| BaekgroudOxidative stress induced by free fatty acids(FFA) plays a key role in the development of cardiovascular diseases in metabolic syndrome.Excessive generation of reactive oxygen species(ROS) can cause tissue injury and cellular dysfunction by directly oxidizing and damaging DNA,proteins,and lipids as well as by activating several cellular stress-sensitive pathways such as nuclear factor-kappa beta,p38 MAPK,and JNK.Reducing ROS production and increasing antioxidant availability are important in preventing diabetic cardiovascular complications.The AMPK pathway responds to energy depletion by stimulating metabolism for ATP generation and plays an important role in controlling metabolism.It has been increasingly recognized that activation of this pathway has vascular protective effects; however,the mechanisms involved are not completely understood.Reactive oxygen species can activate the AMPK pathway.Previous studies have shown that activation of the AMPK pathway reduces intracellular ROS levels.However,it is still unknown whether the activation of the AMPK pathway may affect PA(palmitic acid) induced intracellular ROS levels in human aortic endothelial cells.The regulation of cellular redox balance is critically determined by the activity of Trx(thioredoxin) system.Some studies have shown that the expression of Trx was dramatically increased by several stimuli,especially the oxidative stress.It seems that Trx is an important antioxidant protein and protect the tissues and cells from several oxidative stress injuries.It is unclear that whether Trx is involved in the AMPK pathway reduced intracellular ROS production and how the AMPK pathway regulates the expression of Trx.FOXO transcription factors are important regulators of metabolism,cell-cycle progression,DNA repair,apoptosis,oxidative stress resistance,and longevity.The role of FOXO in regulating cellular functions under oxidative stress has been partially clarified.Recent findings found that FOXOs could regulate the expression of Gadd45 and MnSOD(manganese superoxide dismutase) directly and protect cell from oxidative injure,thus promote the DNA damage repair.This suggests that ROS can activate FOXO.Although FOXOs mediate ROS-induced apoptosis under lethal conditions,they can increase cell survival in response to physiologic oxidative stress, a function that is required for long-term regenerative potential and cell longevity. However,whether FOXO is the transcriptional factor of Trx and whether the AMPK pathway affects the activity of FOXO are still unclear.In the present study,we hypothesized that the activation of AMPK pathway may reduced the intracellular ROS levels by increasing the expression of antioxidant Trx. FOXO may be the transcriptional factor of Trx.Thus,the AMPK-FOXO pathway may be an important defense mechanism against excessive ROS levels induced by metabolic stress and could be a therapeutic target in treating cardiovascular diseases in metabolic syndrome.Methods1.Cell culture:Primary human aortic endothelial cells(HAECs) were cultured at 37℃in 5%CO2 in endothelial cell growth medium-2(EGM-2).The cells were transfected with siRNAs,treated with the AICAR or PA at various concentrations for the time periods indicated in the text.2.Preparation of fatty acid-albumin complexes:Saturated palmitic acid(PA) was dissolved in ethanol at 200 mM and then combined with 10%FFA-free, low-endotoxin BSA to final concentrations of 1-5 mM.The pH of all solutions was adjusted to approximately 7.5,and the stock solutions were filter-sterilized and stored at-20℃until used.Control solutions containing ethanol and BSA were prepared similarly.Working solutions were prepared fresh by diluting the stock solution(1:10) in 2%fetal calf serum-EBM.There was 1%BSA in all PA media;however,the PA/BSA ratio varied with the PA concentrations.3.siRNA-indueed gene silencing:Silencing gene expression was achieved using specific siRNA including,AMPK siRNA,Trx siRNA and FOXO3a siRNA. Transfection of HAECs with siRNAs was carried out using LipofectAMINETM 2000, according to the manufacturer's instruction.Transfected cells were then treated with PA and AICAR at the designated concentrations for the time periods indicated in the text.4.In Vitro Transfection of Plasmid DNA:HAECs were transfected with 0.5ug of FOXO3a plamid DNA including wide type(HA-FOXO3A WT),constitutively active(HA-FOXO3a TM) and dominant-negative(HA-FOXO3a TM deltaDM) The transfection was carried out using LipofectAMINETM 2000 according to the manufacturer's instructions.Transfected cells were then treated with PA and AICAR at the designated concentrations for the indicated time periods.5.Immunofluorescent staining and microscopy:Cells were grown on glass coverslips and treated with PA and AICAR.The coverslips were blocked with 1% BSA,incubated with the primary antibody,washed with PBS,and then incubated with Texas Red-labeled secondary antibody.The cells were incubated for 15 minutes with the DNA stain,4',6-diamidino-2-phenylindole dihydrochloride(DAPI;0.1 ug/mL).The slides were examined with a Leica DMLS epifluorescence microscope equipped with a Leica DC 100 digital camera,and the data were analyzed with Image-Pro Plus V4.5 software.6.Western blot analysis:Cell extracts were prepared with lysis buffer.Protein samples(15μg per lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes.The membranes were blocked,treated with primary antibody,washed,and then incubated with the secondary horseradish peroxidase-labeled antibody.Bands were visualized with Enhanced Chemiluminescence.The expression of cytokine protein was demonstrated by the ratio of integral optical density(IOD) between cytokine andβ-actin.7.Real-time quantitative PCR:Total RNA from treated cells was extracted with Trizol,according to the manufacturer's protocol.The mRNAs were reverse-transcripted into cDNAs using iScript cDNA synthesis kit.Real-time PCR was performed using iCycler iQ real-time PCR detection system.The mRNA levels were estimated from the value of the threshold cycle(Ct) of the real-time PCR adjusted by that ofβ-actin through the formula 2ΔCt(ΔCt=β-actin Ct-gene of interest Ct).8.Intracellular ROS level detection:Intracellular ROS level was determined using the oxidant-sensitive fluorogenic probe CM-H2DCFDA(5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate,acetyl ester).The slides were examined with a Leica DMLS epifluorescence microscope equipped with a Leica DC 100 digital camera and the data were analyzed with Image-Pro Plus V4.5 software.9.Trx Activity Assay:Trx activity was measured using the insulin disulfide reduction assay.The absorption at 412 nm was measured spectroscopically.T10.Chromatin immunoprecipitation assay:We used the ChIP assay kit, according to the Upstate Chromatin Immunoprecipitation Protocol.In brief,treated HAECs were first incubated with formaldehyde to cross-link DNA-protein complexes. Protein-DNA complex was immunoprecipitated with antibody-protein A-agarose slurry.(IgG served as the negative control.) The immunocomplex beads were washed, eluted and reversed the cross-link.The DNA was recovered by extraction with the phenol/chloroform/isoamyl alcohol mixture.The immunoprecipitated DNA was used as a template for PCR.The PCR products were separated by 1.5%agarose gel.11.Immunoprecipitation:Treated cells were lysed for 60 min in ice-cold extraction buffer.For immunoprecipitation,cleared cell lysates were incubated with the appropriate antibody precoupled to protein A/G-agarose beads at 4℃overnight. The beads were washed twice with extraction buffer and twice with extraction buffer containing 0.5 M LiCl.Proteins were eluted directly in SDS sample buffer for Western blot analysis.12.Kinase assays:AMPK was precipitated from cell lysate using an anti-AMPK antibody.AMPK-containing beads were incubated with recombinant FOXO 3 in kinase assay buffer supplemented with 100μM ATP for 20 minutes at 30℃.Samples were separated on a 10%SDS-PAGE and transferred to PVDF membranes. Anti-serine and anti-threonine antibodies were used to detect phosphorylated serines and threonines incorporated into the FOXO 3.Results1.AMPK reduces ROS levels induced by PA in endothelial cells:AICAR by itself had minimal effects on basal ROS levels.Palmitic acid significantly increased intracellular levels of ROS,an observation consistent with previous reports.The PA-induced increase of intracellular ROS levels was reduced by AICAR in a dose-dependent manner with up to a 60%reduction at the highest dose 500μM.This result indicates that activation of AMPK can reduce intracellular ROS levels. Additionally,suppression of AMPK by specific siRNA not only increased basal ROS levels,but also augmented PA-induced increase of ROS levels.Furthermore,AICAR induced reduction of ROS was abolished by AMPK siRNA.2.AMPK increases the expression of the antioxidant Trx:Activation of the AMPK pathway by AICAR,significantly up-regulated the expression of Trx in a dose-dependent manner in the absence and the presence of PA.Prolonged PA exposure decreased Trx expression.Importantly,knockdown of AMPK by its specific siRNA inhibited the basal Trx expression and the upregulation of Trx by AICAR, implicating the involvement of the AMPK pathway in Trx upregulation.3.Trx silencing prevents AICAR-induced ROS reduction:Trx siRNA not only increased basal ROS levels,but also amplified PA-induced increase of ROS levels.Furthermore,AMPK siRNA prevented AICAR induced reduction of ROS.4.AMPK regulates Trx expression at the mRNA level:Using quantitative RT-PCR,we found that AICAR significantly increased Trx mRNA in a dose-dependent manner.Knockdown of AMPK by its specific siRNA reduced basal Trx expression.Moreover,the AICAR-induced Trx mRNA was reduced in the presence of AMPK siRNA5.FOXO 3a is required for AMPK-induced upregulation of Trx:We identified FOXO 3a as one of these transcriptional factors that may mediate AMPKinduced Trx transcription.Silencing FOXO 3 with siRNA significantly prevented AICAR induced expression of Trx at both protein and mRNA levels.6.FOXO 3a binds directly to the Trx promoter:The promoter region in the Trx gene contains 6 putative FOXO 3a binding sites(tgAAAGAgtga at-1346/-1342, tgAAAGAagga at-1339/-1335,gaAAACAcaga at-1236/-1232,caAAATAccgc at -859/-855,ggAAACActga at-807/-803,and tgAAAGAacag at-613/-609).Results of the ChIP assay performed with a FOXO 3a antibody showed that FOXO 3a weakly binds sites 4 and 5,but strongly binds site 6.Importantly,the binding of FOXO 3a to site 6 was significantly increased by AICAR treatment,indicating that FOXO 3a may mediate AMPK-induced Trx transcription.7.AMPK increases FOXO 3a nuclear translocation:The immunostaining assay shows that AICAR significantly increased the nuclear translocation of FOXO 3a, which was prevented by AMPK siRNA.The in vitro kinase assay with purified AMPK as the kinase and recombinant FOXO 3a as the substrate showed that AMPK directly phosphorylated FOXO 3a at serine and threonine sites and that AICAR increased phosphorylation of FOXO 3a.8.FOXO 3a recruits p300 and forms a transcription activator complex on the Trx promoter:Using the ChIP assay,we found that AICAR treatment significantly increased p300 binding to the Trx promoter at consensus site 6.Using coimmunoprecipitation assay,we observed that FOXO 3a was associated with p300 and that the association was increased by AICAR treatment,indicating that p300 recruitment to the Trx promoter may be mediated at least in part by FOXO 3a.The double-ChIP assay showed that the Trx promoter sequence could be recovered from the immunocomplexes precipitated by FOXO 3a and p300 antibodies,indicating the simultaneous association of FOXO 3a and p300 within that region of the Trx promoter.Conclusions1.PA significantly increased intracellular ROS levels in human aortic endothelial cells(HAECs).2.The ativation of the AMPK pathway significantly reduced PA-induced intracellular ROS levels by increasing the expression of the antioxidant Trx.3.Transcriptional factor FOXO 3a mediated AMPK's effect on Trx expression.4.AMPK upregulated Trx transcription by increasing FOXO3a nuclear translocation and promoting its DNA binding to the Trx promoter.The activated FOXO3a may recruit p300 and form a transcription activation complex in the Trx promoter.5.AMPK-FOXO3a pathway has protective effects against cellular superoxide levels induced by metabolic stress and could be a therapeutic target in treating cardiovascular complications in diabetes.
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