title: The RelA/cRel nuclear factor-κB (NF-κB) dimer, crucial for inflammation resolution, mediates the transcription of the key enzyme in melatonin synthesis in RAW 264.7 macrophages.
abstract: Lipopolysaccharide (LPS) modulates the transcription of the gene that codifies the enzyme arylalkylamine-N-acetyltransferase (AA-NAT) through nuclear translocation of the transcription factor nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB). AA-NAT converts serotonin to N-acetylserotonin, the ultimate precursor of melatonin. Activation of kappa B elements (aa-nat-κB), localized in the promoter (nat-κB1 and nat-κB2), leads to Aa-nat transcription in RAW 264.7 macrophages. Competitive electrophoretic mobility shift assay (EMSA) with oligonucleotide probes corresponding to each of the two elements, as well as a NF-κB consensus corresponding probe, revealed different specificities for each κB element. In addition, activator protein-1 (AP-1) as well as signal transducers and activator of transcription-1 and 3 (STAT-1; STAT-3) competed with NF-κB for binding to nat-κB1, while only STAT-3 competed with NF-κB for binding to nat-κB2. According to co-immunoprecipitation (ChiP) assays, these two sites are able to distinguish NF-κB subunits. The sequence nat-κB1 bound dimers containing p52, RelA, and cRel, while nat-κB2 bound preferentially p50, p52, and RelA, and did not bind cRel. The expression of RelA and cRel is essential for the induction of Aa-nat expression and melatonin synthesis. Considering that the expression of cRel is induced by the earlier expressed p50/RelA, the differential effects of NF-κB dimers may be intimately associated with the temporal regulation of inflammatory responses, with the resolution phase being associated with paracrine and autocrine melatonin effects. Such data suggest that the proven effects of exogenous melatonin in the resolution phase of inflammation are paralleled by the effects of locally synthesized melatonin in immune cells.
authors.affiliation: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Sandra Marcia
authors.initials: SM
authors.lastname: Muxel
authors.affiliation: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Laranjeira-Silva
authors.affiliation: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Claudia Emanuelle
authors.initials: CE
authors.lastname: Carvalho-Sousa
authors.affiliation: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Lucile Maria
authors.initials: LM
authors.lastname: Floeter-Winter
authors.affiliation: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Regina P
authors.initials: RP
authors.lastname: Markus
mesh_terms: D000818:Animals; D015153:Blotting, Western; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D047468:Immunoprecipitation; D007249:Inflammation / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D008550:Melatonin / Q000096:biosynthesis*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D060888:Real-Time Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D016328:NF-kappa B; D008550:Melatonin
keywords: aryl-alkyl-N-acetyltransferase (AA-NAT); macrophages; melatonin; nuclear factor kappa B (NF-κB)