Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling*
- Huiming Xu‡,§,
- Kam Sze Tsang¶,
- Yonghui Wang‡,§,
- Juliana CN Chan‖,**,
- Gang Xu‖,‡‡,**1 and
- Wei-Qiang Gao‡,§2
- From the ‡State Key Laboratory of Oncogenes and Related Genes, Renji-MedX Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine and
- §School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200127, China and
- the ¶Department of Anatomical and Cellular Pathology,
- ‖Department of Medicine and Therapeutics,
- **Li Ka Shing Institute of Health Sciences and
- ‡‡Shenzhen Research Institute, The Chinese University of Hong Kong, The Prince of Wales Hospital, Shatin, Hong Kong, China
- ↵1 To whom correspondence may be addressed: Rm. 605, Li Ka Shing Institute of Health Science, The Chinese University of Hong Kong, The Prince of Wales Hospital, Hong Kong, China. Tel.: 85237636074; Fax: 85221446365; E-mail: gangxu{at}cuhk.edu.hk.
- ↵2 To whom correspondence may be addressed: Renji-MedX Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Rd., Shanghai 200127, China. Tel.: 86-21-68383917; Fax: 86-21-6838-3916; E-mail: gao.weiqiang{at}sjtu.edu.cn.
Abstract
Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of β-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-β inhibitor SB431542 and the Wnt/β-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and β-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.
- Cell Culture
- Cell Signaling
- Development
- Differentiation
- Embryonic Stem Cell
- Endoplasmic Reticulum Stress (ER Stress)
Footnotes
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↵* This work was supported by grants from the National Basic Research Program of China (2012CB967903, 2012CB966800, and 2013CB945600), National Natural Science Foundation of China (81270438, 81130038, and 81372189), Science and Technology Commission of Shanghai Municipality (Pujiang program), Shanghai Health Bureau Key Discipline and Specialty Foundation (J50208), Shanghai Education Committee Key Discipline and Specialty Foundation, and KC Wong Foundation.
- Received April 11, 2014.
- Revision received August 3, 2014.
- © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
