Takahashi H, Iizuka H
Department of Dermatology, Asahikawa Medical College, Japan.
J Invest Dermatol. 1993 Jan;100(1):10-5. doi: 10.1111/1523-1747.ep12349867.
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.
兜甲蛋白是角质形成细胞角化包膜的前体蛋白之一。尽管促肿瘤佛波酯可诱导角化包膜的形成，但12 - O -十四酰佛波醇-13 -乙酸酯（TPA）对兜甲蛋白基因表达的影响仍不清楚。我们分离出了人兜甲蛋白基因的5'上游区域，并检测了其依赖TPA的启动子活性。将带有未翻译的第一外显子的兜甲蛋白上游区域连接到氯霉素乙酰转移酶（CAT）-兜甲蛋白启动子表达载体（INV - CAT）上，并转染到胎鼠角质化表皮（FRSK）细胞中。转染了INV - CAT的FRSK细胞表现出相当高的CAT活性，用TPA处理细胞后该活性显著增强。用反向的5'上游序列转染的FRSK细胞几乎没有CAT活性，且对TPA无反应。蛋白激酶C抑制剂1 -（5 -异喹啉磺酰基）-2 -甲基哌嗪二盐酸盐（H - 7）显著抑制了TPA的作用。其他蛋白激酶C激活剂（1 -油酰-2 -乙酰甘油和卫矛醇）也诱导了INV - CAT启动子活性，而4 - O -甲基佛波醇肉豆蔻酸酯，一种非常弱的蛋白激酶C激活剂，只有轻微作用。对5'上游区域核苷酸序列的分析检测到几个5'-TGANTCAA-3'序列，它们是高度保守的TPA反应元件（TRE）。将c - jun和c - fos表达载体与INV - CAT载体共转染到FRSK细胞中导致CAT活性增加。单独将c - jun或c - fos载体与INV - CAT载体转染到FRSK细胞中作用可忽略不计。地塞米松显著抑制了转染了INV - CAT的FRSK细胞中TPA诱导的启动子活性。这些结果表明，TPA通过激活蛋白激酶C/TRE系统正向调控兜甲蛋白基因表达。
