Characterization of DNA-binding proteins required for glucocorticoid induction of CYP3A23.
Characterization of DNA-binding proteins required for glucocorticoid induction of CYP3A23.
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糖皮质激素诱导 CYP3A23 所需的 DNA 结合蛋白的表征。
DOI：
10.1006/abbi.1997.0467
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发表时间：
1998
影响因子：
3.9
通讯作者：
Guzelian,PS
中科院分区：
文献类型：
--
作者：
Quattrochi,LC;Yockey,CB;Barwick,JL;Guzelian,PS
细胞色素P450（CYP）3A 23在大鼠肝脏中受糖皮质激素如地塞米松（DEX）和抗糖皮质激素如双烯醇酮16α-甲腈（PCN）的转录调节。基于对成年大鼠肝细胞原代培养物中表达的CYP 3A 23基因片段的研究以及对DNA-蛋白质相互作用的检测，我们已经提出，这些类固醇激素诱导CYP 3A 23的机制涉及糖皮质激素受体或糖皮质激素诱导的蛋白质与组成性结合到增强子元件的蛋白质间接相互作用，所述增强子元件由7-在CYP 3A 23基因的5′-侧翼区（L. Quattrochie等人，J. Biol. Chem.270，28917，1995）。在本研究中，我们制备并瞬时表达在培养的大鼠肝细胞20-bp的双链（DS）-寡核苷酸含有这个直接重复或插入到氯霉素乙酰转移酶（CAT）报告质粒的这种直接重复的各种突变。我们发现，这两个重复是必要的诱导CAT的DEX或PCN。通过使用UV交联分析与CYP 3A 23增强子结合的蛋白质，发现分子量约为130和100 kDa的两种大鼠肝核蛋白，以及分子量在45和60 kDa之间的几种蛋白质，其特异性结合20-bp双寡核苷酸CYP 3A 23增强子。甲基化干扰测定确定，该寡核苷酸的直接重复序列内的所有鸟嘌呤残基对于蛋白质结合都是重要的。这些鸟嘌呤残基的突变废除核蛋白的结合，消除DEX或PCN诱导的CAT。这些数据表明，组成性结合的蛋白质，与CYP 3A 23增强子可能作为一个异二聚体复合物相互作用，发挥作用，在糖皮质激素诱导的CYP 3A 23。
Cytochrome P450 (CYP) 3A23 is transcriptionally regulated in rat liver by such glucocorticoids as dexamethasone (DEX) and by such antiglucocorticoids as pregnenolone 16α-carbonitrile (PCN). Based on studies of CYP3A23 gene fragments expressed in primary cultures of adult rat hepatocytes and tested for DNA–protein interactions, we have proposed that the mechanism of CYP3A23 induction by these steroid hormones involves the glucocorticoid receptor or a protein induced by glucocorticoids indirectly interacting with proteins constitutively bound to an enhancer element consisting of a direct repeat of 7-bp separated by two nucleotides in the 5′-flanking region of the CYP3A23 gene (L. Quattrochiet al., J. Biol. Chem.270, 28917, 1995). In the present study, we prepared and transiently expressed in cultured rat hepatocytes 20-bp double-stranded (ds)-oligonucleotides containing this direct repeat or various mutations of this direct repeat inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid. We found that both repeats were necessary for induction of CAT by either DEX or PCN. Analysis of proteins bound to CYP3A23 enhancer through the use of uv cross-linking revealed two rat liver nuclear proteins with molecular masses of approximately 130 and 100 kDa, as well as several proteins of molecular masses between 45 and 60 kDa, that specifically bind to the 20-bp ds-oligonucleotide CYP3A23 enhancer. Methylation interference assays determined that all guanine residues within the direct repeats of this oligonucleotide are important for protein binding. Mutations of these guanine residues abolished binding of nuclear proteins and eliminated DEX or PCN inducibility of CAT. These data suggest that constitutively bound proteins, interacting with the CYP3A23 enhancer possibly as a heterodimeric complex, play a role in the glucocorticoid inducibility of CYP3A23.