title: Dual function of protein kinase C (PKC) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced manganese superoxide dismutase (MnSOD) expression: activation of CREB and FOXO3a by PKC-alpha phosphorylation and by PKC-mediated inactivation of Akt, respectively.
abstract: 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA in human lung carcinoma cells, A549, mediated by a protein kinase C (PKC)-dependent activation of cAMP-responsive element-binding protein (CREB)-1/ATF-1-like factors. In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-α, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment of the PKC inhibitor BIM and NADPH oxidase inhibitor DPI. Small interfering RNA (siRNA) experiments indicated that knocking down the NADPH oxidase components e.g. Rac1, p22(phox), p67(phox), and NOXO1 in A549 cells impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-α and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 and most likely mediated by PKC-α-, and -ε-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA up-regulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-α catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser(473) which in turn leads to FOXO3a Ser(253) dephosphorylation and its activation.
authors.affiliation: Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8012, USA.
authors.forename: Youn Wook
authors.initials: YW
authors.lastname: Chung
authors.forename: Ha Kun
authors.initials: HK
authors.lastname: Kim
authors.forename: Ick Young
authors.initials: IY
authors.lastname: Kim
authors.forename: Moon B
authors.initials: MB
authors.lastname: Yim
authors.forename: P Boon
authors.initials: PB
authors.lastname: Chock
mesh_terms: D002273:Carcinogens / Q000494:pharmacology*; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000502:physiology; D055785:Gene Knockdown Techniques; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: C115163:CREB1 protein, human; D002273:Carcinogens; D017362:Cyclic AMP Response Element-Binding Protein; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D016877:Oxidants; C495901:TP53 protein, human; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D051057:Proto-Oncogene Proteins c-akt; D051571:Protein Kinase C-alpha; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate