Régulation de l'activité transcriptionnelle de Fox01 par O-N- acétylglucosaminylation (O-GIcNAc)

Chez les patients diabétiques, l'hyperglycémie perse a des effets délétères, conduisant à une aggravation de l'intolérance au glucose (glucotoxicité). La O-N-acétylglucosaminylation (O-GIcNAc) des protéines cytosoliques ou nucléaires pourrait être impliquée dans la glucotoxicité. Nous avons montré que la O-GIcNAc de FoxO1 entraîne une augmentation de l'expression d'un gène rapporteur de la GSPase dans les cellules HEK 293 et HepG2. L'expression de la G6Pase et d'autres gènes cibles de FoxO1 est également induite par la O-GIcNAc. Par ailleurs, le niveau de O-GIcNAc de FoxO1 est augmenté dans le foie de rats diabétiques GK comparé aux rats témoins, suggérant que la O-GIcNAc de FoxO1 pourrait être impliquée dans la physiopathologie du diabète. Dans la mesure où la G6Pase est essentielle à la production de glucose par le foie, une O-GIcNAc anormale de FoxO1 pourrait jouer un rôle clé dans la production hépatique excessive de glucose observée dans les situations d'hyperglycémie chronique.In diabetic patients, hyperglycaemia per se has deleterious effects, leading to worsening of glucose intolerance (glucotoxicity). The O-GlcNAcylation (O-GlcNAc) of cytosolic or nuclear proteins appears to play an important role in the glucotoxicity. We showed that the O-GlcNAcylation of FoxO1 leads to increased expression of a reporter gene containing the human G6Pase promoter in HEK 293 and liver-derived HepG2 cells. The expression of G6Pase and other target genes of FoxO1 is also induced by O-GlcNAc. Furthermore, the level of O-GlcNAc of FoxO1 is increased in the liver of diabetic Goto-Kakizaki rats compared to the non-diabetic rats. This result suggests that O-GlcNAc of FoxO1 could be involved in the pathophysiology of diabetes. Since the G6Pase is essential for the glucose production by the liver, an abnormal O-GlcNAc of FoxO1 may play an important role in the excessive hepatic production of glucose observed in the situations of chronic hyperglycaemia, such as type 2 diabetes.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Development of a human breast-cancer derived cell line stably expressing a bioluminescence resonance energy transfer (BRET)-based phosphatidyl inositol-3 phosphate (PIP3) biosensor.

International audienceStimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP2 to generate PIP3, which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP3 production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP3 production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP3 and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer

The PI3K stimulatory activity present in human serum can be evaluated using the MCF-7/B2 cells.

<p>(A, B) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with PBS containing 0%, 1%, 2%, 5% or 10% human serum that had been previously heated or not at 95°C during 1 h, and light emission acquisition started immediately. (A) A typical real-time BRET experiment is shown. (B) Means ± SEM of BRET values at the plateau of 3 to 7 independent experiments are shown. (C) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with 5% human serum previously submitted or not to centrifugation on a centrifugal filter device with a molecular weight cut-off of 3 kDa. A typical real-time BRET experiment (left panel) and means ± SEM of BRET values at the plateau (right panel) are shown (n = 3). (D) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with 5% human serum that had been pre-incubated for 1 h in presence of 50 μM IGFBP1. A typical real-time BRET experiment (left panel) and means ± SEM of BRET values at the plateau (right panel) are shown (n = 3). Statistical analysis was performed using ANOVA followed by Tukey’s test. *, P<0.05; **, P<0.01; ***, P<0.001; NS, Non significant.</p

Effect of inhibitors of the PI3K/Akt signaling pathway on BRET signal in MCF-7/B2 cells.

<p>(A) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 20 ng/μl of the humanized anti-EGFR antibody Cetuximab. Cells were then stimulated with EGF (32 nM), and light emission acquisition started immediately. A typical real-time BRET experiment (left panel) and the mean ± SEM of BRET values at the peak (right panel) are shown (n = 3). (B) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 25 μM of the tyrphostin AG1024. Cells were then stimulated with 100 nM insulin, and light emission acquisition started immediately. A typical real-time BRET experiment (left panel) and the mean ± SEM of BRET values at the plateau (right panel) are shown (n = 3). (C) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 25 μM of the PI3K inhibitor LY294002. Cells were then stimulated with 10 nM IGF1 and light emission acquisition started immediately. A typical real-time BRET experiments (left panel) and the mean ± SEM of BRET values at the plateau (right panel) of 3 independent experiments are shown. (D, E) MCF-7/B2 cells were preincubated for 4 h in absence or presence of 10 μM of the inhibitors of Akt-PH/PIP₃ interaction PIT-1 (D) and DMPIT-1 (E). Cells were then stimulated with 10 nM IGF1, and light emission acquisition started immediately. Typical real-time BRET experiments (left panels) and the mean ± SEM of BRET values at the plateau (right panels) of 3 to 4 independent experiments are shown. Statistical analysis was performed using ANOVA followed by Tukey’s test. *, P<0.05; **, P<0.01; ***, P<0.001; NS, Non significant.</p

Development of a human breast cancer-derived clone stably expressing a BRET-based PIP₃ biosensor.

<p>(A) Principle of the BRET-based assay used to monitor PIP₃ production in living cells. Activation of the PI-3 kinase by tyrosine kinase receptors induces phosphorylation of PIP₂ into PIP₃ and recruitment of Akt to the plasma membrane through its PH domain. To monitor PIP₃ production, MCF-7 cells were stably transfected with the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to a membrane localization sequence (YFP-membrane). Recruitment of Luc-Akt-PH to the plasma membrane by PIP₃ results in energy transfer between Luc-Akt-PH and YFP-membrane. This permits to study the pharmacological properties of ligands that activate this pathway, and to evaluate the effects of inhibitory molecules acting on (I) the extracellular part of receptors, (II) the tyrosine kinase activity of the receptors, (III) the catalytic activity of the PI3K, and (IV) the interaction between PIP₃ and the PH domain of Akt. (B) Transfection of a MCF-7 clone stably expressing Luc-Akt-PH with YFP-membrane cDNA gave rise to 4 sub-clones stably expressing both constructs. Ligand-induced BRET could be detected in only 3 sub-clones (B2, D2, C2). Preliminary BRET experiments indicated that insulin-induced BRET was higher with clone B2. (C) Surface expression of pEYFP-Mem in MCF-7/B2 cells was studied by fluorescence microscopy. YFP fluorescence was detected using a FITC filter and nuclei were visualized using a DAPI filter. The image was obtained by deconvolution analysis. (D) Western-blotting experiment showed that expression of Luc-Akt-PH alone or together with YFP-membrane does not affect insulin-induced phosphorylation of endogenous Akt. (E) Insulin dose-dependently stimulated PIP₃ production in MCF-7/B2 cells. Left panel: typical real-time experiment showing basal and insulin-stimulated BRET. Right panel: insulin-induced BRET (BRET above basal at the plateau) was determined for each insulin concentration to establish dose-response curves. Results are means ± S.E.M. (standard error of the mean) of 6 to 11 independent experiments. The freeze-thaw cycle did not affect the sensitivity of the cells to insulin stimulation.</p

Effect of IGF1, IGF2, EGF and glargine on PIP₃ production in MCF-7/B2 cells.

<p>(A) Typical experiments showing real-time effects of IGF1, IGF2, EGF and glargine on PIP₃ production in MCF-7/B2 cells. (B) Dose-dependent effect of IGF1, IGF2, EGF, glargine and insulin on PIP₃ production in MCF-7/B2 cells. Ligand-induced BRET (BRET above basal) at the plateau (IGF1, IGF2, Insulin, Glargine) or at the peak (EGF) was determined for each ligand concentration and was used to establish dose-response curves. Results are means ± S.E.M. of 3 to 8 independent experiments. EC50 for insulin, IGF1, IGF2, EGF and glargine are given in the result section.</p

EV71 infection induces ROS in neural cells in a time-dependent manner.

<p>SF268 cells were infected with EV71 at m.o.i. of 1.25, 1.5 and 2 for 0, 12, 24, 36, 48, 60 and 72 hr, and were subject to H₂DCFDA staining and flow cytometric analysis. (A) Representative histograms of cell counts (<i>counts</i>) vs. DCF fluorescence (<i>FL1-H</i>) for cells infected at an m.o.i. of 1.25 at indicated times are shown. (B) The mean fluorescence intensity (MFI) of DCF of infected cells is expressed as fold change relative to that of uninfected cells. The results are presented as mean±SD of three separate experiments.</p

Mitochondrial mass increases and expression of mitochondrial proteins changes in response to EV71 infection.

<p>(A) SF268 cells were mock- (−) or infected (+) with EV71 at an m.o.i. of 1.25 for 48 hr, and were subject to Mitotracker dye staining and flow cytometric analysis as described in <i>Materials and Methods</i>. The MFI of the stained cells is expressed relative to that of control cells. Results are mean ± SD, n = 3. *p<0.05 vs. uninfected cells. (B & C) Cells were un- or infected under the similar condition, and mitochondria were isolated for SDS-PAGE electrophoresis and silver staining (B). In the silver-stained gel, the leftmost lane corresponds to protein markers with respective molecular weights indicated alongside the bands. (C) Cells similarly infected were harvested for western blotting with indicated antibodies. A representative experiment out of three is shown here.</p

Mitochondrial ROS are essential to EV71 replication.

<p>(A) SF268 cells were mock- (−) or infected (+) with EV71 at an m.o.i. of 1.25, and treated without (<i>Con</i>) or with indicated concentrations of Mito-TEMPO. Forty-eight hours later, cells were subject to MitoSOX Red staining and flow cytometric analysis. The mean fluorescence intensity (MFI) of MitoSOX of mock- and infected cells is expressed as the percentage of that of uninfected cells. The results are presented as mean ± SD, n = 3. *p<0.05 vs. infected Con group. (B) SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25, and treated without or with 200 µM of Mito-TEMPO. Forty-eight hours later, cells were harvested for western blotting with antibodies to phosphorylated eIF2α and total eIF2α, viral protein 3D, and actin. A representative experiment out of three is shown here. (C) SF268 cells were mock- (−) or infected (+) with EV71 at an m.o.i. of 1.25, and treated without (<i>Con</i>) or with 200 µM of Mito-TEMPO. Forty-eight hours later, cells were analyzed for levels of EV71 genomic RNA. The results are presented as means ± SD n = 3. *p<0.05 vs. infected Con group.</p

EV71 infection-induced oxygen consumption is associated with a reduction in respiratory efficiency.

<p>(A) SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25 for indicated times. Oxygen concentration was assayed with Clark oxygen electrode, and oxygen consumption rate (10⁻² µg O₂/5×10⁵ cells/min) was calculated accordingly. Results are mean ± SD, n = 6. *p<0.05 vs. uninfected cells. (B) SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25, and were treated without or with oligomycin. Oxygen concentration was assayed with Clark oxygen electrode, and oxygen consumption rate (10⁻² µg O₂/5×10⁵ cells/min) was calculated. Results are mean ± SD, n = 6. *p<0.05 vs. uninfected cells; ^#p<0.05, oligomycin-treated vs. untreated cells. (C) Oligomycin-sensitive oxygen consumption rate was calculated as the difference in the absence and presence of oligomycin. (D & E) The oxygen consumption was measured as described in (A) and (B). Data were normalized to the relative mitochondrial mass unit (MMU) of control and infected cells. Results are mean ± SD, n = 6. *p<0.05 vs. uninfected cells. (F) Oligomycin-sensitive oxygen consumption rate was calculated as described in (C), and normalized to the relative mitochondrial mass unit (MMU) of control and infected cells. (G–I) SF268 cells were mock- (−) or infected (+) with EV71 at an m.o.i. of 1.25 for 48 hr, and mitochondria were isolated and assayed for oxygen consumption rates (10⁻¹ µg O₂/mg mitochondrial protein/min) during state 3 (G) and 4_o (H) respiration as described in <i>Materials and Methods</i>. RCRs were calculated accordingly, and are shown (I). Results are mean ± SD, n = 6. *p<0.05 vs. uninfected cells.</p