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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 promoter activation in reprogrammed cells. (A) The qRT-PCR analysis of EpCAM expression during porcine fibroblast reprogramming by hOSKM for 14 days. (B) Transduction of pTRIP-EpCAM-EGFP and pTRIP-CAGG-EGFP (Ctrl) into PEF, PK-15, and piPS cells, respectively, to determine activation of the EpCAM promoter. Scale bar, 100 μm. (C) Luciferase assay. Reporter pGL3-EpCAM-Pro (Exp) and pGL3-Basic (Ctrl) were transfected into PEF and PK-15 cells, respectively, for 36 h. (D) Luciferase assay of EpCAM promoter activation regulated by human pluripotent factor alone (left), or combination (right) in 293 T cells. Ctrl, cells were transfected with pGL3-EpCAM-Pro and pMXs-EGFP. (E) Luciferase assay of EpCAM promoter activation that was regulated by porcine pluripotent factors. Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.
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f2: EpCAM promoter activation in reprogrammed cells. (A) The qRT-PCR analysis of EpCAM expression during porcine fibroblast reprogramming by hOSKM for 14 days. (B) Transduction of pTRIP-EpCAM-EGFP and pTRIP-CAGG-EGFP (Ctrl) into PEF, PK-15, and piPS cells, respectively, to determine activation of the EpCAM promoter. Scale bar, 100 μm. (C) Luciferase assay. Reporter pGL3-EpCAM-Pro (Exp) and pGL3-Basic (Ctrl) were transfected into PEF and PK-15 cells, respectively, for 36 h. (D) Luciferase assay of EpCAM promoter activation regulated by human pluripotent factor alone (left), or combination (right) in 293 T cells. Ctrl, cells were transfected with pGL3-EpCAM-Pro and pMXs-EGFP. (E) Luciferase assay of EpCAM promoter activation that was regulated by porcine pluripotent factors. Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.

Mentions: To further investigate the regulation of EpCAM in piPSCs, we detected the expression of EpCAM during porcine cell reprogramming. In the first four days post transfection of human OCT4, SOX2, KLF4, and c-MYC (hOSKM), the endogenous EpCAM was at very low level. After six days post transfection, EpCAM expression was significantly increased (Fig. 2A). To explore how EpCAM was activated, a 3.8 kb DNA fragment (GenBank accession number KY218795) that contains promoter region, 5′ UTR sequence, and ATG codon of EpCAM was cloned, confirmed by DNA sequencing, and subcloned into reporter vectors pTRIP-EGFP and pGL3-basic to form pTRIP-EpCAM-EGFP and pGL3-EpCAM-Pro constructs (Table S2). Results showed that a strong EGFP fluorescence was observed in PK-15 and piPSC cells, but the fluorescent signal was undetectable in control PEF cells (Fig. 2B). Luciferase assay showed that EpCAM promoter was significantly activated in epithelia PK-15 versus fibroblasts PEF (Fig. 2C), indicating the cell-specificity of cloned EpCAM promoter.


EpCAM Intracellular Domain Promotes Porcine Cell Reprogramming by Upregulation of Pluripotent Gene Expression via Beta-catenin Signaling
EpCAM promoter activation in reprogrammed cells. (A) The qRT-PCR analysis of EpCAM expression during porcine fibroblast reprogramming by hOSKM for 14 days. (B) Transduction of pTRIP-EpCAM-EGFP and pTRIP-CAGG-EGFP (Ctrl) into PEF, PK-15, and piPS cells, respectively, to determine activation of the EpCAM promoter. Scale bar, 100 μm. (C) Luciferase assay. Reporter pGL3-EpCAM-Pro (Exp) and pGL3-Basic (Ctrl) were transfected into PEF and PK-15 cells, respectively, for 36 h. (D) Luciferase assay of EpCAM promoter activation regulated by human pluripotent factor alone (left), or combination (right) in 293 T cells. Ctrl, cells were transfected with pGL3-EpCAM-Pro and pMXs-EGFP. (E) Luciferase assay of EpCAM promoter activation that was regulated by porcine pluripotent factors. Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385527&req=5

f2: EpCAM promoter activation in reprogrammed cells. (A) The qRT-PCR analysis of EpCAM expression during porcine fibroblast reprogramming by hOSKM for 14 days. (B) Transduction of pTRIP-EpCAM-EGFP and pTRIP-CAGG-EGFP (Ctrl) into PEF, PK-15, and piPS cells, respectively, to determine activation of the EpCAM promoter. Scale bar, 100 μm. (C) Luciferase assay. Reporter pGL3-EpCAM-Pro (Exp) and pGL3-Basic (Ctrl) were transfected into PEF and PK-15 cells, respectively, for 36 h. (D) Luciferase assay of EpCAM promoter activation regulated by human pluripotent factor alone (left), or combination (right) in 293 T cells. Ctrl, cells were transfected with pGL3-EpCAM-Pro and pMXs-EGFP. (E) Luciferase assay of EpCAM promoter activation that was regulated by porcine pluripotent factors. Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.
Mentions: To further investigate the regulation of EpCAM in piPSCs, we detected the expression of EpCAM during porcine cell reprogramming. In the first four days post transfection of human OCT4, SOX2, KLF4, and c-MYC (hOSKM), the endogenous EpCAM was at very low level. After six days post transfection, EpCAM expression was significantly increased (Fig. 2A). To explore how EpCAM was activated, a 3.8 kb DNA fragment (GenBank accession number KY218795) that contains promoter region, 5′ UTR sequence, and ATG codon of EpCAM was cloned, confirmed by DNA sequencing, and subcloned into reporter vectors pTRIP-EGFP and pGL3-basic to form pTRIP-EpCAM-EGFP and pGL3-EpCAM-Pro constructs (Table S2). Results showed that a strong EGFP fluorescence was observed in PK-15 and piPSC cells, but the fluorescent signal was undetectable in control PEF cells (Fig. 2B). Luciferase assay showed that EpCAM promoter was significantly activated in epithelia PK-15 versus fibroblasts PEF (Fig. 2C), indicating the cell-specificity of cloned EpCAM promoter.

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.