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.