Wang D P, Stroup D, Marrapodi M, Crestani M, Galli G, Chiang J Y
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown 44272, USA.
J Lipid Res. 1996 Sep;37(9):1831-41.
A stable HepG2 cell line harboring a human cholesterol 7 alpha-hydroxylase (CYP7A) minigene/luciferase reporter gene construct was selected for studying transcriptional regulation of CYP7A gene promoter. Insulin and phorbol 12-myristate-13-acetate (PMA) strongly repressed the promoter activity as measured with luciferase activity expressed in the cells. The promoter activity of the 5' progressive deletion/luciferase reporter gene constructs was studied in a transient transfection assay in HepG2 cells. PMA represses the promoter activity and the response elements were localized in the -184/-151 and -134/-81 regions. Insulin also represses the promoter activity and response element was mapped in the -298/-81 region. Surprisingly, glucocorticoid receptor (GR) strongly inhibited promoter activity in the presence of dexamethasone, and response elements were localized in the -298/-151 and the -150/+24 regions. Thyroid hormone receptor also repressed promoter activity and response elements were localized in the -150/+24 and upstream regions. Cotransfection of CYP7A chimeric constructs with an expression vector carrying liver-enriched transcription factor HNF3 alpha stimulated the reporter gene activity, but cotransfection with GR plasmid interfered with the HNF3 alpha-stimulated activity possibly through competition for binding to overlapping GR/HNF3 binding sites. Thus, human cholesterol 7 alpha-hydroxylase gene promoter is strongly repressed by insulin, PMA, and steroid/thyroid hormones and results in the low level of cholesterol 7 alpha-hydroxylase expression in the human liver.
为了研究胆固醇7α-羟化酶（CYP7A）基因启动子的转录调控，我们筛选出了一个稳定转染人CYP7A小基因/荧光素酶报告基因构建体的HepG2细胞系。通过检测细胞中表达的荧光素酶活性发现，胰岛素和佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）强烈抑制启动子活性。我们在HepG2细胞的瞬时转染实验中研究了5'端逐步缺失/荧光素酶报告基因构建体的启动子活性。PMA抑制启动子活性，其反应元件定位于-184/-151和-134/-81区域。胰岛素也抑制启动子活性，反应元件定位于-298/-81区域。令人惊讶的是，在存在地塞米松的情况下，糖皮质激素受体（GR）强烈抑制启动子活性，反应元件定位于-298/-151和-150/+24区域。甲状腺激素受体也抑制启动子活性，反应元件定位于-150/+24区域及上游区域。将CYP7A嵌合构建体与携带肝脏富集转录因子HNF3α的表达载体共转染可刺激报告基因活性，但与GR质粒共转染可能通过竞争重叠的GR/HNF3结合位点干扰HNF3α刺激的活性。因此，人胆固醇7α-羟化酶基因启动子受到胰岛素、PMA以及类固醇/甲状腺激素的强烈抑制，导致人肝脏中胆固醇7α-羟化酶表达水平较低。
