人CYP3A5基因中一种新的地塞米松反应增强子的鉴定及其在人和大鼠肝细胞中的激活。
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Schuetz JD, Schuetz EG, Thottassery JV, Guzelian PS, Strom S，孙D
人CYP3A5基因中一种新的地塞米松反应增强子的鉴定及其在人和大鼠肝细胞中的激活。
Mol Pharmacol. 1996 Jan;49(1):63-72。
- PubMed ID
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8569713 (查看PubMed］
- 摘要
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人肝细胞色素p4503a (CYP3As)，与大鼠糖皮质激素诱导形式同源，由至少4个差异表达成员组成。为了开始研究CYP3A5在糖皮质激素调控中的分子事件，我们将CYP3A5的5'序列与含有单纯疱疹病毒胸苷激酶启动子的载体中的氯霉素乙酰转移酶基因融合。在HepG2细胞中，最大的5' CYP3A5基因片段(1.4 kb)抑制TK启动子。然而，添加10 μ m地塞米松后，抑制作用被克服。一系列的单向缺失发现了一个独特的219 bp片段(从转录起始位点上游-891到-1109 bp)，无论与启动子的距离或方向如何，它都使TK启动子具有地塞米松响应性，因此它似乎是一个增强子。该CYP3A5增强子的核苷酸序列分析显示没有一致的15 bp糖皮质激素响应元件(GRE) (GGTACANNNTGTTCT);然而，发现两个GRE“半位点”(TGTTCT)相距160 bp。尽管地塞米松对HepG2细胞中CYP3A5增强子的刺激仅为3-4倍，但在永生的原代人肝细胞和原代大鼠肝细胞培养物中，CYP3A5增强子的刺激分别为7-和12倍。糖皮质激素受体(GCR)似乎在这一过程中不可或缺，因为1)地塞米松诱导可被抗糖皮质激素RU-486阻断，2)HepG2细胞中依赖地塞米松的CYP3A5增强子转录激活需要共转染含有完整GCR的表达载体，然而3)在地塞米松存在的情况下，与含有GCR配体结合域突变的质粒共转染不会激活CYP3A5增强子。 To further localize the dexamethasone responsive region of the 219-bp CYP3A5 enhancer, it was subdivided and fused to the TKCAT expression vector. Transfection analysis in HepG2 cells demonstrated that neither GRE half-site can independently confer dexamethasone responsiveness on the TK promoter. Block mutations of either of the two GRE half-sites or point mutations at specific GCR binding sites eliminates dexamethasone inducibility, demonstrating the half-sites need to interact. Electromobility shift assays indicate that the CYP3A5 5'-GRE half-site 1) specifically binds purified GCR, 2) can displace binding of the GCR to a consensus GRE, and 3) shifts a protein in HepG2 nuclear extracts that is supershifted by GCR antibody, demonstrating that this enhancer is an authentic GRE. This is the first study to demonstrate that a member of the human CYP3A gene family contains an enhancer that binds the GCR and that this binding is critical to transcriptional activation by dexamethasone.