Eaf2 Protects Human Lens Epithelial Cells Against Oxidative Stress-induced Apoptosis By Wnt Signaling

1 IntroductionCurrently,cataract induced blindnessaccounts for about 50%of blindness cases worldwide.It is recognizedthat damage resulting from oxidative stress in the ocular lens is a major cause ofcataracts.The lens are the main target of oxidative stress.Research shows that oxidative stress induced by H2O2 can stimulate the apoptosis of human lens epithelial cells,and apoptosis of lens epithelial cells is considered as the the cytological basis of all types of cataract except for congenital cataract.Therefore,a better understanding of the mechanisms which drive H2O2-induced apoptosis in lens cells should provide clues regarding the cause of cataract formation.2PurposeIn this study,we investigated the role of Eaf2 in human lens epithelial(HLE)cells undergoingH2O2-induced apoptosis,as well as theassociated molecular mechanism.And investigate how Eaf2 protects against H2O2-induced cell death by inhibitingcaspase-3 enzyme activity and activating the Wnt3 signaling pathwayin HLE cells.3 Methods3.1 Cell culture.The HLE-B3 cells wereculturedin aMEM medium(Gibco,Carlsbad,CA,USA)supplemented with 20%FBS in a humidified atmosphere containing 5%CO2at 37?.3.2H2O2-induced apoptosis in HLE-B3 cells.HLE-B3 cells used in H2O2-induced apoptosisstudieswere placed into50?M ofH2O2 for different time periods(4 h,8 h,and 12 h).3.3 Vector construction and cell transfection.To induce Eaf2 overexpression,the fragment of Eaf2cDNA containing the full-length cDNA forthe human Eaf2gene wasisolated and cloned into pCDNA3.0 to generate pcDNA-Eaf2.The Wnt3a shRNA plasmid used to knockdown Wnt3a was purchased from Santa Cruz Biotechnology(Santa Cruz,CA,USA)and designatedas shWnt3a.Next,cells which overexpressed Eaf2 were identifiedand transfected with shWnt3a and shCon,respectively.3.4 Analysis of apoptotic cells.The H2O2-induced cell wereanalyzed by flow cytometry and calculated the ratio of cell apoptosis.3.5 RNA isolation and RT-PCR analysisTotal RNA was extracted from cells according to aprotocol included withthe TRIZOL Reagent.The cDNA was then synthesized.The 2-??Ct method was used to computethe amounts of human?-catenin,caspase 3,Eaf2,and Wnt3a mRNA formed.3.6 Western blot analysis.The protein level of?-catenin,caspase 3,Eaf2,and Wnt3a was detected by Western blot method.3.7 immunocytochemistryimmunofluorescence staining was used to detect the expression of Wnt3a in the cells.4 Results4.1 Construction of an apoptoticHLE cellmodelinduced byH2O2.Theanalysis showedthat,when compared with the control cells,cell populations exposed to 50 ?M of H2O2 for 4h,8h,and 12 h all hada significantly higher percentage of apoptotic cells,and the apoptosis rate increased depend on the time exposed to H2O2.Our results showed thatthe levels of caspase 3mRNAand protein in HLE-B3 cells becamesignificantly up-regulated when the cells were exposed toH2O2.In contrast,HLE-B3 cells exposed to H2O2had significantly reduced levels of Eaf2mRNA and protein,as confirmed by RT-PCR and western blot analyses.4.2 Overexpressed Eaf2 alleviatesH2O2-induced apoptosis in HLE cells.Our results showed that a significantly lower percentage ofcells with high levels of overexpressed Eaf2 had entered apoptosis(p<0.001).The overexpression of Eaf2 also decreased the activity of LDH(p<0.05).RT-PCR and western blot results showed that,when compared with the control cells,the levels of caspase 3?Bax mRNA and protein were significantly decreased;the levels of Bcl-2 mRNA and protein were significantly increased,indicating that Eaf2 inhibitH2O2-induced apoptosis by regulating the expression of apoptosis related proteins.And the levels ofWnt3a??-catenin mRNA and protein were significantly increased;the levels ofp-GSK3(3 protein were significantly increased the levels ofGSK3? protein were significantly decreased in the cells withhigh levels of overexpressed Eaf2,indicating that Eaf2 has a close relation to Wntsignaling.An immunofluorescence assay study further confirmed the up-regulation ofWnt3a in Eaf2-overexpressing cells when compared with the control cells4.3 Eaf2 inhibitesH2O2-induced apoptosis in HLE cells by regulating Wnt3a.A statistical analysis indicatedthat the knockdown of Wnt3a significantlyincreasedthe overall percentage of Eaf2 overexpressingHLE-B3 cells undergoingapoptosis.Additionally,the suppressed release of LDH in Eaf2 overexpressed cells was induced by the knockdown of Wnt3a.The RT-PCR and western blotresults showed that,when compared with the control cells,the levels of caspase 3?Bax mRNA and protein were significantly increased;the levels ofBcl-2mRNA and protein were significantly decreased;the levels of?-catenin?p-GSK3P protein were significantly decreased;the levels ofGSK3(3 protein were significantly increased in the cells knockdown of Wnt3a.That indicate knockdown of Wnt3a may impact the protection of Eaf2 and Eaf2 protects human lens epithelial cells against oxidative stress-induced apoptosis by Wnt signaling.5 conclusion5.1 H2O2can induce the apoptosis of lens epithelialcells.5.2The Eaf2 OE plasmid and shWnt3a plasmid can be effectively transfected in lens epithelial cells.5.3 Eaf2 protein may inhibit the level of lens epithelial cell apoptosis induced by H2O2.5.4 These data are the first to demonstrate that Eaf2gene transcription products can protect HLE cells from oxidative damage caused by exposure to H2O2.5.5 Eaf2 can reduce cell death induced by H2O2 by regulating caspase 3,Baxand Bcl-2 expression levels.5.6 Eaf2 protects human lens epithelial cells against oxidative stress-induced apoptosis by Wnt signaling.