Human CYP1B1 is regulated by estradiol via estrogen receptor
Human CYP1B1 is regulated by estradiol via estrogen receptor
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DOI：
10.1158/0008-5472.can-04-0166
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发表时间：
2004-05-01
期刊：
影响因子：
11.2
通讯作者：
Yokoi, T
中科院分区：
文献类型：
--
作者：
Tsuchiya, Y;Nakajima, N;Yokoi, T
人细胞色素P450（CYP）1111是17 β-雌二醇（E2）代谢的关键酶。CYP 1B 1主要表达于内分泌调节的组织，如乳腺、子宫和卵巢。由于许多CYP 1B 1酶可能是由底物本身诱导的，因此我们在本研究中检测了人CYP 1B 1表达是否受E2调节。实时荧光定量RT-PCR分析显示，10 nm E2处理12 h诱导雌激素受体（ER）阳性MCF-7细胞中CYP 1B 1 mRNA的表达。使用MCF-7细胞的荧光素酶报告基因测定显示，使用含有人CYP 1B 1基因-152至+25区域的报告基因质粒，E2可显著反式激活高达7倍。计算机辅助同源性检索表明，在CYP 1B 1启动子区-63和-49之间存在一个推定的雌激素反应元件（ERE）。通过染色质免疫沉淀试验和凝胶位移分析证明ER α与推定的ERE的特异性结合。使用含有CYP 1B 1基因上的野生型或突变型推定ERE的报告质粒和野生型或突变型ER α表达载体，使用石川细胞的荧光素酶测定证明推定ERE和ER α对于E2的反式激活是必需的。由于子宫内膜组织高度受雌激素的调节，CYP 1B 1蛋白在人子宫内膜标本中的表达模式通过免疫组化检测。CYP 1B 1的染色在腺上皮细胞增殖期比分泌期强，与雌激素分泌模式一致。这些发现清楚地表明，人CYP 1B 1是通过ER α受雌激素调节的。由于CYP 1B 1对雌激素的4-羟基化导致雌激素活性降低，但产生的代谢产物具有毒理学活性，因此我们的研究结果表明，雌激素调节的CYP 1B 1表达对雌激素的稳态以及雌激素依赖性致癌作用具有临床意义。
Human cytochrome P450 (CYP) 1111 is a key enzyme in the metabolism of 17beta-estradiol (E2). CYP1B1 is mainly expressed in endocrine-regulated tissues, such as mammary, uterus, and ovary. Because many CYP enzymes are likely to be induced by the substrates themselves, we examined whether the human CYP1B1 expression is regulated by E2 in the present study. Real-time reverse transcription-PCR analysis revealed that treatment with 10 nm E2 for 12 h induced CYP1B1 mRNA expression in estrogen receptor (ER)-positive MCF-7 cells. Luciferase reporter assays using MCF-7 cells showed a significant transactivation up to 7-fold by E2 with a reporter plasmid containing a region from -152 to +25 of the human CYP1B1 gene. A computer-assisted homology search indicated a putative estrogen response element (ERE) between -63 and -49 in the CYP1B1 promoter region. Specific binding of ERalpha to the putative ERE was demonstrated by chromatin immunoprecipitation assays and gel shift analyses. With reporter plasmids containing the wild or mutated putative ERE on the CYP1B1 gene and the wild or mutated ERa expression vectors, luciferase assays using Ishikawa cells demonstrated that the putative ERE and ERa are essential for the transactivation by E2. Because endometrial tissue is highly regulated by estrogens, the expression pattern of CYP1B1 protein in human endometrial specimens was examined by immunohistochemistry. The staining of CYP1B1 was stronger in glandular epithelial cells during a proliferative phase than those during a secretory phase, consistent with the pattern of estrogen secretion. These findings clearly indicated that the human CYP1B1 is regulated by estrogen via ERalpha. Because 4-hydroxylation of estrogen by CYP1B1 leads to decrease of the estrogenic activity but the produced metabolite is toxicologically active, our findings suggest a clinical significance in the estrogen-regulated CYP1B1 expression for the homeostasis of estrogens as well as estrogen-dependent carcinogenesis.