FOXO3 Is a Glucocorticoid Receptor Target and Regulates LKB1 and Its Own Expression Based on Cellular AMP Levels via a Positive Autoregulatory Loop

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是一种参与调控多种生理过程的转录因子，涵盖细胞周期阻滞、细胞凋亡、氧化应激应答与能量代谢等多个环节。尽管学界对其翻译后修饰已有较为深入的研究，但FOXO3的转录调控机制，以及转录事件与翻译后修饰事件之间的交叉串扰，仍尚未被充分阐明。本研究证实，FOXO3属于即刻早期糖皮质激素受体（GR）靶基因，其转录过程甚至可被模拟代谢应激的条件进一步增强。由GR结合的类固醇激素所诱导的FOXO3转录，可通过GR拮抗剂RU-486的共处理予以逆转，但可被激活腺苷酸活化蛋白激酶（AMPK）的刺激进一步提升。通过基因组DNA分析、染色质免疫沉淀实验以及荧光素酶报告基因检测，研究人员在FOXO3启动子区域内发现了两个功能性糖皮质激素反应元件。此外，本研究还提供了磷酸化开关的功能性证据，该机制可解释糖皮质激素如何先诱导该基因的转录激活，随后通过位点特异性磷酸化使对应的FOXO3蛋白失活。仅当AMPK被激活时，预先存在的FOXO3才会重新恢复为激活状态。因此，能量匮乏条件可在转录与翻译后两个层面激活FOXO3。借此，FOXO3可作为代谢应激传感器，根据细胞的能量状态协调调控LKB1的表达——LKB1是参与代谢感知的核心上游激酶。另外，本研究还证实FOXO3可通过正向自身调控反馈环结合并激活自身的启动子。综上，本研究数据阐明了分解代谢类糖皮质激素与高水平细胞内AMP如何协同诱导FOXO3转录，并激活其编码的蛋白。