Hepatocyte nuclear factor-4α is a central transactivator of the mouse Ntcp gene
Hepatocyte nuclear factor-4α is a central transactivator of the mouse Ntcp gene
复制标题
DOI：
10.1152/ajpgi.00012.2008
复制
发表时间：
2008-08-01
影响因子：
4.5
通讯作者：
Ananthanarayanan, Meenakshisundaram
中科院分区：
文献类型：
--
作者：
Geier, Andreas;Martin, Ina V.;Ananthanarayanan, Meenakshisundaram
牛磺胆酸钠共转运多肽（Ntcp）是结合胆汁酸的主要摄取系统。删除肝细胞核因子 (HNF)-1 α 和类视黄醇 X 受体-α：小鼠 5' 侧翼区域中的视黄酸受体-α 结合位点（对应于大鼠 Ntcp 的假定中央调节元件）不会显着降低启动子活性。我们假设 HNF-4 α（越来越多地被认为是肝细胞功能的中央调节因子）可能会直接反式激活小鼠 (mNtcp)。克隆了包含小鼠 Ntcp 启动子的 1.1 kb 5'-上游区域，并与大鼠启动子进行了比较。与 HNF-1 α 对 mNtcp 的中等 3.5 倍激活相反，HNF-4 α 共转染导致 mNtcp 的强烈激活 20 倍。小鼠和大鼠 Ntcp 启动子的删除分析分别在 -345/-326 和 -335/-316 bp 处定位了保守的 HNF-4 α 共有位点。 p-475bpmNtcpLUC 不被 HNF-1 α 反式激活，但在与 HNF-4 α 共转染后显示出 50 倍的增强活性。凝胶迁移率变化分析表明，HNF-4 α 元素与肝核提取物形成的复合物被 HNF-4 α 特异性抗体阻断。通过染色质免疫沉淀证实 HNF-4 α 结合。使用 Hepa 1-6 细胞，HNF-4 α 敲低导致 NTCP mRNA 显着减少 95%。总之，小鼠 Ntcp 通过独立于 HNF-1 α 的保守远端顺式元件受到 HNF-4 α 的调节。
Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1 alpha and retinoid X receptor-alpha: retinoic acid receptor-alpha binding sites in the mouse 5'-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF-4 alpha, which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse (mNtcp). A 1.1-kb 5'-upstream region including the mouse Ntcp promoter was cloned and compared with the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF-1 alpha, HNF-4 alpha cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4 alpha consensus site at -345/-326 and -335/-316 bp, respectively. p-475bpmNtcpLUC is not transactivated by HNF-1 alpha but shows a 50-fold enhanced activity upon cotransfection with HNF-4 alpha. Gel mobility shift assays demonstrated a complex of the HNF-4 alpha-element formed with liver nuclear extracts that was blocked by an HNF-4 alpha specific antibody. HNF-4 alpha binding was confirmed by chromatin immunoprecipitation. Using Hepa 1-6 cells, HNF-4 alpha-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4 alpha via a conserved distal cis-element independently of HNF-1 alpha.