Department of Medical Research, National Taiwan University Hospital, Taipei 100, Taiwan.
J Biol Chem. 2011 Sep 23;286(38):33520-32. doi: 10.1074/jbc.M111.256164. Epub 2011 Jul 28.
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.
上皮细胞黏附分子 (EpCAM) 是一种跨膜糖蛋白，在胚胎干细胞 (ESCs) 中高度表达，其在维持多能性中的作用先前已被提出。在上皮癌细胞中，EpCAM 表面到核信号转导途径的激活涉及许多膜蛋白。然而，它们在体细胞重编程中的作用仍然未知。在这里，我们证明 EpCAM 及其相关蛋白 Cldn7 在重编程中起着关键作用。定量 RT-PCR 分析 Oct4、Sox2、Klf4 和 c-Myc (OSKM) 感染的小鼠胚胎成纤维细胞 (MEFs) 表明，EpCAM 和 Cldn7 在重编程过程中上调。碱性磷酸酶和 Nanog 阳性克隆数量分析以及多能性相关基因的表达水平表明，抑制 EpCAM 或 Cldn7 的表达均可导致重编程效率受损，而 EpCAM、EpCAM 加 Cldn7 或 EpCAM 细胞间结构域 (EpICD) 的过表达则显著增强 MEFs 中的重编程效率。此外，EpCAM 或 EpICD 的过表达显著抑制了重编程 MEFs 中 p53 和 p21 的表达，EpCAM 和 EpICD 均激活了 Oct4 的启动子活性。这些观察结果表明，EpCAM 信号可能通过上调 Oct4 并可能抑制 p53-p21 途径来增强重编程。体内和体外表征表明，EpCAM 重编程的 iPSCs 表现出与小鼠 ESCs 相似的分子和功能特征。总之，我们的研究为重编程的分子机制提供了更多的见解，并提出了一种更有效的诱导多能干细胞生成方法。
