A NF-κB p65 subunit is indispensable for activating manganese superoxide: Dismutase gene transcription mediated by tumor necrosis factor-α

Expression of the manganese superoxide dismutase (Mn-SOD) is induced by tumor necrosis factor-α (TNF-α), 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-κB-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-α-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-α stimulation, p50 and p65 NF-κB subunits bound specifically to the newly identified NF-κB transcription factor-binding site, distinct from the previously described NF-κB site, within the intronic enhancer region. In addition, site-directed mutagenesis and cotransfection studies demonstrated that the NF-κB p65 subunit enhanced the transcriptional activity of the Mn-SOD gene through the newly identified NF-κB site. These results show that a NF-κB p65 subunit is mainly involved in the molecular mechanisms controlling TNF-α-mediated Mn-SOD gene transcription. J. Cell. Biochem. 77:474–486, 2000. © 2000 Wiley-Liss, Inc.