Regulation of Embryonic Stem Cell Self-renewal by Phosphoinositide 3-Kinase-dependent Signaling

Abstract

The maintenance of murine embryonic stem (ES) cell self-renewal is regulated by leukemia inhibitory factor (LIF)-dependent activation of signal transducer and activator of transcription 3 (STAT3) and LIF-independent mechanisms including Nanog, BMP2/4, and Wnt signaling. Here we demonstrate a previously undescribed role for phosphoinositide 3-kinases (PI3Ks) in regulation of murine ES cell self-renewal. Treatment with the reversible PI3K inhibitor, LY294002, or more specific inhibition of class I A PI3K via regulated expression of dominant negative ⌬p85, led to a reduction in the ability of LIF to maintain self-renewal, with cells concomitantly adopting a differentiated morphology. Inhibition of PI3Ks reduced basal and LIF-stimulated phosphorylation of PKB/Akt, GSK3␣/␤, and S6 proteins. Importantly, LY294002 and ⌬p85 expression had no effect on LIFinduced phosphorylation of STAT3 at Tyr 705 , but did augment LIF-induced phosphorylation of ERKs in both short and long term incubations. Subsequently, we demonstrate that inhibition of MAP-Erk kinases (MEKs) reverses the effects of PI3K inhibition on self-renewal in a time-and dose-dependent manner, suggesting that the elevated ERK activity observed upon PI3K inhibition contributes to the functional response we observe. Surprisingly, upon long term inhibition of PI3Ks we observed a reduction in phosphorylation of ␤-catenin, the target of GSK-3 action in the canonical Wnt pathway, although no consistent alterations in cytosolic levels of ␤-catenin were observed, indicating this pathway is not playing a major role downstream of PI3Ks. Our studies support a role for PI3Ks in regulation of self-renewal and increase our understanding of the molecular signaling components involved in regulation of stem cell fate.

Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells

“…20 Inhibition of PI3K/Akt signaling with LY-294002 reduces mouse ES proliferation, pluripotency, and Nanog expression. 18,19,21,22 Those effects are similar to the effects of overexpressing DLK in mouse ES cells ( Fig. 3C and D).…”

“…[18][19][20][21][22] Many proteins and signaling pathways involved in proliferation or pluripotency are modulated by PI3K/Akt including Gsk3b/Myc, FoxO, mTOR and Tbx3/Nanog pathways. 8,21,22,41-43, In this study, for the first time, DLK was demonstrated to be an Akt substrate.…”

“…16,17 In mouse ES cells, PI3K/Akt signaling is essential for self-renewal and Nanog expression. 8,[18][19][20][21][22] Inhibition of Akt activity with LY-294002 reduces cellular growth, pluripotency and expression amounts of Nanog protein. 18,19,21,22 On the other hand, the increase of Akt activity by overexpression of ERas or knockout of PTEN promotes cell survival and tumorigenic properties of mouse ES cells.…”

“…8,[18][19][20][21][22] Inhibition of Akt activity with LY-294002 reduces cellular growth, pluripotency and expression amounts of Nanog protein. 18,19,21,22 On the other hand, the increase of Akt activity by overexpression of ERas or knockout of PTEN promotes cell survival and tumorigenic properties of mouse ES cells. 23,24 Even in the absence of LIF, Akt overexpression maintains the undifferentiated status of ES cells and Nanog expression levels.…”

β-Catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells

“…The pathway from LIF stimulation to the inactivation of GSK-3β through the phosphoinositide 3-kinase (PI3K)/Akt pathway has been described in ES cells [20]. However, neither stimulation with LIF or short-term treatment with a PI3K inhibitor, LY294002, showed any effect on the stability of β-catenin, although long-term treatment (around five days) of LY294002 did reduce β-catenin phosphorylation.…”