The Pathway Of Oxidative Stress Promoting Lens Epithelial Cell Apoptosis

Objective:Cataract is one of the common age-related diseases.The unclear pathogenesis of cataract has become the bottleneck of its nonoperative treatment.The onl y consensus is that lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals.A signfficant proportion of lenses and aqueous humor taken from cataract patients have elevated H 2O2 levels.Because H2O2,at concentrations found in cataract,can cause lens opacification and produces a pattern of oxidation similar to that found in cataract,it is concluded that H2O2 is the major oxidant involved in cataract formation.SUMO,the small ubiquitin-related modifier was initiall y discovered to be a reversible post-translational modifier in yeast about a decade ago.Until now,at least four distinct SUMO proteins,SUMO1,2,3 and 4,have been identified in mammalian cells.Lens epithelium derived growth factor is a n ubiquitous and stress-inducible transcriptional nuclear protein that has important functions in regulating cell proliferation,differentiation,and apoptosis.PSIP1 was originall y identified as a transcriptional coactivator as well as a transactivator of stress response genes,thereby providing cytoprotection against various stressors.However,little is known about the role of SUMOylation and PSIP1 in the progression of lens disease,including cataract.This study found,for the first time,the crucial rol e of PSIP1,SUMO and SUMOylation of PSIP1 in response to oxidative stress in lens epithelial cell.Methods:Human lens epithelial cell line(SRA01/04 cell line)were obtained from Provincial Key Laboratory of Lens Research,Liaoning,China.Cultured SRA01/04 cells exposed to different concentrations(100μM,200μM,400μM,800μM and 1m M)of H2O2 for 1h and were incubated in a bumidified 37 ℃ incubator containing 5% CO2.Intracellular ROS level was measured by use of fluorescent dye dichlorofluorescin diacetate(H2-DCF-DA).A colorimetric MTS assay(Promega)was performed to measure cellular proliferation/viabilit y.RT-q PCR,Western blot and immunohistochemistr y detected t he level of SUMO1 and PS IP1 an d the differential expression in oxidative group.The sumoylation of PS IP1 was anal ysed b y co-immunoprecipitations.Construction of p EGFP-PSIP1 and p GL3Hsp27 luc plasmids.Mutation in SUMO1 binding sites of PS IP1 was made by site-directed mutagenesis.Luciferase activit y was anal yzed 72 h post-transfection using Dual-Luciferase Reporter Assay S ystem according to the manufacturer’s instructions.All experiments were repeated at least three times.All data were reported as mean wit h standard deviation(SD).Differences were assessed by an independent samples t-test.P<0.05 was considered statisticall y significant.SPSS 18.0 statistical software was used for statistical analysis.Results:1.Levels of ROS,cell survival and PCNA were elevated under oxidative stress.A significant increase of ROS as quantified by H2 DCF dye,decreased cell viabilit y and the higher expression of PCNA by Western blot was observed in human lens epithelial cells treated with 0.1 to 1 m M H2O2.2.Levels of SUMO1,SUMOylation and PS IP1 were elevated under oxidative stress.Our results indicated that SUMO1 and PS IP1 m RNA and proteins were increased significantly in h LECs cultured with hydrogen peroxide,and that significantl y higher than that of the control(P<0.001).Data also indicated a significant increase in Sumo1 conjugates was observed in cells treated with 0.4m M H2O2.3.PS IP1 is modified by Sumo1 under normal physiological conditions.Human LECs were transientl y transfected with EGFP-Sumo1,48 h later nuclear extracts were processed for co-immunoprecipitation.Data indicated endongenous PS IP1 was Sumoylated in vivo.4.Construction of p EGFP-PS IP1 plasmid.The plasmid was amplified,and the DNA was confirmed by sequencing.Data indicated p EGFP-PSIP1 eukaryotic expression vector can well over express EGFP-PSIP1 fusion protein in human lens epithelial cells.5.SUMOylation of PSIP1 under oxidative stress.Human LECs were transientl y co-transfected with His-Sumo1 and p EGFP-PS IP1,were anal yzed by immunoprecipitation a ssa y using antibody specific to PS IP1,immunoblotting with Sumo1 antibody.Data indicated the SUMOylation of PS IP1 decreased under oxidative stress.6.Construction of p EGFP-K364 R plasmid.We made mutation in SUMO1 binding sites of PS IP1.Briefl y,amino aci d exchanges were generated b y point mutations in the p EGFP-PSIP1.The plasmid was amplified,and the mutation was confirmed by sequencing.The mutated plasmid was transfected and intracellular translocation was determined with fluorescent microscop y and Western blot.Data indicated p EGFP-K364 R eukaryotic expression vector can well over express EGFP-PS IP1 fusion protein in human lens epithelial cells.7.Conserved K364 of PS IP1 is subject to Sumoylation.Coexpression of EGFP-Sumo1 and l ysine mutated forms of EGFP-PS IP1(K364R)showed that EGFP-K364 R was not Sumoylated,and thus could not be detected by anti-Sumo1 antibody.8.Sumoylation/de Sumoylation affects transactivation capacit y of PSIP1.Cells were cotransfected either with p EGFP-C1,p EGFP-PS IP1(WT)or p EGFP-K364R(mutant)plasmid along with reporter plasmid Hsp27 vector,and cell extracts were anal yzed with Dual-Luciferase Reporter Assay S ystem.However,the transactivation potential of p EGFP-K364 R in Hsp27 promoter activit y was significantl y greater than t hat of p EGFP-PS IP1(P<0.001).9.Sumoylation/desumoylation of PSIP1 affected growth and survival of cells.Cells transfected with wild t ype PS IP1,mutant PS IP1 p EGFP-K364 R or p EGFP-vector were submitted to normal physiological conditions.Cells overexpress ing mutant PS IP1 p EGFP-K364 R at Sumo1 conjugation site showed better growth than wild t ype PSIP1 under normal physiological conditions(P<0.001).10.Sumoylation/desumoylation of PSIP1 affected survival of cells against environmental stressors.Cells transf ected with wild t ype PS IP1,mutant PSIP1 p EGFP-K364 R or p EGFP-vector were submitted to oxidative stress.The cells with mutant PS IP1 p EGFP-K364 R had resistance against oxidative stress compared to wild t ype PS IP1 transfected cells(P<0.05).Conclusion: Taken together,it is attempting to speculate that the activity of PSIP1 is kept at basal level under normal conditions by SUMOyl ation.In the presence of stress stimuli,such as oxidative stress,SUMOylation of PS IP1 will be relieved,and thus enhanced expr ession of some heat shock proteins may neutralize cellular responses provoked by the stress conditions.