The Orphan Nuclear Receptor NR4A1 Enhances The Expression Of GPX1 In Pancreatic ? Cell And The Related Mechanisms

Diabetes is a metabolic disease that seriously endangers human health,and islet?-cell apoptosis plays a critical role in the pathogenesis of both type 1 and type 2 diabetes.Our previous studies showed that orphan nuclear receptor NR4A1 is able to resist pancreatic ?-cell apoptosis induced by oxygen free radicals(ROS)or oxidative stress.It is known that glutathione peroxidase 1(GPX1),the most common antioxidant enzyme in the glutathione peroxidase class,playing an important role in resistant to apoptosis induced by oxygen free radicals(ROS)or oxidative stress.Whether or not the orphan nuclear receptor NR4A1 protects pancreatic ?-cells from apoptosis by up-regulating the expression of GPX1,and the related molecular mechanisms needs to be further explored.Therefore,we overexpressed NR4A1 in pancreatic ? cell(MIN6),detected the expression of GPX1 by different methods at different levels,and examined the effect of NR4A1 on GPX1 promoter transcription activity by exploiting dual luciferase assay.Then,overexpressing GPX1 in the MIN6 cells,these cells were treated with different dose of H2O2 or with the same dose of H2O2 but with different time duration,the samples were collected for further analysis.The changes of activation level of Caspase3 under the above conditions were detected.The results showed that overexpression of NR4A1 in MIN6 cells can increase the expression of GPX1 at protein level.The result of dual luciferase assay showed that NR4A1 was able to enhance the transcriptional activity of GPX1 promoter.We further found that there is a potential binding site for NR4A1 on the GPX1 promoter(-273 to-268)by checking the related database.ChIP assay result showed that NR4A1 was able to physically associate with this binding site.And we also found overexpression of GPX1 in MIN6 cells decreased the level of active form of Caspase3 induced by H2O2.In conclusion,NR4A1 has the potential role to resist ?-cell apoptosis induced by oxidative stress via enhancing GPX1 expression.Aim:The aim of this study is to explore if NR4A1 is able to enhance the expression of GPX1 in pancreatic beta cells and the related mechanisms.Methods:1.MIN6 cells overexpressing NR4A1 and the control cells were labled as OV cells and NC cells respectively.After cultured,OV cells and NC cells were harvested for Western Blot analysis to verify NR4A1 overexpression in OV cells.2.Both NC and OV cells being cultured under the same conditions,were harvested for whole transcriptome analysis,Microarray analysis,to find the differentially expressed genes.3.The protein and mRNA extracted from NC or OV cells was apllied for Western Blot and qPCR analysis to verify the results of Microarray.4.Each of the two reporter plasmids with different length of GPX1 promoter was co-transfected with the sea cucumber luciferase plasmid into the NC or OV cells to test the luciferase activity.Thus to verify the effect of NR4A1 on GPX1 promoter transcription activity.5.Predicting the binding site of NR4A1 on GPX1 promoter by software.After infected MIN6 cells with Ad-NR4A1-HA and Ad-GFP,the cell smaples were treated for Chromatin immunoprecipitation(ChIP)analysis,anti-HA monoclonal andibody was applied to precipitate and enrich the gene fragment that binds to the NR4A1 protein,then PCR amplification of the fragment was performed to detect if NR4A1 was able to bind to the predicted or putative binding site.6.The GPX1 overexpression lentivirus and its control lentivirus were used to infect MIN6 cells,to obtain GPX1-overexpressing cell clone(OV-GPX1)and its control cell clone(CON).The protein and mRNA of CON and OV-GPX1 cells were analysed to make sure GPX1 overexpressed in OV-GPX1 cells.7.The OV-GPX1 cells or CON cells were treated with different concentrations of H2O2(0 ?M,100?M,200 ?M,400 ?M,600 ?M)for 12 hours,or the cells treated with 100 ?M H2O2 for 0 h,12 h,16 h,20 h and 24 h;the cells with above treatment were harvested and lysed for the protein extract.The active form of caspase3 was detected with Western Blot.Results:1.Western Blot results showed that the expression of NR4A1 in OV cells was significantly higher than that in NC cells.2.cDNA Microarray results showed that overexpression NR4A1 in MIN6 cells resulted in the increased mRNA expression of genes related to oxidative stress and apoptosis,such as SOD2,BIRC5,BCL2,GPX1 and CAT,among these genes,the expression of GPX1 was the most significant,namely,the expression of GPX1 was significantly increased after overexpression of NR4A1.3.Western Blot results showed that the expression level of GPX1 in OV cells was significantly higher than that in NC cells at both protein and mRNA levels,which was consistent with the cDNA Microarray results.4.We successfully constructed the luciferase reporter plasmids,which contains 0 to-2000 bp fragment or 0 to-4000 bp fragment of the GPX1 promoter;and the sequences of the two fragments were confirmed by doing sequnceing.The results of dual luciferase assay showed that overexpression of NR4A1 enhanced the transcriptional activity of GPX1 promoter with the two different length of fragments,while,the longer fragment(4000 bp)did not show higher transctiption activity compared to the shorter one(2000 bp).These data suggests that NR4A1 exerts its effect mainly in the region of 0 to-2000 bp.5.Analysis of the GPX1 promoter sequence revealed that there are three potential binding sites for NR4A1(TGACCT)on the 4000 bp length promoter sequence.Given the effect of NR4A1 on the GPX1 transcriptional activity of the two different lengths of GPX1 promoter,we speculated that the effect of NR4A1 should occur within 0 to-2000 bp with a potential binding site of-273 to-268.The potential binding sites of NR4A1 were considered to design PCR primers for ChIP analysis.The final ChIP results showed that overexpression of NR4A1 enriched the fragment containing the binding site,while the control group showed no fragment enrichment,which indicats that NR4A1 is able to physically bind to this binding site(-273 to-268)on the GPX1 promoter.6.GPX1 protein and mRNA levels were examined in OV-GPX1 cells and CON cells.The results showed that the expression level of GPX1 in OV-GPX1 cells was significantly higher than that in CON cells at both the protein level and mRNA level.This confirmed that we successfully constructed the MIN6 cell line overexpressing GPX1.7.The result of Western Blot showed that with the increase of H2O2 concentration,the expression of Caspase3 in CON or OV-GPX1 cells also increased.However,the level of active Caspase3 in OV-GPX1 cells treated with different concentrations of H2O2(0 ?M,100 ?M,200 ?M)was significantly lower than that in CON cells.When the concentration of H2O2 was over 400 ?M,the cells tended to die.With the increased duration of H2O2,the level of active form Caspase3 in CON and OV-GPX1 cells also increased,but the level of Caspase3 activity in OV-GPX1 cells at different time points after H2O2 treatment was significantly lower than that in CON cells.When the time was too long,after about 20 h,the cells tended to die.Conclusions:1.NR4A1 increases the expression of GPX1 by enhancing the transactivation of GPX1 promoter through binding to the putative binding site on GPX1 promoter.2.NR4A1 potentially protects pancreatic ?-cells against oxidative stress-mediated apoptosis by modulating GPX1 expression.