The Mechanism Research Of ATF6 On The Endoplasmic Reticulum Stress-induced Vascular Endothelial Cell Apoptosis

Background and ObjectiveAs a barrier between vascular lumen and surrounding tissues,vascular endothelial cell(VEC)plays a key role in vascular remodeling,blood flow regulation,material exchange,lipid leakage prevention,platelet aggregation inhibition,thrombosis and so on.Almost all vascular diseases and their treatments involve vascular endothelial cells,such as thrombosis,inflammation,balloon dilatation,stent implantation and so on,which are closely related to vascular endothelial cells.It is of great significance to study the various mechanisms of vascular endothelial cell injury.Endothelial cells have highly developed endoplasmic reticulum(ER),under the condition of cell injury,such as hypoxia,injury,thrombosis and infection,which can easily lead to the accumulation of a large number of misfolded proteins,resulting in endoplasmic reticulum stress(ERS).Ers mainly includes three signaling pathways: unfolded protein response(UPR),sterol regulatory cascade reaction and endoplasmic reticulum overload response(EOR),which can help maintain intracellular homeostasis.But persistent ERS will lead to apoptosis.UPR is the most important and most clearly studied one among three pathways.In the early stage of ER stress,through UPR pathway,misfolded proteins are eliminated,and correct protein folding is promoted to recover cellular physiological function and reconstruct ER homeostasis.UPR is mediated by a binding immunoglobulin heavy chain protein(BiP)/glucose-regulated protein 78(GRP78)along with three ER stress sensor proteins including inositol requiring kinase(IRE),PRER and activating transcription factor 6(ATF6).ATF6 is a type 2 transmembrane protein kinase outside of ER membrane.In response to ER stress,ATF6 is transported from ER to Golgi apparatus and cleaved into transcriptionally active p50ATF6.Then p50ATF6,combining with promoter of ER stress response elements in cell nucleus,induces expression of ER stress related genes such as C/EBP homologous protein(CHOP)/growth arrest and DNA damage inducible gene 153(GADD153),BiP/GRP78,X-box-binding protein 1(XBP1),calretinin etc.,consequently to regulate cell survival or apoptosis.Recent studies have made some progress in the regulatory mechanism of ATF6,and it is found that the regulatory pathways of activated ATF6 in different cells are different,but there are few studies on the specific mechanism of apoptosis of vascular endothelial cells in ERS state,so we decided to explore the related mechanisms.Material and methods1.Endoplasmic reticulum stress was induced by thapsigargin in vascular endothelial cells,and then transferred into vascular endothelial cells with ATF6 plasmid.2.To detect the effect of ATF6 on the proliferation and apoptosis of vascular endothelial cells under endoplasmic reticulum stress.3.To detect the effect of ATF6 on the expression of caspase-3,caspase-4,caspase-9,caspase-12,CHOP,Cyt C in vascular endothelial cells under endoplasmic reticulum stress.4.To detect the effect of ATF6 on the expression of Bax,Bcl-2,phosphorylated-JNK,JNK and NF-kappa B in vascular endothelial cells under endoplasmic reticulum stress.5.To detect the effect of ATF6 on the expression of pro-caspase-8,cleaved-caspase-8 and Fas in vascular endothelial cells under endoplasmic reticulum stress.Results1.High expression of ATF6 detected in ATF6(1?366aa)?transfected cells.In testing the transfection efficiency,expression of ATF6 protein was detected through western blotting of ATF6(151-3aa)and ATF6(1-366aa)groups.The ATF6 protein was five times more highly expressed in cells that were transfected with the ATF6(1-366aa)plasmid than in other groups(P<0.01).2.TG treatment for 48 h decreased viability of VEC.Cell viabilities in TG,ATF6(151-366aa)+ TG and ATF6(1-366aa)+ TG groups were inhibited under TG-induced ER stress compared to that inthe control group.ATF6(1-366aa)+ TG cells were the most sensitive to ER stress,showing the lowest viability.Cell viability after TG treatment decreased in a time-dependent manner.Furthermore,there was a significant difference among groups when the cells were treated with TG for 48 h(P<0.01 or P<0.05).3.TG treatment significantly increased cell apoptosis rate.Compared to that in the control group,cell apoptosis rates in three TG-treated groups increased,particularly in ATF6(1-366aa)-transfected cells,with the highest significant increase from 5.50±0.43 to 37.41±2.99 5%(P<0.01).Moreover,the apoptosis rate in ATF6(1-366aa)+ TG group was 2.5 times higher than that in the TG only and ATF6(151-366aa)+ TG groups.This reflects that high expression of active ATF6 induces cell apoptosis under ER stress(P<0.01).4.High expression of active ATF6 increased activation of caspase?3,caspase?4,caspase?9 and caspase?12 and cleavage of PARP.TG treatment increased cleavage of caspase-3,caspase-4,caspase-9,caspase-12,and PARP in VEC,when compared to that in the control group(P<0.01).In ATF6(1?366aa)+ TG group where active ATF6 was over-expressed,the protein levels of cleaved caspase-3,caspase-4,caspase-9,caspase-12,and PARP were significantly higher than those in TG only and ATF6(151?366aa)+ TG groups(P<0.01).5.High expression of active ATF6 increased expression of CHOP,cyt c,and Bax but led to decreased Bcl?2 protein levels.RT-PCR and western blotting were conducted to analyse the expression levels of apoptosis-related proteins,including CHOP,cyt c,Bax,and Bcl-2.TG treatment of VECs for 48 h in TG,ATF6(151-366aa)+ TG,and ATF6(1-366aa)+ TG groups increased mRNA and protein levels of CHOP and cyt c in comparison with that in the control group(P<0.05).Expression of these mRNA and proteins in ATF6(1?366aa)+ TG group was significantly different from that in the TG and ATF6(151?366aa)+ TG groups(P<0.01).Expression levels of Bax mRNA and protein were up-regulated,while those of Bcl-2 were down-regulated by TG treatment(P<0.01).mRNA expression and protein levels of CHOP and Bax were nearly double in ATF6 over-expressing cells,while cyt c levels were approximately three times higher.Up-regulation of ATF6 led to a decreased expression of Bcl-2.6.High expression of active ATF6 activated JNK/NF??b pathway.The protein levels of total and phosphorylated JNK as well as total NF-?B were detected by western blotting.The ratio of p-JNK/JNK dramatically increased upon TG treatment(P<0.01).Phosphorylation levels of JNK was further elevated when active ATF6 was up-regulated in ATF6(1-366aa)+ TG(P<0.01).High expression of NF??B was also observed in TG-treated groups,especially in active ATF6-up-regulated cells where the protein levels of NF-?B nearly doubled in comparison to that in the TG and ATF6(151-366aa)+ TG groups(P<0.01).7.No significant effect of TG treatment and up?regulation of ATF6 was found on expression of death receptor-related genes.In the present study,Fas mRNA and protein were determined using PCR and western blotting.The results showed no significant difference in its expression among control,TG,ATF6(151-366aa)+ TG and ATF6(1-366aa)+ TG groups(P>0.05).Moreover,the protein levels of pro-caspase-8 and cleaved-caspase-8 were also found to be not affected by TG treatment and ATF6 expression(P>0.05).ConclusionHigh expression of ATF6 decreased viability and aggravated ER stress-induced apoptosis in VECs.Increased expression of apoptosis-related genes,including those encoding caspase-3,caspase-9,C/EBP homologous protein(CHOP),cytochrome c and B-cell lymphoma-associated protein X(Bax)/B-cell lymphoma(Bcl-)2,was detected by polymerase chain reaction and western blotting in the ATF6(1?366aa)+ TG group.No significant effect of TG treatment and high ATF6 expression was indicated on the expression of death receptor-related genes,including those encoding caspase-8 and Fas.The results demonstrated that high expression of activated ATF6 aggravates ER stress-induced VEC apoptosis through the mitochondrial apoptotic pathway.Furthermore,in response to ER stress,ATF6 upregulates the expression of caspase-3,caspase-9,CHOP,cytochrome c and Bax/Bcl-2.