Luo Yi, Dixon C Jane, Hall John F, White Pamela J, Boarder Michael R
The Cell Signaling Laboratory, The Hawthorn Building, Leicester School of Pharmacy, De Montfort University, Leicester LE1 9BH, UK.
J Pharmacol Exp Ther. 2007 Jun;321(3):884-91. doi: 10.1124/jpet.107.121061. Epub 2007 Mar 19.
Epidermal growth factor (EGF) stimulation of cell cycle progression in cultured primary hepatocytes has previously been reported to be dependent on the mammalian target of rapamycin (mTOR) elements of the phosphoinositide 3-kinase (PI3K) signaling cascade and not the Akt pathway. Here we have established conditions of combined treatment of rat hepatocytes with insulin and EGF that favor cell cycle progression. The resulting cell population expresses albumin and retains receptor regulation of the signaling pathways leading to glycogen phosphorylase activation. We then investigated the hypothesis that the Akt limb of the PI3K pathway plays a central role in this insulin/EGF enhancement of cell cycle progression. The phosphorylation of Akt, central to the PI3K pathway, was increased by both insulin (sustained) and EGF (transient). The stimulation of Akt phosphorylation was inhibited in a concentration-dependent manner by the PI3K inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Cell cycle progression in these cultures was reduced, but not abolished, by this inhibitor. The mTOR inhibitor, rapamycin, also inhibited entry into S phase. The novel Akt inhibitor A-443654 [(S)-1-(1H-indol-3-ylmethyl)-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine] blocked both EGF-stimulated cell cycle progression and phosphorylation of the Akt substrate glycogen synthase kinase-3. Infection of cells with an adenoviral vector expressing a constitutively active form of Akt but not a kinase-dead form increased hepatocyte proliferation probably through enhanced cell cycle progression and reduced apoptosis. These results show that the Akt element of the PI3K cascade is necessary for EGF-stimulated cell cycle progression and provide evidence that the sustained elevation of Akt alone generates a hyperproliferative window in hepatocyte cultures.
先前有报道称，表皮生长因子（EGF）刺激培养的原代肝细胞的细胞周期进程依赖于磷酸肌醇3激酶（PI3K）信号级联的哺乳动物雷帕霉素靶蛋白（mTOR）元件，而非Akt途径。在此，我们建立了用胰岛素和EGF联合处理大鼠肝细胞以促进细胞周期进程的条件。所得细胞群体表达白蛋白，并保留了导致糖原磷酸化酶激活的信号通路的受体调节。然后，我们研究了PI3K途径的Akt分支在胰岛素/EGF增强细胞周期进程中起核心作用这一假说。PI3K途径的核心Akt磷酸化，在胰岛素（持续性）和EGF（短暂性）作用下均增加。PI3K抑制剂2-(4-吗啉基)-8-苯基-4H-1-苯并吡喃-4-酮（LY294002）以浓度依赖性方式抑制Akt磷酸化的刺激。该抑制剂使这些培养物中的细胞周期进程减慢，但未完全消除。mTOR抑制剂雷帕霉素也抑制进入S期。新型Akt抑制剂A-443654 [（S）-1-（1H-吲哚-3-基甲基）-2-[5-（3-甲基-1H-吲唑-5-基）-吡啶-3-基氧基]-乙胺]可阻断EGF刺激的细胞周期进程以及Akt底物糖原合酶激酶-3的磷酸化。用表达组成型活性形式的Akt而非激酶失活形式的腺病毒载体感染细胞，可能通过增强细胞周期进程和减少凋亡来增加肝细胞增殖。这些结果表明，PI3K级联的Akt元件对于EGF刺激的细胞周期进程是必需的，并提供证据表明仅Akt的持续升高在肝细胞培养物中产生了一个增殖窗口。
