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...

The ability to propagate embryonic stem cells (ESCs) while maintaining their pluripotency is critical if their potential use in regenerative medicine is to be realized. The mechanisms controlling ESC self-renewal are under intense investigation, and glycogen synthase kinase 3 (GSK-3) has been implicated in regulating both self-renewal and differentiation. To clarify its role in ESCs we have used chemical genetics. We synthesized a series of bisindolylmaleimides, a subset of which inhibit GSK-3 in murine ESCs and robustly enhance self-renewal in the presence of leukemia inhibitory factor (LIF) and serum, but not in the absence of LIF. Importantly, these molecules appear selective for GSK-3 and do not perturb other signaling pathways regulating self-renewal. Our study clarifies the functional importance of GSK-3 in regulation of ESC self-renewal and provides tools for investigating its role further.

The discovery, understanding and synthetic exploitation of the photochemical transformation of pyridinium salts are described. The investigations surrounding the remarkable transformation of pyridinium salts into a host of structurally complex motifs have helped extend the comprehension of aromatic and heteroaromatic photochemistry. The synthetic community has, in recent years, recognised the potential inherent in these compounds and has since exploited the irradiation of variously substituted pyridinium salts as key steps in the preparation of advanced intermediates in numerous synthetic programmes.

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