Transcriptional activation of the human manganese superoxide dismutase gene mediated by tetradecanoylphorbol acetate

Transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA induced by a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) , was examined to identify the responsive transcriptional regulator. The ef fect of various deletions and mutations within the 5'-flanking region of th e human MnSOD gene promoter was evaluated using the luciferase reporter sys tem in A549 human lung carcinoma cells. Deletion of a region between -1292 and -1202 nucleotides upstream of the transcription start site abolished TP A-responsive induction, whereas deletion of the putative binding sequence f or NF-kappa B or AP-1 did not. The region between -1292 and -1202 contains a cAMP-responsive element-like sequence, TGACGTCT, which we identified as t he manganese Superoxide dismutase TPA-responsive element, MSTRE. Site-speci fic mutation of the MSTRE abolished the TPA-responsive induction, validatin g the critical role of this sequence. We detected specific MSTRE activity f rom nuclear extracts and demonstrated by antibody supershift assay that thi s activity is closely related to CREB-1/ATF-1. TPA treatment rap idly induc ed phosphorylation of the CREB-1/ATF-1-like factor via the protein kinase C pathway. These results led us to conclude that the human MnSOD gene having the promoter construct used in this study is induced by TPA via activation of a CREB-1/ATF-1-like factor and not via either NF-kappa B or AP-1, In ad dition, we found that this induction was blocked by inhibitors of flavoprot eins and NADPH oxidases, indicating involvement of enhanced generation of s uperoxide radical anion as an upstream signal.