5-Lipoxygenase-activating Protein Gene Expression

KEY ROLE OF CCAAT/ENHANCER-BINDING PROTEINS (C/EBP) IN CONSTITUTIVE AND TUMOR NECROSIS FACTOR (TNF) alpha -INDUCED EXPRESSION IN THP-1 CELLS*

K. Veera Reddy, Kenneth J. Serio, Craig R. Hodulik, and Timothy D. BigbyDagger

From the Department of Medicine, Veterans Affairs San Diego Healthcare System and the University of California, San Diego, California 92161

Received for publication, October 30, 2002, and in revised form, February 3, 2003

    ABSTRACT
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We examined expression of the 5-lipoxygenase activating protein (FLAP), which is critical for inflammatory cell leukotriene synthesis. A 3.4-kb segment of the FLAP gene 5'-untranslated region accounted for a 22-fold increase in promoter activity when transfected into the monocyte-like cell line, THP-1, and demonstrated no activity in non-inflammatory cells. Virtually all of the promoter activity was mediated by the first 134 bp upstream of the transcription start site, a region that contains CCAAT/enhancer-binding proteins (C/EBP) consensus binding sites, at -36 to -28 bp (distal) and -25 to -12 bp (proximal). DNase I footprint analyses demonstrated THP-1 nuclear extract proteins bind to the proximal site. Electrophoretic mobility shift assay analyses revealed that C/EBPalpha , delta , and epsilon  bind to the proximal site and C/EBPalpha and epsilon  bind to the distal site, constitutively. Transfection studies indicated that mutation of both the proximal and distal sites decreased constitutive FLAP promoter activity. Overexpression of C/EBPalpha , beta , and delta  transactivated promoter activity and increased native FLAP mRNA accumulation. Mutation of both C/EBP sites essentially abolished promoter induction by C/EBP overexpression. Tumor necrosis factor (TNF) alpha  induced FLAP mRNA expression, FLAP promoter activity, and C/EBPalpha , delta , and epsilon  binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBPalpha , delta , and epsilon  bound to this region of the 5'-untranslated region, whereas C/EBPbeta does not bind even under conditions of overexpression and stimulation. We conclude that the FLAP gene is transactivated by members of the C/EBP family of transcription factors in inflammatory cells and that these factors play an important role in FLAP gene induction by TNFalpha .

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INTRODUCTION
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Leukotrienes, products of the 5-lipoxygenase (5-LO)1 pathway, are potent inflammatory mediators involved in many diseases, such as asthma, allergic rhinitis, glomerulonephritis, rheumatoid arthritis, and inflammatory bowel disease (1-4). The soluble enzyme, 5-LO, and the integral membrane protein, 5-lipoxygenase activating protein (FLAP), are required for the cellular synthesis of leukotrienes in intact cells (5). 5-LO translocates to the nuclear envelope in response to a variety of stimuli and catalyzes the oxygenation of arachidonic acid to 5-HPETE and the subsequent dehydration of 5-HPETE to leukotriene (LT) A4. LTA4 can be metabolized to LTB4 by LTA4 hydrolase or to the cysteinyl leukotrienes LTC4, LTD4, and LTE4, by the action of LTC4 synthase (6, 7).

FLAP is a member of the membrane-associated proteins in eicosanoid and glutathione metabolism family of proteins (8). Other members of this family include LTC4 synthase, prostaglandin E2 synthase, and the microsomal glutathione-S-transferases (8). The exact function of the FLAP enzyme remains controversial, but previous studies suggest that it acts as an arachidonic acid transfer protein for 5-LO (9). In support of this role, MK-886, a specific inhibitor of FLAP, has been shown to inhibit the binding of arachidonic acid to FLAP and the resultant synthesis of leukotrienes (9).

In several types of inflammatory cells, FLAP mRNA can be induced by dexamethasone, interleukin (IL) 3, and granulocyte-monocyte colony-stimulating factor (10-12), suggesting that gene expression is regulated. Cloning of FLAP gene by Kennedy et al. and computer-assisted analysis of this sequence suggested the existence of a possible TATA box 22 bp upstream of the transcription start site, as well as AP-2, NFkappa B, and glucocorticoid receptor binding sites (13). A promoter analysis, using a FLAP gene promoter-chloramphenicol acetyltransferase (CAT) reporter gene construct in the mouse macrophage cell line, P388 D1, indicated the presence of enhancer elements and cell-specific activity (13). They also found a restriction site polymorphism in the second intron and that this polymorphism is present in the normal population at a fairly high frequency (13). A FLAP gene polymorphism in the proximal 5'-UTR region has been identified 94 bp upstream of the transcription start site (14). They found that a hetero- or homozygous poly(A) sequence of 21 bp in the proximal FLAP promoter is present in a higher frequency in asthmatics (73.2%), as compared with normal subjects (54.9%) (14). The functional significance of this finding is unclear.

The CCAAT/enhancer-binding protein (C/EBP) family members, of which six have been identified, are transcription factors that regulate cellular differentiation and the inflammatory response (15, 16). C/EBP family members have been identified as mediating IL-6 signaling and are known to bind to promoter elements within the genes of cyclooxygenase-2, tumor necrosis factor alpha  (TNFalpha ), IL-8, granulocyte-colony stimulating factor, CD14, and inducible nitric oxide synthase (15, 17-21). While C/EBPalpha , beta , and delta  are expressed in liver and lung, C/EBPepsilon expression is believed to be confined to cells of myeloid and lymphoid lineage (15). C/EBPalpha expression has also been reported in peripheral blood mononuclear cells (15). The C/EBPalpha , beta , delta , and epsilon  proteins are similar in their C-terminal DNA-binding basic region and leucine zipper dimerization domains, with a higher degree of diversity in their N-terminal transactivation domain (15, 22, 23). The dimerization domain is highly conserved and is believed to be required for DNA binding (15). C/EBP family members form homo- and heterodimers, the formation of which is required for DNA binding (24). Cell-specific gene regulation by C/EBP proteins has been shown to be dependent upon interactions with other transcription factors, including NFkappa B, Sp1, and Fos/Jun family members (25, 26).

In this report, we investigate the role of cis-acting elements that are located in the first 134 bp of the FLAP promoter that are important in the transcriptional regulation of the FLAP gene in inflammatory cells. Our findings indicate that the alpha , delta , and epsilon  members of the C/EBP family of transcription factors bind to these elements and that the alpha  and delta  isoforms, at least, function to up-regulate FLAP gene expression in mononuclear phagocytes.

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Cell Culture-- THP-1 and HeLa cells were obtained from American Type Culture Collection (Manassas, VA). The monocyte-like cell line, THP-1, constitutively expresses 5-LO and FLAP and has been extensively utilized as a model to study leukotriene metabolic pathways (11, 27). THP-1 cells were grown at 37 °C with 5% CO2 in RPMI 1640 medium (BioWhittaker, Walkersville, MD), supplemented with 10% heat-treated fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamicin. HeLa cells were grown in Eagle's minimum essential medium, supplemented with 10% FCS, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 1% non-essential amino acids. The media were changed every two to three days for all experiments.

Construction of Luciferase Promoter-Reporter Constructs-- A 3.4-kb segment of the FLAP 5'-UTR, ligated into a pCAT vector (Promega, Madison, WI), was generously provided by Dr. Brian Kennedy (Merck Frosst, Kirkland, Québec, Canada). The -3368FLAP-pGL3 construct was created by release of the 3.4 kb-segment from the pCAT vector by restriction digest with SacI, with subsequent ligation into the firefly luciferase pGL3 Basic vector (Promega, Madison, WI). The -3368FLAP-pGL3 construct was used as a PCR template using primers with a common NheI restriction site engineered on the 3' end (5'-ccgctagcggaaggggaagtggagc-3'). The following forward primers were used to create the designated deletion promoter-reporter constructs: 5'-gaataccaggcagccac-3' (-965FLAP-pGL3), 5'-atgccactctgtctgac-3' (-371FLAP- pGL3), 5'-gacacactgaaccacag-3' (-134FLAP-pGL3), and 5'-ctgaaagagygcaagctctcacttccccttccg-3' (-19FLAP-pGL3). Thirty six cycles of PCR were performed, with each cycle consisting of denaturation at 94 °C for 45 s, annealing at 57 °C for 30 s, and extension at 72 °C for 60 s. The promoter segment for the -1192FLAP-pGL3 construct was created by restriction digest of the 3.4-kb pCAT construct with RsaI. All fragments were subsequently subcloned into the pGEM-T vector (Promega). The FLAP promoter segments were released by restriction digest with SacI and NheI and directionally subcloned into the pGL3 Basic vector. Mutant constructs (containing mutations at the C/EBP consensus sites) were created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Sequences of the wild-type and mutant constructs were subsequently confirmed by the dideoxy chain termination method. All constructs were purified using the EndoFree Maxi-prep kit (Qiagen; Chatsworth, CA).

Transient Transfection-- THP-1 cells (1 × 106) were transiently co-transfected with 450 ng of the FLAP promoter-pGL3 firefly luciferase vector and 50 ng of the pRL-TK Renilla luciferase vector (Promega) or a pCMV-beta -galactosidase vector (generously provided by Dr. Kenneth Chien, University of California, San Diego, CA) using Effectene transfection reagent (Qiagen), as has been previously described (27). For overexpression experiments, DNA mixtures consisted of 225 ng of the wild-type or mutant -134FLAP-pGL3 construct, 50 ng of the Renilla luciferase pRL-TK construct, and 225 ng of the C/EBPalpha , beta , delta , and epsilon  expression vectors to a total of 500 ng of DNA per condition. For TNFalpha experiments, the cells were subsequently treated with TNFalpha (at 10 ng/ml) for 24 h. HeLa cells were transfected using Lipofectin reagent (Invitrogen) per the manufacturer's instructions. The cells were incubated for 24 h at 37 °C with 5% CO2 in their respective media supplemented with 10% FCS.

Luciferase activities were measured by the Dual Luciferase Assay Reporter System (Promega) per the manufacturer's instructions. beta -Galactosidase activity was measured using the Tropix beta -galactosidase assay system (Tropix, Bedford, MA) per the manufacturer's instructions. Measurements were made using an Optocomp I luminometer (MGM Instruments, Hamden, CT). Firefly luciferase values were normalized to either Renilla luciferase values or beta -galactosidase values to control for transfection efficiency. Data are expressed as values normalized to the activity of the promoter less pGL3 Basic vector or to the activity of the SV40-driven pGL3 Control vector (Promega).

DNase I Footprint Analysis-- Nuclear extracts were prepared from THP-1 and HeLa cells as previously described (28). The promoter region of the FLAP gene from -134 to +12 bp (with respect to the transcription start site) was prepared by restriction digest of the -134FLAP-pGL3 construct with KpnI and NcoI. The probe was labeled on the 3' end with [32P]ATP using T4 polynucleotide kinase (Promega). DNase I footprinting was performed in 100-µl reaction volumes, with 4 ng (~1 × 104 cpm) of labeled probe and 40 µg of nuclear extract. The reaction conditions consisted of 5% glycerol, 10 mM HEPES, 50 mM KCl, 1 mM dithiothreitol, 1 µg of poly(dI-dC) (Amersham Biosciences), and 1 µg of bovine serum albumin. After a 20-min incubation at room temperature, CaCl2 (1 mM) and MgCl2 (0.5 mM) were added and the reaction was incubated for 1 min. DNase I (1 unit) (Promega) was added, and the reaction was digested for 60 or 120 s. The reaction was inactivated, and the DNA was extracted by phenol:chloroform, followed by ethanol precipitation. The DNA was then analyzed on an 8% PAGE and 50% urea gel. A G + A ladder was prepared by using the same end-labeled fragment and was run on the same gel.

Electrophoretic Mobility Shift Assays (EMSAs)-- EMSA reactions were performed in 20 µl final volumes under the identical conditions as described for footprinting. The probe was labeled with [32P]ATP using T4 polynucleotide kinase (Promega). Each reaction contained 10 µg of nuclear protein extracts from control or TNFalpha -conditioned THP-1 cells and ~3 × 104 cpm of duplexed, labeled nucleotide probe. The reactions were incubated at room temperature for 20 min. Supershift analyses were conducted with antibodies against C/EBP family proteins (Santa Cruz Biotechnology, Santa Cruz, CA) added 5 min before the addition of the radiolabeled probe. The samples were subsequently electrophoresed on a 5% nondenaturing acrylamide gel containing 1% glycerol.

Northern Blot Analysis-- THP-1 cells were treated with TNFalpha (10 ng/ml) or transiently transfected with expression vectors for C/EBPalpha , beta , delta , and epsilon  (450 ng × 106 cells) as has been previously described (27). Following 24 h of incubation, total cellular RNA was extracted and subjected to electrophoresis on a 1% agarose/2.2 M formaldehyde gel. The RNA was then blotted overnight onto a Zeta-Probe nylon membrane (Bio-Rad). The blot was probed with a 32P-labeled full-length cDNA probe for FLAP, washed under high-stringency conditions, and exposed to autoradiographic film. Loading equivalency and transfer efficiency were assessed by probing with a 32P-labeled full-length cDNA probe for beta -actin (Clontech, Palo Alto, CA).

Chromatin Immunoprecipitation (ChIP) Assays-- ChIP assays were performed by a modification of a previously described method (29). Briefly, THP-1 cells (at 1 × 107 cells/ml) were treated with 1% formaldehyde for 30 min at 37 °C and lysed in SDS buffer (50 mM Tris-HCl/1% SDS/10 mM EDTA, pH 8.1). The chromatin samples were sonicated for 5-10 s (to reduce the DNA length to ~200-500 bp) and were precleared with protein A-agarose beads to minimize nonspecific binding of proteins in protein A-agarose. Antibodies (2 µg) were added to the chromatin samples, and the samples were incubated overnight at 4 °C. To minimize nonspecific binding of DNA to protein A-agarose, salmon sperm DNA and protein A-agarose beads were incubated in 1% BSA for 4 h at 4 °C. These beads were then washed repeatedly and resuspended in binding buffer. Subsequently, 50 µl of the treated beads were added to the chromatin samples and incubated for 6 h at 4 °C. The beads were then washed, eluted, and cross-linking-reversed by protease digestions using proteinase K as previously described (30). After proteinase K treatment, the DNA was extracted with phenol-chloroform and precipitated with ethanol. The DNA was dissolved in 20 µl of Tris-EDTA buffer, and 5 µl of the DNA was used for the PCR reaction. PCR was performed using a pair of primers (forward -134 bp to -118 bp; reverse -9 bp to -25 bp, sequence as noted above) that generated a product spanning -134 to -9 bp of the FLAP promoter (126 bp). PCR was performed for 30 cycles under the following conditions: denaturation at 94 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 45 s. The PCR products were electrophoresed through an agarose gel and visualized by ethidium bromide staining.

Materials-- FCS, penicillin, streptomycin, and gentamicin were obtained from the Cell Culture Facility, University of California. RPMI 1640 medium was obtained from BioWhittaker (Walkersville, MD). All restriction enzymes were obtained from Invitrogen. All synthesized oligonucleotides and primers were obtained from Operon Technologies, Inc. (Alameda, CA). The C/EBPalpha expression vector was obtained from Dr. Steve McKnight, University of Texas Southwestern Medical Center (Dallas, TX) (31). The C/EBPbeta expression vector was obtained from Dr. Mario Chojkier, Veterans Affairs San Diego Healthcare System (San Diego, CA) (32, 33). The C/EBPdelta expression vector was obtained from Dr. Jan Trapman, Erasmus University (Rotterdam, Holland) (34). The C/EBPepsilon expression vector was obtained from Dr. Julie Lekstrom-Himes, NIAID National Institutes of Health (Bethesda, MD). TNFalpha was obtained from Calbiochem (La Jolla, CA). Autoradiographic film was purchased from Eastman Kodak Co. (Rochester, NY). The Qiagen-tip 500 column was purchased from Qiagen. All other reagents were from Sigma and were of the finest grade available.

Data Analysis-- Data are expressed as the mean ± S.E. in all circumstances where mean values are compared. Data were analyzed by unpaired Student's t test (InStat, version 2.03, GraphPad Software, San Diego, CA). Differences were considered significant when p < 0.05.

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Functional Analysis and Cell-specific Activity of the FLAP Promoter-- The full-length -3368FLAP-pGL3 construct was assessed for promoter activity in THP-1 cells. Following transient transfection, the cells were assayed for luciferase activity. The -3368FLAP-pGL3 construct exhibited 22-fold higher activity than that of the promoterless pGL3 Basic vector (Fig. 1). In the non-inflammatory cell lines, HeLa and COS-1, which do not constitutively express FLAP protein, the -3368FLAP-pGL3 construct exhibited minimal promoter activity (data not shown). These results are consistent with the known cell-specific pattern of FLAP gene expression in inflammatory cells.


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Fig. 1.   Functional analysis of the FLAP promoter. Schematic representation of FLAP promoter-luciferase reporter constructs that possess a common 3' end at +12 bp from transcription start site and unique 5' ends as indicated. FLAP promoter constructs and the pCMV-beta -galactosidase construct were co-transfected into THP-1 cells, and luciferase activities were normalized to beta -galactosidase activity. Data are presented as the percentage of the SV40-driven pGL3 control vector (n = 3, ± S.E.). The first 134 bp of the FLAP 5'-UTR mediate almost all of the promoter activity observed with the full-length (3368 bp) FLAP promoter.

A serial deletion analysis demonstrated that the first 134 bp of the FLAP promoter (as represented by the activity of the -134FLAP-pGL3 construct) mediated a 5-fold increase in promoter activity over that of the minimal promoter construct, -19FLAP-pGL3 (Fig. 1). Notably, the -134FLAP-pGL3 construct accounted for almost all of the observed full-length (3.4 kb) promoter activity.

THP-1 Nuclear Extract Proteins Bind to the FLAP Promoter-- Because the -134FLAP-pGL3 construct demonstrated maximal promoter activity, we attempted to identify the transacting factors that bind to this region of the FLAP promoter. DNase I footprint analysis using a probe from -134 to +12 bp demonstrated that THP-1 nuclear extract binds to the FLAP promoter region corresponding to approximately -30 to -10 bp (Fig. 2A, lane 3). In similar DNase I footprint assays, HeLa nuclear extract did not demonstrate binding to the FLAP promoter (Fig. 2B, lane 3). Data base analysis revealed that this region of the FLAP promoter contains a proximal C/EBP consensus site (located at -25 to -12 bp), a distal C/EBP consensus site (located at -36 to -28 bp), and an Octomer-1 consensus site (located at -29 to -15 bp).


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Fig. 2.   THP-1 nuclear extract proteins bind to the FLAP promoter. The FLAP promoter segment (from -134 to +12 bp) was radiolabeled with [32P]ATP on the 3' end and used as a probe in DNase I footprint analysis. A, a G + A (C + T) ladder (lane 1), control reaction without THP-1 nuclear extract (lane 2), and reaction with 40 µg of THP-1 nuclear extract (lane 3) are displayed. The proximal and distal consensus binding sites for C/EBP family transcription factors are denoted (boxed). B, similar DNase I footprint experiment using HeLa nuclear extract.

C/EBPalpha , delta , and epsilon  Proteins Bind to the Proximal C/EBP Consensus Site in the FLAP Promoter-- Wild-type and mutant duplexed oligonucleotide probes (Fig. 3) were synthesized for EMSAs to identify the transcription factor(s) that bind to the FLAP promoter region that demonstrated binding in the prior footprint assay. In the presence of nuclear extract from unconditioned THP-1 cells, the EMSA probe from -25 to -9 bp, containing the wild-type proximal C/EBP consensus site (located at -25 to -12 bp), exhibited a 3-band complex (Fig. 4A, lane 1). Supershifted bands were observed in the conditions using antibodies against C/EBPalpha (lane 2), delta  (lane 4), and epsilon  (lane 5). Our data indicate the binding of C/EBPalpha , delta , and epsilon  to the proximal C/EBP consensus site in unconditioned THP-1 cells.


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Fig. 3.   FLAP promoter EMSA probes. Duplexed oligonucleotide probes were synthesized and utilized for EMSAs. A 17-bp probe (from -25 to -9 bp of the FLAP promoter) containing either the wild-type (WT) or mutant (Mut) proximal C/EBP consensus site (underlined) was used. A 20-bp probe (from -39 to -20 bp of the FLAP promoter) containing either the wild-type (WT) or mutant (Mut) distal C/EBP consensus site (underlined) was used. Mutated bases within the probes are denoted in lowercase.


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Fig. 4.   EMSA analysis of the proximal C/EBP consensus site of the FLAP promoter. Nuclear extract (10 µg) from THP-1 cells was incubated with a [32P]ATP-labeled duplexed segment of the FLAP promoter (from -25 to -9 bp) containing the proximal C/EBP site (located at -25 to -12 bp). A, EMSA and supershift assays were performed with wild-type probe using THP-1 nuclear extract (lane 1) and nuclear extract with antibodies against C/EBP proteins alpha , (lane 2), beta  (lane 3), delta  (lane 4), and epsilon  (lane 5). B, EMSA and supershift assays were performed with a probe containing a mutation of the proximal C/EBP site, using THP-1 nuclear extract (lane 2) and nuclear extract with antibodies against C/EBPalpha (lane 3) and beta  (lane 4). C, EMSA and supershift assays were performed with wild-type probe using HeLa nuclear extract (lane 1) and nuclear extract with antibodies against C/EBPalpha (lane 3), beta  (lane 4), delta  (lane 5), and epsilon  (lane 6).

Mutation of the Proximal C/EBP Consensus Site in the FLAP Promoter Abolishes C/EBP Protein Binding-- To identify the critical base pairs within the proximal C/EBP consensus binding site, we used the -25 to -9 bp EMSA probe containing a 4-bp mutation within the proximal C/EBP site at -15 to -12 bp (Fig. 3). An EMSA performed with the probe containing a mutation of the C/EBP site demonstrated a loss of specific binding (Fig. 4B, lane 2), and antibodies against C/EBPalpha and beta  failed to produce supershifted bands (Fig. 4B, lanes 3 and 4). These results confirmed that the proximal consensus site bound C/EBP proteins in a site-specific manner.

HeLa Nuclear Extract Does Not Bind to the Proximal C/EBP Consensus Site in the FLAP Promoter-- When similar EMSAs were performed using HeLa nuclear extract and the wild-type -25 to -9-bp EMSA probe, only nonspecific gel-shifted bands were observed. Supershift assays performed with this probe, HeLa nuclear extract, and C/EBP antibodies against alpha , beta , delta , and epsilon  (Fig. 4C, lanes 3-6) failed to produce supershifted bands, suggesting that these proteins were not constitutively present or did not demonstrate binding in the HeLa cell line.

C/EBPalpha and epsilon  Proteins Bind to the Distal C/EBP Consensus Site in the FLAP Promoter-- To identify the transcription factors that bind to the distal C/EBP consensus site (located at -36 to -28 bp), EMSAs were performed using a probe extending from -39 to -20 bp. Nuclear extracts from unconditioned THP-1 cells produced multiple bands on this EMSA (Fig. 5A, lane 2). C/EBPalpha and epsilon  antibodies produced supershifted bands (Fig. 5A, lanes 3 and 6), but C/EBPbeta and delta  did not (Fig. 5A, lanes 4 and 5). A 3-bp mutation of the distal C/EBP binding site abolished the supershifted bands observed with antibodies of C/EBPalpha and epsilon . (Fig. 5B, lanes 3-7). These data demonstrate that C/EBPalpha and epsilon  bind to the distal C/EBP consensus site in unconditioned THP-1 cells.


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Fig. 5.   EMSA analysis of the distal C/EBP consensus site of the FLAP promoter. Nuclear extract (10 µg) from THP-1 cells was incubated with a [32P]ATP-labeled duplexed segment of the FLAP promoter (from -39 to -20 bp) containing the distal C/EBP site (located at -36 to -28 bp). A, EMSA and supershift assays were performed with wild-type probe, using THP-1 nuclear extract (lane 2) and nuclear extract with antibodies against C/EBPalpha (lane 3), beta  (lane 4), delta  (lane 5), epsilon  (lane 6), and alpha /epsilon (lane 7). B, EMSA and supershift assays were performed with probe containing a mutation of the distal C/EBP site using THP-1 nuclear extract (lane 2) and nuclear extract with antibodies against C/EBPalpha (lane 3), beta  (lane 4), delta  (lane 5), epsilon  (lane 6), and alpha /epsilon (lane 7).

Proximal and Distal C/EBP Consensus Sites Mediate Constitutive FLAP Promoter Activity-- To elucidate the functional importance of the proximal and distal C/EBP consensus sites for FLAP promoter activity, these specific sequences were mutated by site-directed mutagenesis within the -134FLAP-pGL3 construct (at the sequences designated in Fig. 3). In transiently transfected THP-1 cells, mutation of the distal and proximal C/EBP sites reduced FLAP promoter activity by 33 and 81%, respectively (Fig. 6A). Moreover, mutation of both consensus sites further reduced luciferase activity by 89% (Fig. 6A). These results demonstrate that the C/EBP sites function as positive constitutive regulatory elements, with the proximal site serving a critical role.


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Fig. 6.   Proximal and distal C/EBP consensus binding sites mediate constitutive FLAP promoter activity. A, the -134FLAP-pGL3 constructs, consisting of wild-type (Control) or mutated at the proximal (Proximal Mut) and/or distal (Distal Mut) C/EBP consensus sites were transiently co-transfected with the pRL-TK Renilla luciferase vector into THP-1 cells. Luciferase activities were measured and expressed relative to the activity of the pGL3 Basic vector. Mutation of the proximal and distal C/EBP sites results in loss of constitutive FLAP promoter activity. Data represent mean ± S.E., n = 3. B, wild-type -134FLAP-pGL3 (closed bars) and combined proximal and distal C/EBP mutant -134FLAP-pGL3 constructs (open bars) were transiently co-transfected with the pRL-TK Renilla luciferase vector and expression vectors for C/EBPalpha , beta , delta , and epsilon  into THP-1 cells. Luciferase activities were measured, expressed relative to the activity of the pGL3 Basic vector, and normalized to the luciferase activity of wild-type -134FLAP-pGL3 construct. C/EBPalpha , beta , and delta  differentially induce FLAP promoter activity. Overexpression of C/EBPepsilon had no effect on promoter activity. Induction by C/EBPalpha , beta , and delta  is abolished in the presence of combined mutations of the proximal and distal C/EBP consensus sites. Data represent mean ± S.E., n = 3.

Overexpression of C/EBP Transcription Factors Enhances FLAP Promoter Activity-- To further define the role of the C/EBP family members in modulating gene transcription through the proximal and distal consensus sites, C/EBP expression vectors for alpha , beta , delta , and epsilon  were transiently co-transfected with the wild-type -134FLAP-pGL3 construct into THP-1 cells. Overexpression of the C/EBPalpha , beta , and delta  proteins up-regulated FLAP gene activity by 5-, 5.2-, and 14.2-fold, respectively (Fig. 6B). Overexpression of C/EBPepsilon had no effect on promoter activity (Fig. 6B). Mutation of both the proximal and distal C/EBP sites of the -134FLAP-pGL3 construct essentially abolished the C/EBP-induced enhancement of promoter activity (Fig. 6B). Co-transfection of the C/EBPalpha expression vector into HeLa cells did not significantly induce promoter activity (data not shown), consistent with the lack of FLAP gene expression in this cell line.

Overexpression of C/EBPalpha , beta , and delta  Enhances FLAP mRNA Accumulation-- To further define the role of the C/EBP family members in modulating native gene transcription through the proximal and distal FLAP promoter consensus sites, THP-1 cells were transiently transfected with expression vectors for C/EBPalpha , beta , delta , and epsilon , incubated for 24 h, and subjected to Northern blot analysis. The results demonstrate that C/EBPalpha , beta , and delta  can up-regulate FLAP mRNA expression by 6.6-, 6.2-, and 3.3-fold, respectively (Fig. 7, lanes 1-5). Overexpression of C/EBPepsilon had no effect on FLAP mRNA expression (Fig. 7, lanes 6 and 7).


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Fig. 7.   Overexpression of C/EBPalpha , beta , and delta  enhances FLAP mRNA accumulation. THP-1 cells were transiently co-transfected with expression vectors for C/EBPalpha , beta , delta , and epsilon . Total RNA was extracted and subjected to Northern blot analysis. A, representative Northern blots probed for FLAP and beta -actin with their respective empty vector controls. B, densitometric analysis of Northern blots for FLAP, relative to beta -actin and normalized to their control. Overexpression of C/EBPalpha , beta , and delta  increases native FLAP mRNA accumulation. Overexpression of C/EBPepsilon has no effect on FLAP mRNA.

TNFalpha Induces FLAP mRNA Accumulation-- Prior research has demonstrated that TNFalpha can induce the expression of 5-LO activity (35) and FLAP (36). To examine the role of C/EBP in TNFalpha -induced expression of FLAP we conditioned THP-1 cells with TNFalpha , followed by Northern blot analysis. The results demonstrate that TNFalpha up-regulates FLAP mRNA expression more than 6-fold (Fig. 8, B and C).


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Fig. 8.   TNFalpha induces FLAP mRNA accumulation and promoter activity. A, THP-1 cells were conditioned with TNFalpha (10 ng/ml) for 24 h. Total RNA was extracted and subjected to Northern blot analysis. Representative Northern blot probed for FLAP and beta -actin. B, densitometric analysis of Northern blot for FLAP, relative to beta -actin and normalized to control. TNFalpha treatment increases native FLAP mRNA accumulation. Data represent mean ± S.E., n = 4. C, THP-1 cells were transiently transfected with the -3368FLAPpGL3 construct and were subsequently conditioned with TNFalpha for 24 h. Luciferase activities were measured and expressed relative to the activity of the pGL3 Basic vector. TNFalpha treatment induces FLAP promoter activity. Data represent mean ± S.E., n = 3.

TNFalpha Induces FLAP Promoter Activity-- To determine whether TNFalpha induces FLAP promoter activity, THP-1 cells were transiently transfected with the -3368FLAP-pGL3 construct and were conditioned with TNFalpha for 24 h. In the presence of TNFalpha , promoter activity was increased, as compared with control (60.4 ± 5.9 versus 21.4 ± 1.0, respectively; mean ± S.E., n = 3) (Fig. 8C). The results demonstrate that the increase in FLAP mRNA induced by TNFalpha is mediated, at least in part, by an increase in FLAP gene transcription.

TNFalpha Induces C/EBP Family Member Binding to the Proximal Consensus Site in the FLAP Promoter-- To determine the role of TNFalpha in the induction of C/EBP family member binding to the FLAP promoter, THP-1 cells were conditioned with TNFalpha (for various time periods), and the nuclear extracts were used to perform EMSAs using the proximal FLAP promoter probe (-25 to -9 bp). In the presence of nuclear extracts obtained from TNFalpha -treated THP-1 cells, the proximal EMSA probe exhibited a time-dependent increased binding pattern, with peak binding occurring at 4-8 h (Fig. 9, lanes 1-5). Supershift assays identified the bands as being due to C/EBPalpha (lane 6), delta  (lane 8), and epsilon  (lane 9). Our data indicate that TNFalpha induces increased binding of C/EBPalpha , delta , and epsilon  to the proximal C/EBP consensus site in the FLAP promoter.


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Fig. 9.   TNFalpha -induces C/EBP family member binding to the proximal consensus site of the FLAP promoter. TNFalpha -treated (10 ng/ml) nuclear extract (10 µg) from THP-1 cells was incubated with a [32P]ATP-labeled duplexed segment of the FLAP promoter (from -25 to -9 bp) containing the proximal C/EBP site (located at -25 to -12 bp). A, EMSAs were performed using THP-1 nuclear extracts treated with TNFalpha for periods ranging from 0 to 8 h (lanes 1-5). TNFalpha induces time-dependent binding of C/EBPalpha , delta , and epsilon  to the proximal FLAP promoter consensus site. B, EMSAs were performed using THP-1 nuclear extracts treated with TNFalpha for 2 h and antibodies against C/EBP proteins alpha  (lane 6), beta  (lane 7), delta  (lane 8), and epsilon  (lane 9). C/EBPalpha , delta , and epsilon  antibodies supershift these proteins present in the TNFalpha -induced complexes.

TNFalpha Induces C/EBP Family Member Binding to the Distal Consensus Site in the FLAP Promoter-- To determine the role of TNFalpha in the induction of C/EBP family member binding to the FLAP promoter, THP-1 cells were conditioned with TNFalpha (for various time periods), and the nuclear extracts were used to perform EMSAs using the distal FLAP promoter probe (-39 to -20 bp). In the presence of nuclear extracts obtained from TNFalpha -treated THP-1 cells, the proximal EMSA probe exhibited a time-dependent increase in binding pattern, with peak binding occurring at 2-4 h (Fig. 10, lanes 1-5). Supershift assays identified the bands as being due to C/EBPalpha (lane 6), delta  (lane 8), and epsilon  (lane 9). Our data indicate that TNFalpha induces increased binding of C/EBPalpha and epsilon  to the distal C/EBP consensus site in the FLAP promoter and also induces the new binding of C/EBPdelta to this site.


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Fig. 10.   TNFalpha induces C/EBP family member binding to the distal consensus site of the FLAP promoter. TNFalpha -treated (10 ng/ml) nuclear extract (10 µg) from THP-1 cells was incubated with a [32P]ATP-labeled duplexed segment of the FLAP promoter (from -39 to -20 bp) containing the distal C/EBP site (located at -36 to -28 bp). A, EMSAs were performed using THP-1 nuclear extracts treated with TNFalpha for periods ranging from 0 to 8 h (lanes 1-5). TNFalpha induces time-dependent binding of C/EBPalpha , delta , and epsilon  to the distal FLAP promoter consensus site. B, EMSAs were performed using THP-1 nuclear extracts treated with TNFalpha for 2 h and antibodies against C/EBP proteins alpha  (lane 6), beta  (lane 7), delta  (lane 8), and epsilon  (lane 9). C/EBPalpha , delta , and epsilon  antibodies supershift these proteins present in the TNFalpha -induced complexes.

Chromatin Immunoprecipitation (ChIP) Assays Demonstrate Binding of C/EBPalpha , delta , and epsilon  to the FLAP Promoter-- To determine whether C/EBP isoforms are bound to native chromatin in THP-1 cells, we performed ChIP assays on chromatin obtained from THP-1 cells (Fig. 11A). When the PCR reaction was run in the absence of a template or in the presence of an irrelevant antibody (beta  actin), no product was observed. When the PCR reaction was run in the presence of FLAP cDNA corresponding to the DNA sequence of interest containing the two C/EBP sites (-134 to -9) an appropriate product was generated (126 bp). Likewise, when chromatin was incubated with antibodies to C/EBPalpha , delta  , and epsilon  , a PCR product corresponding to this segment was also detected indicating that these isoforms of C/EBP were bound to the C/EBP consensus binding sites. However, when PCR reactions were run on immunoprecipitates generated using the C/EBPbeta antibody, no cDNA product was detected, indicating the absence of binding of C/EBPbeta to either C/EBP site (Fig. 11A). To further clarify whether C/EBPbeta could bind to this segment of the FLAP 5'-UTR, C/EBPbeta was overexpressed in THP-1 cells transiently transfected with a C/EBPbeta expression construct (Fig. 11B). No PCR product (no binding) was detected when immunoprecipitation was performed with C/EBPbeta antibody. To determine whether activation with TNFalpha would modify this outcome, THP-1 cells transfected with C/EBPbeta were activated with 10 ng/ml of TNFalpha for 2 h. Again, no PCR product was detected when chromatin was immunoprecipitated with C/EBPbeta antibody (Fig. 11B).


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Fig. 11.   C/EBP family member binding to native chromatin as demonstrated by ChIP assay. THP-1 cells were fixed with 1% formaldehyde, lysed with SDS, and subjected to immunoprecipitation. DNA was released from immunoprecipitates by proteinase K treatment and the DNA was amplified by PCR using primers corresponding to -134 to -9 of the FLAP gene. A, lane 1, 100 bp ladder. Lane 2, PCR reaction mix, negative control. Lane 3, cDNA template corresponding to region of interest, positive control. Lane 4, ChIP performed on THP-1 cells with beta -actin antibody as an irrelevant control antibody. Lanes 5-8, ChIP assay performed on THP-1 cells with antibody to C/EBPalpha , beta , delta  , and epsilon  , respectively. When fixed chromatin was treated with an irrelevant antibody (beta -actin), no product was detected. Fixed chromatin immunoprecipitated with antibodies to C/EBPalpha , delta , and epsilon  yielded a PCR product corresponding to this segment of the FLAP gene, but when using antibodies to C/EBPbeta no product was detected. B, to further examine the role of C/EBPbeta , THP-1 cells were studied further. Lane 1, ladder. Lane 2, negative control. Lane 3, positive control. Lane 4, irrelevant antibody (beta -actin). Lane 5, ChIP assay performed on THP-1 cells with C/EBPbeta antibody. Lane 6, ChIP with THP-1 cells transfected with an expression construct encoding for C/EBPbeta . Lane 7, ChIP with THP-1 cells transfected with an expression construct encoding for C/EBPbeta and conditioned with TNFalpha (10 ng/ml) for 2 h. Even under these highly induced conditions, binding of C/EBPbeta to native chromatin could not be detected.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our findings demonstrate that C/EBP family members play a role in constitutive expression of the FLAP gene in the human monocyte-like cell line, THP-1. Our findings identify the presence of cis-acting regulatory elements that are contained within the first 134 bp region of the 5'-UTR of this gene. This region accounts for the majority of FLAP promoter activity and appears to confer gene expression in THP-1 cells. Expression of FLAP in inflammatory cells can be attributed, at least in part, to the proximal and distal C/EBP sites contained in this segment of the promoter. Moreover, these sites also play a role in TNF-induced expression of FLAP.

FLAP is a pivotal protein in the 5-LO pathway of leukotriene metabolism. Studies indicate that the concomitant expression of FLAP is required for 5-LO activity in stimulated, intact inflammatory cells (5). Moreover, a variety of studies have suggested that levels of the FLAP protein are regulated and that modulation of its expression may play a key role in altering the capacity of inflammatory cells for 5-lipoxygenation of arachidonic acid. The original description of the FLAP gene suggested that it may possess a TATA box in its proximal promoter, and a deletion analysis was performed in a mouse macrophage cell line (13). These studies suggested that the promoter region contained probable enhancer elements further upstream of the -134 bp position (13), although specific characterization of these sites was not attempted. In the current study, performed in a human monocyte-like cell line, we demonstrate that the full-length FLAP promoter (consisting of 3368 bp) mediates a 22-fold increase in activity over that of the promoterless pGL3 Basic construct. Our findings demonstrate that almost all of the observed constitutive activity is mediated by the first 134 bp of the promoter. This region contains the proximal and distal C/EBP consensus sites and confers gene expression in THP-1 cells. The noted differences between our findings and those reported previously could be explained by the use of more sensitive luciferase constructs in our experiments and the use of a human, rather than mouse, monocytic cell line.

We demonstrate that C/EBPalpha , delta , and epsilon  bind to the proximal consensus site and that C/EBPalpha and epsilon  bind to the distal consensus site within the FLAP promoter under constitutive conditions. C/EBPbeta does not appear to bind to the FLAP proximal or distal promoter sequences in THP-1 cells, consistent with the findings of other investigators that this protein is not expressed in unstimulated cells (20). Previous studies suggest that C/EBPalpha , beta , delta , and epsilon  are similar in their C-terminal basic region and leucine zipper domains, accounting for the interaction of these proteins with virtually identical DNA sequences (22, 23, 37). However, our finding of differential binding of the C/EBPdelta protein to the proximal but not the distal site under constitutive conditions may be accounted for by the fact that the C/EBP proteins can also bind in a sequence-specific fashion. This has been reported to occur for adjacent C/EBP sites within the Clara cell secretory protein gene promoter (38). The differential binding of C/EBP family members may be related to DNA bending, known to be induced by C/EBPalpha or delta  or other basic region-leucine zipper (bZIP) proteins that bind to adjacent sequence elements (38-40).

The C/EBP family members are known to exhibit a variety of protein-protein interactions. C/EBP proteins interact with each other to form homo- and heterodimers, and this dimerization is believed to be required for DNA binding (24). The individual C/EBP dimer components may function to synergistically activate gene transcription (38). The binding of multiple C/EBP proteins, likely present as complexed dimers, occurs at both the proximal and distal domains in the FLAP promoter. The possibility that other transcription factor proteins may be present in this complex cannot be specifically excluded based upon our data. Previous studies indicate that cell-specific gene regulation by C/EBP proteins may also involve interactions with other transcription factors, including NFkappa B, Sp1, and Fos/Jun (25, 26). The binding of C/EBP members to consensus sites that exist in close association with NFkappa B sites has been reported for multiple genes (16). Notably, the FLAP gene possesses an NFkappa B consensus site located adjacent to the C/EBP consensus sites at -43 to -34 bp. Interactions between C/EBP proteins and NFkappa B proteins may enhance or antagonize gene transcription (16), depending on promoter structure and the cellular milieu. Exploration of the role of potential interactions between proteins binding to the adjacent C/EBP and NFkappa B consensus sites in the FLAP promoter remains a subject for future investigation.

The functional consequence of C/EBPalpha and delta  protein binding to consensus sites within the FLAP promoter is to stimulate gene transcription. Oddly, C/EBPdelta binds to the distal site only when cells are conditioned with TNFalpha . We have not found that C/EBPepsilon independently plays a role in FLAP transcription, although it is present and binds to both proximal and distal sites. Moreover, the overexpression of C/EBPbeta can increase promoter activity and induce increased expression of native mRNA encoding for FLAP. The nuclear extracts from control or TNFalpha -conditioned THP-1 cells do not contain C/EBPbeta . More importantly, C/EBPbeta does not bind to native chromatin corresponding to FLAP in these regions under any circumstances, as shown by ChIP assay. These data indicate that C/EBP may play an indirect role in regulating the FLAP gene but has no direct effect.

Mutation of both the proximal and distal C/EBP sites results in an approximate 90% decrement in promoter activity, suggesting that these two consensus sites account for the vast majority of the observed function within the first 134 bp of the FLAP promoter. C/EBP family members have been described to function as positive and negative regulators of gene transcription (16). Our findings also suggest that the C/EBP family members exhibit differential capabilities to induce promoter activity. This difference in transactivation potential has been previously reported (22, 38), with C/EBPalpha being a more potent transcriptional activator than C/EBPbeta (41). The observed differences in the potential for activation by each of these proteins in our studies may be related to differential binding affinity, cooperative or antagonistic interaction with other transcription factors, the differential expression of C/EBP proteins in the THP-1 cell line, or differential phosphorylation of the C/EBP proteins (41). In addition, C/EBPalpha and beta  mRNAs can give rise to truncated forms of the proteins, which may act as inhibitors or weak activators of gene transcription (42, 43). The findings from Northern blot analyses establish the biological relevance of our findings by demonstrating that overexpression of the C/EBPalpha and delta  proteins can positively regulate native gene expression in intact mononuclear phagocytes. Although C/EBPbeta also induces mRNA encoding for FLAP, this is not a direct effect mediated through binding of the FLAP 5'-UTR.

The constitutive or induced expression of C/EBP family members may account for the differential expression of genes in which C/EBP proteins play a regulatory role. The C/EBPalpha and epsilon  proteins are known to be constitutively expressed in mononuclear cells (15, 44-47), and C/EBPdelta expression has been described in myeloid cell lines (48). Although prior studies suggest that C/EBPbeta is not expressed in THP-1 cells or in the premonocytic cell line, U937, under unstimulated conditions (20), this factor is believed to be involved in macrophage activation (49) and has been constitutively detected in RAW264 murine macrophages (17). Our study specifically addresses the TNFalpha -induced binding of C/EBP proteins to the FLAP promoter. While previous studies indicate that TNFalpha induces the expression of C/EBPbeta (50) and delta  (51), we have found that TNFalpha induces the binding of C/EBPalpha , delta , and epsilon , but not beta  to the both the proximal and distal FLAP promoter consensus sites. This is confirmed by our ChIP assays. Notably, TNFalpha treatment results in induction of C/EBPdelta binding to the distal consensus site, which was not present in the absence of TNFalpha conditioning. These findings suggest that TNFalpha induction of FLAP gene expression is mediated by a transcriptional mechanism.

Prior research in our laboratory has demonstrated that FLAP expression can be induced by corticosteroids (11). This is in sharp contrast to the knowledge that the 5-LO pathway is a potent proinflammatory mediator pathway and the finding that FLAP can be induced by proinflammatory cytokines like TNFalpha . However, the induction of C/EBP family members may represent a common mediating pathway for these disparate stimuli. Glucocorticoids are known to increase the expression of C/EBPbeta (52) and C/EBPdelta (47, 53, 54). In addition, the induction of expression or activation of C/EBPbeta may occur in response to IL-1, IL-6, and lipopolysaccharide (15, 18, 55, 56). Similarly, C/EBPdelta expression also is induced by lipopolysaccharide (17, 56). The induction of C/EBP family members by inflammatory stimuli, as has been demonstrated for other mediator genes (15, 17-21), may serve to further modulate FLAP gene transcription. In addition to the established effects of C/EBP family members on inflammatory mediator expression and cell differentiation, our data further define their role in the constitutive and induced expression of a gene of the 5-LO pathway of leukotriene metabolism.

In conclusion, we demonstrate that transcriptional expression of the FLAP gene is constitutively regulated by members of the C/EBP family of transcription factors in the monocyte-like cell line, THP-1. Our data indicate a critical role for C/EBP proteins in modulating expression of this gene in the 5-LO pathway in mononuclear phagocytes. The induction of C/EBP family member binding to the FLAP promoter by TNFalpha (and potentially other inflammatory stimuli) contributes to the modulation of FLAP gene expression.

    ACKNOWLEDGEMENT

We are grateful to Scott Johns (Veterans Affairs San Diego Healthcare System, San Diego, CA) for his expert technical assistance in the performance of the studies.

    FOOTNOTES

* This research was supported by a grant from the Merit Review Board of the Department of Veterans Affairs (to T. D. B.) and Grant 8KT-0126 from the University of California Tobacco-related Disease Research Program (to K. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Mail Code 111-J, Veterans Affairs San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, CA 92161. Tel.: 858-552-8585 (ext. 3541); Fax: 858-546-1754; E-mail: tbigby@UCSD.edu.

Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M211102200

    ABBREVIATIONS

The abbreviations used are: 5-LO, 5-lipoxygenase; C/EBP, CCAAT enhancer-binding protein; ChIP, chromatin immunoprecipitation; FLAP, 5-lipoxygenase activating protein; 5-HPETE, 5-hydroperoxy eicosatetraenoic acid; LT, leukotriene; CAT, chloramphenicol acetyltransferase; UTR, untranslated region; IL, interleukin; EMSA, electrophoretic mobility shift assays; TNFalpha , tumor necrosis factor alpha ; FCS, fetal calf serum.

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ABSTRACT
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RESULTS
DISCUSSION
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