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 effect of various deletions and mutations within the 5′-flanking region of the human MnSOD gene promoter was evaluated using the luciferase reporter system in A549 human lung carcinoma cells. Deletion of a region between −1292 and −1202 nucleotides upstream of the transcription start site abolished TPA-responsive induction, whereas deletion of the putative binding sequence for NF-κB or AP-1 did not. The region between −1292 and −1202 contains a cAMP-responsive element-like sequence, TGACGTCT, which we identified as the m anganese s uperoxide dismutase T PA- r esponsive e lement, MSTRE. Site-specific mutation of the MSTRE abolished the TPA-responsive induction, validating the critical role of this sequence. We detected specific MSTRE activity from nuclear extracts and demonstrated by antibody supershift assay that this activity is closely related to CREB-1/ATF-1. TPA treatment rapidly induced 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-κB or AP-1. In addition, we found that this induction was blocked by inhibitors of flavoproteins and NADPH oxidases, indicating involvement of enhanced generation of superoxide radical anion as an upstream signal.