Paling Nicholas R D, Wheadon Helen, Bone Heather K, Welham Melanie J
Department of Pharmacy and Pharmacology, Centre for Regenerative Medicine, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
J Biol Chem. 2004 Nov 12;279(46):48063-70. doi: 10.1074/jbc.M406467200. Epub 2004 Aug 24.
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.
小鼠胚胎干细胞（ES细胞）自我更新的维持受白血病抑制因子（LIF）依赖的信号转导及转录激活因子3（STAT3）激活和包括Nanog、BMP2/4及Wnt信号在内的LIF非依赖机制调控。在此，我们证明了磷酸肌醇3激酶（PI3Ks）在调控小鼠ES细胞自我更新中存在此前未被描述的作用。用可逆性PI3K抑制剂LY294002处理，或通过显性负性Deltap85的调控表达对I（A）类PI3K进行更特异性抑制，均导致LIF维持自我更新的能力下降，细胞同时呈现分化形态。PI3Ks的抑制降低了蛋白激酶B/蛋白激酶B（PKB/Akt）、糖原合成酶激酶3α/β（GSK3α/β）及核糖体蛋白S6（S6）蛋白的基础磷酸化水平和LIF刺激后的磷酸化水平。重要的是，LY294002和Deltap85的表达对LIF诱导的酪氨酸（Tyr）705位点STAT3磷酸化无影响，但在短期和长期孵育中均增强了LIF诱导的细胞外信号调节激酶（ERK）磷酸化。随后，我们证明抑制丝裂原活化蛋白激酶/细胞外信号调节激酶（MAP-Erk激酶，MEKs）可在时间和剂量依赖的方式下逆转PI3K抑制对自我更新的影响，提示PI3K抑制后观察到的ERK活性升高促成了我们所观察到的功能反应。令人惊讶的是，在长期抑制PI3Ks后，我们观察到经典Wnt途径中GSK-3作用靶点β-连环蛋白的磷酸化水平降低，尽管未观察到β-连环蛋白胞质水平的一致变化，表明该途径在PI3Ks下游未起主要作用。我们的研究支持PI3Ks在自我更新调控中的作用，并增进了我们对参与干细胞命运调控的分子信号成分的理解。
