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.


Knockdown EpCAM expression in piPSCs.(A,B) Quantitative RT-PCR (A) and western blotting (B) analyses of EpCAM expression knockdown by shRNAs in PK-15 cells for 60 h. b-act, beta-actin was used as internal control. (C) Schematic diagram of cell reprogramming by hOSKM and shRNA treatment. (D) Alkaline phosphatase staining of cells that were reprogrammed with shRNAs for 13 days (upper) and the morphology of reprogrammed cell colony (lower). (E) The number of AP positive colonies after shRNA treatment for 13 days. (F) Morphology of piPSCs that were transfected with shRNA. (G) qRT-PCR analysis of pluripotent gene expressions. DOX-iPSCs were transfected with shRNAs for 60 h. Ctrl, cells were treated without shRNA. Scale bar, 50 μm. 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

f3: Knockdown EpCAM expression in piPSCs.(A,B) Quantitative RT-PCR (A) and western blotting (B) analyses of EpCAM expression knockdown by shRNAs in PK-15 cells for 60 h. b-act, beta-actin was used as internal control. (C) Schematic diagram of cell reprogramming by hOSKM and shRNA treatment. (D) Alkaline phosphatase staining of cells that were reprogrammed with shRNAs for 13 days (upper) and the morphology of reprogrammed cell colony (lower). (E) The number of AP positive colonies after shRNA treatment for 13 days. (F) Morphology of piPSCs that were transfected with shRNA. (G) qRT-PCR analysis of pluripotent gene expressions. DOX-iPSCs were transfected with shRNAs for 60 h. Ctrl, cells were treated without shRNA. Scale bar, 50 μm. Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.

Mentions: To knock down EpCAM expression, we designed three shRNAs that targeted to EpCAM extracellular domain sequence, and constructed three lentiviral shRNA vectors (Fig. S1). When PK-15 cells were transfected by lentiviral shRNA, the expression of endogenous EpCAM was down 70–90 percent compared to the control in both RNA level (Fig. 3A) and protein level (Fig. 3B). A diagram of cell reprogramming by four factors hOSKM and shRNA treatments was illustrated in Fig. 3C. PEF cells that were transfected with lentiviral shRNAs were reprogrammed by hOSKM (Fig. 3C). For two weeks induction and shRNA treatment, the reprogrammed cells showed the alkaline phosphatase (AP) staining positive colonies (Fig. 3D, upper), and the morphology of EpCAM knockdown cells showed much incompact (Fig. 3D lower). The quantification of AP-positive colonies showed that the reprogramming efficiency was significantly reduced when EpCAM expression was knocked down by shRNA (Fig. 3E). We then detected the effect of EpCAM in piPSCs. The shRNA constructs were transfected into DOX-iPSCs, a DOX induced piPSC line described previously11, for 60 h, respectively. The morphology of shRNA treated iPSC clones presented much looser and flat comparing to control clones (Fig. 3F). Quantitative RT-PCR analysis showed that in EpCAM-knockdown cells, the expression level of pluripotent genes including maternal factors OCT4, Sox2, and SALL4 and zygote activated factors LIN28 and ESRRB were significantly decreased (Fig. 3G), suggesting that EpCAM as an key surface-to-nucleus factor plays an important role to regulate porcine pluripotent gene expression.


EpCAM Intracellular Domain Promotes Porcine Cell Reprogramming by Upregulation of Pluripotent Gene Expression via Beta-catenin Signaling
Knockdown EpCAM expression in piPSCs.(A,B) Quantitative RT-PCR (A) and western blotting (B) analyses of EpCAM expression knockdown by shRNAs in PK-15 cells for 60 h. b-act, beta-actin was used as internal control. (C) Schematic diagram of cell reprogramming by hOSKM and shRNA treatment. (D) Alkaline phosphatase staining of cells that were reprogrammed with shRNAs for 13 days (upper) and the morphology of reprogrammed cell colony (lower). (E) The number of AP positive colonies after shRNA treatment for 13 days. (F) Morphology of piPSCs that were transfected with shRNA. (G) qRT-PCR analysis of pluripotent gene expressions. DOX-iPSCs were transfected with shRNAs for 60 h. Ctrl, cells were treated without shRNA. Scale bar, 50 μm. 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

f3: Knockdown EpCAM expression in piPSCs.(A,B) Quantitative RT-PCR (A) and western blotting (B) analyses of EpCAM expression knockdown by shRNAs in PK-15 cells for 60 h. b-act, beta-actin was used as internal control. (C) Schematic diagram of cell reprogramming by hOSKM and shRNA treatment. (D) Alkaline phosphatase staining of cells that were reprogrammed with shRNAs for 13 days (upper) and the morphology of reprogrammed cell colony (lower). (E) The number of AP positive colonies after shRNA treatment for 13 days. (F) Morphology of piPSCs that were transfected with shRNA. (G) qRT-PCR analysis of pluripotent gene expressions. DOX-iPSCs were transfected with shRNAs for 60 h. Ctrl, cells were treated without shRNA. Scale bar, 50 μm. Data are presented as mean ± S.D., *P < 0.05, **P < 0.01, n = 3.
Mentions: To knock down EpCAM expression, we designed three shRNAs that targeted to EpCAM extracellular domain sequence, and constructed three lentiviral shRNA vectors (Fig. S1). When PK-15 cells were transfected by lentiviral shRNA, the expression of endogenous EpCAM was down 70–90 percent compared to the control in both RNA level (Fig. 3A) and protein level (Fig. 3B). A diagram of cell reprogramming by four factors hOSKM and shRNA treatments was illustrated in Fig. 3C. PEF cells that were transfected with lentiviral shRNAs were reprogrammed by hOSKM (Fig. 3C). For two weeks induction and shRNA treatment, the reprogrammed cells showed the alkaline phosphatase (AP) staining positive colonies (Fig. 3D, upper), and the morphology of EpCAM knockdown cells showed much incompact (Fig. 3D lower). The quantification of AP-positive colonies showed that the reprogramming efficiency was significantly reduced when EpCAM expression was knocked down by shRNA (Fig. 3E). We then detected the effect of EpCAM in piPSCs. The shRNA constructs were transfected into DOX-iPSCs, a DOX induced piPSC line described previously11, for 60 h, respectively. The morphology of shRNA treated iPSC clones presented much looser and flat comparing to control clones (Fig. 3F). Quantitative RT-PCR analysis showed that in EpCAM-knockdown cells, the expression level of pluripotent genes including maternal factors OCT4, Sox2, and SALL4 and zygote activated factors LIN28 and ESRRB were significantly decreased (Fig. 3G), suggesting that EpCAM as an key surface-to-nucleus factor plays an important role to regulate porcine pluripotent gene expression.

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.