Abstract
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.
Highlights
Forkhead box transcription factors constitute a family of evolutionary well conserved proteins that share a common DNA-binding domain, the so-called forkhead box [1,2,3]
Beside NF-Y and Sp1, representing two of the most prominent transcription factors involved in the regulation of a large number of genes [56,57], we showed that FOXO3 acts as key regulatory factor in Liver Kinase B1 (LKB1) transcription
We demonstrated that a serum and glucocorticoid-inducible kinase 1 (SGK-1) phosphorylation site deficient mutant of FOXO3 (FOXO3 A3: triple mutant T32A/S253A/S315A) was more potent in inducing LKB1 promoter activity than the wild type form, suggesting that SGK-1 might be a negative regulator of LKB1 expression [33]
Summary
Forkhead box transcription factors constitute a family of evolutionary well conserved proteins that share a common DNA-binding domain, the so-called forkhead box [1,2,3]. FOXO3 belongs to subfamily O of forkhead box transcription factors (FOXO) [4], whose members induce cell cycle arrest [5,6], DNA damage repair [7,8] and apoptosis [9,10]. The identification of chromosomal breakpoints within FOXO genes, producing hybrid proteins in human tumours further supports this idea [14,15,16] In addition to their role in cell cycle regulation, FOXO transcription factors regulate glucose metabolism in various organs [17,18,19] and increase the resistance to oxidative stress [20,21,22]. The FOXO orthologue DAF-16 controls life span extension in Caenorhabditis elegans together with homologues of the human
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