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 When LIF is withdrawn from culture medium, the mTOR activity rapidly increases as detected by phosphorylation of its targets – ribosomal protein S6 and translation factor 4EBP1.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.
Highlights
MTOR signaling pathway has an important role in a variety of cell processes including cell growth, translation regulation, cell metabolism, cell cycle regulation and autophagy
We examined the activity of mTOR signaling pathway under conditions that permit self-renewal of mESCs, and under conditions that promote differentiation. mTOR activity in the mTORC1 and mTORC2 complexes increases after LIF withdrawal from the medium indicating that LIF/STAT3 signaling negatively effects the activity of mTOR
It turned out that in LIF-depleted cells the activated ERK is involved in phosphorylation of TSC2 protein thereby relieving its negative effect on mTOR activity. qRT-PCR analysis showed that the activation of mTOR upon LIF withdrawal occurs simultaneously in parallel with a decrease in transcription of genes oct[4], nanog, klf[4] and sox[2] and by an increase of fgf[5] gene expression – the marker of post-implantation epiblast and primed pluripotent state
Summary
MTOR (mammalian target of rapamycin) signaling pathway has an important role in a variety of cell processes including cell growth, translation regulation, cell metabolism, cell cycle regulation and autophagy. Received 02.11.15; revised 29.11.15; accepted 02.12.15; Edited by G Raschellà suggest that mTOR-mediated activation of p70-S6K induces differentiation.[12,13] DEPTOR, a negative regulator of mTOR signaling, has an important role in maintenance of pluripotent state of embryonic stem cells and its level dramatically decreases with differentiation of mouse ESCs.[14]. Treatment with MEK1,2 inhibitor PD0325901 canceled the mTOR activation thereby implying the involvement of MEK-ERK pathway in mTOR activation Together, these data indicate that LIF depletion of mouse ESCs induces a transition from LIF/STAT3-supported pluripotency to FGFR/ERK-committed primed-like state mediated through activation of mTOR signaling
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