TUMOR-NECROSIS-FACTOR-ALPHA AND INTERLEUKIN-1-BETA REGULATE THE MURINE MANGANESE SUPEROXIDE-DISMUTASE GENE THROUGH A COMPLEX INTRONIC ENHANCER INVOLVING C EBP-BETA AND NF-KAPPA-B/

Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)- inducible reactive oxygen-scavenging enzyme, protects cells from TNF-m ediated apoptosis. To understand how MnSOD is regulated, transient tra nsfections of promoter-reporter gene constructions, in vitro DNA bindi ng 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 -1 beta (IL-1). This TNF response element (TNFRE) had the properties o f 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-kappa B and C/EB P. The 5' portion of the TNFRE bound C/EBP-beta in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promote r or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE bound both NF-KB 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 en hancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one t hat mediates induction when it is downstream of a promoter.