title: NFKB1 Promoter DNA from nt+402 to nt+99 Is Hypomethylated in Different Human Immune Cells.
abstract: Sepsis, with a persistently high 90-day mortality of about 46%, is the third most frequent cause of death in intensive care units worldwide. Further understanding of the inflammatory signaling pathways occurring in sepsis is important for new efficient treatment options. Key regulator of the inflammatory response is the transcription factor NFκB. As we have recently shown, the -94 Ins/Del NFKB1 promoter polymorphism influences sepsis mortality. However, a molecular explanation is still missing. Thus, promoter activity might be varying depending on the NFKB1 genotype, explaining the genotype dependent mortality from sepsis, and one likely mechanism is the degree of promoter methylation. Therefore, we tested the hypothesis that NFκB mRNA expression is regulated by promoter methylation in human cell lines and primary immune cell cultures. First, we examined the methylation of the NFKB1 promoter in U937, REH and HL-60 cells. In the promoter region of nt+99/+229 methylation in all analyzed cell lines was below 1%. Following incubation with bacterial cell wall components, no significant changes in the frequency of promoter methylation in U937 and REH cells were measured and the methylation frequency was under 1%. However, NFκB1 mRNA expression was two-fold increased in U937 cells after 24 h incubation with LPS. By contrast, demethylation by 5-Aza-2'-deoxycytidine incubation enhanced NFκB1 expression significantly. In addition, we analyzed NFKB1 promoter methylation in primary cells from healthy volunteers depending on the NFKB1-94 Ins/Del genotype. Methylation in the promoter region from nt+402 to nt+99 was below 1%. Genotype dependent differences occurred in neutrophil cells, where DD-genotype was significantly more methylated compared to II genotype at nt+284/+402. Besides in the promoter region from nt-227/-8 in ID-genotypes methylation of neutrophils was significantly decreased compared to lymphocytes and in II-genotypes methylation in neutrophils was significantly decreased compared to lymphocytes and monocytes. In addition, CHART-PCR showed that the hypomethylated promoter regions are highly accessible. Therefore we assume that the demethylated regions are very important for NFKB1 promoter activity.
authors.affiliation: Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum-Langendreer der Ruhr-Universität Bochum, In der Schornau 23-25, 44892 Bochum, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Unterberg
authors.affiliation: Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum-Langendreer der Ruhr-Universität Bochum, In der Schornau 23-25, 44892 Bochum, Germany.
authors.forename: Maxmiliane Julia
authors.initials: MJ
authors.lastname: Kreuzer
authors.affiliation: Klinik für Anaesthesiologie, Klinikum der Universität Ludwig-Maximilians Universität München, München, Germany.
authors.forename: Simon Thomas
authors.initials: ST
authors.lastname: Schäfer
authors.affiliation: Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum-Langendreer der Ruhr-Universität Bochum, In der Schornau 23-25, 44892 Bochum, Germany.
authors.forename: Zainab
authors.initials: Z
authors.lastname: Bazzi
authors.affiliation: Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum-Langendreer der Ruhr-Universität Bochum, In der Schornau 23-25, 44892 Bochum, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Adamzik
authors.affiliation: Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum-Langendreer der Ruhr-Universität Bochum, In der Schornau 23-25, 44892 Bochum, Germany.
authors.forename: Katharina
authors.identifier: http://orcid.org/0000-0003-2220-519X
authors.initials: K
authors.lastname: Rump
mesh_terms: D002460:Cell Line; D004247:DNA / Q000235:genetics*; D019175:DNA Methylation; D006801:Humans; D016328:NF-kappa B / Q000235:genetics*; D011401:Promoter Regions, Genetic
chemical_list: D016328:NF-kappa B; D004247:DNA