SENP6 De-SUMOylation Promotes Annexin-A1 Nuclear Translocation And Triggers OGD/R-induced Neuronal Apoptosis

Objective:Annexin A1(ANXA1),a member of the Annexin protein superfamily,plays an important role in neuronal apoptosis after ischemic stroke.Our previous studies demonstrated that ANXA1 phosphorylation increased nuclear translocation of ANXA1 and participated in the process of ischemia-induced neuronal apoptosis.We also found that ANXA1 could combine and be modified by small like ubiquitin protein modifying molecule(small ubiquitin-like modifier,the SUMO),which is named SUMOylation modification.ANXA1 SUMOylation modification was significantly weakened after cerebral ischemia reperfusion injury,and increasing ANXA1 SUMOylation reduced significantly neurons apoptosis and nervous impairment after cerebral ischemia reperfusion injury.Based on the above,with significantly reduced SUMOylation modification of ANXA1 after neurons oxygen deprivation sugar/reoxygenation(oxygen and glucose Deprivation/reperfusion,OGD/R)as the breakthrough point,this article will detect the relationship of ANXA1 SUMOylation level with SUMO specific protease(SUMO-specific protease,SENP).On this basis,this research aims to study the effect of SENP and ANXA1 de SUMOylation on neuronal apoptosis induced by OGD/R mechanism.Methods:(1)Using primary neurons,HEK293 T and N2 a cell lines and constructing OGD/R cell model,the change of ANXA1 SUMOylation modification were detected by Ni2+-NTA agarose affinity pull-down assay.Co-immunoprecipitation technique(Co-IP)was used to screen proteases in the SENPs family that interact with ANXA1.Immunoprecipitation technique(Co-IP)and immunohistochemical methods were used to determine the interaction between ANXA1 and sentrin/SUMO-specific proteases(SENPs)and their localization in brain slices.(2)The plasmids expressing ANXA(WT,wild-type),ANXA1-SUMO2(SUMO2 fusion protein)and ANXA1mutant(K113/161/257 R,3KR)were constructed,and using western blotting and immunofluorescence to detect nucleus distribution of ANXA1 in N2 a cells.(3)Using IP and Co-IP techniques to detect phosphorylation and SUMOylation of ANXA1 and the interaction between ANXA1 and phosphate kinase PKC and TRPM7.(4)Detecting the transcriptional activity of p53,Bid m RNA level and protein expression of Bid and t Bid by luciferase reporter gene?real-time fluorescence quantitative PCR(RT-q PCR)and western blot.(5)Infecting neurons with adenovirus expressing ANXA1-WT,ANXA1-SUMO2 and ANXA1-3KR,and using western blotting to detect the effect of ANXA1 SUMOylation on the expression of apoptotic related proteins including cleaved caspase-3,cleaved caspase-9 and cleaved PARP.(6)Detecting neural apoptosis by TUNEL staining and Lactate Dehydrogenase(LDH)release assay.Results:1.SENP6 induced de-SUMOylation of ANXA1 after OGD/R in neurons.OGD/R cell model was constructed,and the level of ANXA1 de SUMOylation after OGD/R was detected by Co-IP and Ni2+-NTA affinity chromatography.The results showed that the level of ANXA1 SUMOylation decreased significantly after OGD/R.To investigate the reason why ANXA1 SUMOylation level reduced,we respectively overexpressed SENP in HK293 T cells to explore the enzyme molecule that mediates ANXA1 de-SUMOylation in SENPs family(SENP1?SENP2?SENP3?SENP4?SENP5?SENP6?SENP7)by Co-IP technology,and the results show that the overexpression of de-SUMOylation enzyme SENP6 cause ANXA1 SUMOylation level declined obviously,and transfection SENP6 mutant(-C1030S)or SENP6 sh RNA increased ANXA1 SUMOylation levels significantly.Immunofluorescence showed that ANXA1 and SENP6 co-localized in mouse cerebral cortex neurons,and Co-IP showed that the binding of ANXA1 and SENP6 was significantly increased after OGD/R.These results suggest that SENP6 is involved in the development of OGD/R in eurons,and this process is associated with ANXA1 de SUMOylation.2.De-SUMOylation of ANXA1 induced by SENP6 promoted ANXA1 nuclear transposition after OGD/R.Over-expression of ANXA1-WT,ANXA1-SUMO2 and ANXA1-3KR in N2 a neurons,using immunofluorescence double standard technique to detect ANXA1 nuclear distribution after OGD/R,the result showed that ANXA1-WT accumulate significantly in the nucleus after OGD/R,and ANXA1-3KR mutant mainly located in the nucleus,ANXA1-SUMO2 tend to locate in the cytoplasm;In order to detect the effect of SENP6 on endogenous ANXA1 nuclear localization after OGD/R,we overexpressed SENP6-WT,SENP6-C1030 S or SENP6 sh RNA in N2 a cells,the fluorescence double-label technique showed that the localization of ANXA1 in the nucleus increased after OGD/R,in contrast,the nuclear localization of ANXA1 induced by OGD/R was further increased after transfection with SENP6-WT,while the localization of ANXA1 in the nucleus was significantly decreased in SENP-C1030 S and SENP6 sh RNA transfection group.Western blotting results also showed that transfection of SENP6-WT enhanced nuclear translocation of ANXA1 after OGD/R,while the transfection of SENP-C1030 S and SENP6 sh RNA reduced nuclear translocation of ANXA1 induced by OGD/R.These results suggest that SENP6 induces ANXA1 de-SUMOylation after OGD/R,then promotes the nuclear translocation of ANXA1.3.SENP6 mediating ANXA1 de-SUMOylation increased PKC and TRPM7 kinase dependent ANXA1 phosphorylation of serine residues.In order to discuss SENP6 mediated ANXA1 de-SUMOylation promoting its nuclear transfer whether has relationship with ANXA1 phosphorylation after OGD/R,we first tested the changes of ANXA1 phosphorylation and SUMOylation levels after OGD/R,the results showed that after 1h of oxygen sugar deprivation,and reoxygenation respective after 3h?6h?12h and 24 h,ANXA1 serine residues phosphorylation level increased with the extension of reoxygenation time and eached the highest when 24 h.ANXA1 SUMOylation modification level decreased gradually with the prolongation of reoxygenation time(all OGD/R subsequent were treated with oxygen and sugar deprivation for 1h and reoxygenation for 24h).Subsequently,the primary neurons were infected with SNEP6-WT,SENP6-C1030 S and SENP6 sh RNA respectively,and the phosphorylation level of ANXA1 serine residues was detected.The results showed that the phosphorylation level of ANXA1 serine residues induced by OGD/R was significantly decreased after SENP6-C1030 S infection compared with the SENP6-WT infection group.Similarly,infection with SENP6 sh RNA also resulted in down-regulation of phosphorylation of ANXA1 after OGD/R.The Co-IP technique was used to study the relationship between ANXA1 and PKC/TRPM7 kinase.The results showed that after OGD/R,the overexpression of SENP6-WT significantly increased the binding of ANXA1 to PKC and ANXA1 to TRPM7,while the overexpression of SENP6-C1030 S and SENP6 sh RNA decreased the binding.These results suggest that after OGD/R,SENP6 increases the phosphorylation of ANXA1 serine residues by de-SUMOylation of ANXA1,and enhances the nuclear translocation of ANXA1 induced by OGD/R.Furthermore,this phosphorylation may be PKC and TRPM7 kinase dependent.4.SENP6 mediating ANXA1 de-SUMOylation enhanced p53 transcriptional activity and Bid protein expression after OGD/R.We respectively use the luciferase report gene ? RT-q PCR and WB technology to detect p53 transcriptional activity?Bid m RNA level and Bid?t Bid protein expression.First of all,we transfected HEK293 T cells with ANXA1-WT,ANXA1-SUMO2 and ANXA1-3KR to explore the effect of ANXA1 SUMOylation on p53 transcriptional activity and Bid protein expression,the results showed that compared with ANXA1-WT and ANXA1-SUMO2 overexpression groups,ANXA1-3KR transfection significantly enhanced the transcriptional activity of p53 after OGD/R and increased the m RNA and protein expression of Bid.In rder to detect whether SENP6 involves that or not,we transfected HEK293 T cells with SNEP6-WT,SENP6-C1030 S and SENP6 sh RNA,then detected the changes of p53 transcriptional activity and Bid expression after OGD/R,the results showed that transfection with SENP6-WT significantly increased the transcriptional activity of p53,Bid m RNA and Bid?t Bid protein expressions after OGD/R,importantly,the transfection with SENP6-C1030 S and SENP6 sh RNA significantly reduced the transcriptional activity of p53 induced by OGD/R,and the m RNA expression of Bid and the protein level of Bid were also significantly reduced.These results indicated that SENP6 enhanced p53 transcriptional activity and the m RNA and protein expression of Bid through ANXA1de-SUMOylation after OGD/R.5.SENP6 de-SUMOylation ANXA1 promoted the activation of apoptotic proteins caspase-3,caspase-9 and PARP after OGD/R.First,using adenovirus packaging ANXA1-WT?ANXA1-SUMO2 and ANXA1-3KR plasmids to infect primary cultured neurons,and the changes of apoptotic proteins including caspase-3,caspase-9 and PARP spliceosomes were detected by Western blot to determine the influence of ANXA1 SUMOylation on the activation of apoptotic proteins.The results showed that compared with ANXA1-WT infection group,the activation of apoptosis-related caspase-3,caspase-9 and PARP was significantly decreased in ANXA1-SUMO2 infection group,while ANXA1-3KR infection promoted the activation of apoptosis-related proteins after OGD/R.In order to detect the effect of SENP6 on the activation of apoptosis-related proteins,we infected primary neurons with adenovirus-wrapped SNEP6-WT?SENP6-C1030 S and SENP6 sh RNA plasmids.The results showed that after OGD/R,the expression of apoptosis-related proteins was significantly increased in SENP6-WT infection group,while the activation of apoptotic proteins was decreased when SENP6-C1030 S and SENP6 infected.In order to investigate whether the effect of SENP6 on apoptosis-related proteins activation was related o ANXA1 or not,we infected neurons with adenovirus of SENP6 and ANXA1 or ANXA1 sh RNA,and western blot results showed that compared with SENP6 and ANXA1 or ANXA1 sh RNA infection alone,neurons infected with SENP6 and ANXA1 sh RNA significantly increased apoptotic proteins activation,conversely,neurons infected with SENP6 and ANXA1 sh RNA significantly decreased the expression of cleaved caspase-3? cleaved caspase-9 and cleaved PARP.In addition,neurons were infected with SENP6-WT or SENP6 sh RNA on the basis of the infection of ANXA1-WT,ANXA1-SUMO2 and ANXA1-3KR,and western blotting results showed that after OGD/R,SENP6-WT infection significantly increased the expression of apoptotic proteins in the ANXA1-WT group compared with the Vector group,while SENP6-C1030 S inhibited the activation of apoptotic proteins in the ANXA1-WT group,but had little effect on ANXA1-SUMO2 and ANXA1-3KR groups,suggesting that the effect of SENP6 on apoptotic proteins was achieved through its enzymatic activity on ANXA1.These results indicated that SENP6 promoted the level of apoptosis-related caspase-3,caspase-9 and PARP spliceosome proteins after OGD/R,and this effect was realized by its de-SUMOylation enzyme activity on ANXA1.6.SENP6 de-SUMOylation of ANXA1 promoted OGD/R-induced neurons apoptosis.First,we infected neurons with adenovirus expressing ANXA1-WT,ANXA1-SUMO2 and ANXA1-3KR.TUNEL staining and LDH release test were used to detect the effect of ANXA1 SUMOylation on neuron apoptosis after OGD/R.The results showed that after OGD/R,compared with ANXA1-WT infection group,ANXA1-SUMO2 infection decreased the number of TUNEL+cells and the release of LDH,while TUNEL+ cells and LDH release significantly increased when neurons were infected with ANXA1-3KR.To investigate the effect of SENP6 on neuronal apoptosis,we infected neurons with adenovirus of SNEP6-WT,SENP6-C1030 S and SENP6 sh RNA,the results showed that compared with the Control group,OGD/R treatment upregulated the umber of TUNEL+ cells and the release of LDH,importantly,the number of TUNEL+ cells and the release of LDH were further increased by SNEP6-WT infection after OGD/R,while SENP6-C1030 S and SENP6 sh RNA infection inhibited the number of TUNEL+ cells and the release of LDH.These results indicate that SENP6 de-SUMOylation of ANXA1 enhances neuronal apoptosis under OGD/R conditions.To investigate whether SENP6-mediated neurons apoptosis depends on its de-SUMOylation enzyme activity on ANXA1 after OGD/R,we infected neurons with adenovirus packaging SENP6 and ANXA1 or ANXA1 sh RNA plasmid.TUNEL staining and LDH release assay results showed that compared with SENP6 and ANXA1 or ANXA1 sh RNA infection alone,the number of TUNEL+ cells and the release of LDH were significantly increased by infection of SENP6 and ANXA1,whereas the number of TUNEL+cells and the release of LDH were significantly decreased by infection of SENP6 and ANXA1 sh RNA.In addition,SENP6-WT or SENP6 sh RNA were infected on the basis of the infection of ANXA1-WT,ANXA1-SUMO2 and ANXA1-3KR,and the results showed that compared with the Vector group,SENP6-WT significantly increased neuronal apoptosis in the ANXA1-WT infection group after OGD/R,while SENP6-C1030 S inhibited neuronal apoptosis in the ANXA1-WT infection group,but had no significant effect on ANXA1-SUMO2 and ANXA1-3KR infection groups,suggesting that SENP6 influence neuron-apoptosis through its effect on ANXA1 de-SUMOylation.In summary,these results indicate that SENP6 promotes OGD/R-induced neuronal apoptosis through its de-SUMOylation enzyme activity on ANXA1.Conclusion:1.OGD/R treatment induced an increase in the binding of SENP6 to ANXA1,which resulted in ANXA1 de-SUMOylation and induced a decrease in ANXA1 SUMOylation modification.2.The increased nuclear translocation of ANXA1 induced by SENP6-mediating NXA1 de SUMOylation after OGD/R may be related to the enhanced phosphorylation of OGD/R-induced TRPM7 and PKC kinase-dependent ANXA1 serine residues.3.SENP6-mediating ANXA1 de SUMOylation enhanced p53 activity after OGD/R,increasing the expression level of Bid protein,activating the apoptotic proteins caspase-3?caspase-9 and PARP cleavage,thus inducing neuronal apoptosis.