5-Lipoxygenase-activating Protein Gene Expression

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.