Nuclear factor κB-dependent mechanisms coordinate the synergistic effect of PMA and cytokines on the induction of superoxide dismutase 2

Manganese superoxide dismutase (MnSOD) serves a protective role under conditions of oxidative stress mediated by such diverse agents as adriamycin, radiation, chemical hypoxia and ischaemia and might act as a newly recognized type of tumour-suppressor. MnSOD is an inducible enzyme; however, the signalling molecules and pathways involved in its induction have not been fully elucidated. Recently we reported the identification of a 342 bp enhancer within the second intron (I2E) of the human gene encoding MnSOD (SOD2), which contains sites for binding nuclear factor κB (NF-κB), CCAAT-enhancer-binding protein (C/EBP) and nuclear factor 1 (NF-1). Using a human fibroblast cell line transformed by simian virus 40, we have identified the I2E fragment as being responsive to PMA. Furthermore, simultaneous treatment with PMA and cytokines (tumour necrosis factor α and interleukin 1β) synergistically increases MnSOD induction. The use of mutant constructs identified the NF-κB element within the enhancer fragment as being essential for the PMA and PMA/cytokine effect. Mutations in the C/EBP- and NF-1-binding sites revealed a potential cooperation between proteins that bind to these sites and the NF-κB element. Evaluation of inhibitory κB (IκB)-α and IκB-β proteins reveals agent-specific differences in their turnover kinetics. Both C/EBP and NF-κB DNA-binding activities were increased in cells receiving a combination of cytokine and PMA. Supershift and immunoprecipitation studies suggest a physical interaction between C/EBP and NF-κB proteins. Taken together, these studies suggest the activation of multiple transcription factors as well as pathways leading to increased NF-κB activity as being the mechanisms responsible for the synergistic induction of MnSOD by PMA and cytokines.