Glucocorticoid-induced transcriptional activation of FOXO3 is further enhanced by AMPK activation.

<p>(A) MCF-10A cells were treated with 1 mM of specific AMPK activator AICAR in combination with either vehicle (Ctrl), 1 µM dexamethasone (DEX) or a mixture of 1 µM dexamethasone and 1 µM RU-486 (DEX/RU) for 3, 6, 12 and 24 h. (B) MCF-10A cells were treated with vehicle (Ctrl), 1 µM dexamethasone (DEX) or a combination of 1 µM dexamethasone and 1 mM AICAR (DEX+AICAR) in the presence or absence of 10 µg/ml cycloheximide (CHX) for 6 h. Relative mRNA levels of FOXO3 were analysed by quantitative real-time PCR (mean ±SD, n = 3). Relative protein levels of FOXO3 in comparison to actin as internal control were analysed by western blotting in the presence or absence of cycloheximide.</p

SGK-1 inhibits FOXO3 transcriptional activity and subsequently LKB1 promoter activation.

<p>(<b>A</b>) Illustration of the LKB1 promoter region, extending from nucleotide position -2537 to +727 relative to the transcription start site. Positions of the transcription start site (TSS) and binding sites of transcription factors involved in LKB1 regulation (FOXO3, NF-Y and Sp1) as well as their positions relative to the transcription start site are indicated in brackets. (<b>B</b>) LKB1 reporter activity in HEK293 cells after ectopic expression of SGK-1 (dark grey) is compared to the activity obtained from co-transfections with the empty vector (Ctrl, light grey). Reporter activity is expressed as fold of luciferase activity (relative light units normalized to renilla luciferase activity) obtained from co-transfection of the plasmid containing the LKB1 promoter (nucleotides −2537 to +727) together with the empty expression vector (pcDNA3). Instead of the empty vector the same amount of either a FOXO3 wild-type expression plasmid (FOXO3 WT) or a SGK-1 phosphorylation site deficient triple mutant (T32A/S253A/S315A) of FOXO3 (FOXO3 A3) has been co-transfected. Each bar represents the means ± standard deviation of three measurements. (<b>C</b>) Western blot analysis of the same lysates used in the LKB1 reporter assay described in (B) using antibodies against phospho-FOXO3 (T32), total FOXO3 and actin. The ratios of p-FOXO3/FOXO3 for each experimental condition are indicated. The shown blot is a representative of three independent experiments.</p

Transcriptional activation of Foxo3 by glucocorticoids in mice.

<p>Semi-quantitative (left panel) and quantitative real-time PCR analysis (right panel) of Foxo3 mRNA levels in livers of C57Bl6 mice treated with dexamethasone (DEX) (1 mg/kg/day) or saline (Ctrl) for a period of (<b>A</b>) 3 h or (<b>B</b>) 3 weeks. Bars represents the means ± standard deviation (n: number of animals; panel A, n = 6; panel B, n = 7).</p

Oligonucleotides used in EMSA.

<p>FOXO3 core binding motive is indicated in bold. Parts of adjacent FOXO3 binding sites within the same oligonucleotide are underlined.</p

AMPK is essential for FOXO3 induced activation of LKB1 and FOXO3 transcription.

<p>(<b>A</b>) MCF-10A cells were either treated with vehicle (Ctrl) and 1 µM dexamethasone (DEX) or in combination with either 1 mM AICAR (left panel) or 50 mM 2-deoxyglucose (right panel) in the presence or absence of 20 µM of the specific AMPK inhibitor “Compound C” for 18 h. Relative mRNA levels of LKB1, FOXO3 and GAPDH as internal control were analysed by semi-quantitative RT-PCR in a 1% agarose gel after ethidium bromide staining. The data shown is representative of three independent experiments. (<b>B</b>) Illustration of metabolic regulation of FOXO3’s transcriptional activity and subsequent effects on FOXO3 and LKB1 expression. Activating events are indicated in green colour, inhibiting events in red. High glucocorticoid levels immediately activate FOXO3 and SGK-1. Following stimulation of FOXO3 transcription, the protein is phosphorylated at threonine 32 (Thr 32), leading to its inactivation. Treatment with AMPK activating stimuli (metabolic stress) triggers phosphorylation of FOXO3 by AMPK at position 413 of the serine residue (Ser 413), thereby counteracting inactivation of FOXO3 by SGK-1. This results in a temporally delayed further enhancement of FOXO3 transcription since the FOXO3 protein can bind and activate its own gene promoter via a positive autoregulatory feedback loop in the presence of glucocorticoids. Functional FOXO3 in turn induces the master upstream kinase that further actives AMPK by threonine phosphorylation at position 172 (Thr 172). Restored ATP levels or inhibition of AMPK by compound C interrupts this circuit.</p

Glucocorticoid receptor directly binds and activates the FOXO3 promoter.

<p>(A) Illustration of the FOXO3 promoter region, extending from nucleotide position −4255 to +91 relative to the transcription start site, matched with deletion constructs generated by the indicated restriction enzymes. The 5′-extension relative to the transcription start site of each construct in base pairs (bp) is given on the right side (full length: −4255 to NotI: −11 respectively). The 3′-end at +91 is identical for each construct. The position of the TATA box, the transcription start site (TSS) and the potential glucocorticoid response elements (GRE 1, 2, 3) are indicated. The PCR products amplified in the ChIP assay (ChIP A, B, C) as well as the sequence of the potential GREs are marked above the illustration. (B) Not I restriction digestion of all constructed FOXO3 promoter deletion constructs separated in a 1% agarose gel. (C) FOXO3 promoter reporter assay in A549 cells treated with either vehicle (Ctrl, dark grey) or 1 µM dexamethasone (DEX, light grey) for 12 h. Luciferase activity of all FOXO3 promoter deletion constructs (relative light units normalized against renilla luciferase activity) is expressed as fold of the signal obtained with the plasmid containing the full length promoter (−4255 to +91) without dexamethasone treatment. Assays were performed in triplicates. The error bars denote mean ± standard deviation. (D) ChIP assay in MCF-10A cells treated with either 1 µM dexamethasone (DEX) or vehicle (Ctrl) for 3 h. DNA was sonicated to an average size of <500 bp and run on a 1% (wt/vol) agarose gel (upper panel). PCR products specific for the different potential GREs within the FOXO3 promoter (ChIP A, B, C) were amplified from sonicated DNA of 1/10 of the starting material (Input, positive control) as well as from sonicated DNA after ChIP with either a non-specific antibody (IgG, negative control) or with an antibody against the glucocorticoid receptor (α-GR). Products were visualized within the linear range of the reaction by ethidium bromide staining on a 1% (wt/vol) agarose gel (lower panel). The figure shows a representative of three independent experiments.</p

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

<div><p>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.</p> </div

Glucocorticoid-mediated SGK-1 induction inhibits LKB1 transcription through FOXO3 (T32) phosphorylation.

<p>MCF-10A cells were treated with either vehicle (ethanol) (Ctrl), 1 µM dexamethasone (DEX) or a combination of 1 µM dexamethasone and 1 µM of the GR antagonist RU-486 (DEX/RU) for 0, 6, 24, and 48 h. (<b>A</b>) Relative mRNA levels of SGK-1, FOXO3 target genes (LKB1, TRAIL, IGFBP3) and the transcription factors involved in LKB1 regulation (Sp1, NF-YB, FOXO3). β-tubulin was used as internal control. RT-PCR products were analysed in a 1% agarose gel after ethidium bromide staining. (<b>B</b>) Relative protein levels of phospho-FOXO3 (T32), total FOXO3 and actin as internal control were analysed by western blotting. The ratios of p-FOXO3/FOXO3 for each experimental condition are indicated.</p

FOXO3 coordinates LKB1 gene expression depending on the metabolic status of the cell.

<p>MCF-10A cells were treated with AMPK activating stimuli (1 mM AICAR, 50 mM 2- Deoxyglucose or 2 µM Oligomycin) in combination either with vehicle (Ctrl), 1 µM dexamethasone (DEX) or a mixture of 1 µM dexamethasone and 1 µM RU-486 (DEX/RU) for 18 hours. (<b>A</b>) Relative mRNA levels of SGK-1, LKB1, FOXO3 and GAPDH as internal control were analysed by semi-quantitative RT-PCR and separated in a 1% agarose gel after ethidium bromide staining. (<b>B</b>) Relative mRNA levels of LKB1 and FOXO3 were analysed by quantitative real time PCR and normalized to GAPDH (mean ±SD, n = 3). (<b>C</b>) Western blot analyses of the relative protein levels of phospho-AMPK (T172), phospho-FOXO3 (S413), phospho-FOXO3 (T32), total FOXO3 in comparison to actin as internal control. The ratios of p-FOXO3/FOXO3 for each experimental condition are indicated. The data shown is representative of three independent experiments. (<b>D</b>) Schematic illustration underlying the glucocorticoid-dependent modulation of FOXO3 transcriptional activity.</p