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 kappaB (NF-kappaB), 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 alpha and interleukin 1beta) synergistically increases MnSOD induction. The use of mutant constructs identified the NF-kappaB 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 co-operation between proteins that bind to these sites and the NF-kappaB element. Evaluation of inhibitory kappaB (IkappaB)-alpha and IkappaB-beta proteins reveals agent-specific differences in their turnover kinetics. Both C/EBP and NF-kappaB 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-kappaB proteins. Taken together, these studies suggest the activation of multiple transcription factors as well as pathways leading to increased NF-kappaB activity as being the mechanisms responsible for the synergistic induction of MnSOD by PMA and cytokines.