Hoekman M F, Rientjes J M, Twisk J, Planta R J, Princen H M, Mager W H
Department of Biochemistry and Molecular Biology, Vrije Universiteit, Amsterdam, Netherlands.
Gene. 1993 Aug 25;130(2):217-23. doi: 10.1016/0378-1119(93)90422-y.
The cytochrome P450 enzyme, cholesterol 7 alpha-hydroxylase (CYP7A), catalyses the first and rate-limiting step in the conversion of cholesterol to bile acids. Expression of the CYP7A gene is under complex physiological control, encompassing amongst others a feedback down-regulation by bile acids. Using the CYP7A cDNA of the rat as a probe, we isolated a rat genomic clone containing the 5' part of the gene, including approximately 3.6 kb of upstream sequences. Sequence analysis revealed the presence of several putative regulatory elements. Transient expression analyses of transfected primary hepatocytes demonstrated that the major transcription-activating region is located in the proximal 145 nucleotide (nt). Upon addition of taurocholate to the culture, a significant reduction of the transcriptional activity was observed, suggesting the presence of a bile acid-responsive element in the proximal region of the CYP7A promoter. In addition, evidence was obtained for the presence of a thyroxine-responsive site further upstream. After addition of taurocholate, steady-state CYP7A mRNA levels, as judged by Northern analysis of hepatocyte RNA, are eightfold reduced. On the other hand, the transcriptional activity of CYP7A, as shown both in CAT assays and run-on experiments, revealed only a threefold decrease. These experiments suggest that both transcriptional control and regulation of CYP7A mRNA stability play an important part in the feedback regulation of CYP7A activity in the rat.
细胞色素P450酶，胆固醇7α-羟化酶（CYP7A），催化胆固醇转化为胆汁酸过程中的第一步且是限速步骤。CYP7A基因的表达受复杂的生理控制，其中包括胆汁酸的反馈下调。我们以大鼠的CYP7A cDNA为探针，分离出一个大鼠基因组克隆，该克隆包含该基因的5'部分，包括约3.6 kb的上游序列。序列分析显示存在几个推定的调控元件。对转染的原代肝细胞进行的瞬时表达分析表明，主要的转录激活区域位于近端145个核苷酸（nt）处。在培养物中添加牛磺胆酸盐后，观察到转录活性显著降低，这表明在CYP7A启动子的近端区域存在胆汁酸反应元件。此外，还获得了在更上游存在甲状腺素反应位点的证据。添加牛磺胆酸盐后，通过对肝细胞RNA进行Northern分析判断，稳态CYP7A mRNA水平降低了八倍。另一方面，如在CAT分析和连续转录实验中所示，CYP7A的转录活性仅降低了三倍。这些实验表明，转录控制和CYP7A mRNA稳定性的调节在大鼠CYP7A活性的反馈调节中都起着重要作用。
