Novel CAR-mediated mechanism for synergistic activation of two distinct elements within the human cytochrome P4502B6 gene in HepG2 cells
Novel CAR-mediated mechanism for synergistic activation of two distinct elements within the human cytochrome P4502B6 gene in HepG2 cells
复制标题
DOI：
10.1074/jbc.m411318200
复制
发表时间：
2005-02-04
影响因子：
4.8
通讯作者：
Negishi, M
中科院分区：
文献类型：
--
作者：
Swales, K;Kakizaki, S;Negishi, M
组成型活性受体 (CAR) 通过苯巴比妥型诱导剂（例如 1,4 双[2-(3,5-二氯吡啶氧基）]苯 (TCPOBOP)）通过远端苯巴比妥响应增强子模块（PBREM，-1732/-1685 bp）调节细胞色素 P450 2B6 (CYP2B6) 基因的诱导。 TCPOBOP 激活 PBREM 在稳定表达小鼠 CAR (Ym17) 的 HepG2 细胞中产生 10 倍的 CYP2B6 mRNA 诱导。与蛋白磷酸酶抑制剂冈田酸 (OA) 共同治疗可协同地将这种诱导作用提高 100 倍以上，而无需直接激活 CAR 或 PBREM。尽管 OA 协同作用需要 PBREM 的存在，但删除分析描绘了 CYP2B6 启动子中近端 24 bp (-256/-233) 序列 (OARE) 的 OA 响应活性。在电泳迁移率变动分析中，CAR 不直接与 OARE 结合。然而，DNA亲和力和染色质免疫沉淀分析均显示，在TCPOBOP和OA共同处理后，CAR与OARE的关联显着增加，表明CAR与OARE的间接结合。染色质结构内的两个顺式作用元件，即远端 PBREM 和近端 OARE，均由 CAR 分别响应 TCPOBOP 和 OA 进行调节，以最大限度地诱导 CYP2B6 启动子。两个位点之间的这种功能性相互作用扩展了目前对 CAR 介导的诱导转录机制的理解。
The constitutive active receptor (CAR) regulates the induction of the cytochrome P450 2B6 (CYP2B6) gene by phenobarbital-type inducers, such as 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) via the distal phenobarbital-responsive enhancer module (PBREM, at -1732/-1685 bp). Activation of the PBREM by TCPOBOP generated a 10-fold induction of CYP2B6 mRNA in HepG2 cells stably expressing mouse CAR (Ym17). Co-treatment with the protein phosphatase inhibitor okadaic acid (OA) synergistically increased this induction over 100-fold without directly activating CAR or the PBREM. Although OA synergy required the presence of PBREM, deletion assays delineated the OA-responsive activity to a proximal 24-bp (-256/-233) sequence (OARE) in the CYP2B6 promoter. CAR did not directly bind to the OARE in electrophoretic mobility shift assays. However, both DNA affinity and chromatin immunoprecipitation assays showed a significant increase in CAR association with the OARE after co-treatment with TCPOBOP and OA, indicating the indirect binding of CAR to the OARE. The two cis-acting elements, the distal PBREM and the proximal OARE, within the chromatin structure are both regulated by CAR in response to TCPOBOP and OA, respectively, to maximally induce the CYP2B6 promoter. This functional interaction between the two sites expands the current understanding of the mechanism of CAR-mediated inducible transcription.