Eltom S E, Zhang L, Jefcoate C R
Center for Environmental Toxicology and Department of Pharmacology, University of Wisconsin Medical School, Madison 53706, USA.
Mol Pharmacol. 1999 Mar;55(3):594-604.
Cytochrome P-450 (CYP) 1B1 expression in mouse hepatoma (Hepa-1) wild-type (WT) cells was compared with responses in Hepa-1 variants LA1 and LA2, which, respectively, exhibit low aryl hydrocarbon receptor (AhR) level and defective AhR nuclear translocator (ARNT) protein. 10T1/2 mouse embryo fibroblasts express predominantly CYP1B1 and at a 100 times higher level than in Hepa-1 cells, whereas they express about 300-fold lower CYP1A1 than Hepa-1 cells. The expression of CYP1B1 in WT and LA1 variant, although at a much lower level, follows that of CYP1A1, reflecting their common regulation through the AhR. The LA2 (ARNT-defective) cells showed a major difference between CYP1B1 and CYP1A1 expression. Although CYP1A1 mRNA levels in LA2 were extremely low and unresponsive to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), basal CYP1B1 mRNA and protein were expressed at levels similar to those seen in TCDD-induced WT. The elevated basal CYP1B1 mRNA in LA2 cells decreased by 50% after transient transfection of ARNT cDNA, in parallel with substantial restoration of CYP1A1 induction. This implicates ARNT as a suppressor of CYP1B1 basal expression in Hepa cells. In transient CYP1B1-luciferase constructs in LA2 cells, ARNT shows stimulatory effects in the enhancer region but an inhibitory effect on the proximal promoter. Two CYP1B1 enhancer elements [xenobiotic-responsive element (XRE) 1/2 and XRE4] formed TCDD-unresponsive complexes of similar mobility to TCDD-stimulated AhR-ARNT complex with XRE5. However, because these two complexes were formed to the same extent in LA2 as in WT cells, they cannot be due to ARNT or contribute to ARNT-regulated suppression.
将小鼠肝癌（Hepa-1）野生型（WT）细胞中的细胞色素P-450（CYP）1B1表达与Hepa-1变体LA1和LA2中的反应进行比较，这两种变体分别表现出低芳烃受体（AhR）水平和有缺陷的AhR核转运体（ARNT）蛋白。10T1/2小鼠胚胎成纤维细胞主要表达CYP1B1，其水平比Hepa-1细胞高100倍，而它们表达的CYP1A1比Hepa-1细胞低约300倍。WT和LA1变体中CYP1B1的表达，尽管水平低得多，但遵循CYP1A1的表达模式，反映了它们通过AhR的共同调节。LA2（ARNT缺陷）细胞在CYP1B1和CYP1A1表达之间表现出主要差异。尽管LA2中CYP1A1 mRNA水平极低且对2,3,7,8-四氯二苯并对二恶英（TCDD）无反应，但基础CYP1B1 mRNA和蛋白的表达水平与TCDD诱导的WT细胞中所见水平相似。LA2细胞中基础CYP1B1 mRNA的升高在瞬时转染ARNT cDNA后降低了50%，同时CYP1A1诱导也有大量恢复。这表明ARNT是Hepa细胞中CYP1B1基础表达的抑制因子。在LA2细胞中的瞬时CYP1B1-荧光素酶构建体中，ARNT在增强子区域显示出刺激作用，但对近端启动子有抑制作用。两个CYP1B1增强子元件[异生素反应元件（XRE）1/2和XRE4]形成了与TCDD刺激的AhR-ARNT复合物与XRE5迁移率相似的TCDD无反应复合物。然而，由于这两种复合物在LA2中与在WT细胞中形成的程度相同，它们不可能是由于ARNT或促成ARNT调节的抑制作用。
