Induction of 12-lipoxygenase expression by phorbol 12-myristate 13-acetate in human epidermoid carcinoma A431 cells.
Phorbol 12-myristate 13-acetate (PMA) increased the expression of 12-lipoxygenase activity and mRNA in a time-dependent manner in human epidermoid carcinoma A431 cells. The increase of 12-lipoxygenase was accompanied by the increase in protein level in microsomes prepared from A431 cells. The PMA-induced expression of 12-lipoxygenase activity and mRNA was inhibited by the treatment of cells with a protein kinase C inhibitor GF 109203X. Promoters of different DNA lengths for human 12-lipoxygenase gene were used to prepare the luciferase fusion vectors. These plasmid constructs were transiently transfected into A431 cells. Following treatment of PMA for 18 h, a 4- to 5-fold increase in luciferase reporter activity was observed in plasmids with the 5'-flanking region length of -951 bp and that of -224 bp upstream from translation starting site. A time-dependent induction of luciferase activity by PMA was found to parallel the PMA-induced enzyme activity and mRNA expression. Transient transfection with a series of 5'-deletion constructs showed that the 5'-flanking region spanning from -224 to -100 bp from translation starting site played an important role for PMA response. Gel mobility shift assay and site-directed mutagenesis indicated that two Sp1 binding sequences residing at -158 to -150 bp and -123 to -114 bp were responsible for the PMA response in activating the transcription of human 12-lipoxygenase gene.
Liaw
Y W
YW
Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Liu
Y W
YW
Chen
B K
BK
Chang
W C
WC
D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell; D004268:DNA-Binding Proteins / Q000378:metabolism; D004790:Enzyme Induction; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D007211:Indoles; D008301:Maleimides; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D001092:Arachidonate 12-Lipoxygenase; D008156:Luciferases; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate