Regulation of Nanog Expression by Phosphoinositide 3-Kinase-dependent Signaling in Murine Embryonic Stem Cells

Abstract

Embryonic stem (ES) cell pluripotency is regulated by a combination of extrinsic and intrinsic factors. Previously we have demonstrated that phosphoinositide 3-kinase (PI3K)-dependent signaling is required for efficient self-renewal of murine ES cells. In the study presented here, we have investigated the downstream molecular mechanisms that contribute to the ability of PI3Ks to regulate pluripotency. We show that inhibition of PI3K activity with either pharmacological or genetic tools results in decreased expression of RNA for the homeodomain transcription factor Nanog and decreased Nanog protein levels. Inhibition of glycogen synthase kinase 3 (GSK-3) activity by PI3Ks plays a key role in regulation of Nanog expression, because blockade of GSK-3 activity effectively reversed the effects of PI3K inhibition on Nanog RNA, and protein expression and self-renewal under these circumstances were restored. Furthermore, GSK-3 mutants mimicked the effects of PI3K or GSK-3 inhibition on Nanog expression. Importantly, expression of an inducible form of Nanog prevented the loss of self-renewal observed upon inhibition of PI3Ks, supporting a functional relationship between PI3Ks and Nanog expression. In addition, expression of a number of putative Nanog target genes was sensitive to PI3K inhibition. Thus, the new evidence provided in this study shows that PI3K-dependent regulation of ES cell self-renewal is mediated, at least in part, by the ability of PI3K signaling to maintain Nanog expression. Regulation of GSK-3 activity by PI3Ks appears to play a key role in this process.Embryonic stem cell pluripotency underpins their potential utility as a source of differentiated progeny for use in regenerative medicine. Leukemia inhibitory factor (LIF) 2 plays an important role in maintaining self-renewal of murine ES (mES) cells (1, 2) via activation of STAT3 (3-6) and induction of c-Myc (7). LIF also activates additional signals including the Ras/ERK kinase pathway (8, 9), ribosomal S 6 kinases (10), phosphoinositide 3-kinases (11), and Src kinases (12). However, whereas LIF-induced STAT3 activation promotes self-renewal, LIF-induced ERK activation appears to promote differentiation (8), leading to the proposal that the balance between STAT3 and ERK signals contributes to the determination of mES cell fate (13).Other extrinsic factors that also play a role in maintenance of mES cell self-renewal include bone morphogenic protein 4 (BMP4), which acts in synergy with LIF to maintain self-renewal via Smad-mediated induction of Id transcriptional repressor expression (14). A further report suggests BMP4 inhibition of p38 mitogen-activated protein kinase (MAPK) may also contribute to maintenance of self-renewal (15). Wnt signaling has been implicated in regulation of pluripotency of both mES and human ES (hES) cells, work stemming largely from use of the GSK-3 inhibitor 5-bromoindirubin-3-oxime (BIO) (16,17).A number of intrinsic regulators in the form of transcription factors have been identified that play important roles in regulatio...

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.…”

“…[7][8][9][10] Expression level of Nanog is regulated by PI3K/Akt signaling, 8,18,22 and DLK (Figs. 1, 3 and 6).…”

“…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.…”