Zheng Wenchao, Brake Paul B, Bhattacharyya Kalyan K, Zhang Leying, Zhao Dong, Jefcoate Colin R
Department of Pharmacology, Medical Science Center, University of Wisconsin, 1300, University Avenue, Madison, WI 53706, USA.
Arch Biochem Biophys. 2003 Aug 1;416(1):53-67. doi: 10.1016/s0003-9861(03)00282-0.
CYP1B1 is unique among P450 cytochromes in exhibiting inductive responses mediated by both the Ah receptor (AhR) and cAMP. cAMP induction was mediated either by a 189bp far upstream enhancer region (FUER, -5110 to -5298) or by a 230bp AhR-responsive enhancer region (AhER) (-797 to -1026). CYP1B1 luciferase reporters respond selectively to cAMP and TCDD in adrenal Y-1 cells (only cAMP), testis MA10 cells (cAMP>TCDD), and C3H10T1/2 mouse embryo fibroblasts (only TCDD). In Y-1 cells, which lack AhR, cAMP induction is totally dependent on the FUER, including absolute requirements for upstream and downstream halves of this region, and for CREB activity at a CRE sequence located at the 3(')-end. cAMP stimulation of the FUER was remarkably high (27-fold) and equally effective when linked to an HSV-TK promoter, indicating direct cAMP activation of the FUER. Binding of CREB to the essential CRE was demonstrated along with dominant negative effects of functionally impaired mutants. cAMP induction in MA10 cells was partially mediated by the FUER mechanism but was regulated additionally by AhER through AhR activity. MA10 cells also exhibit cAMP-dependent AhR down-regulation and AhR/Arnt complex formation. Mutations in AhER including XRE5 were similarly inhibitory to cAMP stimulation in MA10 cells and to TCDD stimulation in C3H10T1/2 cells. Transfection of AhR into the AhR-deficient Y-1 cells did not introduce this second mechanism, which indicated a need for additional components that are present in MA10 cells.
细胞色素P450 1B1（CYP1B1）在P450细胞色素中独具特色，它能展现出由芳烃受体（AhR）和环磷酸腺苷（cAMP）介导的诱导反应。cAMP诱导作用可由一个位于上游远侧189bp的增强子区域（FUER，-5110至-5298）介导，也可由一个230bp的AhR反应性增强子区域（AhER）（-797至-1026）介导。CYP1B1荧光素酶报告基因在肾上腺Y-1细胞（仅对cAMP有反应）、睾丸MA10细胞（cAMP>TCDD）和C3H10T1/2小鼠胚胎成纤维细胞（仅对TCDD有反应）中对cAMP和2,3,7,8-四氯二苯并对二恶英（TCDD）有选择性反应。在缺乏AhR的Y-1细胞中，cAMP诱导完全依赖于FUER，包括对该区域上游和下游两半部分以及位于3′端的CRE序列处的CREB活性的绝对需求。当与单纯疱疹病毒胸苷激酶（HSV-TK）启动子相连时，FUER对cAMP的刺激作用非常高（27倍）且同样有效，表明cAMP对FUER有直接激活作用。证明了CREB与必需的CRE结合以及功能受损突变体的显性负效应。MA10细胞中的cAMP诱导部分由FUER机制介导，但还通过AhR活性由AhER额外调节。MA10细胞还表现出cAMP依赖性的AhR下调和AhR/芳烃受体核转运蛋白（Arnt）复合物形成。包括XRE5在内的AhER突变对MA10细胞中的cAMP刺激以及C3H10T1/2细胞中的TCDD刺激同样具有抑制作用。将AhR转染到缺乏AhR的Y-1细胞中并未引入这第二种机制，这表明需要MA10细胞中存在的其他成分。
