Lipopolysaccharide induces 5-lipoxygenase-activating protein gene expression in THP-1 cells via a NF-kappaB and C/EBP-mediated mechanism.
We examined induced expression of the 5-lipoxygenase-activating protein (FLAP), which is critical for leukotriene synthesis in mononuclear phagocytes. Prolonged exposure to the bacterial component, lipopolysaccharide (LPS), increased FLAP gene transcription, mRNA expression, and protein expression in the human monocyte-like THP-1 cell line. Activation and inhibition of the NF-kappaB pathway modulated LPS induction of FLAP gene expression. An NF-kappaB-mediated mechanism of action was supported by overexpression of dominant-negative IkappaBalpha and p50/p65 proteins. EMSA/supershift and DNase I footprint analyses revealed that p50 binds to an NF-kappaB site located in the proximal FLAP promoter, while chromatin immunoprecipitation assays demonstrated that LPS induced binding of p50 but not of p65. Moreover, EMSA/supershift analyses demonstrated that LPS induced time-dependent binding of THP-1 nuclear extracts (containing p50) to this promoter region. Mutation of the NF-kappaB site decreased basal promoter activity and abolished the p50- and p65-associated induction. EMSA/supershift analyses also demonstrated that LPS induced binding of THP-1 nuclear extracts [containing CCAAT/enhancer binding protein (C/EBP)-alpha, -delta, and -epsilon] to a C/EBP site located adjacent to the NF-kappaB site in the FLAP promoter. We conclude that LPS enhances FLAP gene expression via both NF-kappaB- and C/EBP-mediated transcriptional mechanisms in mononuclear phagocytes.
Serio
Kenneth J
KJ
Dept. of Veterans Affairs San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, CA 92161, USA.
Reddy
K Veera
KV
Bigby
Timothy D
TD
D058927:5-Lipoxygenase-Activating Proteins; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology; D006801:Humans; D015289:Leukotrienes / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D015289:Leukotrienes; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D016328:NF-kappa B