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EpCAM Intracellular Domain Promotes Porcine Cell Reprogramming by Upregulation of Pluripotent Gene Expression via Beta-catenin Signaling

View Article: PubMed Central - PubMed

ABSTRACT

Previous study showed that expression of epithelial cell adhesion molecule (EpCAM) was significantly upregulated in porcine induced pluripotent stem cells (piPSCs). However, the regulatory mechanism and the downstream target genes of EpCAM were not well investigated. In this study, we found that EpCAM was undetectable in fibroblasts, but highly expressed in piPSCs. Promoter of EpCAM was upregulated by zygotic activated factors LIN28, and ESRRB, but repressed by maternal factors OCT4 and SOX2. Knocking down EpCAM by shRNA significantly reduced the pluripotent gene expression. Conversely, overexpression of EpCAM significantly increased the number of alkaline phosphatase positive colonies and elevated the expression of endogenous pluripotent genes. As a key surface-to-nucleus factor, EpCAM releases its intercellular domain (EpICD) by a two-step proteolytic processing sequentially. Blocking the proteolytic processing by inhibitors TAPI-1 and DAPT could reduce the intracellular level of EpICD and lower expressions of OCT4, SOX2, LIN28, and ESRRB. We noticed that increasing intracellular EpICD only was unable to improve activity of EpCAM targeted genes, but by blocking GSK-3 signaling and stabilizing beta-catenin signaling, EpICD could then significantly stimulate the promoter activity. These results showed that EpCAM intracellular domain required beta-catenin signaling to enhance porcine cell reprogramming.

No MeSH data available.


EpCAM expression in porcine tissues and iPSCs.(A) EpCAM (EPC) expression in porcine tissues and epithelial cell PK-15. GAPDH (GAP) was used as internal control. (B) Expression profile of vital genes for the pluripotency and three germ layers (upper), and MET/EMT markers (lower) in porcine somatic and pluripotent cells. (C) Expression profile of EpCAM and pluripotent genes in porcine oocytes and early stage embryos maturated in vivo and in vitro. (D) Immunofluorescences of EpCAM (green) in piPS, PK-15, and PEF cells. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 μm.
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f1: EpCAM expression in porcine tissues and iPSCs.(A) EpCAM (EPC) expression in porcine tissues and epithelial cell PK-15. GAPDH (GAP) was used as internal control. (B) Expression profile of vital genes for the pluripotency and three germ layers (upper), and MET/EMT markers (lower) in porcine somatic and pluripotent cells. (C) Expression profile of EpCAM and pluripotent genes in porcine oocytes and early stage embryos maturated in vivo and in vitro. (D) Immunofluorescences of EpCAM (green) in piPS, PK-15, and PEF cells. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 μm.

Mentions: The expression profile of EpCAM in porcine tissues from newborn piglet was conducted by RT-PCR analysis. As described previously2526, EpCAM is highly expressed in epithelial cells. In our study, EpCAM message was detectable in all tested samples, which may be due to the widespread epithelial cells in most of organs. In those epithelia enriched organs, for instance lung, kidney, and small intestine, EpCAM was relatively abundant than in other tissues (Fig. 1A). The heatmap of microarray data (note: NANOG and SOX2 genes were not included in the Affymetrix Pig GeneChipe13) of eight piPSC lines and two primary porcine skin fibroblasts showed that EpCAM and core pluripotent genes, such as OCT4, LIN28, UTF1, and ZFP42, were highly expressed, and the expression of lineage-specific genes were downregulated (Fig. 1B), suggesting that EpCAM might play an important role during porcine cell reprogramming. Additionally, the expression level of EpCAM and many epithelial genes were clearly higher in piPSCs than in fibroblasts, and the expression level of mesenchymal markers was clearly downregulated in piPSCs (Fig. 1B). These results indicated that a mesenchymal to epithelial transition was occurred during the porcine cell reprogramming, which was an important event during cell reprogramming as reported in mouse and human iPSCs2728.


EpCAM Intracellular Domain Promotes Porcine Cell Reprogramming by Upregulation of Pluripotent Gene Expression via Beta-catenin Signaling
EpCAM expression in porcine tissues and iPSCs.(A) EpCAM (EPC) expression in porcine tissues and epithelial cell PK-15. GAPDH (GAP) was used as internal control. (B) Expression profile of vital genes for the pluripotency and three germ layers (upper), and MET/EMT markers (lower) in porcine somatic and pluripotent cells. (C) Expression profile of EpCAM and pluripotent genes in porcine oocytes and early stage embryos maturated in vivo and in vitro. (D) Immunofluorescences of EpCAM (green) in piPS, PK-15, and PEF cells. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5385527&req=5

f1: EpCAM expression in porcine tissues and iPSCs.(A) EpCAM (EPC) expression in porcine tissues and epithelial cell PK-15. GAPDH (GAP) was used as internal control. (B) Expression profile of vital genes for the pluripotency and three germ layers (upper), and MET/EMT markers (lower) in porcine somatic and pluripotent cells. (C) Expression profile of EpCAM and pluripotent genes in porcine oocytes and early stage embryos maturated in vivo and in vitro. (D) Immunofluorescences of EpCAM (green) in piPS, PK-15, and PEF cells. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 μm.
Mentions: The expression profile of EpCAM in porcine tissues from newborn piglet was conducted by RT-PCR analysis. As described previously2526, EpCAM is highly expressed in epithelial cells. In our study, EpCAM message was detectable in all tested samples, which may be due to the widespread epithelial cells in most of organs. In those epithelia enriched organs, for instance lung, kidney, and small intestine, EpCAM was relatively abundant than in other tissues (Fig. 1A). The heatmap of microarray data (note: NANOG and SOX2 genes were not included in the Affymetrix Pig GeneChipe13) of eight piPSC lines and two primary porcine skin fibroblasts showed that EpCAM and core pluripotent genes, such as OCT4, LIN28, UTF1, and ZFP42, were highly expressed, and the expression of lineage-specific genes were downregulated (Fig. 1B), suggesting that EpCAM might play an important role during porcine cell reprogramming. Additionally, the expression level of EpCAM and many epithelial genes were clearly higher in piPSCs than in fibroblasts, and the expression level of mesenchymal markers was clearly downregulated in piPSCs (Fig. 1B). These results indicated that a mesenchymal to epithelial transition was occurred during the porcine cell reprogramming, which was an important event during cell reprogramming as reported in mouse and human iPSCs2728.

View Article: PubMed Central - PubMed

ABSTRACT

Previous study showed that expression of epithelial cell adhesion molecule (EpCAM) was significantly upregulated in porcine induced pluripotent stem cells (piPSCs). However, the regulatory mechanism and the downstream target genes of EpCAM were not well investigated. In this study, we found that EpCAM was undetectable in fibroblasts, but highly expressed in piPSCs. Promoter of EpCAM was upregulated by zygotic activated factors LIN28, and ESRRB, but repressed by maternal factors OCT4 and SOX2. Knocking down EpCAM by shRNA significantly reduced the pluripotent gene expression. Conversely, overexpression of EpCAM significantly increased the number of alkaline phosphatase positive colonies and elevated the expression of endogenous pluripotent genes. As a key surface-to-nucleus factor, EpCAM releases its intercellular domain (EpICD) by a two-step proteolytic processing sequentially. Blocking the proteolytic processing by inhibitors TAPI-1 and DAPT could reduce the intracellular level of EpICD and lower expressions of OCT4, SOX2, LIN28, and ESRRB. We noticed that increasing intracellular EpICD only was unable to improve activity of EpCAM targeted genes, but by blocking GSK-3 signaling and stabilizing beta-catenin signaling, EpICD could then significantly stimulate the promoter activity. These results showed that EpCAM intracellular domain required beta-catenin signaling to enhance porcine cell reprogramming.

No MeSH data available.