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.
Paling
Nicholas R D
NR
Department of Pharmacy and Pharmacology, Centre for Regenerative Medicine, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
Wheadon
Helen
H
Bone
Heather K
HK
Welham
Melanie J
MJ
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