Research Program Infections and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.
PLoS One. 2012;7(7):e42166. doi: 10.1371/journal.pone.0042166. Epub 2012 Jul 27.
FOXO3 is a transcription factor involved in the regulation of multiple physiological processes including cell cycle arrest, apoptosis, oxidative stress-response and energy metabolism. Although much is known about its post-translational modification, the transcriptional regulation of FOXO3, as well as the cross-talk between transcription and post-translational events, is still poorly understood. In the present study, we show that FOXO3 is an immediate early glucocorticoid receptor (GR) target, whose transcription is even further enhanced by conditions that mimic metabolic stress. Induction of FOXO3 transcription by GR-binding steroids was reversed by concomitant treatment with the GR antagonist RU-486, but further enhanced by stimuli that activate the AMP-activated protein kinase (AMPK). Analysis of genomic DNA and chromatin immunoprecipitation, as well as luciferase reporter assays, revealed two functional glucocorticoid responsive elements within the FOXO3 promoter. Furthermore, we provide functional evidence for a phosphorylation switch that explains how glucocorticoids induce transcriptional activation of the gene but subsequently inactivate the corresponding protein by site-specific phosphorylation. Only when AMPK is stimulated, pre-existing FOXO3 becomes reverted toward an active form. Energy deprived conditions thus activate FOXO3 on two different levels, namely transcriptional and post-translational. In that way, FOXO3 acts as a metabolic stress sensor that coordinates expression of LKB1, the master upstream kinase involved in metabolic sensing, depending on the energy status of the cell. Additionally, we show that FOXO3 binds and activates its own promoter via a positive autoregulatory feedback loop. In conclusion, our data explain how catabolic glucocorticoid hormones and high intracellular AMP levels cooperate in inducing FOXO3 transcription and in activating the corresponding protein.
FOXO3 是一种转录因子，参与多种生理过程的调节，包括细胞周期停滞、细胞凋亡、氧化应激反应和能量代谢。尽管人们对其翻译后修饰了解很多，但 FOXO3 的转录调控以及转录和翻译后事件之间的串扰仍知之甚少。在本研究中，我们表明 FOXO3 是糖皮质激素受体 (GR) 的即时早期靶标，其转录甚至在模拟代谢应激的条件下进一步增强。GR 结合甾体诱导的 FOXO3 转录被 GR 拮抗剂 RU-486 逆转，但被激活 AMP 激活蛋白激酶 (AMPK) 的刺激进一步增强。基因组 DNA 和染色质免疫沉淀分析以及荧光素酶报告基因检测表明，FOXO3 启动子内存在两个功能性糖皮质激素反应元件。此外，我们提供了功能证据，证明了一种磷酸化开关如何解释糖皮质激素如何诱导基因的转录激活，但随后通过特定的磷酸化使相应的蛋白失活。只有当 AMPK 受到刺激时，预先存在的 FOXO3 才会恢复为活性形式。因此，能量剥夺条件在转录和翻译后两个不同水平上激活 FOXO3。通过这种方式，FOXO3 作为一种代谢应激传感器，根据细胞的能量状态协调参与代谢感应的主要上游激酶 LKB1 的表达。此外，我们还表明 FOXO3 通过正反馈自动调节回路结合并激活其自身的启动子。总之，我们的数据解释了分解代谢的糖皮质激素和细胞内高 AMP 水平如何协同诱导 FOXO3 转录并激活相应的蛋白。
