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.
|Number of pages||11|
|Journal||Biochimica et Biophysica Acta - Lipids and Lipid Metabolism|
|Publication status||Published - 1998 Jan 5|
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