Peng H M, Coon M J
Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, Michigan 48109, USA.
Mol Pharmacol. 1998 Oct;54(4):740-7.
The regulation of cytochrome P450 (CYP) 2E1, the ethanol-inducible isoform, is particularly complex. The level is affected by a variety of other foreign compounds, by insulin (as studied in several laboratories), and by triiodothyronine (T3), which has not been previously examined at the molecular level. In the present investigation, a stably transfected HepG2 cell line harboring a rabbit CYP2E1 minigene containing the coding sequence together with 1.6 kilobases of the 5' flanking region and the untranslated region (UTR), as well as 0.5 kilobases of the 3' UTR, was established. Western blot analysis showed that 1 microM insulin decreased the CYP2E1 protein level in a dose- and time-dependent manner, whereas 1 microM T3 increased the level 2-fold in 1 day and 8-fold in 5 days. Similarly, steady state CYP2E1 mRNA levels were decreased by insulin but were increased by T3. Neither hormone affected the transcription rate of the CYP2E1 5' flanking region with an UTR/luciferase fusion gene, indicating that the regulation is post-transcriptional in this system under our experimental conditions. When the CYP2E1 3' UTR was removed from the minigene, CYP2E1 mRNA and protein were up-regulated by insulin but were not affected by T3. These findings, including mRNA half-life determinations, indicate that the effects of insulin and T3 are a result of altered mRNA stability and that the 3' UTR of CYP2E1 contains regulatory information for these hormone-mediated effects.
细胞色素P450（CYP）2E1是乙醇诱导的同工酶，其调控尤为复杂。该酶水平受多种其他外来化合物、胰岛素（多个实验室已对此进行研究）以及三碘甲状腺原氨酸（T3）的影响，此前尚未在分子水平上对T3进行研究。在本研究中，构建了一种稳定转染的HepG2细胞系，该细胞系携带一个兔CYP2E1小基因，其包含编码序列以及1.6千碱基的5'侧翼区和非翻译区（UTR），还有0.5千碱基的3'UTR。蛋白质免疫印迹分析表明，1微摩尔胰岛素以剂量和时间依赖性方式降低CYP2E1蛋白水平，而1微摩尔T3在1天内使该水平增加2倍，在5天内增加8倍。同样，CYP2E1 mRNA的稳态水平受胰岛素降低，但受T3升高。两种激素均不影响带有UTR/荧光素酶融合基因的CYP2E1 5'侧翼区的转录速率，这表明在我们的实验条件下，该系统中的调控是转录后调控。当从小基因中去除CYP2E1 3'UTR时，CYP2E1 mRNA和蛋白受胰岛素上调，但不受T3影响。这些发现，包括mRNA半衰期的测定，表明胰岛素和T3的作用是mRNA稳定性改变的结果，并且CYP2E1的3'UTR包含这些激素介导作用的调控信息。
