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Biology of the Cell (2010) 102, (549–560) (Printed in Great Britain)
Inhibition of GSK3β enhances both adhesive and signalling activities of β-catenin in mouse embryonic stem cells
Galina S. Sineva1 and Valery A. Pospelov
Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia
Key words: adhesion, β-catenin-dependent transcription, glycogen synthase kinase 3β (GSK3β) inhibitor, mouse embryonic stem cell, proliferation. Abbreviations used: Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-7-amino4-methylcoumarin; BIO, 6-bromoindirubin-3′-oxime; Cdc, cell division cycle; CHIR, CHIR99021; DAPI, 4′,6-diamidino-2-phenylindole; DTT, dithiothreitol; EMT, epithelial–mesenchymal transition; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK3β, glycogen synthase kinase 3β; GST, glutathione transferase; ESC, embryonic stem cell; mESC, mouse ESC; LIF, leukaemia inhibitory factor; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-2H-tetrazolium bromide; Oct3/4, octamer-binding transcription factor 4; PI3K, phosphoinositide 3-kinase; RT–PCR, reverse transcription–PCR; qRT–PCR, quantitative RT–PCR; RA, retinoic acid; Tcf, T-cell factor. 1To whom correspondence should be addressed (email galsin2209@gmail.com). Background. GSK3β (glycogen synthase kinase 3β) regulates the expression level and activity of various target proteins, including β-catenin. β-Catenin is a co-activator of Wnt-dependent genes as well as a partner for transmembrane cadherins to mediate cell-to-cell adhesion. In some cases, inhibition of GSK3β activity was shown to promote self-renewal of ESCs (embryonic stem cells), but immediate effects of GSK3β inhibitors in these cells still remain elusive. Results. Here, we address the effects of GSK3β inhibitors BIO (6-bromoindirubin-3′-oxime) and CHIR99021 on mESCs (mouse ESCs), focusing on modulation of β-catenin activities. We found that, upon GSK3β inhibition, the colonies of undifferentiated mESCs acquire a more compact morphology. This change is paralleled by two somewhat polar effects: (i) the accumulation of the β-catenin, which is co-localized with E-cadherin at the plasma membrane, and the cytoplasmic, tyrosine unphosphorylated β-catenin, which is able to bind the GST (glutathione transferase)-fused cytoplasmic domain of E-cadherin; and (ii) the accumulation of the tyrosine phosphorylated β-catenin and its nuclear translocation that is accompanied by activation of the Tcf (T-cell factor)/β-catenin-dependent transcription of Top-Flash reporter. The Tcf-mediated activation, however, does not affect most of the analysed Wnt-responsive genes involved in EMT (epithelial–mesenchymal transition) or cell-cycle progression, suggesting that the adhesive function of β-catenin is dominant over transcription in undifferentiated mESCs. Treatment with BIO decreases proliferation rates of mESCs. This is not due to apoptosis, but rather to accumulation of cells in G1 phase of the cell cycle and is accompanied by down-regulation of the c-myc mRNA content. Conclusion. Our results suggest that inhibition of GSK3β activity in mESCs enhances both the β-catenin/E-cadherin-mediated adhesion and the Tcf/β-catenin-dependent transcription, but does not activate transcription in most of the examined genes involved in EMT and cell cycle progression. Received 10 February 2010/14 June 2010; accepted 14 July 2010 Published as Immediate Publication 14 July 2010, doi:10.1042/BC20100016 © The Authors Journal compilation © 2010 Portland Press Limited |
 
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