Advanced Oxidation Protein Products Induce Chondrocyte Apoptosis Via Receptor For Advanced Glycation End Products-mediated Redox-dependent Intrinsic Apoptosis Pathway

BackgroudArticular cartilage is the tissue that lines the surfaces of diarthrodial joints,which allows a smooth,painless,low-frictional movement of synovial joints.The function of articular cartilage is maintained by a limited number of chondrocytes.Chondrocytes apoptosis plays a key role in the process of cartilage destruction in rheumatoid arthritis(RA).Oxidative stress(OS)occurs due to an imbalance between reactive oxygen species(ROS)generating and scavenging activities.Increase level of ROS is easy to damage proteins and other cellular components.Extensive OS is prevalent in several chronic inflammatory diseases including RA.Increasing ROS generation can disrupt the mitochondrial membrane potential(??m)and activate caspases family,result in apoptosis of chondrocytes in the pathologic process of RA.Therefore,ROS plays an important role in the pathogenesis of cartilage damage in RA.Advanced oxidation protein products(AOPPs),are dityrosine-containing and cross-linking protein products formed by the reaction of proteins with chlorinated compounds during OS.The accumulation of AOPPs can be found in OS-related diseases,such as diabetes mellitus,chronic kidney disease,coronary artery disease,and RA.AOPPs represent not only a marker of oxidative stress but also an inflammatory mediator,which is involved in a number of pathophysiological conditions,mainly by the nicotinamide adenine dinucleotide phosphate(NADPH)oxidases-dependent redox mechism.Recently,we have been demonstrated that AOPPs can induce intestine epithelial cell apoptosis,inhibit proliferation and differentiation of rat osteoblast-like cells,and stimulate inflammatory response of rat fibroblast-like synoviocytes.As a receptor of AOPPs,receptor for advanced glycation end products(RAGE)regulates a number of cell processes of pivotal importance like apoptosis,inflammation,proliferation and autophagy.RAGE was demonstrated as a receptor for AOPPs by several recent studies in which AOPPs can induce vascular endothelial dysfunction,promote podocyte apoptosis and depletion via a RAGE mediated signaling.Intriguingly,AOPPs can also upregulate the expression levels of almost all components of the renin-angiotensin system(RAS)in cultured proximal tubular epithelial cells via the scavenger receptor CD36.Therefore,these above findings suggested that AOPPs can stimulate cell signals via different receptors in a cell-type-specific manner.It has been reported that AOPPs can induce apoptosis in a variety of cell types,such as podocyte and cardiomyocyte.Although accumulation of AOPPs is prevalent in patients with RA,the effect of AOPPs accumulation on chondrocytes remains unclear.It was demonstrated that chondrocytes from newborn murine provide a valid model of the representative human chondrocytes.Hence,the current study was designed to investigate whether AOPPs have an effect on chondrocytes apoptosis in vitro model of newborn rat chondrocytes.Matedals and methods1.AOPPs-RSA preparation and determinationAOPPs-Rat Serum Albumin(AOPPs-RSA)was prepared according to previous described procedure.Briefly,Rat Serum Albumin(RSA)solution(20 mg/ml,Sigma,St Louis,MO,USA)was exposed to 40mmol/L of HOCL(Fluke,Buchs,Switzerland)for 30 mins at room temperature and then dialyzed against PBS four times at 4? for 24 h to remove free HOCL.All the preparations were passed through a Detoxi-Gel column(Thermo,Massachusetts,USA)to remove endotoxin.An amebocyte lysate assay kit(Sigma,USA)was used to determine the level of endotoxin in both AOPPs-RSA and unmodified RSA group,as well as the concentration of endotoxin in those below 0.025 EU/ml.AOPPs contents in the preparations were determined with an OxiSelect AOPP Assay Kit(Cell Biolabs,San Diego,CA,USA).AOPPs content in the AOPPs-RSA and unmodified RSA were 51.10±4.79 ?mol/g protein and 0.35±0.81 ?mol/g protein respectively.AOPPs content in the AOPPs-RSA and unmodified RSA were 51.10±4.79 ?mol/g protein and 0.35±0.81 ?mol/g protein respectively.2.Rat chondrocyte culture and treatmentIt was demonstrated that chondrocyte from newborn murine provide a valid model of the representative human chondrocyte.Fresh cartilage was isolated aseptically from both knee joints of newborn Sprague-Dawley rats and washed with phosphate-buffered saline(PBS).After that it was minced and digested in 0.25%trypsin(Gibco,Life Technologies,California,USA)for 30 mins,the tissues were further digested in a solution of 0.2%collagenase ?(Sigma,St Louis,MO,USA)in DMEM(Gibco,Life Technologies,California,USA)at 37? for 3 h and then the cells were centrifuged at 1000g for 5 mins.The released articular chondrocytes were resuspended in DMEM containing 10%FBS(Gibco,Life Technologies,California,USA)and antibiotics(100 IU/ml penicillin,100 IU/ml streptomycin,Gibco,Life Technologies,California,USA),then seeded onto a 25cm2 culture flask at 37 ? in a humidified atmosphere of 5%CO2-95%air.Up to approximately 80-90%confluency,chondrocytes were passaged at a ratio of 1:3.To avoid the phenotype loss,only passages 1 chondrocytes were used in our study.This study was approved by the Institutional Animal Care and Use Committee of Southern Medical University(Guangzhou,China).In most of the experiments,chondrocytes were extensively washed with PBS,cultured in DMEM without serum for 12h,and then stimulated by adding control medium,various concentrations of AOPPs-RSA or unmodified RSA in the presence or absence of catalase(100U/ml),superoxide dismutase(SOD,100U/ml),Rotenone(100?mol/l),TIFA(10?mol/l),L-NAME(10?mol/l),Z-VAD-FMK(20?M,Cell permeable pan caspase inhibitor)were all purchased from Beyotime(China),diphenyleneiodonium(DPI,10?mol/l),apocynin(1000?mol/l)were purchased from Sigma-Aldrich(St.Louis,MO,USA).Anti-RAGE neutralizing antibodies(20 mg/ml for 2 h;R&D Systems,Minneapolis,MN,USA),soluble RAGE peptide and Anti-CD3 6 neutralizing antibodies(20 mg/ml for 2 h;Abcam,Cambridge,UK)were also pretreatment in chondrocyte cultures before AOPP-RSA added.3.Cell apoptosisThe chondrocytes were placed in 6-well culture plates at 5×105 cells per well.The apoptosis rate of chondrocytes was detected by Annexin V/PI double staining kit(KeyGen Biotech,China)according to the manufacturer's instructions.Briefly,the cells of different groups were collected by trypsinization and centrifugation,then washed witih ice-cold PBS twice and resuspended in 500 ?l binding buffer.5?l Annexin V and 5?l PI were added and further incubated in the dark for 15 min at room temperature.The apoptosis rate was analyzed by flow cytometry(BD,LSRFortessa,USA).The cells staining positive for annexin V and negative for propidium iodide and those positive for double staining were identifed as apoptotic cells in each sample.They were counted and represented as a percentage of the total cell populations.Cells were cultured,treated,and stained as stated above.Sequentially,the stained cells were observed under a confocal microscope(Olympus,Center Valley,PA,USA).We also analyzed cell apoptosis using the Cell Death Detection ELISA kit(Roche Applied Science).The assay is based on a quantitative sandwich-enzyme-immunoassay principle,using rat monoclonal antibodies directed against DNA and histones.This allows the specific detection and quantitation of mono-and oligonucleosomal fragmented DNA that released into the cytoplasm of cells undergoing apoptosis.4.Determination of intracellular ROS generationThe level of intracellular ROS was assessed by fluorescence microplate reader with the probe 2',7'-dichlorofluorescein diacetate(DCFH-DA Sigma,USA),which oxidizes to fluorescent dichlorofluorescein(DCF)in the presence of ROS,as described previously.Briefly,chondrocyte were pre-incubated for 30 min with 1 nmol/L DCFH-DA in PBS lacking Ca2+ and Mgt+.The cells were then incubated with various concentrations of AOPPs-RSA at indicated times in darkness.Fluorescence intensity was measured on a SpectraMax M5 system(Molecular Devices,California,USA).The excitation and emission wavelength were 488nm and 525nm respectively.Sequentially,The chondrocyte were seeded into sterile Petri dish(diameter of 35 mm)special for the detection of laser scan confocal microscopy,at a density of 1×104 cells per well.Cells were cultured and treated as stated above.Then the fluorescence intensity was observed under a confocal microscope.5.Evaluation of mitochondrial membrane potential(??m)To assess changes of the ??m,we used the fluorescent dye JC-1(Beyotime,China).The chondrocytes were seeded into sterile Petri dish(diameter of 35 mm)special for the detection of laser scan confocal microscopy,at a density of 2×104 cells per well.Following the treatment of 100ug/ml AOPPs-RSA on chondrocyte,the cells were incubated with the mixture of 500?l JC-1 staining fluid and 500?l cell culture medium in the dark at 37? for 30 min.Subsequently,the cells were washed twice with the staining buffer preserved in 40C.Eventually,lml cell culture medium was added into the each specimen and the cells were observed under the laser scan confocal microscope.The level of ??m was represented by red fluorescence ratio to green fluorescence.6.Western blot analysisAt the indicated time points,stimulated or control chondrocyte monolayers were washed three times with PBS solution and whole cell proteins were extracted by incubation with lysis buffer(pH 7.4,50 mm Tris,150 mm NaCl,1%NP-40,0.25%deoxycholic acid,1%protease inhibitor cocktail and 1%DTT)on ice for 15 min before cell debris was removed by centrifugation.After their amounts(10-50 ?g of protein per lane)were adjusted equal,total proteins were separated by SDS-PAGE(10-15%gels)under reducing conditions.After the separated proteins were transferred onto polyvinylidene difluoride(PVDF)membranes,they were pre-incubated in blocking buffer(5%BSA)for 60 min at room temperature,followed by incubation with primary antibodies against p22phox,p47phox(1:200,Santa Cruz,CA,USA),?-actin,GAPDH,Nox2,Nox4(1:1000,Abcam Cambridge,UK),Bcl-2,Bax,cytochrome c,chop,caspase-12,caspase-9,and cleaved caspase-3(1:500,Cell Signaling Technology,Beverly,MA,USA)overnight at 4?.Membranes were washed three times with TBST(TBS with Tween),and subsequently incubated with the DyLight 800-Labeled antibody(1:15,000)for 1 h at 37?.They were finally washed three times with TBST.The blots were visualized by enhanced chemiluminescence using LI-COR Odyssey(Lincoln,Nebraska USA).Densitometric measurements of the scanned bands were performed using Gel-Pro analyzer software.Each band was scanned three times,and the mean band intensity was obtained.Data were expressed as mean(S.D.).7.Immunoprecipitation and immunoblotting analysisImmunoprecipitation and immunoblotting were performed respectively to analyze the phosphorylation of p47phox and interaction of p22phox with p47phox,NOX2 or NOX4 as described previously.Briefly,the cell lysates were preabsorbed with protein A/G agarose beads(Santa Cruz Biotechnology)and incubated with anti-p47phox or anti-p22phox antibody.The immunocomplexes were resolved on SDS-PAGE and then transferred to PVDF membranes.The membranes were then incubated with primary anti-phosphoserine antibody(1:1000,Abcam Cambridge,UK)to analyze the phosphorylation of p47phox;and the primary antibodies against p47phox,NOX2 or NOX4 respectively to analyze interaction of p22phox with p47phox,Nox2 and Nox4.The values were normalized to the amount of total p47phox or p22phox per sample.8.Statistical analysisResults were expressed as mean ± standard deviation.Statistical differences between means for different groups were compared using one-way ANOVA(analysis of variance).Multiple comparisons were performed using the LSD method or Dunnett's C method.Comparing several treatments with control group was evaluated by the Dunnett's T3 procedure.Statistical analyses were conducted with SPSS 13.0 software(SPSS Inc.,Chicago,IL,USA).Results:1.Increased extracellular AOPPs triggered chondrocyte apoptosis in vitroThe effect of AOPPs on the apoptosis of rat chondrocyte was examined by AnnexinV/PI dual staining.Flow cytometry revealed that AOPPs induced apoptosis of chondrocyte in a dose-dependent manner.An evident elevation of apoptosis rate at different concentration(25-200ug/ml)was noted following the treatment of AOPPs for 24h.Unmodified RSA(200ug/ml),under similar conditions,failed to trigger chondrocyte apoptosis.Increased AnnexinV/PI-labeled cells after AOPPs stimulation was also shown by confocal microscopic analysis.Moreover,AOPPs-induced chondrocyte apoptosis could be largely blocked by pretreatment of NADPH oxidase inhibitor apocynin for 1h.AOPPs-induced apoptosis in chondrocyte was also confirmed by the increased levels of mono-and oligonucleosomal fragmented DNA in cytoplasm in a dose-and time-dependent manner.2.AOPPs activated NADPH oxidase via RAGE dependent manner,instead of CD36,in chondrocyteAOPPs have been shown to activate cell signals via RAGE and/or CD36.Previous studies demonstrated that NADPH oxidase activation triggered ROS production mediate AOPPs-induced cell apoptosis.In this study,chondrocyte treated with AOPPs(100ug/ml)showed significantly increased expression of NADPH oxidase subunits p22phox,p47phox,Nox2,and Nox4 compared with chondrocyte incubated with medium alone and native RSA(100ug/ml).Then,we evaluated NADPH oxidase activity in chondrocyte cultures stimulated with AOPPs.p47phox phosphorylation and the amount of p22phox-p47phox,p22phox-Nox2,and p22phox-Nox4 complex formation rapidly increased in chondrocyte treated with AOPPs(100ug/ml).Eventually,AOPPs-induced increased expression of NADPH oxidase subunits and NADPH oxidase activation was significantly suppressed by blockade of RAGE,instead of CD36,which support that these effects was mediated by RAGE but not CD36.3.AOPPs induced ROS generation was mediated by NADPH oxidase and RAGE but not CD36Incubation of chondrocyte cultures with AOPPs induced dose-and time-dependent increase in ROS production.The increased ROS generation was also observed by a confocal microscope.Further,AOPPs-induced ROS production could be largely blocked by pretreatment of NADPH oxidase inhibitor apocynin.To further verify the sources of ROS generation,chondrocyte cultures were pretreated with the inhibitors of various enzymatic systems involved in ROS generation before AOPPs stimulation.AOPPs-induced ROS production was significantly suppressed by ROS scavenger(SOD and catalase)and the NADPH oxidase inhibitor(DPI and apocynin),but not by inhibitor of nitric oxide synthase(L-NAME)and mitochondria inhibitor(rotenone and TIFA).Lastly,we verified that AOPPs-induced ROS production was significantly abolished by blockade of RAGE,but not CD36.4.AOPPs activated intrinsic apoptosis pathway in chondrocyteDiverse stimuli that affect either mitochondrial function or that cause reticulum(ER)stress can initiate apoptosis.To examine the potential pro-apoptotic effectors of AOPPs-induced apoptosis,we analyzed the mitochondrial dysfunction related mediators(Bax,Bcl-2 and cytochrome c)and ER stress-induced effectors C/EBP homologous protein(chop)and caspase12.Besides,we investigated common downstream effectors of these two cascades including caspase9,cleaved caspase3 and poly ADP-ribose polymerase(PARP).AOPPs treatment significantly increased in the amounts of Bax,cytochrome c,chop,cleaved caspase12,cleaved caspase-9,cleaved caspase-3,cleaved PARP and decreased the amounts of Bcl-2,full length caspase12,caspase9 and PARP in chondrocyte cytoplasm.To further investigate mitochondrial dysfunction was induced by AOPPs,the level of mitochondrial membrane potential(??m)was analyzed by confocal microscopy.AOPPs treatment reduced the level of ??m in chondrocyte,and this effect was attenuated by pretreatment with apocynin.5.AOPPs-triggered intrinsic apoptosis pathway was mediated via RAGE and NADPH oxidase-dependent ROS generation but not CD36To examine whether AOPPs-triggered activation of intrinsic apoptosis signal was transduct by RAGE and/or CD36,we preincubated chondrocyte with anti-RAGE neutralizing antibody,soluble RAGE peptide and anti-CD36 neutralizing antibody for 2h before AOPPs exposure(100ug/ml),and subsequently examined the expression of intrinsic apoptosis effectors.AOPPs-triggered intrinsic apoptosis pathway was dramatically inhibited by blockade of RAGE but not CD36,which support that the signal was transduct by RAGE yet not CD36.Moreover,pretreatment of scavenger of ROS(SOD and catalase)and NADPH oxidase inhibitor(DPI and apocynin)1h before treated with AOPPs(100ug/ml)in chondrocyte attenuated the activation of intrinsic apoptosis signal either,which suggest that NADPH oxidase-dependent ROS generation is the upstream of these cascades.6.AOPPs-induced chondrocyte apoptosis was mediated by RAGE,NADPH oxidase-dependent ROS production and caspases but not CD36Blockade of RAGE on chondrocyte before exposure to AOPPs(200ug/ml 24h),markedly attenuated AOPPs-induced chondrocyte apoptosis.However,Blockade of CD36 failed to have this effect.Further,AOPPs-triggered apoptosis was largely abolished in the presence of apocynin(a NADPH oxidase inhibitor),SOD(a ROS scavenger)and Z-VAD-FMK(a casepase inhibitor)(Figure.6B).Besides,these effects were further supported by tested the level of mono-and oligonucleosomal fragmented DNA in cytoplasm.These findings show that AOPPs-induced apoptosis of chondrocyte was mediated by NADPH oxidase,ROS,caspases and RAGE,but not CD36.Conclusions:In conclusion,our results show that AOPPs can induce the apoptosis of chondrocyte via RAGE,NADPH oxidase-dependent ROS generation and activation of intrinsic apoptosis pathway in vitro.This information implicates that accumulation of AOPPs might be involved in chondrocyte apoptosis of RA.AOPPs may represent a novel pathogenic factor that contributes to RA progression.Targeting AOPPs-related cellular mechanisms might emerge as a promising therapeutic option for patients with RA.