity was measured using an O-nitrophenyl beta-D-galactopyranoside assay (Invitrogen Life Technologies) per the instructions of the manufacturer. Protein concentrations were determined using the Bradford reagent (Bio-Rad). Spectrophotometric measurements were made on a Molecular Dynamics 96-well plate reader. Luciferase activity is represented as relative luciferase units of firefly per beta-galactosidase activity. Statistically significant differences between groups vs control were obtained with a Student t test and significant differences are indicated by asterisks (Figs. 3B and 6B).


Figure 3
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  		FIGURE 3. Promoter activity of COX-2 deletion and mutation constructs. A, A nested set of deletions was prepared from the native –966/+23 murine COX-2 promoter. RAW264.7 cells were transiently cotransfected with 2.5 µg of luciferase promoter reporter plasmids containing the indicated regions of the COX-2 promoter and 0.5 µg of a CMV beta-galactosidase-expressing plasmid. Twenty-four hours posttransfection, the cells were stimulated for 12 h with LPS (200 ng/ml). Measurements of relative light intensities from the luciferase activity (relative luciferase units (RLU)) were normalized to the units of beta-galactosidase activity as described in Materials and Methods. Bars represent the average fold increase in promoter activity between unstimulated control and LPS-stimulated RAW264.7 cells for each of the COX-2 promoter reporter plasmid. The error bars represent the SD relative to the average fold increase in promoter activity observed across three independent experiments. B, Reporter activities of mCOX-2 promoter reporter constructs with individual cis-elements mutated. RAW264.7 cells were transiently cotransfected with 3 µg of luciferase promoter-reporter plasmids containing mutations in the indicated elements of the mCOX-2 promoter (–966/+23) and 0.5 µg of a pSV beta-galactosidase expression plasmid. LPS treatment and quantification of transfection data were performed as above. Data represent the promoter activity from cells transfected with reporters driven by either the WT COX-2 promoter or promoters with the indicated mutations, normalized to the beta-galactosidase activity, with and without LPS treatment. Error bars represent the SD from the average values obtained from three independent experiments performed in duplicate. *, p < 0.05 and **, p < 0.01 for native (WT) plasmid-transfected LPS-treated cells vs mutants plasmid-transfected LPS-treated cells.

 

Figure 6
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  		FIGURE 6. Functional assays of COX-2 transcriptional regulation in LPS-treated RAW264.7 cells. RAW264.7 cells were transiently cotransfected with the indicated amounts of ACREB expression plasmid (A), I{kappa}B expression plasmid (C), CBP expression plasmid (D), or p300 expression plasmid (E) as well as 1 µg of COX-2 luciferase promoter reporter plasmid and 0.5 µg of the Renilla (A, C, and E) or beta-galactosidase expression plasmid (D). ACREB which cannot bind to DNA and I{kappa}B S/R which traps p65/p50 heterodimer in the cell cytoplasm suppressed COX-2 promoter activity (A and C, respectively) and CBP and p300 overexpression increased COX-2 promoter activity (D and E) in LPS-treated RAW264.7 cells. Cells were stimulated with 200 ng/ml LPS for 12 h after 24 h of transfection and cell extracts were prepared and processed to measure luciferase activity. Data represent two independent experiments performed in duplicate. B, Both CRE-1 and CRE-2 require CREB binding for LPS-stimulated COX-2 transcription in RAW264.7 cells. RAW264.7 cells were transiently cotransfected with 1 µg of ACREB expression vector and 1 µg of WT, mutant CRE-1, or mutant CRE-2 COX-2 promoter construct and 0.5 µg of the beta-galactosidase expression plasmid for measuring transfection efficiency. Data represent three independent experiments performed in duplicate. Numbers next to the error bars represent the average fold induction in promoter activity by LPS treatment. Error bars represent the SD of the average values obtained from all three experiments. *, p < 0.05 and **, p < 0.01 for WT plasmid-transfected LPS-treated cells vs mutant COX-2 construct and/or ACREB expression plasmid-transfected LPS-treated cells.

 
Northern blot analysis

Total cellular RNA was isolated from cells using TRIzol RNA isolation reagent (Invitrogen Life Technologies). Total cellular RNA (15 µg) was electrophoresed on a 3.7% formaldehyde, 0.8% agarose gel in TAE (10 mM Tris-acetate (pH 8–8.1) and 10 mM EDTA) and transferred to a nitrocellulose membrane. The membrane was prehybridized for 1 h at 65°C in prehybridization buffer (5x SSC, 50% formamide, 5x Denhardt’s, 1% SDS, and sheared salmon sperm DNA (100 µg/ml)) and hybridized with a probe in TES/NaCl solution (10 mM Tris-HCl (pH 7.4), 10 mM EDTA, 0.2% SDS, and 0.6 M NaCl). Following hybridization, the membranes were washed twice in 2x SSC at 65°C for 20 min. A 1.8-kb NotI fragment of pSVLN mCOX-2 and a 1.3-kb EcoR1, XhoI fragment of a beta-actin plasmid (Stratagene) were labeled by random priming using a Mega Prime Labeling kit (Amersham Biosciences) with [{alpha}-32P]CTP (New England Nuclear). The membranes were exposed to a phosphoimaging screen and densitometry was performed using ImageQuant Software (Molecular Dynamics).

EMSAs

Nuclei were isolated from RAW264.7 cells that had been stimulated for 1 or 12 h with LPS (200 ng/ml). Cell lysis and nuclear isolations were performed using NE-PER reagents (Pierce) per the instructions of the manufacturer in the presence of 2 mM Pefabloc and 1x complete protease inhibitor mixture. Oligonucleotide probes (Michigan State University Macromolecular Structure Facility and Invitrogen Life Technologies) were annealed in T4 PNK buffer and end-labeled with T4 polynucleotide kinase (New England Biological) and [{gamma}-32P]ATP (New England Nuclear). The sequences of the probes are summarized in Table I. The probes were electrophoresed on a TAE 10% acrylamide gel. The double-stranded probes were excised and eluted for 2 h at 37°C in 0.5 M sodium acetate, 10 mM MgCl2, 1 mM EDTA, and 0.1% SDS, ethanol precipitated, and resuspended in Tris-EDTA. Binding reactions were performed with nuclear extract (5 µg of protein) and probe in the presence of 100 mM KCl, 20 mM HEPES (pH 7.9), 1 mM EDTA, 10% glycerol, 2 mM Pefabloc, 1x complete protease inhibitor mixture, 2 mM DTT, and 2 µg of poly(dI-dC)-poly(dI-dC) and electrophoresed on 5% Tris borate-EDTA acrylamide gels at 150 volts for 1.5 h. For EMSAs, the probes were incubated with the nuclear extracts for 1 h at 25°C, and for supershift assays, the nuclear extracts were combined with the probes for 15 min at 25°C and then incubated with antisera for 4–6 h at 2–8°C. EMSA gels were dried on 3M filter paper and exposed to a phosphoimaging screen and densitometry was performed using ImageQuant software (Molecular Dynamics).

Nuclear run-on assays

RAW264.7 cells (~107) were stimulated with LPS (200 ng/ml) for 0.25, 0.5, 1, 3, 6, 9, or 12 h. The cells were rinsed twice with ice-cold PBS and were scraped into 1 ml of PBS. The cells were collected by centrifugation and lysed by resuspension in 1 ml of 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Igepal CA-650 (Sigma-Aldrich) for 5 min. The nuclei were collected by centrifugation for 10 min at 1000 rpm in a minicentrifuge and resuspended in 500 ml of freeze buffer (50 mM Tris-HCl (pH 8.3), glycerol (40% v/v), 5 mM MgCl2, and 0.1 mM EDTA). Nuclei were either used fresh or stored at –80°C. To begin a run-on reaction, 225 ml of suspended nuclei (~107) was combined with 60 ml of run-on buffer (25 mM Tris-HCl (pH 8.0), 12.5 mM MgCl2, 750 mM KCl, 1.25 mM ATP, GTP, CTP, 2 mM DTT), 120 U of RNase-OUT (Invitrogen Life Technologies), and 100 mCi [{alpha}-32P]UTP. After 15 min at 37°C, 1 ml of TRIzol was added, followed by 200 ml of chloroform. The mixture was vortexed vigorously and transferred to a prespun Phase Lock Gel tube (Beckman/Eppendorf) and centrifuged for 10 min at 10,000 x g. The aqueous phase was removed and combined with 500 ml of isopropanol. The RNA was precipitated at –80°C for 15 min then centrifuged for 20 min at 10,000 x g and resuspended in RNase-free H2O. The RNA was further purified and genomic DNA was fragmented with RNase-free DNase I (Invitrogen Life Technologies) at 37°C for 5 min, then chilled on ice for 5 min before the addition of 1 M NaOH for exactly 2 min, followed by the addition of 1 M HEPES (free acid). The RNA was then precipitated by the addition of isopropanol. After centrifugation, the pellets were resuspended in diethyl pyrocarbonate (DEPC)-treated water. The activity was determined by scintillation counting. Slot blots were prepared on nitrocellulose membranes (Schleicher & Schuell) with 10 µg of denatured, linearized murine COX-2 expression plasmid (pSVLN-mCOX-2), beta-actin plasmid (Stratagene), empty vector, or vehicle without plasmid. After baking, the membranes were prehybridized in 5x SSC, 50% formamide, 5x Denhardt’s solution, 1% SDS, and sheared salmon sperm DNA (100 µg/ml) for 1 h at 65°C. Hybridization of the labeled RNAs with membranes was performed at 65°C in TES/NaCl Solution (10 mM Tris-HCl (pH 7.4), 10 mM EDTA, 0.2% SDS, and 0.6 M NaCl). Following hybridization, the membranes were rinsed twice in 10x SSC, then washed twice in 2x SSC at 65°C for 20 min. The dried membranes were exposed to a phosphoimaging screen and densitometry was performed using ImageQuant software (Molecular Dynamics).

Chromatin immunoprecipitation (ChIP) assay

LPS treated RAW264.7 cells were treated with 1% aqueous formaldehyde at room temperature for 20 min. Cells were washed twice with 10 ml of ice-cold PBS and scraped into 10-ml conical tubes. The suspended cells were then washed with 10 ml of ice-cold buffer A (0.25% Nonidet P-40, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES (pH 6.5), and 0.5 mM PMSF) and buffer B (20 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES (pH 6.5), and 0.5 mM PMSF) and lysed with a lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8), 1x complete protease inhibitor mixture, and 2 mM Pefabloc); 1 ml of the lysis buffer was used per 108 cells. Cell lysates were sonicated seven times for 15 s each at setting 3 on a Misonix sonicator to shear the DNA to lengths between 200 and 1000 bp. Samples were then centrifuged for 10 min at 12,000 rpm at 4°C. The supernatant fraction was diluted 10-fold in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8), 1x complete protease inhibitor mixture, and 2 mM Pefabloc) and 2 µg of p50, p65, CREB-1, c-Jun, p300, USF-1, or USF-2 Ab was added for each immunoprecipitation. After an overnight incubation with the Ab, 20 ml of 50% slurry of protein A/G agarose, which had been washed three times with dilution buffer, was added to the sample and the samples were incubated for 4 h at 4°C with gentle mixing. Immunoprecipitated materials were washed once with buffer TSE (0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 20 mM Tris (pH 8.0)), once with buffer TSE plus 250 mM NaCl, once with buffer TSE plus 500 mM NaCl, once with buffer C (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, and 10 mM Tri-HCl (pH 8)), and three times with Tris-EDTA buffer. Protein-DNA complexes were eluted with 300 ml of elution buffer (1% SDS and 0.1 M NaHCO3) for 30 min at room temperature. NaCl (200 mM) was added and the samples were incubated at 65°C for 16 h. After reverse cross-linking, 3 ml of protease K (20 mg/ml) and 1 ml of an aqueous glycogen solution (20 mg/ml) were added and the samples were incubated at 42°C for 2 h. DNA was extracted with phenol/chloroform. The pellet was resuspended in 40 ml of water. For PCRs, 5 ml of each sample was used and 20 ml of the 50-ml PCR product was loaded onto agarose gels. To obtain conditions where the signal intensity was linear with the input, various numbers of cycles of PCR were performed: 28 cycles (26 cycles for CBP ChIP assay) of reaction were used in the experiments depicted in the figures. PCR primers were as follows: for GAPDH, 5'-GCTGACATCAACTCCCAGGT-3', 5'-TTCCGTTCTCAGCCTTGACT-3'; for COX-2 (distal region), 5'-TCCCGGGATCTAAGGTCCTA-3' and 5'-CAGATGTGGACCCTGACAGA-3'; for COX-2 (proximal region), 5'-TCCTTCGTGAGCAGAGTCCT-3' and 5'-CGCAACTCACTGAAGCAGAG-3'. PCR products were electrophoresed on 2% agarose gels and visualized by staining with Cyber Green. PCR products for GAPDH, COX-2 (distal region), and COX-2 (proximal region) are 470, 159, and 248 bp, respectively.

RT-PCR COX-2 RNA segments

Total RNA isolated using an RNeasy Mini kit (Qiagen) from RAW264.7 cells treated for various times with LPS was used for RT-PCR using a One-Step RT-PCR System (Invitrogen Life Technologies) to determine the relative levels of COX-2 "intron 2" (newly formed, unprocessed mRNA) and "exon 3" (processed mRNA). PCR primers for intron 2 were: 5'-AGGACTCTGCTCACGAAGGA-3' (bridges exon 2/intron 2 boundary) and 5'-CCTTGAAGTGGGTCAGGATG-3' (located in exon 3) which permit amplification of a 332-bp sequence containing all of exon 2 and portions of introns 2 and 3. PCR primers for exon 3 were: 5'-TGTGAACAACATCCCCTTCC-3' (bridges exons 3 and 4) and 5'-GGCAAAGAATGCAAACATCA-3' (located in exon 4) which permit amplification of a 304-bp sequence containing portions of exons 3 and 4 with no intervening intron.

In separate experiments, RT-PCR was also used to determine the effects of JNK and NF-{kappa}B p50 inhibitors on RNA accumulation by RAW264.7 cells treated for various times with LPS. Total RNA was isolated and subjected to RT-PCR as described above. COX-2 primers used to determine COX-2 mRNA levels in these experiments were: 5'-ACACTCTATCACTGGCATCC-3' and 5'-GAAGGGACACCCTTTCACAT-3' which permit amplification of a 585-bp fragment. PCR products were electrophoresed on 2% agarose gels and visualized by staining with Cyber Green.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LPS causes sustained, phasic increases in the rate of COX-2 gene transcription in RAW264.7 cells

Stimulation of macrophage and macrophage-like cells with LPS results in the synthesis of prostaglandins and sustained increases in the expression of COX-2 protein and other inflammation-related proteins (8, 20, 21, 22, 23). It has been hypothesized that the sustained increase in COX-2 expression is a result of increased transcription of the COX-2 gene, but there has been no direct demonstration of this. We performed Northern blot analyses (Fig. 2A) and nuclear run-on assays (Fig. 2B) that suggest that LPS treatment of RAW264.7 cells does cause a rapid and prolonged increase in the rate of synthesis of COX-2 mRNA. In the run-on assays, there is a gradual increase in the rate of mRNA synthesis during the first hour after LPS treatment. This appears to be followed by a pause in the rate of transcription and another increase in the transcription rate that occurs 6–9 h after adding LPS and continues to increase at 12 h. As an additional test of this concept, we performed semiquantitative RT-PCR using primers for mature mRNA transcripts having contiguous exon 3 and 4 sequences and for primary transcripts containing portions of introns 2 and 3 (and all of exon 3) (Fig. 2C). The results are consistent with the results of the Northern blot (Fig. 2A) and nuclear run-on (Fig. 2B) experiments and clearly indicate that there are at least three distinct phases to LPS-induced COX-2 gene expression in RAW264.7 cells: an early (~0–4 h), a middle (~4–8 h), and a late (~8–12 h) phase. Further support for this hypothesis was obtained using inhibitors of JNK and NF-{kappa}B p50 and these data are presented later in this section.


Figure 2
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  		FIGURE 2. Nuclear run-on, Northern blot, and COX-2 RT-PCR analysis of mCOX-2 gene expression in LPS-stimulated RAW264.7 cells. A, Northern blot analysis was performed with RNA isolated from RAW264.7 cells that had been treated with LPS (200 ng/ml) for the indicated times. Total RNA (15 µg) was separated on a 0.8% agarose, 4% formaldehyde gel, transferred to a nitrocellulose membrane and hybridized to COX-2 or beta-actin probes as detailed in Materials and Methods. Similar results were obtained in five independent experiments. The graph presents densitometry data. The value for relative COX-2 transcripts was calculated using intensity data ((COX-2-empty vector)/(actin-empty vector)) + 1. B, Nuclear run-on analyses of two separate experiments with duplicate samples. Nuclei were isolated from RAW264.7 cells stimulated with LPS (200 ng/ml) for the indicated times and incubated with [{gamma}-32P]UTP to label newly synthesized RNA transcripts. RNA was isolated and blotted onto a nitrocellulose membrane with vehicle, COX-2 cDNA, beta-actin cDNA, or an empty vector DNA control. Four sets of data were used except for the 0.25-h time point where only three points were available. The data are the ratios of the densities of COX-2 minus those of empty vector to the densities of the actin control minus those of empty vector. Values are plotted as the means ± SE. Multiple comparisons analyzed with Student’s t test (JMP 6.02; SAS Institute) was used to determine significance. Means not sharing a letter are significantly different (p > 0.05). C, RT-PCR of COX-2 RNA. RAW264.7 cells were treated with LPS (200 ng/ml) for indicated times. Total RNA was isolated and was reverse transcribed and amplified using primers specific for COX-2 primary transcripts (intron 2/3), COX-2 mRNA (exon 3), or beta-actin as a control. Descriptions of the primers are presented in Materials and Methods. Amplicons were visualized on an agarose gel stained with SYBR green I.

 
Characterization of new cis-acting elements in the murine mCOX-2 promoter associated with LPS-induced COX-2 gene expression

Several cis-acting elements in the mCOX-2 promoter, including an NF-{kappa}B site at –400/–392, a C/EBP response element at –136/–128, and a CRE-1 at –56/–51, have been shown previously to be required for LPS-induced COX-2 transcription in RAW264.7 cells (24, 25, 26). To further elucidate the transcriptional regulatory components of the mCOX-2 promoter, deletion constructs of the promoter driving a luciferase reporter were transfected into RAW264.7 cells (Fig. 3A). Transfected cells were treated for 12 h with or without LPS to determine which general regions of the mCOX-2 promoter are necessary for maximal LPS responses (Figs. 1 and 3). Deletion of a segment from –459 to –414 removes a previously uncharacterized CRE (CRE-2) without eliminating the adjoining, conserved NF-{kappa}B response element (Fig. 1). Deletion of this CRE-2 site resulted in an ~50% reduction in the LPS-induced COX-2 expression (Fig. 3A). Larger deletions that removed the NF-{kappa}B and C/EBP-1 response elements caused no further decreases in the LPS-induced promoter responses, suggesting that the CRE-2 site is an important regulatory component of LPS-mediated COX-2 induction. Additional deletion analyses of proximal regions of murine COX-2 were not performed in this study, as this region had been characterized previously in LPS-treated RAW264.7 cells and shown to be important in regulating COX-2 mRNA accumulation (24, 27).

To investigate the relative contribution of the CRE-2 and other putative cis-acting elements of the COX-2 promoter, we engineered and analyzed mutations in the CRE-1 (–56/–51), E-box (–52/–47), AP-1 (–67/–62), CRE-2 (–433/–428), and c-Ets (–314/–309) sites in the mCOX-2 promoter (–966 to + 23) fused to a luciferase reporter gene (Table I; Fig. 3B). Consistent with the deletion analysis (Fig. 3A), mutation of the CRE-2 resulted in a loss of about half of the LPS-stimulated luciferase activity. The AP-1 and CRE-1 mutations eliminated ~15 and 60% of the reporter activity, whereas the c-Ets mutation did not cause any change in luciferase activity when compared with the wild-type (WT) promoter (Fig. 3 and data not shown). Our data indicate that in addition to the previously reported cis-acting elements (i.e., NF-{kappa}B, C/EBP, and CRE-1), the CRE-2 and the AP-1 elements are required for maximal mCOX-2 promoter activity elicited by LPS treatment of RAW264.7 cells. Interestingly and in contrast to the reduced activity resulting from mutation of the other regulatory elements of COX-2 promoter, mutation of the E-box caused a significant increase in luciferase activity (Fig. 3B), suggesting that the E-box is involved in inhibition of mCOX-2 gene transcription in LPS-stimulated RAW264.7 macrophages.

Transcription factor binding to cis-acting elements in the mCOX-2 promoter

EMSA supershift assays were performed to identify proteins binding to the COX-2 cis-acting elements. To examine binding to the CRE-2, a [32P] 26-bp dsDNA probe containing the CRE-2 (5'-ACGTCA-3') (Table I) was incubated with nuclear extracts from either control RAW264.7 cells or cells that had been treated with LPS for 0, 1, or 12 h (Fig. 4, A and B, lanes 1–3). The mobility and intensities of the radioactive CRE-2 protein complexes with control IgG were similar when using nuclear extracts from both treated and untreated cells. That is, the amount of CRE-2 bound by nuclear proteins does not appear to be substantially up or down-regulated by LPS. Supershift assays with Abs against CREB, ATF-1, ATF-2, or phospho-c-Jun (Fig. 4, A and B) indicated that CRE-2 is bound constitutively by members of the CREB/ATF transcription factor family (i.e., CREB and ATF-2) and by phosphorylated c-Jun.


Figure 4
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  		FIGURE 4. EMSA supershift assays analyzing transcription factor binding to mCOX-2 promoter elements. A and B, Transcription factor binding to CRE-2. EMSA supershift assays were performed as described in Materials and Methods. The double-stranded [32P]CRE-2 probe was incubated with a nuclear extract from RAW264.7 cells stimulated with LPS (200 ng/ml) for 0, 1, or 12 h and either an IgG control Ab or an Ab to CREB or ATF-2 (A) or to phosphorylated c-Jun or ATF-1 (B). CREB, ATF-2, and P-c-Jun supershifted complexes are denoted with open arrows and the CRE-2 complex is indicated with the closed arrows. C, p65 and p50 binding to a mCOX-2 NF-{kappa}B probe. EMSA supershift assays were performed as detailed in Materials and Methods. The double-stranded [32P]NF-{kappa}B probe was incubated with a nuclear extract from RAW264.7 cells stimulated with LPS (200 ng/ml) for 0, 1, or 12 h and either an IgG control Ab or an Ab to p50 or p65. Complexes of the NF-{kappa}B probe and p50/p65 heterodimers or p50/p50 homodimers are indicated with closed arrows. The supershifted (SS) complexes bound to Ab to p50 or p65 are indicated with open arrows. D, AP-1 transcription factor binding to a COX-2 AP-1 probe. EMSA supershift assays were performed using a double-stranded [32P]AP-1 probe. The probe was incubated with nuclear extracts from RAW264.7 cells stimulated with LPS (200 ng/ml) for 0, 1, or 12 h and either an IgG control Ab or an Ab to phosphorylated c-Jun or c-fos. The AP-1 complex is indicated with closed arrows and the phospho-c-Jun and c-fos supershifted complex is indicated with open arrows. E, CREB, USF-1, USF-2, and phosphorylated c-Jun binding to a COX-2 probe with the overlapping CRE-1/E-box probe. EMSA supershift assays were performed with a double-stranded [32P]COX-2 probe containing the overlapping CRE/E-box. The probe was incubated with a nuclear extract from RAW264.7 cells stimulated with LPS (200 ng/ml) for 0, 1, or 12 h and either an IgG control Ab or an Ab to CREB, USF-1, USF-2, or phosphorylated c-Jun. The CRE-1 and E-box complexes are indicated with closed arrows and the supershifted complexes are indicated with open arrows.

 
The NF-{kappa}B response element (5'-GGGATTCCC-3'; Table I) is necessary for maximal mCOX-2 promoter activity in RAW264.7 cells (7, 28), but the nuclear proteins bound to this response element had not been characterized (Fig. 4C). p65 and p50 are known to be involved in distinct dimeric NF-{kappa}B complexes (29). To determine the proteins bound to the NF-{kappa}B site, supershift assays were performed using nuclear proteins from untreated cells or cells treated with LPS for either 1 or 12 h. Two distinct complexes were observed with the NF-{kappa}B probe and these nuclear extracts. Anti-p50 Ab caused shifts in both the high and low mobility complexes, whereas the anti-p65 Ab shifted only the low mobility complex (Fig. 4C). This indicates that the low mobility complex includes a p50/p65 heterodimer while the high mobility complex includes a p50/p50 homodimer. Interestingly, our data revealed that the NF-{kappa}B site is primarily occupied by the p65/p50 heterodimer during the early phase (1 h) of the LPS response and predominantly occupied by the p50 homodimer at the late phase (12 h) of COX-2 transcriptional activation (Fig. 4C). It was after 4 h of exposure of RAW264.7 cells to LPS that we observed a shift in the composition of the nuclear complex bound to the NF-{kappa}B site from predominantly p50/p65 heterodimers to predominantly p50 homodimers (data not shown). This result is despite the fact that COX-2 transcription is up-regulated for at least 12 h by LPS treatment (Fig. 2) and that the p50 homodimer is typically associated with inhibition of gene expression.

Gel shift analysis revealed that the binding of nuclear proteins to the AP-1 site (5'-GAGTCA-3'; Table I) is induced by LPS stimulation but that the extent of binding then remains about the same 1 and 12 h after initiating LPS treatment (Fig. 4D). To identify the transcription factors bound to the AP-1 site, supershift assays were performed using Abs reactive with phosphorylated c-Jun, c-fos, ATF-1, ATF-2, and CREB. This analysis indicated that the AP-1 site is bound primarily by phosphorylated c-Jun and c-fos (Fig. 4D). No binding of members of the CREB/ATF transcription factor family to the AP-1 site was observed (data not shown). Specific binding activity of phosphorylated c-Jun was almost undetectable in untreated control cells but was increased considerably after stimulation of macrophages with LPS. Binding of c-fos to the AP-1 site was increased at 1 h of treatment of LPS but was absent at 12 h of LPS treatment, suggesting that the c-fos is specifically playing an important role in the early phase of COX-2 induction.

To identify nuclear proteins that bind to the overlapping CRE-1 (5'-ACGTCA-3'; Table I) and E-box element (5'-CACGTG-3'; Table I), a [32P] 25-bp oligonucleotide containing the overlapping CRE-1 and E-box was prepared for EMSA supershift assays. As was observed at the NF-{kappa}B probe, two distinct complexes were formed with the 25-bp CRE-1/E-box probe (Fig. 4E, lanes 1–3). The higher mobility complex was shifted by anti-CREB Ab (Fig. 4E, lanes 4–6) and the lower mobility complex was shifted by USF-1 (Fig. 4E, lanes 7–9) or USF-2 Abs (Fig. 4E, lanes 10–12). Transcription factor recruitment to this element appears to be constitutive, as similar levels of binding are observed in untreated RAW264.7 cells and after 1 and 12 h of LPS stimulation. Although mutation of the E-box lead to a significant increase in COX-2 transcription, overexpression of USF-1 or USF-2 failed to repress COX-2 promoter activity (data not shown) suggesting that the repression does not directly involve USF-1 or USF-2 binding to the E-box.

In vivo association of transcription factors with the mCOX-2 promoter in LPS-stimulated RAW264.7 cells

Results of the EMSA supershift assays suggest that CREB, p65, and p50, phosphorylated c-Jun and c-fos and USF-1/-2 transcription factors can bind to the CRE-1 and CRE-2, NF-{kappa}B, AP-1, and the E-box, respectively. ChIP analysis were performed to determine the composition of trans-acting factors associated with the mCOX-2 promoter in intact RAW264.7 cells after 0, 1, or 12 h of LPS stimulation (Fig. 5). In agreement with the EMSA supershift results, ChIP assays revealed that CREB and USF-1/-2 are constitutively associated with the COX-2 promoter (Fig. 5, C and F). As predicted by the observed changes in the composition of the NF-{kappa}B dimer by EMSA supershifts, p65 binding is increased at 1 h and decreased after 4 h whereas p50 association increases from 0 to 12 h (Fig. 5B). Similar to the in vitro pattern of c-Jun recruitment, c-Jun binding in vivo is increased 1 h after LPS treatment is decreased at 4 h and then is increased again at 12 h (Fig. 5D). Consistent with increased COX-2 gene transcription (Fig. 2), the transcriptional coactivators CBP and p300 as well as RNA polymerase II associate with the COX-2 promoter in response to LPS treatment (Fig. 5E).


Figure 5
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  		FIGURE 5. Chromatin immunoprecipitation (ChIP) assays to identify transcription factors associated with the mCOX-2 promoter in LPS-stimulated RAW264.7 cells. A, RAW264.7 cells were stimulated with LPS for various times (0, 1, 4, or 12 h) and PCR analyses were performed on 3-fold serial dilutions of the input chromatin to establish that the amounts of PCR products are proportional to the amounts of starting material. B–F, PCR analysis of DNA from the ChIP assays. Mock immunoprecipitation with IgG (B–F) was performed as a negative control and PCR was performed using the same primers. Chromatin from the RAW264.7 cells was diluted 1/10 and the samples were processed through the ChIP protocol as described in Materials and Methods. Abs to p65 or p50 were used for B; Ab to CREB-1 was used for C; c-Jun Ab was used for D; Abs to CBP, p300, RNA polymerase II were used for E; and USF-1 and USF-2 for F. GAPDH primers were used as negative controls for the PCR. G, Primers used for PCR. Closed arrows are primers used for the distal regions and open arrows indicate proximal regions.

 
Collectively, the results of the EMSA supershift assays and the ChIP analyses are qualitatively and semiquantitatively consistent and suggest that there is a distinct temporal pattern to the binding of different transcription factors to different cis-acting elements in the COX-2 promoter in response to LPS stimulation.

Effect of dominant-negative transcription factors on COX-2 transcriptional activation

To investigate the mechanism whereby CREB regulates COX-2 transcription, a dominant-negative CREB called ACREB, which cannot bind DNA, was cotransfected into RAW264.7 cells along with the COX-2 promoter luciferase reporter construct (Fig. 6A). Transfection of increasing amounts of ACREB lead to a reduction in LPS-inducible luciferase activity (Fig. 6A), suggesting that recruitment of endogenous cofactors to the promoter by native CREB is required for LPS-induced COX-2 transcription in RAW264.7 cells. The COX-2 promoter has two CRE sites, both of which can bind CREB (Fig. 4). To determine the impact of the interaction with CREB at each of the CRE sites, the ACREB expression vector was cotransfected with reporter constructs driven by either the native mCOX-2 promoter or a version of the promoter with individual mutations in either CRE-1 or CRE-2 (Fig. 6B). Although expression of the mutant CRE-1 reporter construct lead to a more substantial reduction in reporter activity than expression of the mutant CRE-2 construct, both mutations resulted in a significant reduction in reporter activity compared with intact mCOX-2 promoter. Furthermore, cotransfection of each of the mutant CRE reporters with ACREB resulted in a comparable fold reduction in reporter activity (Fig. 6B). These results indicate that LPS-induced expression of mCOX-2 depends on CREB binding through both the CRE-1 and the CRE-2 and suggests that another transcription factor may also be activating COX-2 expression through the CRE-1.

To evaluate the role of NF-{kappa}B activation in this system, RAW264.7 macrophages were cotransfected with the COX-2 reporter construct and a dominant-negative I{kappa}B (I{kappa}B S/R) that fails to release p65/p50 after activation and thus traps NF-{kappa}B in the cytoplasm (Fig. 6C). Again, transfection of increasing amounts of the dominant-negative expression vector lead to a reduction in mCOX-2 transcriptional activity. This suggests that p65/p50 is also required for COX-2 transcription.

CBP and p300 are both involved in COX-2 transcriptional regulation

CBP and p300 are homologous coactivators that interact with many trans-acting transcription factors including CREB, NF-{kappa}B, and c-Jun (30, 31). To determine the influence of CBP and p300 on LPS-induced COX-2 transcriptional regulation, we overexpressed each of these coactivators and evaluated the effect on COX-2 reporter activity. In both cases, LPS-inducible COX-2 luciferase activity was increased with increasing amounts of CBP and p300 expression vector (Fig. 6, D and E). This suggests that transcriptional coactivators CBP and p300 are involved in LPS-induced COX-2 transcriptional activation in RAW264.7 cells.

P50 and phosphorylated c-Jun are uniquely required for the middle phase of COX-2 gene transcription

Consistent with Northern blot analysis and RT-PCR of exons 3 and 4 (Fig. 2), semiquantitative RT-PCR analysis showed that COX-2 mRNA levels increased over the 12 h period of LPS treatment and that there are three phases (Fig. 7, top panel). Our ChIP data suggest that binding of both p50 homodimers and phosphorylated c-Jun to the COX-2 promoter increase from 4 to 12 h of LPS treatment (Fig. 5, B and D). To determine the relative contribution of phosphorylated c-Jun and p50 at the middle and late phases of COX-2 induction, JNK and p50 inhibitors were added separately and in combination 3.5 h after initiating the LPS treatment and the COX-2 mRNA levels were analyzed by semiquantitative RT-PCR at 4, 6, 9, and 12 h (Fig. 7). COX-2 mRNA levels were reduced by about half by the individual inhibitors and was completely abrogated at 6 h after LPS treatment in cells treated with both JNK and p50 inhibitors. However, COX-2 expression begins to recover at the 9 and 12 h time points (Fig. 7) likely as a result of the involvement of other transcription factors such as phosphorylated CREB or C/EBP. This experiment demonstrates that the contribution of different transcription factors varies with time during LPS-induced COX-2 expression.


Figure 7
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  		FIGURE 7. Effects of JNK and p50 inhibitors on COX-2 mRNA induction in LPS-treated RAW264.7 macrophages as determined by semiquantitative RT-PCR. A, Cells were stimulated for 1, 2, 4, 6, 9, or 12 h with or without LPS (200 ng/ml) and JNK inhibitor I (10 µM) or p50 inhibitor (NF-{kappa}B, SN50 (90 µM) were added 3.5 h after initiating the LPS treatment. Total RNA was extracted and subjected to RT-PCR using specific primers for COX-2 and beta-actin (internal control) and agarose gels stained with Cyber Green as described in Materials and Methods. B, Densitometric analysis combining results from two independent experiments. COX-2 values were normalized to beta-actin values in all experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
LPS and certain cytokines and growth factors increase the expression of COX-2 which, in turn, leads to the formation of proinflammatory PGs. Specific COX-2 inhibitors such as celecoxib and rofecoxib are effective anti-inflammatory and analgesic agents. Although the mechanism of COX-2 transcription in macrophage cells is complex, resolving the complicated network of regulatory events controlling COX-2 expression during inflammation will likely uncover new potential clinical targets for lowering COX-2 activity in a cell-specific manner. In this study, we have used LPS-treated RAW264.7 cells as a model to study the role of COX-2 transcription and how it is controlled during an acute inflammatory response. We have 1) identified three previously uncharacterized, functional cis-elements in the COX-2 promoter in addition to the three that had been reported previously, 2) catalogued the binding of transcription factors to each element over a 12 h time course following LPS treatment, 3) showed that there are at least three distinct temporal phases of COX-2 transcriptional activation, and 4) provided evidence that each phase involves the participation of unique combinations of cis elements and trans-activating factors.

LPS treatment of RAW264.7 cells increases the rate of COX-2 transcription

We first established, using nuclear run-on experiments and RT-PCR of an intron-containing primary transcript, that LPS treatment causes an increase in the rate of COX-2 gene transcription that continues for 12 h but occurs in distinct early, middle and late phases. COX-2 transcription is rapid for the first 4 h of LPS treatment (early phase), then there is a slowing of the transcription rate during the next 2–4 h (middle phase), and finally, the rate of COX-2 gene transcription increases again between 8 and 12 h (late phase). Additional indirect evidence for these three distinct phases was obtained in experiments with inhibitors that block c-Jun and NF-{kappa}B actions. These two transcription factors are uniquely functional at ~6 h whereas other transcription factors participate at earlier and later time points. The sustained COX-2 mRNA expression observed in LPS-treated RAW264.7 cells contrasts with the more transient inducible COX-2 expression observed in other cell types and in response to other agonists (e.g., serum-induced COX-2 expression in murine fibroblasts (17, 32)) where gene transcription rates also increase rapidly but with COX-2 mRNA levels peaking within 1 h and then decreasing to basal levels within 2–3 h. Although the rate of COX-2 transcription is significantly increased in LPS-treated RAW264.7 cells, it should also be noted that the increased amount of COX-2 mRNA in response to LPS may also result from a decrease in the rate of posttranscriptional processing (33). Our studies focus on the transcriptionally modulated aspects of COX-2 expression and cannot rule out the participation of mRNA stability in this process.

cis-acting elements functional in LPS-induced COX-2 gene expression

Previously published reports have identified several cis-acting elements of the murine COX-2 promoter that are necessary for a response to LPS. These elements include a CRE (CRE-1) at –56/–51 (6), a C/EBP site at –136/–128 (27), and an NF-{kappa}B response element at –400/–392 (5, 7, 8, 10) (Fig. 1). In addition, it has been shown that inhibition of NF-{kappa}B activation with specific inhibitors or decoy oligonucleotides inhibits COX-2 activation (34, 35, 36, 37, 38). Using promoter reporter assays, we have identified a second CRE (CRE-2) located at –433/–428 and established that the AP-1 site located at –67/–62 is necessary for maximal LPS induction of the mCOX-2 gene (Fig. 1). Additionally, we have provided evidence for a role of the E-box element at –52/–47 in repression of mCOX-2 transcription.

Trans-acting factors functional in LPS-induced COX-2 gene expression

Using EMSA and ChIP assays, we found that AP-1 or CREB bind to the CRE-1 site. Studies with dominant-negative ACREB suggest that CREB binding to both CRE-1 and CRE-2 is important in COX-2 transcriptional activation. USF transcription factors bind to the E-box that overlaps the CRE-1 site. However, binding of AP-1 or CREB to the CRE-1 site and binding of USF-1/2 to the E-box are mutually exclusive. Unlike other systems where the E-box is involved in activation of COX-2 transcription (15, 39, 40), our promoter reporter assays indicate that the E-box in the mCOX-2 promoter is involved in repression in RAW264.7 cells. Mestre et al. (14) reported that the E-box mediates COX-2 transcriptional activation in response to LPS and involves USF-1 binding to the E-box; however, their study used a reporter gene under the control of a short human COX-2 promoter construct (–327/+59) that did not contain the CRE-2 or NF-{kappa}B element. There could also be species differences in COX-2 transcriptional regulation that complicate the interpretation of the results. There are precedents for an E-box mediating repression in other cell types. In mesangial cells, USF-1 and USF-2 trans-repress IL-1beta-induced inducible NO synthase transcription by binding to the E-box element (41). However, in our studies, USF-1 and USF-2 overexpression did not lower COX-2 promoter activity. This suggests that an as yet unidentified repressor complex binds to the E-box in LPS-treated RAW264.7 cells to mediate transcriptional repression.

The AP-1 element has been shown to be required for COX-2 promoter activation in herpes virus 6 (HHV-6)-infected monocytes (42), PMA-treated MC3T3 cells (43), and fluid shear stress-treated osteoblasts (44). However, the AP-1 site in LPS-induced COX-2 transcription in RAW264.7 cells had not previously been studied. Our results establish that mutation of the AP-1 site causes a decrease in promoter activity similar in magnitude to that caused by mutation of CRE-1. Additionally, as shown by EMSA supershift assays, members of the AP-1 transcription factor family, phosphorylated c-Jun and c-fos, bind to the AP-1 site and activate the COX-2 gene at early time points. Phosphorylated c-Jun homodimers are the predominant species bound to this site and play an important role in the middle and late phases of COX-2 transcription.

We have also discovered that the p50/p65 heterodimer and the p50 homodimer bind to the NF-{kappa}B site in a temporally regulated manner following LPS treatment. The p50/p65 dimer predominates in the early phase and the p50 homodimer predominates in the middle and late phases of LPS-induced COX-2 gene transcription. CREB/ATF transcription factors bind to the CRE-2 site and studies with a dominant-negative CREB (ACREB) suggest that the regulatory action of CREB occurs through the CRE-2 site as well as the CRE-1 site. Although not examined in the present studies, others have documented the binding of several C/EBP transcription factors the C/EBP response element of the COX-2 promoter (24, 45).

Putative c-Ets sites at –734/–725 and –329/–320 in the mCOX-2 promoter have been previously described as mediating COX-2 transcriptional activation in response to LPS-induced phosphorylation of PU.1 (46). We were unable to identify a functional c-Ets element using promoter analyses. Sequence analysis of the mCOX-2 promoter using the Genomatrix program (<www.genomatrix.de>) located a potential c-Ets site (5'-GAGGAA-3') at –314/–309, but this site is different from those previously reported. The study that implicated PU.1 in mediation of COX-2 transcription in LPS-treated RAW264.7 cells did not include promoter assays or evidence of PU.1 binding to an c-Ets site. If PU.1 is functional in this system, it is likely binding to an element other than an c-Ets site or activating COX-2 transcription indirectly.

Dynamics of COX-2 transcriptional activation

The results of nuclear run-on assays and RT-PCR of intron-containing primary transcripts indicate that there are three different phases of transcriptional activation. EMSA and ChIP assays showed differences in the composition of the transcription factors bound to cis elements in the COX-2 promoter at different times after LPS treatment. Thus, the process of COX-2 gene activation appears to involve sequential, coordinated events. In further testing this concept, we found that when inhibitors of Jun kinase and p50 translocation were added together to RAW264.7 cells 3.5 h after initiating LPS treatment that transcription was completely blocked at 6 h, but that during the period from 8 to 12 only partial inhibition of transcription was observed. Based on these observations, we developed a model illustrating events that are likely occurring at the COX-2 promoter at different times after LPS treatment (Fig. 8).


Figure 8
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  		FIGURE 8. Model depicting the actions of transcription factors at the COX-2 promoter at different times after beginning LPS-induced COX-2 expression in RAW264.7 macrophages. A, Unstimulated cells; 1 h after LPS addition (B); 4 h after LPS stimulation (C); 6 h after LPS stimulation (D); and 8–12 h after LPS stimulation (E).

 
Even before LPS stimulation, CREB, USF-1, and USF-2 are bound to their cognate CRE-1, CRE-1, or E-box response elements within the mCOX-2 promoter (Fig. 8). In contrast, there is little or no binding of cognate transcription factors to the NF-{kappa}B, C/EBP, or AP-1 sites. CBP, p300, and Pol II are associated with the COX-2 promoter at low levels. The presence of these activating components may permit basal COX-2 expression. We have not examined how these latter factors are recruited to the COX-2 promoter before LPS induction, but unphosphorylated CREB can bind CBP/p300 (31) and such an interaction involving one of the two CREs could recruit CBP/p300 and subsequently Pol II to the promoter.

During the early phase (0–4 h) of LPS treatment (Fig. 8B), we speculate that CREB bound at CRE-1 and CRE-2 becomes phosphorylated which enhances the recruitment of coactivators such as CBP and p300 (31). Additionally, the 65/p50 heterodimer is the major species that binds to the NF-{kappa}B site and p65 is presumably phosphorylated, and the C/EBPbeta homodimer binds to the C/EBP element (24). In the proximal region of the COX-2 promoter, a heterodimer of newly phosphorylated c-Jun/c-fos becomes bound to the AP-1 site. Phosphorylated p65, C/EBPbeta, and phosphorylated c-Jun have all been shown to interact with CBP/p300 (47, 48, 49). This suggests that the various activated transcription factors acting in concert may be recruiting CBP/p300 and general transcription factors to the COX-2 promoter leading to active transcription.

Based on EMSA and ChIP assays and the experiment showing that the combination of a JNK and a p50 inhibitor abolishes COX-2 induction at 4 h of LPS treatment, the middle phase of COX-2 transcription (4–8 h, Fig. 8, C and D) appears to be dominated by the AP-1 and NF-{kappa}B sites and presumably involves phosphorylated c-Jun homodimers bound to the AP-1 site and p50 homodimers bound to the NF-{kappa}B site. p50 Homodimers are typically associated with negative regulation of gene transcription (50), but there are reports suggesting that p50 homodimers can be involved in transcriptional activation (51, 52).

The rate of COX-2 transcription increases again after ~8 h of LPS treatment and after the middle phase of induction and the transcription rate remains elevated at 12 h (Fig. 8E). During the late stage of induction (~8–12 h), there is increased phosphorylation of c-Jun such that the concentration of phosphorylated c-Jun is increased and a relatively higher level of phosphorylated c-Jun homodimer binds to the AP-1 site. Binding of p50 homodimers to the NF-{kappa}B sites also occurs and at levels comparable to those seen during the middle phase of induction. The amount of CREB bound to the CRE-2 and CRE-1 elements remains the same before and throughout LPS treatment, however, CREB may be phosphorylated during the late phase of LPS stimulation. Binding of C/EBP transcription factors to the C/EBP site may also contribute to enhanced transcription during the late phase. Again, all these transcriptional activators are likely to be facilitating transcription by binding to CBP/p300.


   Acknowledgments
 
We thank Drs. David L. DeWitt and Richard Schwartz from Michigan State University and Drs. Roland Kwok and Daniel Bochar from the University of Michigan for helpful advice and discussions. We also thank Dr. Roland Kwok for generously providing ACREB and p300 expression vectors and for help in designing USF-1 and USF-2 expression vectors.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants DK22042 and GM68848 from the National Institutes of Health. Back

2 Y.-J.K. and B.A.W. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. William L. Smith, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109. E-mail address: smithww{at}umich.edu Back

4 Abbreviations used in this paper: COX, cyclooxygenase; mCOX, murine COX; CRE, cAMP response element; USF, upstream stimulating factor; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; WT, wild type. Back

Received for publication December 5, 2005. Accepted for publication September 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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