Sublytic C5b-9 Induces P300 Acetylation Modified ATF3 To Regulate The Mechanism Of GMC Apoptosis In Thy-1 Nephritis Rats

Part I Up-regulation of p300,Egr-1,ATF3 and Gadd45expression in renal tissue of Thy-1N and in GMCstimulated with sublytic C5b-9 including their roles inGMC apoptosis induced by sublytic C5b-9Objective:To investigate the expression of p300,early growth response gene-1(Egr-1),activating transcription factor 3(ATF3)and growth arrest-and DNA-damage-inducible gene 45(Gadd45)both in renal tissues of Thy-1 nephritis(Thy-1N)rats and in glomerular mesangial cell(GMC)stimulated with sublytic C5b-9.And to explore the roles of p300,Egr-1,ATF3 and Gadd45 in GMC apoptosis induced by sublytic C5b-9.Methods:Rat Thy-1N model was reproduced firstly,and the cultured GMC was stimulated with sublytic C5b-9.The expression of p300,Egr-1,ATF3 and Gadd45 in renal tissue of Thy-1N rats(in vivo)and in GMC attacked by sublytic C5b-9(in vitro)was detected using qPCR and western blot(WB)at fixed time and in different treatment groups.Meantime,the morphology of apoptosis GMC in renal tissue of Thy-1N rats was observed by electron microscopy(EM),and the percentage of apoptosis GMC after sublytic C5b-9 stimulation was measured by flow cytometry.Thereafter,the expression plasmids(pIRES2/Egr-1,pIRES2/ATF3,pIRES2/Gadd45a,pIRES2/Gadd45p and pIRES2/Gadd45y)and short hairpin RNAs(shRNAs,namely shp300,shEgr-1,shATF3,shGadd45a,shGadd45p and shGadd45y)of p300,Egr-1,ATF3 and Gadd45 genes were well constructed,and then transfected into GMC respectively for 48h,followed with(or without)sublytic C5b-9 treatment for 3h.Then cells were collected to detect the percentage of apoptosis GMC by flow cytometry analysis.Results:The gene expression of p300,Egr-1,ATF3 and Gadd45 was significantly up-regulated both in renal tissues of Thy-1N rats and in GMC in response to sublytic C5b-9,with Egr-1 protein level peaked at 2h in vivo and 1h in vitro,and p300,ATF3 as well as Gadd45 protein levels peaked at 3h both in vivo and in vitro.Meanwhile,the apoptotic changes of GMC were observed in Thy-1N rats,and the number of apoptosis GMC was markedly increased after sublytic C5b-9 exposure.Furthermore,overexpression of Egr-1,ATF3 and Gadd45?/?/? in GMC dramaticlly increased GMC apoptosis,while knockdown of p300,Egr-1,ATF3 and Gadd45?/?/? obviously reduced the number of GMC apoptosis.Conclusion:In renal tissues of Thy-1N rats and in cultured GMC,sublytic C5b-9 can up-regulate the expression of p300,Egr-1,ATF3 and Gadd45?/?/?,and these molecules can lead to GMC apoptosis.Part ? The relationship between Egr-1 and ATF3 as wellas the effects of Egr-1 and ATF3 on Gadd45 expressionObjective:To explore the upstream and downstream relationship between Egr-1 and ATF3 in the GMC induced by sublytic C5b-9 attack.And to investigate the roles and mechanism in governing Gadd45 genes expression mediated by of Egr-1 and ATF3.Methods:Firstly,qPCR and WB were used to detect the level of ATF3 mRNA and protein in GMC transfected with pIRES2/Egr-1 or shEgr-1.Similarly,the expression of Egr-1 was also measured in GMC after pIRES2/ATF3 or shATF3 transfection.Subsequently,GMC was treated with pIRES2/Egr-1+shATF3(shCTR as the control)or shEgr-1+pIRES2/ATF3+sublytic C5b-9(pIRES2 as the control),and the number of apoptosis GMC was analyzed by flow cytometry.Thereafter,genome walking was used to identify the unknown proximal promoter sequences of ATF3 gene,and ATF3 promoter luciferase reporter plasmid(pGL3-ATF3)was well constructed.pGL3-ATF3 was co-transfected with pIRES2/Egr-1 or shEgr-1 into GMC,then ATF3 promoter activity was measured.Furthermore,in order to explore the potential mechanism by which Egr-1 post-transcriptionally modulates ATF3 protein level,rat GMC was transfected with shEgr-1 and followed with sublytic C5b-9 attack and proteasome inhibitor MG 132 treatment,ubiquitination level of ATF3 was analyzed by immunoprecipitation(IP)with anti-ATF3 or anti-ubiquitin antibodies.Simultaneously,GMC was transfected with shEgr-1,followed with sublytic C5b-9 attack for 1h and actinomycin D treatment for various time points,then cellular transcription was arrested and qPCR was used to detect the mRNA stability of ATF3.In addition,mRNA and protein levels of Gadd45 genes were also examined by qPCR and WB in GMC transfected with Egr-1 or ATF3 overexpression or shRNAs vectors.On the other hand,the full-length(FL)promoter reporter vectors of Gadd45?/?/?(pGL3-Gadd45a-FL,pGL3-Gadd45?-FL and pGL3-Gadd45y-FL)were constructed,and were separately transfected into GMC accompanied with Egr-1 or ATF3 overexpression or shRNAs vectors,then each luciferase activity of Gadd45 gene promoters was measured.With several Egr-1-and ATF3-binding sites on the Gadd45?/? promoters has predicted using TF search software,4 truncated promoter reporter plasmids of Gadd45?(-561~+236nt,-319~+236nt,-146~+236nt and+23~+236nt)as well as 3 truncated promoter reporter plasmids of Gadd45?(-456~+72nt,-211~+72nt and-61~+72nt)were constructed.Subsequently,GMC was transfected with above-mentioned Gadd45?/? promoter reporter plasmids(full-length and different deletion mutants)accompanied with pIRES2/Egr-1 or pIRES2/ATF3,the luciferase activities of Gadd45?/? promoters were detected to speculate the binding regions of Egr-1 and ATF3.Next,according to the results mentioned-above experiments,several corresponding primers were designed to determine the binding sites of Egr-1 and ATF3 to Gadd45p/y promoters by ChIP.Finally,the interaction between Egr-1 and ATF3 in GMC at various time points upon sublytic C5b-9 stimulation was examined by IP.Results:GMC transfected with pIRES2/Egr-1 or shEgr-1 respectively exhibited increased or decreased expression of ATF3 protein but not mRNA.On the contrary,Egr-1 mRNA and protein level had no significant change after ATF3 overexpression or interference.Additionally,ATF3 knockdown abrogated Egr-1's pro-apoptotic effect,while ATF3 overexpression enhanced GMCs apoptosis which was mitigated by shEgr-1.Thereafter,430bp nucleotide sequencing of ATF3 proximal promoter was obtained by using genome walking,and ATF3 promoter reporter plasmid(pGL3-ATF3)was well constructed.Correspondingly,ATF3 promoter activity displayed no markedly changes after Egr-1 overexpression or knockdown in GMC.Next,we found that MG 132 restored the up-regulation of ATF3 which was mitigated by shEgr-1.but ATF3 ubiquitination level was not increased.However,by using actinomycin D,we observed that Hgr-1 intcricrencc markedly destabilized ATF3 mRNA.Afterwards,overexpression or knockdown of Egr-1 or ATF3 remarkably increased or decreased the mRNA and protein levels as well as promoter activities of Gadd45?/?/? genes.As for Gadd45? promoter,fragments(-146~+236nt and+23~+236nt)co-transfected with plRES2/Kgr-1 as well as fragment(+23~+236nt)co-transtected with pIRES2/ATF3displayed notably reduced Gadd45? promoter activity,implving that the Gadd45P promoter region(-319~-146nt and-146~+23nt)might contain Egr-1 binding sites?and region(-146~+23nt)might contain an ATF3 binding site.As to Gadd45y promoter,fragments(-211~+72nt and-61~+72nt)co-transfected with plRES2/Egr-1 or pIRES2/ATF3 both exhibited acute reduction in promoter activity,indicating that the Gadd45y promoter region(-456~-211nt and-211~-61 nt)might contain both Egr-1 and AT3 binding sites.ChlP experiment confirmed that the GMC in response to subiytic C5b-9 could result in the bindinu of Egr-1 and ATF3 to the sequence(-139 to+115nt and-320 to-110nt)of Gadd45? promoter and to the sequence(-198 to+28nt and-467 to-198nt)of Ciadd45? promoter.Finally.IP assay showed that Egr-1 could not bind to ATF3 for their transcription function.Conclusion:Egr-1.which was induced by subiytic C5b-9,can up-regulate ATF3 protein abundance by enhancing the stability of ATF3 mRNA.ATF3 acts as the dowTistream of Egr-1 and can mediate Egr-l's function in GMC apoptosis upon subiytic C5b-9 stimulation.Moreover,Egr-1 and ATF3 can bind to the specific binding elements on Gadd45 promoter,and contribute to Gadd45 genes transcription.Part ? The roles of ATF3 acetylation mediated by p300 inGadd45?/y gene expression and GMC apoptosisObjective:To investigate the roles of p300-dependent ATF3 acetylation in regulating Gadd45p/? expression and GMC apoptosis.Methods:Firstly,IP test was performed to detect the interaction of p300 with Egr-1 and ATF3 including acetylation level of Egr-1 and ATF3 in GMC attacked by sublytic C5b-9 at fixed time and in different treatment groups.Next,to confirm the effect of p300 on ATF3 acetylation,GMC were transfected with shp300 or treated with histone acetyltransferase(HAT)inhibitor(anacardic acid)and histone deacetylase inhibitor(trichostatin A,TSA),followed with sublytic C5b-9 stimulation.Cell lysates were subjected to IP by ATF3 antibody and subsequent IB analysis was used to measure the level of ATF3 acetylation.Meanwhile,IP and WB assays were also used to detect the interaction of ATF3 with CREB-binding protein(CBP)and p300/CBP-associated factor(PCAF)in GMC stimulated with sublytic C5b-9 for 3h.Then mass spectrometry analysis was used to identify the acetylation site of ATF3.According to the site,FLAG-tagged wild type(WT)and acetylation site-mutated(arginine residues,R)ATF3 expression plasmids were constructed.Afterwards,ATF3 expression plasmids(WT and mutant),shp300 and control vectors were transfected into GMC,followed with sublytic C5b-9 attack for 3h.qPCR,WB and luciferase reporter assay were then performed to examine Gadd45?/? gene expression and promoter activity.Furthermore,shATF3,shp300,ATF3 expression plasmids(WT and mutant)were respectively transfected into GMC,followed with sublytic C5b-9 treatment for 3h.ChIP assay was then performed using ATF3 antibody,and PCR was used to measure the binding of ATF3 to Gadd45?/?promoters.Additionally,GMC was exposed to sublytic C5b-9 for 3h,thereafter a ChIP assay was done by using the antibody against ATF3,and subsequently chromatin immunodepletion(ChID)and re-ChIP assay were performed by using the antibody against acetylated-lysine(Ac-K)to detect the binding of ATF3 to Gadd45p/y promoters in different ATF3 acetylation status.On the other hand,GMC was transfected with shATF3 or shCTR,followed with sublytic C5b-9 for 3h.Antibodies against ATF3 and p300 were successively used to performed ChIP and re-ChIP,and the binding of p300 to Gadd45?/? promoters was examined by PCR.Moreover,shp300 and shCTR were transfected into GMC,separately.Then,the cytoplasmic and nuclear extracts were extracted from GMC after sublytic C5b-9 treatment for Oh and 3h,and IB analysis was performed to detect the intracellular localization of ATF3.Finally,flow cytometry was used to calculate the percentage of apoptosis GMC after ATF3 expression vectors(WT and mutant)transfection.Results:Up-regulated p300 in GMC after sublytic C5b-9 attack could interact with ATF3,and acetylation of ATF3 was readily detected,while the interation and acetylation level both reached the maximum at 3h.In the meantime,the interaction of p300 with Egr-1 and acetylation of Egr-1 was not detected.GMC transfected with shp300 or treated with anacardic acid to inhibiting p300 enzymatic activity both exhibited decreased ATF3 acetylation level which was up-regulated after sublytic C5b-9 attack.Enzyme inhibition of histone deacetylase by TSA could dramatically enhance ATF3 acetylation in GMC,whereas p300 gene knockdown abrogated the effect induced by TSA.Besides,IP assay showed that ATF3 could only interact with p300 but CBP nor PCAF after sublytic C5b-9 stimulation.Afterwards,our results from mass spectrometry analysis manifested that lysine 42(K42)of ATF3 was acetylated in GMC upon sublytic C5b-9 attack.FLAG-tagged ATF3 expression plasmids(WT and mutant)were named as pIRES2-FLAG/ATF3(WT)and pIRES2-FLAG/ATF3(K42R),respectively.Mutation of K42 to non-acetylatable arginine residues could markedly attenuated the level of ATF3 acetylation.Further studies demonstrated that shp300 as well as pIRES2-FLAG/ATF3(K42R)not only reduced Gadd45?/? expression and promoter activities but also inhibited ATF3 binding to Gadd45?/? gene promoters.In addition,supernatant DNA of ChID exhibited that depletion of the acetylated protein removed all ATF3-bound Gadd45?/? promoter regions,while immunoprecipitated DNA of re-ChIP assay manifested that reimmunoprecipitation of acetylated ATF3 recovered ATF3-bound Gadd45?/?promoter regions.Furthermore,re-ChIP by p300 antibody confirmed that shATF3 could eliminate the binding of p300 to the Gadd45?/? promoters.Examination of ATF3 protein level in cytoplasmic and nuclear extracts demonstrated that ATF3 was mainly distributed in the nucleus,and the subcellular localization of ATF3 has not been affected after p300 silencing compared with shCTR group.Finally,flow cytometry showed that the number of apoptosis GMC was increased in pIRES2/FLAG-ATF3(WT)group,but pIRES2/FLAG-ATF3(K42R)group manifested no obvious increase in the percentage of GMC apoptosis.Conclusion:Elevated p300 can acetylate ATF3 at lysine 42 in GMC stimulated by sublytic C5b-9,which markedly up-regulated ATF3's transcriptional activity to Gadd45?/? genes.p300 binds with ATF3 to form a complex for binding to the ATF3 binding elements on Gadd45?/? promoters and subsequently enhances Gadd45?/?transcription and GMC apoptosis.