An intronic NF-kappa B element is essential for induction of the human manganese superoxide dismutase gene by tumor necrosis factor-alpha and interleukin-1 beta

Tumor necrosis factor-alpha (TNF) and interleukin-1 beta (IL-1) are cytokin es that induce expression of various genes through activation of the redox- sensitive transcription Factor nuclear factor-kappa B (NF-kappa B), We have previously cloned the entire human MnSOD (SOD2) gene and found several NF- kappa B-binding sites in the 5' and 3' flanking and intronic regions. To te st whether these putative NF-kappa B-binding sites are able to respond to T NF and IL-1, we performed induction analysis using various deletion constru cts ligated to a luciferase reporter gene. We found that the 5' and 3' flan king regions containing several NF-kappa B-binding sites do not mediate MnS OD induction by TNF or IL-1, When a 342-bp intron 2 fragment containing NF- kappa B, C/EBP, and NF-1 binding sites was linked to the basal promoter of the SOD2 gene, transcriptional activities were significantly increased in r esponse to TNF and IL-1 in an orientation- and position-independent manner. To accurately identify the element that is most critical for the enhancer activity, deletions and specific mutations of each individual site were stu died. The results indicated that the NF-kappa B binding site is essential b ut not sufficient for TNF- or IL-1-mediated induction. Furthermore, NF-kapp a B elements in the 5' and 3' flanking regions could be made to function in TNF or IL-1 induction when they were transposed to the intronic fragment. Taken together, these results suggest that an NF-kappa B element and its lo cation in the SOD2 gene is critical for TNF/IL-1-mediated induction. Howeve r, a complex interaction between NF-kappa B and other transcription element s is needed for a high-level induction.