Zhang M, Chiang J Y
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272, USA.
J Biol Chem. 2001 Nov 9;276(45):41690-9. doi: 10.1074/jbc.M105117200. Epub 2001 Sep 4.
Sterol 12alpha-hydroxylase catalyzes the synthesis of cholic acid and controls the ratio of cholic acid over chenodeoxycholic acid in the bile. Transcription of CYP8B1 is inhibited by bile acids, cholesterol, and insulin. To study the mechanism of CYP8B1 transcription by bile acids, we have cloned and determined 3389 base pairs of the 5'-upstream nucleotide sequences of the human CYP8B1. Deletion analysis of CYP8B1/luciferase reporter activity in HepG2 cells revealed that the sequences from -57 to +300 were important for basal and liver-specific promoter activities. Hepatocyte nuclear factor 4alpha (HNF4alpha) strongly activated human CYP8B1 promoter activities, whereas cholesterol 7alpha-hydroxylase promoter factor (CPF), an NR5A2 family of nuclear receptors, had much less effect. Electrophoretic mobility shift assay identified an overlapping HNF4alpha- and CPF-binding site in the +198/+227 region. The human CYP8B1 promoter activities were strongly repressed by bile acids, and the bile acid response element was localized between +137 and +220. Site-directed mutagenesis of the HNF4alpha-binding site markedly reduced promoter activity and its response to bile acid repression. On the other hand, mutation of the CPF-binding site had little effect on promoter activity and bile acid inhibition. A negative nuclear receptor, small heterodimer partner markedly inhibited transactivation of CYP8B1 by HNF4alpha. Mammalian two-hybrid assay confirmed that HNF4alpha interacted with small heterodimer partner. Furthermore, bile acids and farnesoid X receptor reduced the expression of nuclear HNF4alpha in HepG2 cells and rat livers and its binding to DNA. Bile acids and farnesoid X receptor also inhibited mouse HNF4alpha gene transcription. In summary, our data revealed the critical roles HNF4alpha play on CYP8B1 transcription and its repression by bile acids. Bile acids repress human CYP8B1 transcription by reducing the transactivation activity of HNF4alpha through interaction of HNF4alpha with SHP and reduction of HNF4alpha expression in the liver.
甾醇12α-羟化酶催化胆酸的合成，并控制胆汁中胆酸与鹅去氧胆酸的比例。CYP8B1的转录受到胆汁酸、胆固醇和胰岛素的抑制。为了研究胆汁酸对CYP8B1转录的机制，我们克隆并测定了人类CYP8B1 5'-上游核苷酸序列的3389个碱基对。对HepG2细胞中CYP8B1/荧光素酶报告基因活性的缺失分析表明，从-57到+300的序列对基础和肝脏特异性启动子活性很重要。肝细胞核因子4α（HNF4α）强烈激活人类CYP8B1启动子活性，而胆固醇7α-羟化酶启动子因子（CPF），一种NR5A2家族的核受体，作用则小得多。电泳迁移率变动分析在+198/+227区域鉴定出一个重叠的HNF4α和CPF结合位点。人类CYP8B1启动子活性受到胆汁酸的强烈抑制，胆汁酸反应元件定位于+137和+220之间。HNF4α结合位点的定点诱变显著降低了启动子活性及其对胆汁酸抑制的反应。另一方面，CPF结合位点的突变对启动子活性和胆汁酸抑制作用影响很小。一种负性核受体，小异源二聚体伴侣显著抑制HNF4α对CYP8B1的反式激活。哺乳动物双杂交试验证实HNF4α与小异源二聚体伴侣相互作用。此外，胆汁酸和法尼酯X受体降低了HepG2细胞和大鼠肝脏中核HNF4α的表达及其与DNA的结合。胆汁酸和法尼酯X受体也抑制小鼠HNF4α基因转录。总之，我们的数据揭示了HNF4α在CYP8B1转录及其受胆汁酸抑制方面的关键作用。胆汁酸通过HNF4α与小异源二聚体伴侣的相互作用降低HNF4α的反式激活活性并减少肝脏中HNF4α的表达，从而抑制人类CYP8B1转录。
