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 G 1 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/Ecadherin-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.

Leukemia inhibitory factor (LIF) is indispensable to maintain the pluripotent state of mouse embryonic stem cells (ESCs), but the mechanisms underlying the role of LIF/STAT3 pathway are yet poorly understood. Here we first showed that the LIF/STAT3-regulated signaling pathway contributes to the maintenance of self-renewal and pluripotency of mouse ESCs by suppressing mTOR (mammalian target of rapamycin), which is necessary for early differentiation. When LIF is withdrawn from culture medium, the mTOR activity rapidly increases as detected by phosphorylation of its targets – ribosomal protein S6 and translation factor 4EBP1. In turn, suppression of STAT3 phosphorylation on Tyr-705 by a specific small molecule WP1066 also activates phosphorylation of the mTOR target S6 ribosomal protein. LIF removal strongly activates ERK activity indicating that ERK can be involved in either direct phosphorylation of mTOR or phosphorylation of an upstream negative regulator of mTOR – TSC1/TSC2 proteins. According to western blotting data, LIF withdrawal leads to phosphorylation of TSC2 protein thereby relieving its negative effect on mTOR activity. mTOR activation is accompanied by a decrease of pluripotent gene expression Oct-4, Nanog, Sox2 and by an augmentation of fgf5 gene expression – a marker of post-implantation epiblast. Together, these data indicate that LIF-depleted mouse ESCs undergo a transition from the LIF/STAT3-supported pluripotent state to the FGFR/ERK-committed primed-like state with expression of early differentiation markers mediated through activation of mTOR signaling.

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