Cell Cycle and Apoptosis Regulator 1, CCAR1, Regulates Enhancer-Dependent Nuclear Receptor CAR Transactivation
Cell Cycle and Apoptosis Regulator 1, CCAR1, Regulates Enhancer-Dependent Nuclear Receptor CAR Transactivation
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DOI：
10.1124/mol.118.114272
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发表时间：
2019-01-01
影响因子：
3.6
通讯作者：
Inouye, Yoshio
中科院分区：
文献类型：
--
作者：
Kanno, Yuichiro;Zhao, Shuai;Inouye, Yoshio
结构型活性雄烷受体(CAR)控制与异种化学代谢有关的基因。虽然已有大量的辅助因子被报道参与了CAR介导的反式激活，但CAR招募的未知和不明确的蛋白质还没有被表征。在本研究中，通过免疫共沉淀法从表达FLAG表位标记CAR的人肝癌细胞中鉴定了一种新的CAR相互作用蛋白--细胞周期和凋亡调节因子1(CCAR1)。我们证明了CCAR1可以作为CAR的增强子依赖的辅活化子。首先，我们发现CCAR1的过表达增强了CAR诱导的报告基因的活性，其中三重共识直接重复4基序(DR4-LUC)、异种生物反应增强模块(XREM)-CYP3A4的增强子(XREM-LUC)和UDP-葡萄糖醛酸基转移酶1A1的苯巴比妥反应增强模块(UGT1A1)(GtPBREM)-UGT1A1的增强子(gtPBREM-LUC)驱动的报告质粒(gtPBREM-LUC)驱动的报告基因活性，而不是由PBREM-LUC驱动的报告活性(PBREM-Luc)。此外，我们还发现，在HepTFi/CAR和HepaRG细胞中，CCAR1的敲除抑制了CAR诱导的UGT1A1基因的表达，但不影响CAR诱导的CYP2B6基因的表达。此外，CCAR1可以通过CAR募集到UGT1A1增强子的gtPBREM中，而不能募集到CYP2B6增强子的PBREM中。此外，我们还证明了CCAR1可以通过与类固醇受体共激活物(SRCs)的p160家族合作而发挥辅助共激活剂的作用。这些发现证明了CCAR1是CAR的一个新的转录辅助因子，并为CAR介导的基因选择性反式激活的机制提供了洞察力。
The constitutive active/androstane receptor (CAR) controls genes involved in xenochemical metabolism. Although numerous cofactors have been reported to be involved in CAR-mediated transactivation, unknown and poorly defined proteins recruited by CAR have yet to be characterized. In this study, a novel CAR-interacting protein, cell cycle and apoptosis regulator 1 (CCAR1), was identified by coimmunoprecipitation analysis using human hepatocarcinoma HepG2 cells expressing FLAG epitope-tagged CAR. We demonstrated that CCAR1 can act as an enhancer-dependent coactivator of CAR. First, we showed that overexpression of CCAR1 enhanced CAR-induced reporter gene activity with triplicate consensus direct repeat 4 motif (DR4-Luc), xenobiotic-responsive enhancer module (XREM)-enhancer of CYP3A4 (XREM-Luc), and phenobarbital-responsive enhancer module of UDP-glucuronosyltransferases 1A1 (UGT1A1) (gtPBREM)-enhancer of UGT1A1 (gtPBREM-Luc)-driven reporter plasmids but not PBREM-enhancer of CYP2B6 (PBREM-Luc)driven reporter activity. Furthermore, we showed that knockdown of CCAR1 suppressed CAR-induced UGT1A1 mRNA expression but did not affect CAR-induced CYP2B6 mRNA expression in HepTFi/CAR and HepaRG cells. Moreover, CCAR1 could be recruited to the gtPBREM of the UGT1A1 enhancer by CAR but not to the PBREM of the CYP2B6 enhancer. Moreover, we showed that CCAR1 can act as a secondary coactivator by cooperating with the p160 family of steroid receptor coactivators (SRCs). These findings demonstrated CCAR1 to be a novel transcriptional cofactor for CAR and provided insight regarding the mechanism of CAR-mediated gene-selective transactivation.