A NF-kappaB p65 subunit is indispensable for activating manganese superoxide: dismutase gene transcription mediated by tumor necrosis factor-alpha.
Expression of the manganese superoxide dismutase (Mn-SOD) is induced by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and lipopolysaccharide (LPS). Recently, a TNF-responsive element (TNFRE) was identified within the second intron of the murine Mn-SOD gene. The 5' CCAAT/enhancer binding protein (C/EBP)-related region within the TNFRE was responsive to TNF, whereas the 3' NF-kappaB-related region alone was not. This report describes the minimal promoter region of the Mn-SOD gene and investigates the cis-acting elements and trans-acting factors responsible for TNF-alpha-induced Mn-SOD gene expression. Reporter plasmid transfection studies demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the intronic enhancer region. Electrophoretic mobility shift assays demonstrated that after TNF-alpha stimulation, p50 and p65 NF-kappaB subunits bound specifically to the newly identified NF-kappaB transcription factor-binding site, distinct from the previously described NF-kappaB site, within the intronic enhancer region. In addition, site-directed mutagenesis and cotransfection studies demonstrated that the NF-kappaB p65 subunit enhanced the transcriptional activity of the Mn-SOD gene through the newly identified NF-kappaB site. These results show that a NF-kappaB p65 subunit is mainly involved in the molecular mechanisms controlling TNF-alpha-mediated Mn-SOD gene transcription.
Maehara
K
K
Department of Basic Gerontology, National Institute for Longevity Sciences, Obu, Aichi, 474-8522 Japan.
Hasegawa
T
T
Isobe
K I
KI
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D007438:Introns; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D052002:NF-kappa B p50 Subunit; D009687:Nuclear Proteins / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D013482:Superoxide Dismutase