Abstract
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.
Highlights
Using RT-PCR and Q-RT-PCR, we showed that transcripts of Epithelial cell adhesion molecule (EpCAM), Cldn7 (CLDN7), Cd9 (CD9), Adam17 (ADAM17) and Psen2 (PSEN2), but not Cd44v6 (CD44v6) or Tspan8 (TSPAN8) were highly expressed in both mouse ESCs (mESCs) and human ESCs (hESCs)
These results demonstrated that EpCAM and its complex proteins, Cldn7 (CLDN7), Cd9 (CD9), Adam17 (ADAM17), and Psen2 (PSEN2), were co-expressed in the cell membrane of both mESCs and hESCs
A, Q-RT-PCR analysis showed that the expression level of p53 and p21 in mouse embryonic fibroblasts (MEFs) infected by lentivirus encoding EpCAM intercellular domain (EpICD) was significantly down-regulated (*, p Ͻ 0.05) compared with MEFs infected by lentivirus encoding control vector at day 6 of viral infection
Summary
EpCAM is highly expressed in ESCs. the role of EpCAM complex proteins in pluripotency reprogramming is still unknown. Results: Overexpression of EpCAM complex proteins significantly repressed the expression of p53 and enhanced reprogramming efficiency in MEFs. Conclusion: EpCAM signaling enhance reprogramming through suppression of the p53-p21 pathway. Activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. Their role in somatic cell reprogramming is still unknown. 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 Oct and possible suppression of the p53-p21 pathway.
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