Acetylation Control Type And Dissolve C5b Irf - 1-9 Compounds Of The Rat Glomerular Mesangial Cell Apoptosis Induced By Lesions In Mechanism Research

Part I Sublytic C5b-9promotes the apoptosis of rat GMCvia the expression of XAF1geneObjective: To investigate the effect of sublytic C5b-9complexes on the expression ofX-linked inhibitor of apoptosis associated factor1(XAF1) in rat GMC, and explorethe role of XAF1in sublytic C5b-9-mediated GMC apoptosis.Methods: Firstly, the cultured GMC were stimulated with sublytic C5b-9fordifferent time points and then divided into different groups. The protein level ofXAF1was measured by Western blot. Moreover, XAF1eukaryotic expressionplasmid (pEGFP-N1/XAF1) and XAF1short hairpin RNA (shRNA) expressionplasmid (shXAF1) were well constructed. The recombinant plasmids were transfectedinto GMC respectively for48h, followed by sublytic C5b-9treatment for another6h.Thereafter, the protein levels of XAF1in the different groups and the apoptosis ofGMC was measured using Western blot or by Flow cytometry respectively.Results: The protein level of XAF1in the GMC stimulated with sublytic C5b-9wassignificantly up-regulated, and reached the peak at6h after sublytic C5b-9stimulation.Transfection of GMC with pEGFP-N1/XAF1could obviously increase XAF1production and GMC apoptosis. However, shXAF1treatment could effectivelysuppress the protein expression of XAF1and reduce the number of GMC apoptosisinduced by sublytic C5b-9.Conclusion: Sublytic C5b-9can promote the apoptosis of GMC via up-regulating theexpression of XAF1gene. PartⅡ Sublytic C5b-9regulates the expression of XAF1gene through IRF-1in rat GMCObjective: To explore the effect of interferon regulatory factor1(IRF-1) expressionon the XAF1gene transcription in rat GMC stimulated with sublytic C5b-9and itsmechanism, and further evaluate the role of IRF-1expression in the GMC apoptosis.Methods: Rat XAF1promoter (-1490~+157nt) was amplified by PCR and clonedinto the luciferase reporter plasmid (pGL3-basic). The recombinant plasmids(pGL3-XAF1-QC) were transfected into GMC accompanied with IRF-1expressionplasmids (pcDNA3.1/IRF-1) or IRF-1shRNA (shIRF-1) for48h, followed bysublytic C5b-9stimulation for6h, and then the luciferase activity was detected.Meanwhile, the possible IRF-1binding sites within XAF1promoter were predictedby bioinformatics software (TFsearch). Based on the predicted results, the luciferasereporter plasmids of different truncated XAF1gene promotors were constructed,which are named pGL3-XAF1-1, pGL3-XAF1-2, pGL3-XAF1-3and pGL3-XAF1-4.The above-mentioned promoter luciferase reporter plasmids and pcDNA3.1/IRF-1were co-transfected into rat GMC. Thereafter, the luciferase activity was detected toscreen IRF-1binding sites. Afterwards, the corresponding primers were designedaccording to the possible IRF-1binding sites, and ChIP assay was used to determinethe IRF-1binding sites. Additionally, pcDNA3.1/IRF-1and shIRF-1were transfectedinto the GMC for48h respectively, and followed by sublytic C5b-9attack for6h. Thelevel of XAF1protein and GMC apoptosis were measured by Western blot or flowcytometry.Results: The GMC treated with pcDNA3.1/IRF-1transfection could significantlyenhance the XAF1promotor luciferase activity, whereas shIRF-1could obviouslysuppress the XAF1transcription activity in GMC due to sublytic C5b-9attack. Thefurther study with truncated XAF1promotor luciferase reporter plasmids and ChIPassays showed that the region of rat XAF promoter (-337~-47nt) contain a IRF-1binding element. Furthermore, pcDNA3.1/IRF-1plasmids markedly up-regulatedXAF1protein level and increased GMC apoptosis, and shIRF-1plasmids couldobviously inhibited the XAF1expression and GMC apoptosis induced by sublytic C5b-9.Conclusion: Sublytic C5b-9can up-regulate XAF1gene transcription and GMCapoptosis via increasing IRF-1expression. sublytic C5b-9; glomerular mesangial cells (GMC); interferonregulatory factor1(IRF-1); promoter activity Part III Sublytic C5b-9induces XAF1expression and GMCapoptosis through p300-regulated IRF-1acetylationObjective: To observe whether cAMP response element-binding protein (CBP) andits homolog p300can bind to IRF-1and induce IRF-1acetylation, XAF1expressionand GMC apoptosis exposed to sublytic C5b-9.Methods: First of all, we assessed whether CBP/p300could bind to IRF-1andincrease the acetylation of IRF-1in the GMC stimulated with sublytic C5b-9complexes by Co-IP assay. In addition, p300shRNA (shp300) was transfected intothe GMC to estimate the effects of silencing p300gene on the expression of IRF-1and XAF1as well as the acetylation of IRF-1due to sublytic C5b-9stimulation byWestern blot or Co-IP assay. Furthermore, the role of p300in GMC apoptosismediated by sublytic C5b-9was evaluated by flow cytometry.Results: Sublytic C5b-9could induce p300binding to IRF-1and increase IRF-1acetylation. Silencing p300gene obviously inhibited the acetylation of IRF-1, theexpression of XAF1and the apoptosis of GMC due to sublytic C5b-9attack.Conclusion: Sublytic C5b-9complexes induces XAF1expression and GMCapoptosis through p300-regulated IRF-1acetylation.