Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling.
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 Deltap85, 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, GSK3alpha/beta, and S6 proteins. Importantly, LY294002 and Deltap85 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 beta-catenin, the target of GSK-3 action in the canonical Wnt pathway, although no consistent alterations in cytosolic levels of beta-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.
Department of Pharmacy and Pharmacology, Centre for Regenerative Medicine, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
Nicholas R D
NR
Paling
Helen
H
Wheadon
Heather K
HK
Bone
Melanie J
MJ
Welham
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D002867:Chromones / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D053605:Leukemia Inhibitory Factor; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D015534:Trans-Activators / Q000378:metabolism; D051176:beta Catenin
D002867:Chromones; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D050796:STAT3 Transcription Factor; D015534:Trans-Activators; D051176:beta Catenin; C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; C507837:LIF protein, human; C507838:Lif protein, mouse; C494086:STAT3 protein, human; C494087:Stat3 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one