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• W2121071922 abstract "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/EBPα, δ, and ε bind to the proximal site and C/EBPα and ε 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/EBPα, β, and δ 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) α induced FLAP mRNA expression, FLAP promoter activity, and C/EBPα, δ, and ε binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBPα, δ, and ε bound to this region of the 5′-untranslated region, whereas C/EBPβ 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 TNFα. 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/EBPα, δ, and ε bind to the proximal site and C/EBPα and ε 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/EBPα, β, and δ 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) α induced FLAP mRNA expression, FLAP promoter activity, and C/EBPα, δ, and ε binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBPα, δ, and ε bound to this region of the 5′-untranslated region, whereas C/EBPβ 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 TNFα. Leukotrienes, products of the 5-lipoxygenase (5-LO) 1The abbreviations used are: 5-LO5-lipoxygenaseC/EBPCCAAT enhancer-binding proteinChIPchromatin immunoprecipitationFLAP5-lipoxygenase activating protein5-HPETE5-hydroperoxy eicosatetraenoic acidLTleukotrieneCATchloramphenicol acetyltransferaseUTRuntranslated regionILinterleukinEMSAelectrophoretic mobility shift assaysTNFαtumor necrosis factor αFCSfetal calf serum1The abbreviations used are: 5-LO5-lipoxygenaseC/EBPCCAAT enhancer-binding proteinChIPchromatin immunoprecipitationFLAP5-lipoxygenase activating protein5-HPETE5-hydroperoxy eicosatetraenoic acidLTleukotrieneCATchloramphenicol acetyltransferaseUTRuntranslated regionILinterleukinEMSAelectrophoretic mobility shift assaysTNFαtumor necrosis factor αFCSfetal calf serum pathway, are potent inflammatory mediators involved in many diseases, such as asthma, allergic rhinitis, glomerulonephritis, rheumatoid arthritis, and inflammatory bowel disease (1Pauwels R.A. Joos G.F. Kips J.C. Allergy. 1995; 50: 615-622Crossref PubMed Scopus (48) Google Scholar, 2Rifai A. Sakai H. Yagame M. Kidney Int. Suppl. 1993; 39: S95-S99PubMed Google Scholar, 3Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1162) Google Scholar, 4Ford-Hutchinson A.W. Adv. Prostaglandin Thromboxane Leukotriene Res. 1989; 19: 507-510PubMed Google Scholar). 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 (5Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (645) Google Scholar). 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 LTB4by LTA4 hydrolase or to the cysteinyl leukotrienes LTC4, LTD4, and LTE4, by the action of LTC4 synthase (6Samuelsson B. Dahlén S.E. Lindgren J.A. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (1960) Google Scholar, 7Rouzer C.A. Matsumoto T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 857-861Crossref PubMed Scopus (268) Google Scholar). 5-lipoxygenase CCAAT enhancer-binding protein chromatin immunoprecipitation 5-lipoxygenase activating protein 5-hydroperoxy eicosatetraenoic acid leukotriene chloramphenicol acetyltransferase untranslated region interleukin electrophoretic mobility shift assays tumor necrosis factor α fetal calf serum 5-lipoxygenase CCAAT enhancer-binding protein chromatin immunoprecipitation 5-lipoxygenase activating protein 5-hydroperoxy eicosatetraenoic acid leukotriene chloramphenicol acetyltransferase untranslated region interleukin electrophoretic mobility shift assays tumor necrosis factor α fetal calf serum FLAP is a member of the membrane-associated proteins in eicosanoid and glutathione metabolism family of proteins (8Jakobsson P.-J. Morgenstern R. Mancini J.A. Ford-Hutchinson A.W. Persson B. Protein Sci. 1999; 8: 689-692Crossref PubMed Scopus (294) Google Scholar). Other members of this family include LTC4 synthase, prostaglandin E2 synthase, and the microsomal glutathione-S-transferases (8Jakobsson P.-J. Morgenstern R. Mancini J.A. Ford-Hutchinson A.W. Persson B. Protein Sci. 1999; 8: 689-692Crossref PubMed Scopus (294) Google Scholar). 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 (9Abramovitz M. Wong E. Cox M.E. Richardson C.D. Li C. Vickers P.J. Eur. J. Biochem. 1993; 215: 105-111Crossref PubMed Scopus (178) Google Scholar). 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 (9Abramovitz M. Wong E. Cox M.E. Richardson C.D. Li C. Vickers P.J. Eur. J. Biochem. 1993; 215: 105-111Crossref PubMed Scopus (178) Google Scholar). In several types of inflammatory cells, FLAP mRNA can be induced by dexamethasone, interleukin (IL) 3, and granulocyte-monocyte colony-stimulating factor (10Ring W.L. Riddick C.A. Baker J.R. Munafo D.A. Bigby T.D. J. Clin. Invest. 1996; 97: 1293-1301Crossref PubMed Scopus (55) Google Scholar, 11Riddick C.A. Ring W.L. Baker J.R. Hodulik C.R. Bigby T.D. Eur. J. Biochem. 1997; 246: 112-118Crossref PubMed Scopus (94) Google Scholar, 12Coffey M.J. Phare S.M. Cinti S. Peters-Golden M. Kazanjian P.H. Blood. 1999; 94: 3897-3905Crossref PubMed Google Scholar), 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, NFκB, and glucocorticoid receptor binding sites (13Kennedy B.P. Diehl R.E. Boie Y. Adam M. Dixon R.A. J. Biol. Chem. 1991; 266: 8511-8516Abstract Full Text PDF PubMed Google Scholar). 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 (13Kennedy B.P. Diehl R.E. Boie Y. Adam M. Dixon R.A. J. Biol. Chem. 1991; 266: 8511-8516Abstract Full Text PDF PubMed Google Scholar). 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 (13Kennedy B.P. Diehl R.E. Boie Y. Adam M. Dixon R.A. J. Biol. Chem. 1991; 266: 8511-8516Abstract Full Text PDF PubMed Google Scholar). A FLAP gene polymorphism in the proximal 5′-UTR region has been identified 94 bp upstream of the transcription start site (14Koshino T. Takano S. Kitani S. Ohshima N. Sano Y. Takaishi T. Hirai K. Yamamoto K. Morita Y. Mol. Cell. Biol. Res. Commun. 1999; 2: 32-35Crossref PubMed Scopus (30) Google Scholar). 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%) (14Koshino T. Takano S. Kitani S. Ohshima N. Sano Y. Takaishi T. Hirai K. Yamamoto K. Morita Y. Mol. Cell. Biol. Res. Commun. 1999; 2: 32-35Crossref PubMed Scopus (30) Google Scholar). 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 (15Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-29548Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar, 16Poli V. J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). 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 α (TNFα), IL-8, granulocyte-colony stimulating factor, CD14, and inducible nitric oxide synthase (15Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-29548Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar,17Caivano M. Gorgoni B. Cohen P. Poli V. J. Biol. Chem. 2001; 276: 48693-48701Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 18Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1197) Google Scholar, 19Stein B. Baldwin Jr., A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Crossref PubMed Google Scholar, 20Pan Z. Hetherington C.J. Zhang D.E. J. Biol. Chem. 1999; 274: 23242-23248Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 21Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Snyder S.H. Russell S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9730-9734Crossref PubMed Scopus (1001) Google Scholar). While C/EBPα, β, and δ are expressed in liver and lung, C/EBPε expression is believed to be confined to cells of myeloid and lymphoid lineage (15Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-29548Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar). C/EBPα expression has also been reported in peripheral blood mononuclear cells (15Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-29548Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar). The C/EBPα, β, δ, and ε 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 (15Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-29548Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar, 22Cao Z. Umek R.M. McKnight S.L. Gene Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1329) Google Scholar, 23Williams S.C. Cantwell C.A. Johnson P.F. Gene Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (438) Google Scholar). The dimerization domain is highly conserved and is believed to be required for DNA binding (15Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-29548Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar). C/EBP family members form homo- and heterodimers, the formation of which is required for DNA binding (24Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1989; 243: 1681-1688Crossref PubMed Scopus (418) Google Scholar). Cell-specific gene regulation by C/EBP proteins has been shown to be dependent upon interactions with other transcription factors, including NFκB, Sp1, and Fos/Jun family members (25Pope R.M. Lovis R. Mungre S. Perlman H. Koch A.E. Haines III, G.K. Clin. Immunol. 1999; 91: 271-282Crossref PubMed Scopus (30) Google Scholar, 26Lee Y.H. Williams S.C. Baer M. Sterneck E. Gonzalez F.J. Johnson P.F. Mol. Cell. Biol. 1997; 17: 2038-2047Crossref PubMed Google Scholar). 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 α, δ, and ε members of the C/EBP family of transcription factors bind to these elements and that the α and δ isoforms, at least, function to up-regulate FLAP gene expression in mononuclear phagocytes. 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 (11Riddick C.A. Ring W.L. Baker J.R. Hodulik C.R. Bigby T.D. Eur. J. Biochem. 1997; 246: 112-118Crossref PubMed Scopus (94) Google Scholar, 27Serio K.J. Hodulik C.R. Bigby T.D. Am. J. Respir. Cell Mol. Biol. 2000; 23: 234-240Crossref PubMed Scopus (20) Google Scholar). THP-1 cells were grown at 37 °C with 5% CO2in 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. 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 withSacI 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). 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-TKRenilla luciferase vector (Promega) or a pCMV-β-galactosidase vector (generously provided by Dr. Kenneth Chien, University of California, San Diego, CA) using Effectene transfection reagent (Qiagen), as has been previously described (27Serio K.J. Hodulik C.R. Bigby T.D. Am. J. Respir. Cell Mol. Biol. 2000; 23: 234-240Crossref PubMed Scopus (20) Google Scholar). For overexpression experiments, DNA mixtures consisted of 225 ng of the wild-type or mutant -134FLAP-pGL3 construct, 50 ng of theRenilla luciferase pRL-TK construct, and 225 ng of the C/EBPα, β, δ, and ε expression vectors to a total of 500 ng of DNA per condition. For TNFα experiments, the cells were subsequently treated with TNFα (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. β-Galactosidase activity was measured using the Tropix β-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 β-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). Nuclear extracts were prepared from THP-1 and HeLa cells as previously described (28Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). 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. 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 TNFα-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. THP-1 cells were treated with TNFα (10 ng/ml) or transiently transfected with expression vectors for C/EBPα, β, δ, and ε (450 ng × 106 cells) as has been previously described (27Serio K.J. Hodulik C.R. Bigby T.D. Am. J. Respir. Cell Mol. Biol. 2000; 23: 234-240Crossref PubMed Scopus (20) Google Scholar). 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 β-actin (Clontech, Palo Alto, CA). ChIP assays were performed by a modification of a previously described method (29Yan C. Wang H. Toh Y. Boyd D.D. J. Biol. Chem. 2003; 278: 2309-2316Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 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 (30Barre B. Avril S. Coqueret O. J. Biol. Chem. 2003; 278: 2990-2996Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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. 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/EBPα expression vector was obtained from Dr. Steve McKnight, University of Texas Southwestern Medical Center (Dallas, TX) (31Friedman A.D. Landschulz W.H. McKnight S.L. Genes Dev. 1989; 3: 1314-1322Crossref PubMed Scopus (363) Google Scholar). The C/EBPβ expression vector was obtained from Dr. Mario Chojkier, Veterans Affairs San Diego Healthcare System (San Diego, CA) (32Houglum K. Buck M. Adir V. Chojkier M. J. Clin. Invest. 1994; 94: 808-814Crossref PubMed Google Scholar, 33Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). The C/EBPδ expression vector was obtained from Dr. Jan Trapman, Erasmus University (Rotterdam, Holland) (34Cleutjens C.B. van Eekelen C.C. van Dekken H. Smit E.M. Hagemeijer A. Wagner M.J. Wells D.E. Trapman J. Genomics. 1993; 16: 520-523Crossref PubMed Scopus (28) Google Scholar). The C/EBPε expression vector was obtained from Dr. Julie Lekstrom-Himes, NIAID National Institutes of Health (Bethesda, MD). TNFα 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 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. 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. 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. 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). 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/EBPα (lane 2), δ (lane 4), and ε (lane 5). Our data indicate the binding of C/EBPα, δ, and ε to the proximal C/EBP consensus site in unconditioned THP-1 cells.Figure 4EMSA 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 α, (lane 2), β (lane 3), δ (lane 4), and ε (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/EBPα (lane 3) and β (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/EBPα (lane 3), β (lane 4), δ (lane 5), and ε (lane 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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/EBPα and β 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. 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 α, β, δ, and ε (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. 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 e" @default.
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