Tumor Necrosis Factor Alpha and Interleukin-1β Regulate the Murine Manganese Superoxide Dismutase Gene Through a Complex Intronic Enhancer Involving C/EBP-β and NF-κB

Abstract

Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identified a 238-bp region of intron 2 that was responsive to TNF and interleukin-1β (IL-1). This TNF response element (TNFRE) had the properties of a traditional enhancer element that functioned in an orientation- and position-independent manner. IVGF of the TNFRE revealed TNF- and IL-1-induced factor occupancy of sites that could bind NF-κB and C/EBP. The 5′ portion of the TNFRE bound C/EBP-β in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3′ end of the TNFRE bound both NF-κB and C/EBP but was not necessary for TNF responsiveness with the MnSOD promoter. However, this 3′ portion of the TNFRE was required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter.