Inokuchi A, Hinoshita E, Iwamoto Y, Kohno K, Kuwano M, Uchiumi T
Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
J Biol Chem. 2001 Dec 14;276(50):46822-9. doi: 10.1074/jbc.M104612200. Epub 2001 Oct 4.
The enterohepatic circulation is essential for the maintenance of bile acids and cholesterol homeostasis. The ileal bile acid transporter on the apical membrane of enterocytes mediates the intestinal uptake of bile salts, but little is known about the bile salt secretion from the basolateral membrane of enterocytes into blood. In the basolateral membrane of enterocytes, an ATP-binding cassette transporter, multidrug resistance protein 3 (MRP3), is expressed, which has the ability to transport bile salts. We hypothesized that MRP3 might play a role in the enterohepatic circulation of bile salts by transporting them from enterocytes into circulating blood through the up-regulation of MRP3 expression, so we investigated the transcriptional control of MRP3 in response to bile salts. MRP3 mRNA levels were increased about 3-fold in human colon cells by chenodeoxycholic acid (CDCA), in a dose- and time-dependent manner. In the promoter assay, the promoter activity of MRP3 was increased about 3-fold over the basal promoter activity when treated with CDCA, and the putative bile salt-responsive elements exist in the region -229/-138 including two alpha-1 fetoprotein transcription factor (FTF)-like elements. Constructs with a specific mutation in the consensus sequence of FTF elements showed no increase in basal transcriptional activity following CDCA treatment. In electrophoretic mobility shift assay with nuclear extracts, specific binding of FTF to FTF-like elements was observed when treated with CDCA. The expression of FTF mRNA levels were also markedly enhanced in response to CDCA, and overexpression of FTF specifically activated the MRP3 promoter activity about 4-fold over the basal promoter activity. FTF thus might play a key role not only in the bile salt synthetic pathway in hepatocytes but also in the bile salt excretion pathway in enterocytes through the regulation of MRP3 expression. MRP3 may contribute as a plausible bile salt-exporting transporter to the enterohepatic circulation of bile salts.
肠肝循环对于维持胆汁酸和胆固醇的稳态至关重要。肠上皮细胞顶端膜上的回肠胆汁酸转运体介导胆汁盐的肠道摄取,但关于胆汁盐从肠上皮细胞基底外侧膜分泌到血液中的情况却知之甚少。在肠上皮细胞的基底外侧膜中,表达了一种ATP结合盒转运体,即多药耐药蛋白3(MRP3),它具有转运胆汁盐的能力。我们推测MRP3可能通过上调MRP3的表达将胆汁盐从肠上皮细胞转运到循环血液中,从而在胆汁盐的肠肝循环中发挥作用,因此我们研究了MRP3对胆汁盐的转录调控。鹅去氧胆酸(CDCA)可使人类结肠细胞中的MRP3 mRNA水平以剂量和时间依赖性方式增加约3倍。在启动子分析中,用CDCA处理时,MRP3的启动子活性比基础启动子活性增加了约3倍,并且在-229/-138区域存在假定的胆汁盐反应元件,其中包括两个α-1甲胎蛋白转录因子(FTF)样元件。FTF元件共有序列发生特定突变的构建体在CDCA处理后基础转录活性没有增加。在对核提取物进行的电泳迁移率变动分析中,用CDCA处理时观察到FTF与FTF样元件的特异性结合。FTF mRNA水平的表达也因CDCA而显著增强,并且FTF的过表达使MRP3启动子活性比基础启动子活性特异性激活约4倍。因此,FTF不仅可能在肝细胞中的胆汁盐合成途径中起关键作用,还可能通过调节MRP3的表达在肠上皮细胞中的胆汁盐排泄途径中起关键作用。MRP3可能作为一种合理的胆汁盐输出转运体对胆汁盐的肠肝循环起作用。