Limits...
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.


Overexpression of EpCAM improves PEF cell reprogramming.(A) Schematic diagram of lentiviral vectors for expressing EpCAM and ECFP-EpCAM fusion protein (fEpCAM). SP, signal peptide. TM, transmembrane domain. EpEX, extracellular domain. ICD, intracellular domain. (B) Western blotting analysis. Proteins were prepared from PEF (Ctrl) and PEF + EpCAM (Exp) cells that were transfected by pSIN-EpCAM lentivirus for 60 h. Anti-EpCAM antibody and anti-b-actin antibody were applied for immunoblotting. (C) Immunostaining of EpCAM by anti-EpCAM antibody (red) in PK-15 cells that were transfected with pSIN-fEpCAM (green). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 25 μm. (D) AP staining of cells reprogrammed by OSKM for 13 days (upper) and the morphology of derived colonies (lower). AP positive colonies were counted (right panel). Scale bar, 50 μm. (E) qRT-PCR analysis of pluripotent gene expressions in DOX-iPSCs. Ctrl, cells were transfected with pSIN-EGFP. Exp, cells were transfected with pSIN-EpCAM. (F) qRT-PCR analysis of gene markers in piPS cells that were spontaneously differentiated into ectoderm (NESTIN), mesoderm (CD34), and endoderm (FOXA1). Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5385527&req=5

f4: Overexpression of EpCAM improves PEF cell reprogramming.(A) Schematic diagram of lentiviral vectors for expressing EpCAM and ECFP-EpCAM fusion protein (fEpCAM). SP, signal peptide. TM, transmembrane domain. EpEX, extracellular domain. ICD, intracellular domain. (B) Western blotting analysis. Proteins were prepared from PEF (Ctrl) and PEF + EpCAM (Exp) cells that were transfected by pSIN-EpCAM lentivirus for 60 h. Anti-EpCAM antibody and anti-b-actin antibody were applied for immunoblotting. (C) Immunostaining of EpCAM by anti-EpCAM antibody (red) in PK-15 cells that were transfected with pSIN-fEpCAM (green). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 25 μm. (D) AP staining of cells reprogrammed by OSKM for 13 days (upper) and the morphology of derived colonies (lower). AP positive colonies were counted (right panel). Scale bar, 50 μm. (E) qRT-PCR analysis of pluripotent gene expressions in DOX-iPSCs. Ctrl, cells were transfected with pSIN-EGFP. Exp, cells were transfected with pSIN-EpCAM. (F) qRT-PCR analysis of gene markers in piPS cells that were spontaneously differentiated into ectoderm (NESTIN), mesoderm (CD34), and endoderm (FOXA1). Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.

Mentions: To further investigate EpCAM regulation function, we cloned porcine EpCAM coding DNA sequence (GenBank Accession No. KX904866), and constructed two overexpression vectors. The construct pSIN-EpCAM was derived from pSIN lentiviral plasmid with puromycin resistance, and the construct pSIN-fEpCAM was derived from pSIN-EpCAM with ECFP (enhanced cyan fluorescent protein) insertion in N-terminal of EpCAM protein (Fig. 4A). Western blotting analysis of PEF cells that were stably transfected by pSIN-EpCAM and selected by puromycin showed that cloned gene encoded EpCAM protein and overexpressed in transfected PEF cells (Fig. 4B). We then transfected pSIN-fEpCAM into PK-15 cells, and did immunofluorescence staining, which showed that fEpCAM fusion protein and endogenous EpCAM were co-localized in cell plasma membrane (Fig. 4C), indicating that the cloned EpCAM retained the biological function, and the constructs were able to be used for the following experiments.


EpCAM Intracellular Domain Promotes Porcine Cell Reprogramming by Upregulation of Pluripotent Gene Expression via Beta-catenin Signaling
Overexpression of EpCAM improves PEF cell reprogramming.(A) Schematic diagram of lentiviral vectors for expressing EpCAM and ECFP-EpCAM fusion protein (fEpCAM). SP, signal peptide. TM, transmembrane domain. EpEX, extracellular domain. ICD, intracellular domain. (B) Western blotting analysis. Proteins were prepared from PEF (Ctrl) and PEF + EpCAM (Exp) cells that were transfected by pSIN-EpCAM lentivirus for 60 h. Anti-EpCAM antibody and anti-b-actin antibody were applied for immunoblotting. (C) Immunostaining of EpCAM by anti-EpCAM antibody (red) in PK-15 cells that were transfected with pSIN-fEpCAM (green). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 25 μm. (D) AP staining of cells reprogrammed by OSKM for 13 days (upper) and the morphology of derived colonies (lower). AP positive colonies were counted (right panel). Scale bar, 50 μm. (E) qRT-PCR analysis of pluripotent gene expressions in DOX-iPSCs. Ctrl, cells were transfected with pSIN-EGFP. Exp, cells were transfected with pSIN-EpCAM. (F) qRT-PCR analysis of gene markers in piPS cells that were spontaneously differentiated into ectoderm (NESTIN), mesoderm (CD34), and endoderm (FOXA1). 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

f4: Overexpression of EpCAM improves PEF cell reprogramming.(A) Schematic diagram of lentiviral vectors for expressing EpCAM and ECFP-EpCAM fusion protein (fEpCAM). SP, signal peptide. TM, transmembrane domain. EpEX, extracellular domain. ICD, intracellular domain. (B) Western blotting analysis. Proteins were prepared from PEF (Ctrl) and PEF + EpCAM (Exp) cells that were transfected by pSIN-EpCAM lentivirus for 60 h. Anti-EpCAM antibody and anti-b-actin antibody were applied for immunoblotting. (C) Immunostaining of EpCAM by anti-EpCAM antibody (red) in PK-15 cells that were transfected with pSIN-fEpCAM (green). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 25 μm. (D) AP staining of cells reprogrammed by OSKM for 13 days (upper) and the morphology of derived colonies (lower). AP positive colonies were counted (right panel). Scale bar, 50 μm. (E) qRT-PCR analysis of pluripotent gene expressions in DOX-iPSCs. Ctrl, cells were transfected with pSIN-EGFP. Exp, cells were transfected with pSIN-EpCAM. (F) qRT-PCR analysis of gene markers in piPS cells that were spontaneously differentiated into ectoderm (NESTIN), mesoderm (CD34), and endoderm (FOXA1). Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.
Mentions: To further investigate EpCAM regulation function, we cloned porcine EpCAM coding DNA sequence (GenBank Accession No. KX904866), and constructed two overexpression vectors. The construct pSIN-EpCAM was derived from pSIN lentiviral plasmid with puromycin resistance, and the construct pSIN-fEpCAM was derived from pSIN-EpCAM with ECFP (enhanced cyan fluorescent protein) insertion in N-terminal of EpCAM protein (Fig. 4A). Western blotting analysis of PEF cells that were stably transfected by pSIN-EpCAM and selected by puromycin showed that cloned gene encoded EpCAM protein and overexpressed in transfected PEF cells (Fig. 4B). We then transfected pSIN-fEpCAM into PK-15 cells, and did immunofluorescence staining, which showed that fEpCAM fusion protein and endogenous EpCAM were co-localized in cell plasma membrane (Fig. 4C), indicating that the cloned EpCAM retained the biological function, and the constructs were able to be used for the following experiments.

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.