Published online before print February 16, 2009, doi: 10.1161/CIRCULATIONAHA.108.812537
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(Circulation. 2009;119:1124-1134.)
© 2009 American Heart Association, Inc.
Stroke |
Ligand-Activated Peroxisome Proliferator–Activated Receptor-
Protects Against Ischemic Cerebral Infarction and Neuronal Apoptosis by 14-3-3
Upregulation
From the Institute of Biomedical Sciences (J.-S.W., W.-M.C., Y.-T.C., W.-H.F., H.-D.T., Y.-C.C., S.-K.S., J.-J.C., T.-N.L.), Academia Sinica, Taipei, Taiwan; Graduate Institute of Life Sciences (J.-S.W.), National Defense Medical Center, Taipei, Taiwan; Graduate Institute of Clinical Medicine (Y.-S.T.), National Cheng Kung University Medical College, Tainan, Taiwan; National Health Research Institutes (J.-Y.L., K.K.W.), Zhunan, Taiwan; Cardiovascular Center (Y.E.C.), University of Michigan Medical Center, Ann Arbor, Mich; Department of Pathology and Laboratory Medicine (N.M.), University of North Carolina, Chapel Hill, NC; and University of Texas Health Science Center (K.K.W.), Houston, Tex.
Correspondence to Kenneth K. Wu, MD, PhD, National Health Research Institutes, Zhunan Township, Miaoli County 350, Taiwan (e-mail kkgo{at}nhri.org.tw); or Teng-nan Lin, PhD, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan (e-mail bmltn@ibms.sinica.edu.tw).
Received August 3, 2008; accepted December 22, 2008.
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Abstract |
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Background— Thiazolidinediones have been reported to protect against ischemia-reperfusion injury. Their protective actions are considered to be peroxisome proliferator–activated receptor-
(PPAR-)–dependent; however, it is unclear how PPAR- activation confers resistance to ischemia-reperfusion injury.
Methods and Results— We evaluated the effects of rosiglitazone or PPAR- overexpression on cerebral infarction in a rat model and investigated the antiapoptotic actions in the N2-A neuroblastoma cell model. Rosiglitazone or PPAR- overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR- small interfering RNA. In mice with knock-in of a PPAR- dominant-negative mutant, infarct volume was enhanced. Proteomic analysis revealed that brain 14-3-3
was highly upregulated in rats treated with rosiglitazone. Upregulation of 14-3-3 was abrogated by PPAR- small interfering RNA or antagonist. Promoter analysis and chromatin immunoprecipitation revealed that rosiglitazone induced PPAR- binding to specific regulatory elements on the 14-3-3 promoter and thereby increased 14-3-3 transcription. 14-3-3 Small interfering RNA abrogated the antiapoptotic actions of rosiglitazone or PPAR- overexpression, whereas 14-3-3 recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3 enhanced binding of phosphorylated Bad and protected mitochondrial membrane potential.
Conclusions— Ligand-activated PPAR- confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3 transcription. 14-3-3 Upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.
Key Words: apoptosis • infarction • stroke • PPAR gamma
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Introduction |
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Peroxisome proliferator-activated receptor-
(PPAR-), a member of the PPAR nuclear receptor family, is a ligand-activated transcription factor that regulates diverse biological activities and plays major roles in important human diseases such as diabetes mellitus, metabolic syndrome, and atherosclerosis.1 Several classes of PPAR- ligands have been identified. Naturally occurring fatty acid derivatives such as 15 deoxy-
12,14 prostaglandin J2 (15d-PGJ2) bind and activate PPAR- and are thought to mediate the antiinflammatory action of PPAR-.2,3 Synthetic PPAR- ligands such as thiazolidinedione are clinically efficacious in treating type 2 diabetes mellitus.4 Ligand-activated PPAR- forms heterodimers with the retinoid X receptor, which binds PPAR response elements (PPREs) situated at the promoter region of target genes and regulates gene expression.1 Extensive investigations have reported that ligand-activated PPAR- suppresses proinflammatory genes at the transcriptional level.5–8 The antiinflammatory action of PPAR- ligands was considered to contribute to tissue protection. Rosiglitazone was reported to protect against ischemia-reperfusion (I/R)–induced myocardial damage, whereas troglitazone and pioglitazone protect against cerebral infarction in a rat I/R stroke model.9–11 We have previously reported that 15d-PGJ2 was effective in reducing cerebral infarct volume in a rat model.12 Our previous in vitro data suggest that 15d-PGJ2 and thiazolidinediones such as rosiglitazone protect neurons from oxidant-induced apoptosis.12
Clinical Perspective p 1134
The purpose of the present study was to evaluate the effects of rosiglitazone and PPAR- overexpression on neural apoptosis and to identify the downstream effector molecules. The results show that rosiglitazone and PPAR- overexpression protected against I/R damage in a rat stroke model and in mice with knock-in of a PPAR- dominant-negative mutant. Proteomic analysis of ischemic rat brain identified 14-3-3 to be highly elevated in rosiglitazone-treated rats. Results from animal and cell experiments revealed that 14-3-3 was upregulated by ligand-activated PPAR- at the transcriptional level. Knockdown of 14-3-3 with RNA interference abrogated the protective effect of rosiglitazone and PPAR-, whereas ectopic expression of 14-3-3 or infusion of 14-3-3 proteins rescued brains from infarction and neuronal cells from ischemic damage.
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Methods |
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Animal Models
The rat focal cerebral I/R model was described previously13,14 (see Data Supplement). PPAR-
P465L mutant mice were prepared as described previously.15 In brief, the inbred foundation colony was maintained by breeding heterozygous P465L mutant mice with inbred 129/SvEv mice. The production colony for experiments was maintained by breeding heterozygous P465L knock-in mice on the 129/SvEv genetic background with C57BL/6J mice. This produced both wild-type and heterozygous P465L knock-in mice on the same inbred genetic background (129/SvEv x C57BL/6J F1). Littermate wild-type mice served as controls.
Oxygen/Glucose Deprivation Cell Model
Murine N2-A neuroblastoma cells (American Type Culture Collection, Manassas, Va) grown to 70% confluence were treated with rosiglitazone (Cayman Chemical, Ann Arbor, Mich) alone or in combination with GW9662 (Cayman Chemical), washed with deoxygenated glucose-free Hanks’ balanced salt solution, and transferred to an anaerobic chamber (model 1025, Forma Scientific, Marietta, Ohio) that contained a gas mixture of 5% CO2, 10% H2, 85% N2, and 0.02% to 0.1% O2 for 3 hours.16,17 After oxygen/glucose deprivation (OGD), N2-A cells were cultured in glucose-containing Hanks’ balanced salt solution under normoxic conditions in a 5% CO2 incubator for various time periods.
Transient Transfection
Mouse PPAR-1 expression plasmid was prepared by cloning PPAR-1 into pcDNA3.1+ vector. Specific PPAR- small interfering RNA (siRNA) and scrambled RNA (scRNA) were purchased from Ambion (Austin, Tex). 14-3-3 Expression plasmid was prepared as described previously.18 In brief, the complete coding sequence of 14-3-3 was amplified by polymerase chain reaction and cloned into pcDNA3.1+ vector (Invitrogen, Carlsbad, Calif). Specific 14-3-3 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Lipofectamine 2000 (Invitrogen) was used as a transfection carrier according to the manufacturer’s instructions (Data Supplement).
Reporter Assay
PPRE reporter construct, acyl-CoA oxidase (ACO)-Luc, was prepared by cloning luciferase into an ACO vector that contained 4 PPREs and a minimal cytomegalovirus promoter. pCMV-β-galactosidase (β-Gal) plasmid was used as an internal control of transfection. For cloning 14-3-3 promoter, a 1.6-kb (–1625 to +24) 5'-flanking region of human genomic sequence was amplified by polymerase chain reaction and cloned into pGL3 luciferase reporter.18 Transfection was performed as described previously18 and in the Data Supplement.
Western Blot and Immunoprecipitation Analysis
Analysis of proteins in the cortex and N2-A cells by Western blotting12 and immunoprecipitation18 was performed as described previously (Data Supplement).
Flow Cytometry
Flow cytometry was used to analyze apoptosis and mitochondria membrane potential (Data Supplement).
Intraventricular Injection of Rosiglitazone, GW9662, PPAR- siRNA, and 14-3-3 siRNA
The procedure was performed as described previously.12 Briefly, anesthetized rats were placed in a stereotaxic apparatus; 50 ng (0.14 nmol) of rosiglitazone, 165 ng (0.58 nmol) of GW9662, or 0.1 to 2 nmol of siRNA in 10-µL volume was injected into the right lateral ventricle at 2 µL/min at the following coordinates: Anterior, 2.5 mm caudal to the bregma; right, 2.8 mm lateral to the midline; and ventral, 3.0 mm ventral to the dural surface. Periodic confirmation of proper placement of the needle was performed with infusion of fast green FCF (Sigma-Aldrich, St Louis, Mo). Rosiglitazone, PPAR- siRNA, or control was injected into the lateral ventricle for 24 hours immediately after the 30-minute transient occlusion. The extent of PPAR- knockdown by siRNA was quantified by reverse-transcription polymerase chain reaction 24 hours after reperfusion.
Intraventricular Infusion of PPAR- and 14-3-3 Recombinant Proteins
Rats were anesthetized with chloral hydrate (360 mg/kg IP; Sigma-Aldrich). The brain infusion cannula was inserted into the right ventricle of the brain at a point located 2 mm lateral and 2 mm posterior to the bregma and at a depth of 3 mm from the cortical surface. The osmotic pump (ALZET 1007D, DURECT Corp, Cupertino, Calif) with a brain infusion kit II was installed in a subcutaneous pocket on the lateral back of the rats. An instant adhesive gel (Loctite 454, Henkel, Düsseldorf, Germany) was used to affix the infusion cannula to the skull, and the wound was closed with sutures. Recombinant PPAR- (Cayman), His-tagged 14-3-3 (ATGen, Korea), or vehicle was infused continuously for 72 hours (1.2 µg/d at a rate of 0.5 µL/h) via the osmotic pump before I/R.
RNA Isolation, Reverse Transcription, and Polymerase Chain Reaction
Total RNA isolation and reverse-transcription polymerase chain reaction amplification were performed as described previously12 (Data Supplement).
Two-Dimensional Gel Electrophoresis and Mass Spectrometry
Rat brain was homogenized, and 130 µg of the supernatant proteins was subjected to 2D gel electrophoresis analysis (Data Supplement). Gels were stained and analyzed by ImageMaster 2D analysis software (GE Healthcare Bio-Sciences Corp, Piscataway, NJ). The gel images were normalized according to the total quantity in the analysis set. An expression intensity ratio >2.0 was considered a significant change. Spots with significant changes were removed and analyzed by liquid chromatography and tandem mass spectrometry (Data Supplement).
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation assay was performed as described previously18,19 (Data Supplement).
Statistical Analysis
ANOVA was used to compare the expression of proteins or infarct volumes. The level of significance for differences between groups was further analyzed with post hoc Fisher’s protected t tests by GB-STAT 5.0.4 (Dynamic Microsystems, Inc, Silver Springs, Md). P<0.05 was considered significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Rosiglitazone-PPAR-
Protects Brain Against I/R Injury in a Rat ModelTo evaluate the in vivo neuroprotective effect, rosiglitazone was injected intraventricularly immediately after 30-minute ischemia, and the infarct volume at the ipsilateral brain was measured 24 hours later. There was no significant difference in physiological variables between vehicle control and rosiglitazone-treated groups (supplemental Table I). Rosiglitazone at 0.5 to 150 ng significantly reduced the infarct volume. The maximal reduction occurred at 50 ng, whereas at higher concentrations (300 and 500 ng), rosiglitazone no longer had a protective effect (Figure 1A). We used 50 ng of rosiglitazone in the remaining studies. Infusion of rosiglitazone 2 hours after reperfusion was still effective in reducing infarct volume (Figure 1B). The protective effect of rosiglitazone was blocked by GW9662 (Figure 1C). Intraventricular injection of a PPAR-
siRNA for 24 hours suppressed PPAR- mRNA in normal brain tissues (supplemental Figure I) and in ischemic brain tissues compared with scRNA injection (Figure 1D). PPAR- siRNA injection immediately after the 30-minute ischemia abrogated the protective effect of rosiglitazone in a concentration-dependent manner, whereas scRNA had no effect (Figure 1E). Intraventricular infusion of PPAR- proteins (5 µg) for 72 hours before I/R reduced the infarct volume by >60% compared with control (Figure 1F). The essential role of PPAR- was further evaluated in mice with heterozygous knock-in of a PPAR- dominant-negative mutant, P465L (L/+). Wild-type or L/+ mice were subjected to 30 minutes of ischemia followed by 24 hours of reperfusion. Compared with wild-type mice with a mean infarct volume of 8 mm3, the infarct volume in L/+ mice was increased by 
80% (Figure 1G).
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The antiapoptotic effect of rosiglitazone was evaluated in ischemic rat brain tissues. Rosiglitazone injection immediately after 30-minute ischemia reduced cleaved poly(ADP-ribose) polymerase (PARP) and activated caspase 3 and caspase 9 compared with vehicle (DMSO; Figure 2A). PPAR- siRNA abrogated the antiapoptotic action of rosiglitazone (Figure 2A), whereas infusion of recombinant PPAR- proteins suppressed the apoptotic changes (Figure 2b). These results indicate that rosiglitazone protects brain from I/R-induced apoptosis.
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Proteomic Analysis Identified 14-3-3 Upregulation by Rosiglitazone in Ischemic Brain
To identify proteins important in the rosiglitazone–PPAR- signaling pathway, we analyzed ischemic brain tissues by proteomics. Rats were subjected to a 30-minute period of transient ischemia. Rosiglitazone or vehicle was administered immediately after ischemia for 24 hours. Brain was isolated and homogenized, and proteins were analyzed by 2D gel electrophoresis. A spot with a very large increase in density in rosiglitazone versus control (5.5-fold) was identified (Figure 3A). The spot was removed, and the protein identity was analyzed by liquid chromatography and tandem mass spectrometry. The protein matched 14-3-3. Western blot analysis confirmed that rosiglitazone pretreatment restored the suppressed 14-3-3 proteins in ischemic brain tissues (Figure 3B). To determine whether 14-3-3 upregulation in brain tissues is PPAR- dependent, we analyzed 14-3-3 proteins in the ipsilateral brain 24 hours after infusion of rosiglitazone or DMSO. Rosiglitazone increased 14-3-3 proteins by >3 fold (Figure 3C). This increase was inhibited by PPAR- siRNA. Infusion of recombinant PPAR- proteins also increased 14-3-3 (Figure 3D). Furthermore, 14-3-3 proteins in L/+ mouse brain were reduced by 50% compared with wild-type brain (Figure 3E).
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Rosiglitazone Upregulates 14-3-3 Transcription
To elucidate the mechanism by which 14-3-3 expression is upregulated, we used N2-A neuroblastoma cells as a model. Cells were incubated with rosiglitazone at increasing concentrations, and 14-3-3 proteins were analyzed 24 hours later. Rosiglitazone increased 14-3-3 proteins in a concentration-dependent manner (Figure 4A). This increase was blocked by GW9662 (Figure 4B) or PPAR- siRNA (Figure 4C). Transfection with PPAR- plasmids for 24 hours increased 14-3-3 proteins in a concentration-dependent manner (Figure 4D).
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We have previously cloned an 1.6-kb 5'-flanking region of the 14-3-3 gene that harbors 3 PPREs between –1348 and –1625.18 To determine the involvement of PPREs in rosiglitazone-induced 14-3-3 upregulation, we transfected N2-A cells with this promoter fragment (p1625-LUC) or a 5'-deletion mutant in which the PPREs were removed (p1348-LUC). Rosiglitazone increased the p1625 promoter activity in a concentration-dependent manner (supplemental Figure IIa) but did not increase p1348 activity (Figure 4E). Overexpression of PPAR- by transient transfection of PPAR- plasmid (Data Supplement Figure IIb) also increased the p1625 promoter activity (Data Supplement Figure IIc), which was abrogated when the PPRE region was deleted (Figure 4f). Chromatin immunoprecipitation assays showed that rosiglitazone induced binding of PPAR- but not PPAR-β/
to the PPRE-harboring region between –1554 and –1854 of the 14-3-3 promoter, despite expression of both PPAR proteins in this cell line (data not shown; Figure 4G). These results indicate that rosiglitazone selectively induced binding of PPAR- to 14-3-3 PPREs, thereby activating the 14-3-3 promoter and upregulating 14-3-3 protein expression.
14-3-3 Protects Brain From I/R Injury
To determine the role of 14-3-3 upregulation in controlling I/R-induced infarction, we infused rosiglitazone with 14-3-3 siRNA or control scRNA immediately after the 30-minute ischemia. Reduction of the infarct volume by rosiglitazone was abrogated by 14-3-3 siRNA in a concentration-dependent manner (Figure 5A). 14-3-3 mRNA in siRNA-treated brain was reduced compared with scRNA control (Figure 5A, inset). Conversely, overexpression of 14-3-3 (Figure 5B, inset) by infusion of recombinant 14-3-3 proteins for 72 hours before I/R reduced infarct volume in a dose-dependent fashion (Figure 5B).
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Rosiglitazone Prevents Neuronal Apoptosis via PPAR- to 14-3-3 Pathway
To confirm that ligand-activated PPAR- prevents neuronal apoptosis via 14-3-3 upregulation, N2-A cells were subjected to OGD for 3 hours followed by reoxygenation for 24 hours (H3R24), and cells with apoptosis were analyzed by flow cytometry. OGD (H3R24) increased the percentage of apoptotic cells, which was reduced by pretreatment with rosiglitazone (Figure 6A). The effect of rosiglitazone was abrogated by GW9662 (Figure 6A). OGD enhanced PARP cleavage and caspase 3 and 9 activation, which were suppressed by rosiglitazone and reversed by GW9662 (Figure 6B). To ascertain the antiapoptotic action of ligand-activated PPAR-, we transfected N2-A with PPAR- siRNA and analyzed apoptotic changes. PPAR- in N2-A cells was functional, as evidenced by activation by rosiglitazone of a PPAR promoter construct, ACO-(PPRE)4-Luc, which was inhibited by GW9662 (supplemental Figure IIIa). PPAR- siRNA, which suppressed PPAR- proteins (supplemental Figure IIIb) and mRNA (supplemental Figure IIIc), abrogated the ACO-(PPRE)4 promoter activity induced by rosiglitazone (supplemental Figure IIId). The antiapoptotic effect of rosiglitazone was abrogated by PPAR- siRNA but not scRNA (Figure 6C). Treatment of N2-A with PPAR- vectors reduced the number of apoptotic cells (Figure 6D). These results are consistent with inhibition of apoptosis by rosiglitazone via PPAR-.
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To confirm that the neuroprotective effect of rosiglitazone and PPAR- overexpression is mediated by 14-3-3, we evaluated the effect of rosiglitazone on 14-3-3 levels in OGD-injured N2-A cells. 14-3-3 Proteins were detected in N2-A, and this expression was enhanced by rosiglitazone (Figure 7A). 14-3-3 Proteins were suppressed in OGD-treated cells but were restored by rosiglitazone, although this rescue was prevented by GW9662 (Figure 7A). Similarly, restoration of 14-3-3 proteins after OGD insults was abrogated by PPAR- siRNA but not scRNA (Figure 7B). Conversely, reduction of 14-3-3 proteins in OGD-treated cells was attenuated by PPAR- transfection (Figure 7C). These results suggest that rosiglitazone or PPAR- overexpression prevents 14-3-3 suppression by OGD insults.
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To determine whether 14-3-3 upregulation is essential for the antiapoptotic action of rosiglitazone and PPAR-, we treated cells with rosiglitazone in the presence of 14-3-3 siRNA or control scRNA and subjected them to OGD (H3R24). 14-3-3 siRNA suppressed 14-3-3 proteins in N2-A in a concentration-dependent manner (Data Supplement Figure IVa). The antiapoptotic effect of rosiglitazone and PPAR- overexpression was abrogated by 14-3-3 siRNA but not scRNA (Figure 7D and 7E). We next determined whether 14-3-3 overexpression rescued cells from OGD-induced apoptosis. Overexpression of 14-3-3 by transient transfection (Data Supplement Figure IVb) significantly reduced the number of OGD-induced apoptotic cells (Figure 7F).
Ligand-Activated PPAR- Restores Phosphorylated Bad and Its Interaction With 14-3-3
The antiapoptotic action of 14-3-3 is attributed to the binding and sequestration of phosphorylated Bad (p-Bad). To understand how rosiglitazone–PPAR- protects against apoptosis via 14-3-3, we analyzed p-Bad under OGD stress and determined 14-3-3 and p-Bad interaction. p-Bad was detected in control cells but was then suppressed by OGD (H3R24) and partially restored by rosiglitazone (Figure 8A). The decline in p-Bad in OGD-treated cells correlates with that of 14-3-3, and rosiglitazone restored both proteins in a correlative fashion (Figure 8A). The decrease in 14-3-3 and p-Bad by OGD was attenuated by PPAR- overexpression (Figure 8B). To determine whether rosiglitazone enhanced binding of Bad by 14-3-3, we prepared cell lysates of OGD-injured N2-A treated with or without rosiglitazone. The lysate proteins were immunoprecipitated with a 14-3-3 antibody and analyzed by Western blotting. In the presence of rosiglitazone, a large quantity of p-Bad and 14-3-3 proteins was detected in the immunoprecipitate (Figure 8C), consistent with enhanced binding of p-Bad to 14-3-3 by rosiglitazone. To determine whether rosiglitazone and PPAR- attenuated Bad-induced mitochondrial damage, we measured mitochondrial membrane potential (MMP) by flow cytometry using JC-1 fluorescent probe. OGD (H3R12) caused a significant reduction of normal MMP cells that was attenuated by rosiglitazone (Figure 8D) or PPAR- overexpression (Figure 8E). The protective action of either treatment was abrogated by 14-3-3 siRNA and not control scRNA (Figure 8D and 8E). Furthermore, 14-3-3 overexpression attenuated OGD (H3R12)-induced MMP disruption (Figure 8F).
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Discussion |
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In the present study, we identified a novel transcriptional mechanism by which thiazolidinediones such as rosiglitazone protect against ischemic neuronal apoptosis and cerebral infarction. The present results provide strong evidence for an essential role of PPAR-
–mediated 14-3-3 upregulation in neural protection. Suppression of PPAR- with RNA interference or inhibition of PPAR- with pharmacological inhibitors abrogates the protective action of rosiglitazone in vivo and in vitro. Furthermore, PPAR- transfection or infusion of PPAR- proteins alone attenuates neuronal apoptosis and infarct volume in rats, whereas knock-in of a PPAR- mutant (P465L) aggravates infarction in mice. These results suggest that PPAR- activated by exogenous ligands such as rosiglitazone or certain endogenous ligands plays an essential role in protection against ischemic and hypoxic insults. The present data provide strong evidence for direct activation of 14-3-3 transcription by PPAR-. We showed that PPAR- binds to the PPRE-harboring region of 14-3-3 promoter and upregulates 14-3-3 expression. Importantly, rosiglitazone restored 14-3-3 expression in hypoxia-injured cells or brain tissues. Reduction of 14-3-3 expression was detected recently by proteomic analysis in neonatal rat hypoxia/ischemia,20 but to the best of our knowledge, this has not been reported in ischemic stroke. Because reduction of 14-3-3 makes the neural tissues vulnerable to damage, restoration of 14-3-3 by rosiglitazone contributes to its protective action. The effect of rosiglitazone on 14-3-3 restoration is PPAR- dependent. These results indicate that ligand-activated PPAR- drives 14-3-3 expression and increases cellular 14-3-3 proteins in normal and ischemic tissues. The present results further show that the protective actions of rosiglitazone or PPAR- overexpression depend on 14-3-3 upregulation. Knockdown of 14-3-3 renders rosiglitazone or PPAR- ineffective in protecting neural tissues, whereas 14-3-3 overexpression renders cells resistant to apoptosis. Furthermore, direct intraventricular injection of 14-3-3 or PPAR- proteins attenuates the I/R-induced infarction in a dose-dependent manner. Intraventricular injection of growth factors, antibodies, or inhibitory peptides was previously shown to control ischemic brain lesions.21,22 It is unclear how the injected proteins or peptides work. It was proposed that they may enter cells via endocytosis. Taken together, the results indicate that PPAR-–mediated 14-3-3 upregulation plays a crucial role in protection against neuronal apoptosis and cerebral infarction. This transcriptional pathway represents a new paradigm of cell and tissue protection.
Recent reports from our laboratory indicate that PPAR- ligands protect endothelial cells and colon cancer cells from apoptosis by inducing PPAR- binding to PPRE, thereby enhancing 14-3-3 proteins and increasing Bad sequestration.18,23 Promoter analysis reveals that ligand-activated PPAR- binds to a similar PPRE region as ligand-activated PPAR-.18 A C/EBP (CCAA/enhancer binding protein) binding site was also implicated in PPAR-–mediated 14-3-3 expression.24 Thus, the protective actions of these 2 PPAR isoforms are mediated by a similar transcriptional mechanism. We provide evidence in the present study that rosiglitazone selectively activates PPAR-–mediated 14-3-3 upregulation. It does not induce PAPR- binding to PPRE sites. It will be important to determine whether PPAR- ligands such as prostacyclin also selectively activate PPAR-–mediated 14-3-3 upregulation. Another important issue to be investigated is whether there exists cellular and tissue specificity for PPAR-– versus PPAR-–mediated 14-3-3 upregulation by their respective ligands. For example, it is unclear whether PPAR- ligands are capable of promoting endothelial cell survival via the PPAR-
14-3-3 pathway, nor is it clear whether PPAR- ligands protect against I/R injury via PPAR-–mediated 14-3-3 upregulation. Nevertheless, findings from the present study together with results from previous studies suggest a potential synergistic interaction between PPAR- and PPAR- in cytoprotection via similar transcriptional activation of 14-3-3, although this is dependent on their respective ligands.
14-3-3 Is a member of the 14-3-3 protein family,25 which binds diverse proteins and functions as a scaffold to facilitate or attenuate the activities of the binding proteins.26 Recently, 14-3-3 has been shown to colocalize with a variety of specific pathological deposits, such as neurofibrillary tangles, Pick bodies, and Lewy bodies.27 Moreover, upregulation of 14-3-3 immunoreactivity was noted in human brain infarction and was considered to be involved in postischemic cell survival and astrogliosis.28,29 However, the physiological functions or pathological roles of each given 14-3-3 isoform are generally unknown. In the present study, we identified the 14-3-3 isoform as an important protector of brain. 14-3-3 Has previously been shown to bind p-Bad; it sequesters p-Bad in the cytoplasm and thereby prevents Bad-induced apoptosis via the mitochondrial pathway.30–32 In the present study, we demonstrated a quantitative correlation between 14-3-3 proteins and p-Bad and an enhanced binding of p-Bad by PPAR-–mediated 14-3-3 upregulation. Furthermore, elevated 14-3-3 contributes to normalization of MMP. Taken together, these results indicate that 14-3-3 upregulation by ligand-activated PPAR- enhances p-Bad sequestration, thereby reducing Bad translocation to mitochondria to disrupt MMP and induce apoptosis.
Rosiglitazone exerted a dose-related biphasic effect on infarct volume (Figure 1A). The U-shaped dose response, which was named hormesis, has been noted with pharmacologically active compounds, including corticosteroids and receptor agonists.33,34 The underlying mechanisms for these biphasic actions are largely unknown. Because we were interested in understanding how rosiglitazone protects I/R brain injury, we focused in the present study on the concentrations that exert protective actions. It remains unclear why rosiglitazone at high doses (>300 ng) loses its protective effect, nor is it known whether at high doses, rosiglitazone is still active in PPAR-–dependent upregulation of 14-3-3 expression. Further studies are needed to resolve this complex issue.
In summary, we have discovered a transcriptional pathway that is critical for protection against neuronal apoptosis and cerebral infarction. PPAR-–mediated 14-3-3 upregulation represents an important transcription mechanism by which PPAR- agonists protect tissues from I/R injury and is a potential therapeutic target for important human diseases such as ischemic stroke and myocardial infarction.
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Acknowledgments |
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The authors wish to thank the Core Facility Laboratory of Institute of Biomedical Sciences, Academia Sinica for technical assistance with liquid chromatography and tandem mass spectrometry analysis. We thank Dr Chiun-Gung Juo for data consultation.
Sources of Funding
This study was supported by grants from the National Science Council, Academia Sinica, National Health Research Institutes in Taiwan, and the US National Institutes of Health (NS-23327 and HL-50675 to Dr Wu; DK67320 to Dr Maeda; HL-68878, HL-75397 and HL-89544 to Dr Y. Eugene Chen).
Disclosures
None.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.812537/DC1.
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