Hashimoto Tetsuya, Nakamura Takaaki, Maegawa Hiroshi, Nishio Yoshihiko, Egawa Katsuya, Kashiwagi Atsunori
Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Otsu, Japan.
J Biol Chem. 2005 Jan 21;280(3):1893-900. doi: 10.1074/jbc.M410759200. Epub 2004 Nov 4.
We have reported that the combined expression of Pdx-1 (pancreatic duodenal homeobox 1) and Isl-1 (islet 1) enables immature rat enterocytes (IEC-6) to produce and release insulin. A key component regulating the release of insulin is the ATP-sensitive potassium channel subunit Kir6.2. To investigate the regulation of Kir6.2 gene expression, we assessed Kir6.2 expression in IEC-6 cells expressing Pdx-1 and/or Isl-1. We observed that Kir6.2 protein was expressed de novo in IEC-6 cells expressing both Pdx-1 and Isl-1 but not in cells expressing Pdx-1 alone. Next, we analyzed the regions of the Kir6.2 promoter (-1677/-45) by performing a luciferase assay and electrophoretic mobility shift assay. The results have demonstrated that Kir6.2 promoter possesses two regions regulating the promoter activity: a Foxa2-binding site (-1364 to -1210) and an Sp1/Sp3-binding site (-1035 to -939). The additional expression of Isl-1 in IEC-6 cells expressing Pdx-1 attenuated overexpression of Foxa2 protein and enhanced Kir6.2 expression. Finally, knockdown of Isl-1 using the iRNA technique resulted in decreased expression of Kir6.2 protein in a rat pancreatic beta-cell line (RIN-5F cells). These results indicate that expression of Kir6.2 in the rat intestine is moderated by Isl-1.
我们曾报道，胰腺十二指肠同源盒基因1（Pdx-1）和胰岛1（Isl-1）的联合表达可使未成熟的大鼠肠上皮细胞（IEC-6）产生并释放胰岛素。调节胰岛素释放的一个关键成分是ATP敏感性钾通道亚基Kir6.2。为了研究Kir6.2基因表达的调控机制，我们评估了在表达Pdx-1和/或Isl-1的IEC-6细胞中Kir6.2的表达情况。我们观察到，Kir6.2蛋白在同时表达Pdx-1和Isl-1的IEC-6细胞中从头表达，而在仅表达Pdx-1的细胞中不表达。接下来，我们通过进行荧光素酶报告基因检测和电泳迁移率变动分析，对Kir6.2启动子区域（-1677/-45）进行了分析。结果表明，Kir6.2启动子具有两个调节启动子活性的区域：一个叉头框蛋白A2（Foxa2）结合位点（-1364至-1210）和一个特异性蛋白1/特异性蛋白3（Sp1/Sp3）结合位点（-1035至-939）。在表达Pdx-1的IEC-6细胞中额外表达Isl-1可减弱Foxa2蛋白的过表达并增强Kir6.2的表达。最后，使用RNA干扰技术敲低Isl-1导致大鼠胰腺β细胞系（RIN-5F细胞）中Kir6.2蛋白的表达降低。这些结果表明，大鼠肠道中Kir6.2的表达受Isl-1调控。
