Ligand-activated peroxisome proliferator-activated receptor-gamma protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3 epsilon upregulation - PubMed (original) (raw)

Comparative Study

. 2009 Mar 3;119(8):1124-34.

doi: 10.1161/CIRCULATIONAHA.108.812537. Epub 2009 Feb 16.

Wai-Mui Cheung, Yau-Sheng Tsai, Yi-Tong Chen, Wen-Hsuan Fong, Hsin-Da Tsai, Yu-Chang Chen, Jun-Yang Liou, Song-Kun Shyue, Jin-Jer Chen, Y Eugene Chen, Nobuyo Maeda, Kenneth K Wu, Teng-Nan Lin

Affiliations

Comparative Study

Ligand-activated peroxisome proliferator-activated receptor-gamma protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3 epsilon upregulation

Jui-Sheng Wu et al. Circulation. 2009.

Abstract

Background: Thiazolidinediones have been reported to protect against ischemia-reperfusion injury. Their protective actions are considered to be peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-dependent; however, it is unclear how PPAR-gamma activation confers resistance to ischemia-reperfusion injury.

Methods and results: We evaluated the effects of rosiglitazone or PPAR-gamma overexpression on cerebral infarction in a rat model and investigated the antiapoptotic actions in the N2-A neuroblastoma cell model. Rosiglitazone or PPAR-gamma overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-gamma small interfering RNA. In mice with knock-in of a PPAR-gamma dominant-negative mutant, infarct volume was enhanced. Proteomic analysis revealed that brain 14-3-3epsilon was highly upregulated in rats treated with rosiglitazone. Upregulation of 14-3-3epsilon was abrogated by PPAR-gamma small interfering RNA or antagonist. Promoter analysis and chromatin immunoprecipitation revealed that rosiglitazone induced PPAR-gamma binding to specific regulatory elements on the 14-3-3epsilon promoter and thereby increased 14-3-3epsilon transcription. 14-3-3epsilon Small interfering RNA abrogated the antiapoptotic actions of rosiglitazone or PPAR-gamma overexpression, whereas 14-3-3epsilon recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3epsilon enhanced binding of phosphorylated Bad and protected mitochondrial membrane potential.

Conclusions: Ligand-activated PPAR-gamma confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3epsilon transcription. 14-3-3epsilon Upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.

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Figures

Figure 1

Figure 1. Rosiglitazone and PPAR-γ reduce ischemic brain injury in vivo

(a) Rosiglitazone was injected intraventricularly immediately after 30-min ischemia. Infarct volumes were determined after 24 h reperfusion. (b) Rosiglitazone (50 ng) was infused 2 h after a 30-min transient occlusion. (c) Rosiglitazone with & without GW9662 was infused. (d) PPAR-γ siRNA (0.5 nmol) or scRNA (2 nmol) was infused intraventricularly immediately after ischemia and PPAR-γ mRNA of brain tissues was measured 24 h later. The upper panel shows a representative gel and the lower panel, mean ± SD of densitometric analysis of four independent experiments. (e) Rosiglitazone with or without PPAR-γ siRNA was infused immediately after 30-min ischemia, and infarct volume was measured 24 h later. (f) Recombinant PPAR-γ protein (5 μg) was infused intraventricularly for 72 h before a 30-min ischemia. The inset indicates cortical PPAR-γ protein levels at 24 h after reperfusion. (g) PPAR-γ P465L dominant negative mutant mice (L/+) and wild type littermate controls (+/+) were subjected to 30-min ischemia and 24 h reperfusoin. Each bar denotes mean ± SD (n as indicated). *P<0.05. **P<0.01.

Figure 2

Figure 2. Analysis of apoptotic signals in ischemic brain

(a) Rosiglitazone (50 ng) or DMSO was injected with PPAR-γ siRNA or control scRNA, immediately after 30 min ischemia. Active caspases and cleaved PARP were analyzed by Western blotting. (b) Recombinant PPAR-γ proteins were infused for 72 h before I/R. Left panels show representative blots and right panels, densitometry of three experiments. *p<0.05; ** p<0.01.

Figure 3

Figure 3. 14-3-3ε is increased in rosiglitazone-treated ischemic brain

(a) Rats were subjected to I/R with or without Rosi treatment. Proteins in rat brains were analyzed with 2-DGE. The insets show a spot with increased density in Rosi-treated vs. control brain. Analysis by LC-MS/MS identified this spot to be 14-3-3ε. Similar results were obtained in two other experiments. (b) Western blot analysis of 14-3-3ε in I/R vs. sham. Rats were treated with rosiglitazone or DMSO immediately after 30-min ischemia. Upper panel shows a representative blot and the lower panel the error bars from 3 independent experiments. Each bar denotes mean ± SD. *p<0.05, **p<0.01. (c) 14-3-3ε protein levels in brain tissues treated with or without rosiglitazone in the presence or absence of PPAR-γ siRNA or control scRNA. (d) 14-3-3ε proteins in brain tissues treated with recombinant PPAR-γ proteins or vehicle. (e) 14-3-3ε protein levels in wild-type (+/+) and L/+ mutant mouse brain tissues. Similar results were obtained in two other experiments.

Figure 4

Figure 4. Ligand-activated PPAR-γ increases 14-3-3ε transcription

(a-d) 14-3-3ε proteins were analyzed by Western blotting in N2-A cells treated with rosiglitazone (a), rosiglitazone in the presence of GW9662 (b), rosiglitazone in the presence of PPAR-γ siRNA or scRNA (c), or PPAR-γ expression vectors (mPPAR-γ) (d). (e) & (f) N2-A cells transfected with 14-3-3ε promoter constructs p1625 or p1348 were treated with rosiglitazone (e) or PPAR-γ (f). Promoter activity was expressed as relative light unit (RLU) using β-gal (b-Gal) as control to normalize the activity. (g) ChIP analysis of PPAR-γ binding to the PPRE region (upper panel) of 14-3-3ε promoter. Binding of PPAR-β/δ was included as a control. Each bar represents mean ± SD of at least three independent experiments conducted in triplicate. *P<0.05. **P<0.01.

Figure 5

Figure 5. Control of cerebral infarction by 14-3-3ε in vivo

(a) Rosiglitazone with or without 14-3-3ε siRNA or scRNA was injected immediately after 30-min ischemia. Inset shows cortical 14-3-3ε mRNA levels. Similar results were obtained in two other experiments. (b) His-tagged 14-3-3ε recombinant proteins (5~20 μg) were infused 72 h before I/R. Inset shows 14-3-3ε and His analyzed by Western blotting. Each bar denotes mean ± SD. *P<0.05. **P<0.01. Similar results were obtained in two other experiments.

Figure 6

Figure 6. Rosiglitazone attenuates N2-A apoptosis in a PPAR-γ dependent manner

(a) Cells were subjected to OGD for 3h followed by reoxygenation for 24 h (H3R24) with or without rosiglitazone (Rosi) and/or GW9662. Apoptosis was analyzed by flow cytometry. (b) Cells were treated as a) and active caspase 3, 9 and cleaved PARP were determined by Western blotting. A representative blot is shown. (c) N2-A cells transfected with PPAR-γ siRNA or control were subjected to OGD (H3R24) with or without Rosi. (d) Cells transfected with PPAR-γ plasmids were subjected to H3R24. Upper panels show PPAR-γ proteins analyzed by Western blotting. Each bar denotes mean ± SD from at least three independent experiments conducted in triplicate. *P<0.05. **P<0.01.

Figure 7

Figure 7. Rosiglitazone and PPAR-γ restore 14-3-3ε

(a) N2-A cells were subjected to OGD (H3R24) in the presence or absence Rosi and GW9662, and protein levels of 14-3-3ε were measured. (b) Cells transfected with PPAR-γ siRNA or control scRNA were subjected to H3R24 in the presence or absence of Rosi. (c) Cells transfected with PPAR-γ plasmids were subjected to H3R24. The upper panel shows a representative blot and the lower panel, the densitometry analysis. (d) & (e) Cells transfected with 14-3-3ε siRNA or control scRNA were subjected to H3R24 with or without Rosi (d) or PPAR-γ transfection (e). (f) N2-A transfected with 14-3-3ε plasmids were subjected to H3R24. Each bar denotes mean ± SD of at least three independent experiments conducted in triplicate. *P<0.05. **P<0.01.

Figure 8

Figure 8. Interaction between 14-3-3ε and phosphorylated Bad (p-Bad)

(a) & (b) N2-A cells were subjected to H3R24 in the presence or absence of Rosi (a) or PPAR-γ (b). 14-3-3ε and p-Bad were analyzed by Western blotting. (c) Cells treated with H3R24 in the presence or absence of Rosi (R) were lysed and immunoprecipitated with a 14-3-3ε antibody. Proteins in the immunoprecipitate were analyzed by Western blotting using p-Bad or 14-3-3ε antibodies. Upper panels show representative immunoblots and the lower panels densitometry analysis. (d) & (e) Cells were transfected with 14-3-3ε siRNA or control and subjected to H3R12 in the presence or absence of Rosi (d) or PPAR-γ (e). MMP was analyzed by flow cytometry using JC-1 probe. (f) MMP was measured in cells treated with H3R12 in the presence or absence of 14-3-3ε plasmids. Each bar represents mean ± SD of at least three experiments. *P<0.05. **P<0.01.

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