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

• Published in: The Journal of biological chemistry 2004-11, Vol.279 (46), p.48063-48070
• Main Authors: Paling, Nicholas R D, Wheadon, Helen, Bone, Heather K, Welham, Melanie J
• Format: Article
• Language: English
• Subjects: Animals; beta Catenin; Cell Differentiation - physiology; Cell Line; Cell Proliferation; Cell Survival; Chromones - pharmacology; Cytoskeletal Proteins - metabolism; DNA-Binding Proteins - metabolism; Enzyme Activation; Enzyme Inhibitors - metabolism; Extracellular Signal-Regulated MAP Kinases - antagonists & inhibitors; Extracellular Signal-Regulated MAP Kinases - metabolism; Humans; Interleukin-6 - pharmacology; Leukemia Inhibitory Factor; Mice; Morpholines - pharmacology; Phosphatidylinositol 3-Kinases - metabolism; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction - physiology; STAT3 Transcription Factor; Stem Cells - cytology; Stem Cells - drug effects; Stem Cells - physiology; Trans-Activators - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M406467200

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 Δp85, 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, GSK3α/β, and S6 proteins. Importantly, LY294002 and Δp85 expression had no effect on LIF-induced 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 β-catenin, the target of GSK-3 action in the canonical Wnt pathway, although no consistent alterations in cytosolic levels of β-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.