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Disrupted WNT Signaling in Mouse Embryonic Stem Cells in the Absence of Calreticulin

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Abstract

The role of endoplasmic reticulum (ER) homeostasis and protein quality control in the regulation of WNT signaling is not understood. Here we provide evidence for a role of calreticulin in the regulation of WNT signaling. We show that a deficiency in calreticulin disrupted WNT signaling, and prevented cell cycle progression via the miR-302 microRNA family. These effects were dependent on the Ca2+ buffering capacity of calreticulin, as the protein is important in regulating ER Ca2+ release and activation of Ca2+-dependent kinase and phosphatase cascades (including c-Src, Akt, and PTP1B). We also show that calreticulin plays a role in the secretion and ER retention of WNT3a, thereby affecting downstream WNT signaling. In calreticulin-deficient ES cells, the WNT and miR-302 dependent maintenance of the naïve ES cell state and the transition to primed pluripotency transition were lost, preventing cells from undergoing accurate differentiation. Together, these findings demonstrate unexpected roles of calreticulin and ER Ca2+ homeostasis/signaling in the canonical WNT signaling pathway.

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Abbreviations

ER:

Endoplasmic reticulum

BMP:

Bone morphogenetic protein

BAPTA-AM:

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

CaMKII:

Ca2+/calmodulin kinase II

c/EBP:

CCAAT/enhancer binding protein

EpiS:

Epiblast like stem cell

Fgf:

Fibroblast growth factor

GSK3β:

Glycogen synthase kinase 3β

JNK:

c-Jun kinase

Klf4:

Krueppel-like factor 4

MEF2c:

Myocyte enhancer factor 2

αMHC:

α myosin heavy chain

NLK:

Nemo-like kinase

Oct-4:

Octamer-binding protein 4

Otx2:

Orthodenticle homeobox 2

PPAR:

Peroxisome proliferator-activated receptor

PTP1B:

Protein tyrosine phosphatase 1B

SERCA:

Sarco/endoplasmic reticulum Ca2+-ATPase

Smad:

Mothers against decapentaplegic homolog

Sox2:

Sex determining region Y-box 2

TCF:

T-cell factor

TGF:

Transforming growth factor

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Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (MOP-53050, MOP-15415, MOP-15291). We thank M. Opas, University of Toronto, for advice and help with immunocytochemistry (Fig. 4). We are grateful to Lori Cormack and Virginie Martin for invaluable help with initial experiments, advice, and support at the initial stages of this work. We thank Michel Puceat, Joanna Jung and Daniel Prins for critical reading of the manuscript and for very helpful comments.

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The authors indicate no potential conflicts of interest.

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  1. Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2S7

    Jody Groenendyk & Marek Michalak

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  1. Jody Groenendyk
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Correspondence to Marek Michalak.

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Groenendyk, J., Michalak, M. Disrupted WNT Signaling in Mouse Embryonic Stem Cells in the Absence of Calreticulin. Stem Cell Rev and Rep 10, 191–206 (2014). https://doi.org/10.1007/s12015-013-9488-6

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