title: 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.
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/EBP alpha, delta, and epsilon bind to the proximal site and C/EBP alpha 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/EBP alpha, 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/EBP alpha, delta, and epsilon binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBP alpha, delta, and epsilon bound to this region of the 5'-untranslated region, whereas C/EBP beta 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.
authors.affiliation: Department of Medicine, Veterans Affairs San Diego Healthcare System and the University of California, San Diego 92161, USA.
authors.forename: K Veera
authors.initials: KV
authors.lastname: Reddy
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Serio
authors.forename: Craig R
authors.initials: CR
authors.lastname: Hodulik
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Bigby
mesh_terms: D020121:5' Untranslated Regions; D058927:5-Lipoxygenase-Activating Proteins; D001665:Binding Sites; D015152:Blotting, Northern; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D009154:Mutation; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D002843:Chromatin; D008565:Membrane Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D003850:Deoxyribonuclease I