Quattrochi L C, Yockey C B, Barwick J L, Guzelian P S
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262, USA.
Arch Biochem Biophys. 1998 Jan 15;349(2):251-60. doi: 10.1006/abbi.1997.0467.
Cytochrome P450 (CYP) 3A23 is transcriptionally regulated in rat liver by such glucocorticoids as dexamethasone (DEX) and by such antiglucocorticoids as pregnenolone 16 alpha-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. Quattrochi et al., J. Biol. Chem. 270, 28,917, 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.
细胞色素P450（CYP）3A23在大鼠肝脏中受地塞米松（DEX）等糖皮质激素以及孕烯醇酮16α-腈（PCN）等抗糖皮质激素的转录调控。基于对成年大鼠肝细胞原代培养物中表达的CYP3A23基因片段进行的DNA-蛋白质相互作用研究，我们提出这些甾体激素诱导CYP3A23的机制涉及糖皮质激素受体或由糖皮质激素间接诱导的一种蛋白质，该蛋白质与组成型结合于CYP3A23基因5'-侧翼区由两个核苷酸分隔的7碱基对直接重复序列组成的增强子元件的蛋白质相互作用（L. Quattrochi等人，《生物化学杂志》270, 28917, 1995）。在本研究中，我们制备了包含该直接重复序列或其各种突变体的20碱基对双链（ds）寡核苷酸，并在培养的大鼠肝细胞中瞬时表达，这些寡核苷酸插入到氯霉素乙酰转移酶（CAT）报告质粒中。我们发现两个重复序列对于DEX或PCN诱导CAT都是必需的。通过紫外线交联分析与CYP3A23增强子结合的蛋白质，发现了两种分子量约为130和100 kDa的大鼠肝脏核蛋白，以及几种分子量在45至60 kDa之间的蛋白质，它们特异性结合20碱基对ds寡核苷酸CYP3A23增强子。甲基化干扰试验确定该寡核苷酸直接重复序列内的所有鸟嘌呤残基对于蛋白质结合都很重要。这些鸟嘌呤残基的突变消除了核蛋白的结合并消除了CAT的DEX或PCN诱导性。这些数据表明，组成型结合的蛋白质可能作为异二聚体复合物与CYP3A23增强子相互作用，在CYP3A23的糖皮质激素诱导中发挥作用。
