Analysis of the 5'-upstream promoter region of human involucrin gene: activation by 12-O-tetradecanoylphorbol-13-acetate.
Involucrin is one of the precursor proteins of keratinocyte cornified envelope. Although the formation of the cornified envelope is induced by tumor-promoting phorbol esters, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the involucrin gene expression remains unknown. We have isolated a 5'-upstream region of human involucrin gene and examined its TPA-dependent promoter activity. The involucrin upstream region with the untranslated first exon was connected to chloramphenicol acetyltransferase (CAT)-involucrin promoter expression vector (INV-CAT) and was transfected into fetal rat keratinizing epidermal (FRSK) cells. The INV-CAT-transfected FRSK cells showed considerable CAT activity that was significantly augmented by the treatment of cells with TPA. FRSK cells that were transfected with a reversely oriented 5'-upstream sequence revealed little CAT activity and did not respond to TPA. The effect of TPA was significantly inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7). Other protein kinase C activators (1-oleoyl-2-acetylglycerol and mezerein) also induced the INV-CAT promoter activity, whereas 4-O-methyl phorbol myristate acetate, a very weak protein kinase C activator, had only a slight effect. Analysis of the nucleotide sequence of the 5'-upstream region detected several 5'-TGANTCAA-3' sequences that are highly conserved TPA-response elements (TRE). Cotransfection of both c-jun and c-fos expression vectors with the INV-CAT vector into FRSK cells resulted in increased CAT activity. Cotransfection of either the c-jun or c-fos vector singly with the INV-CAT vector into FRSK cells had negligible effects. Dexamethasone significantly inhibited the TPA-induced promoter activity in the INV-CAT-transfected FRSK cells. These results indicate that involucrin gene expression is positively controlled by TPA through the activation of the protein kinase C/TRE system.
Department of Dermatology, Asahikawa Medical College, Japan.
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Takahashi
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Iizuka
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002461:Cell Line, Transformed / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D010879:Piperazines / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013539:Simian virus 40 / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D007546:Isoquinolines; D010879:Piperazines; D011498:Protein Precursors; D012333:RNA, Messenger; C032253:involucrin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015500:Chloramphenicol O-Acetyltransferase; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate