Leukemia inhibitory factor (LIF) withdrawal activates mTOR signaling pathway in mouse embryonic stem cells through the MEK/ERK/TSC2 pathway

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Abstract

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 When LIF is withdrawn from culture medium, the mTOR activity rapidly increases as detected by phosphorylation of its targets – ribosomal protein S6 and translation factor 4EBP1.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. Cell Death and Disease (2016) 7, e2050; doi:10.1038/cddis.2015.387; published online 14 January 2016 Embryonic stem cells (ESCs) are pluripotent cells derived from the early blastocyst that are capable of self-renewing for a long time in vitro. The mechanisms underlying pluripotency and self-renewal are of great interest, and a number of signaling pathways (LIF/STAT3, Ras/MAPK, BMP/Smad, PI3K/Akt and Wnt/β-cathenin) have been shown to contribute to the circuitry of ESCs state. 1–3 LIF/STAT3 (Leukemia Inhibitory Factor/Signal Transducer and Activator of Transcrip-tion-3) signaling pathway is one of the first described pathways regulating pluripotency in mouse ESCs. 4 LIF is a cytokine from the IL-6 family, which acts through the LIFR/gp130 receptor and activates STAT3 transcription factor at Tyr-705 that provides activation of pluripotency genes expression and downregulation of such differentiation markers as Gata3, Gata4, T/brachyury and Eomes. 5 It has been also shown that LIF can activate Ras-MAPK and PI3K-Akt through the involvement of gp130 adaptor protein. 6,7 Thus LIF/STAT3 signaling pathway promotes self-renewal and blocks the differentiation of mouse ESCs. Respectively, LIF removal from culture media is the first step to change mouse ESCs pluripotent state and to direct to differentiation. It is known that suppression of MEK/ERK by pharmacological inhibitors promotes their self-renewal, whereas ERK activation is essential for differentiation induction in embryonic stem cells suggesting a role of LIF pathway in maintaining the low levels of the ERK activity in undifferentiated cells.

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Cherepkova, M. Y., Sineva, G. S., & Pospelov, V. A. (2016). Leukemia inhibitory factor (LIF) withdrawal activates mTOR signaling pathway in mouse embryonic stem cells through the MEK/ERK/TSC2 pathway. Cell Death and Disease, 7, e2050. https://doi.org/10.1038/cddis.2015.387

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