A new mechanism of Peroxisome Proliferator–Activated Receptor-γ (PPAR-γ) for ischemic stroke: PPAR-γ protects ischemic brain against mitochondria induced neuronal Apoptosis through the activation of 14-3-3－phosphorylated Bad (p-Bad) signaling pathway

• Main Authors: Jui-Sheng Wu, 吳瑞昇
• Other Authors: Teng-Nan Lin
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/07016380295013429093

博士 === 國防醫學院 === 生命科學研究所 === 98 === Thiazolidinediones(TZD)s have been reported to protect against ischemia-reperfusion injury. Their protective actions are considered to be peroxisome proliferator–activated receptor-γ (PPAR-γ)– dependent; however, it is unclear how PPAR-γ activation confers resistance to ischemia-reperfusion injury. We evaluated the effects of rosiglitazone or PPAR-γ overexpression on cerebral infarction in a rat model. Rosiglitazone or PPAR-γ overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-γ small interfering RNA. In mice with knock-in of a PPAR-γ dominant-negative mutant, infarct volume was enhanced. Proteomic analysis revealed that brain 14-3-3ε was highly upregulated in rats treated with rosiglitazone. Upregulation of 14-3-3ε was abrogated by PPAR-γ small interfering RNA or GW9662, a PPAR-γ antagonist. Promoter analysis and chromatin immunoprecipitation (ChIP) revealed that rosiglitazone induced PPAR-γ binding to specific regulatory elements on the 14-3-3ε promoter and thereby increased 14-3-3ε transcription. 14-3-3ε small interfering RNA abrogated the antiapoptotic actions of rosiglitazone or PPAR-γ overexpression, whereas 14-3-3ε recombinant proteins rescued brain tissues. To determine the involvement of PPAR-γ in neuronal cytoprotection, we subjected N2-A cells to oxygen–glucose deprivation followed by reoxygenation (H-R). Following H-R insults, H2O2 production was increased while cell viability declined, which was accompanied by loss of mitochondrial membrane potential (MMP), cytochrome c release, caspases 9 and 3 activation, poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis. Rosiglitazone up to 5μM protected cell viability, normalized MMP, and prevented apoptotic signals. The protective effect of rosiglitazone was abrogated by GW9662 or a specific PPAR-γ small interference RNA (siRNA) but not a control scRNA. PPAR-γ overexpression alone was effective in maintaining MMP and preventing apoptosis and its protective effect. To elucidate the mechanism by which PPAR-γ protects MMP and prevents apoptosis, we analyzed 14-3-3ε, Bcl-2, Bcl-xl, and phosphorylated Bad (p-Bad). H-R suppressed them. Rosiglitazone or PPAR-γ overexpression restored them via PPAR-γ. Elevated 14-3-3ε enhanced binding of phosphorylated Bad and protected mitochondrial membrane potential. Rosiglitazone or PPAR-γ overexpression preserved phosphorylated Akt and 3-phosphoinositide-dependent kinase-1 (PDK-1) in a PPAR-γ dependent manner. These results indicate that Ligand-activated PPAR-γ confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3ε transcription. 14-3-3ε upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis. It was also reported that ligand-activated PPAR-γ protects N2-A cells against H-R damage by suppressing H2O2 production、enhancing Bcl-2/Bcl-xl and maintaining p-Bad via preservation of p-Akt.