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.
Highlights
KEY ROLE OF CCAAT/ENHANCER-BINDING PROTEINS (C/EBP) IN CONSTITUTIVE AND TUMOR NECROSIS FACTOR (TNF) ␣-INDUCED EXPRESSION IN THP-1 CELLS*
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
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
Summary
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, electroeases, such as asthma, allergic rhinitis, glomerulonephritis, rheumatoid arthritis, and inflammatory bowel disease [1,2,3,4]. 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 phoretic mobility shift assays; TNF␣, tumor necrosis factor ␣; FCS, fetal calf serum. Our findings indicate that the ␣, ␦, and ⑀ members of the C/EBP family of transcription factors bind to these elements and that the ␣ and ␦ isoforms, at least, function to up-regulate FLAP gene expression in mononuclear phagocytes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
