Paquet Y, Trottier E, Beaudet M J, Anderson A
Centre de recherche en cancérologie de l'Université Laval, L'H otel-Dieu de Québec, Centre hospitalier universitaire de Québec, Québec G1R 2J6, Canada.
J Biol Chem. 2000 Dec 8;275(49):38427-36. doi: 10.1074/jbc.M005776200.
A 163-base pair enhancer in the CYP2B2 5' flank confers phenobarbital (PB) inducibility and constitutes a PB response unit (PBRU). By transfection of primary hepatocytes, we analyzed the function of elements comprising the PBRU and evaluated the role of the constitutive androstane receptor (CAR) in PB responsiveness. A 51-base pair PB-responsive enhancer module (PBREM) within the PBRU confers near-maximal PB response when fused to a tk promoter. However, replacing the PBRU with the PBREM in the CYP2B2 5' flank in the natural sequence context reduced PB responsiveness by approximately 4-fold. Mutational analysis also demonstrated that PBRU sequence elements outside the PBREM are essential for maximal PB responsiveness. The PBRU contains two putative nuclear receptor binding sites, NR1 and NR2. CAR binds to retinoic acid beta2 response elements (betaRARE) and to the NR1 and NR2 sites of the PBRU and activates transcription of reporter genes in cell lines. However, conversion of NR1 into betaRARE was the equivalent of an inactivating mutation, indicating that CAR does not activate PB-dependent transcription via NR1 in the natural sequence context. A betaRAREx2-tk reporter construct was inducible by all-trans-retinoic acid (at-RA) as expected and also responded to PB. The latter can be attributed to nuclear accumulation of CAR after PB exposure. Exogenous CAR increased both the basal and PB-induced response of betaRAREx2-tk but reduced PBRU-dependent PB response. Furthermore, exogenous CAR also reduced the at-RA response of the betaRAREx2-tk construct. Thus, CAR acts negatively on PB responsiveness mediated by the CYP2B2 PBRU just as it prevents maximal at-RA responsiveness mediated by betaRARE.
CYP2B2 5'侧翼区一个163个碱基对的增强子赋予苯巴比妥（PB）诱导性，并构成一个PB反应单元（PBRU）。通过原代肝细胞转染，我们分析了构成PBRU的元件的功能，并评估了组成型雄烷受体（CAR）在PB反应性中的作用。PBRU内一个51个碱基对的PB反应性增强子模块（PBREM）与tk启动子融合时赋予接近最大的PB反应。然而，在天然序列背景下，用PBREM替换CYP2B2 5'侧翼区的PBRU会使PB反应性降低约4倍。突变分析还表明，PBREM之外的PBRU序列元件对于最大PB反应性至关重要。PBRU包含两个假定的核受体结合位点，NR1和NR2。CAR与视黄酸β2反应元件（βRARE）以及PBRU的NR1和NR2位点结合，并激活细胞系中报告基因的转录。然而，将NR1转化为βRARE相当于一个失活突变，表明在天然序列背景下，CAR不会通过NR1激活PB依赖性转录。一个βRAREx2-tk报告构建体如预期那样可被全反式维甲酸（at-RA）诱导，并且也对PB有反应。后者可归因于PB暴露后CAR的核积累。外源性CAR增加了βRAREx2-tk的基础反应和PB诱导反应，但降低了PBRU依赖性PB反应。此外，外源性CAR还降低了βRAREx2-tk构建体的at-RA反应。因此，CAR对由CYP2B2 PBRU介导的PB反应性起负作用，就如同它阻止由βRARE介导的最大at-RA反应性一样。
