Study On The Mechanism By Which Integrin ?4 Associated Apoptosis With Autophagy In Vascular Endothelial Cells

Background and ObjectiveVascular endothelial cells are all throughout the whole body.In addition to the vascular disease,the dysfunction of vascular endothelial cells can also cause neurodegenerative diseases,diabetes,autoimmune diseases and so on.Autophagy and apoptosis are two important factors to influence the function of endothelial cells.There are close relationship between autophagy and apoptosis,however,the molecular mechanisms of their conjunction are not very clear.To make clear their conjunction and its underlying molecular mechanisms as well as find out the key factors and signaling pathway that can inhibit the apoptosis are important strategies for prevention and treatment of vascular related diseases.Three major autophagic pathways have been described,macroautophagy(hereinafter referred to as autophagy),microautophagy and chaperone-mediated autophagy.Macroautophagy(hereinafter referred to as autophagy)plays an important role in controlling cell fate and is closely associated with apoptosis.Autophagy is of obvious cell specific.At present,most studies are focusing on autophagy in carcinoma cells or autophagy induced by starvation.Neither the regulation of autophagy nor the relation of autophagy and apoptosis in vascular endothelial cells are well studied.There are crosstalk between apoptosis and autophagy,which reflect the synergy in autophagy and apoptosis to promote cell death;autophagy roled in antagonism the apoptosis.Some factors can regulate both of these two processes.However,what are the key factors associating autophagy and apoptosis in endothelial cells?What are the molecular switches and signal pathways of autophagy and apoptosis in vascular endothelial cells?These need to be further studied.In our previous studies,we found that during the process of multiple stimuli regulating apoptosis in vascular endothelial cells,the protein level and the subcellular localization of integrin ?4 changed;integrin ?4 was involved in A549 lung cancer cell autophagy and cadmium-induced autophagy in HUVECs.Thus,integrin ?4 could be a linker that associating apoptosis and autophagy in endothelial cells.But the detailed mechanism that integrin ?4 associates apoptosis and autophagy in vascular endothelial cells is unclear and needs to be further studied.That is:under what cases that integrin ?4 roled in promoting autophagy while inhibiting apoptosis or promoting apoptosis.Studies have showed that phosphorylation of the integrin ?4 cytoplasmic domain at Y-1494 plays a critical role in downstream signaling transduction;this site controls overall tyrosine phosphorylation in the cytoplasmic domain of integrin ?4 as well as its subcellular localization.Whether this site is phosphorylated and the position is changed in apoptotic endothelial cells are not known.Sphingosylphosphorylcholine,broken down by sphingomyelin,is an important cardiovascular regulatory factor.In the plasma,SPC is identified as a component of high density lipoprotein.SPC is the endogenous factor that has an important role in the regulation of physiological function of endothelial cells.However,whether SPC can regulate autophagy and apoptosis by integrin ?4 in vascular endothelial cells is not known.Application of chemical biology provides important experimental basis for clarifying the problems of life sciences.We screened a small chemical molecules that could induced nuclear translocation of integrin ?4 and promoted apoptosis in endothelial cells.This provides a new powerful tool for our in-depth study of integrin?4 in the subcellular location as well as regulation of endothelial cell apoptosis.In view of the above problems,here,we studied the role and the action mechanism of integrin ?4 in autophagy induced by sphingosylphosphorylcholine(SPC);Using ECPC,31-S as tools,we further investigate integrin ?4 nuclear translocation and its role in apoptosis regulation.These would provide a new experimental evidence for clarifying the molecular mechanism of SPC in regulation of vascular endothelial cell function;finding out new mechanisms of integrin ?4 in the nuclear translocation and in the regulation of vascular endothelial cell apoptosis,all of which provide scientific basis for new clinical strategies.Study Contents1.Sphingosylphosphorylcholine induced autophagy and inhibited apoptosis in vascular endothelial cells by the deprivation of serum and FGF-2.2.To study the molecular mechanisms of sphingosylphosphorylcholine-induced autophagy.3.Using epoxy alkyl pyrazole derivatives ECPC as a tool,investigating integrin ?4 nuclear translocation as well as the molecular mechanism in regulation of apoptosis in vascular endothelial cells.4.Effects of chiral pyrazole alkylene oxide derivative 31-S on integrin ?4 nuclear translocation.Methods1.Isolation and culture of vascular endothelial cells:According to the methods of Jaffe et al[1].2.Detection of autophagy and autophagic influx:1)Using Western blot to detect the protein level of LC3-II and p62 to analyze the number of autophagosomes and autophagic influx.2)Immunofluorescence to detect the number and distribution of MAPI LC3 protein in HUVECs.3)Using an autophagy inhibitor 3MA to detect autophagy and autophagic influx induced by SPC.4)With ATG5 siRNA to knockdown its expression,SPC treatment and Western blot to analyze the protein level of LC3-?.3.Detection of apoptosis:1)Inverted phase contrast microscope to observe changes in cell morphology.2)MTT and SRB to analyze cell viability.3)Hochest 33258,TUNEL to indicate apoptosis.4)Western blot to detect PARP cleavage.5)Using fluorescence probe JG-1 to detect mitochondrial membrane potential.6)Using fluorescence probe DCHF to detect ROS level.4.Assay the activity of phosphatidylcholine-specific phospholipase C:Accroding to the previous report by Wu et al[2].5.Using RNA interference to knockdown the gene expression:Chemical synthesis of siRNA against ATG5,integrin ?4,FGFR1 to specific inhibit genes expression.6.Overexpression of integrin ?4:HUVECs and HEK 293 cells were transfected with full-length integrin P4-cDNA constructs(pCMV6-XL5-integrin ?4)that were wide type or mutated type with tyrosine 1494 to phenylalanine.7.Microarray analysis to detect changes of genes.8.Immunoprecipitation to determine the interaction of integrin ?4 and FGFR1.9.Detection of the distribution and the expression of proteins.1)Immunofluorescence staining with confocal microscopy to detect protein level and distribution of p53 and integrin ?4.2)qPCR to analyze the mRNA of KITLG,PTGS2,ATF3,IL-8,TGF-?2 and GAPDH.3)Western blot to detect the protein level of LC3,p62,ATG5,p53,integrin ?4,FGFR1,KITLG,PTGS2,ATF3,IL-8,phospho-Y-1494 integrin ?4,PARP,GAPDH,?-actin.4)Nuclear and non-nuclear cellular components were extracted,with Western blot to detect the distribution of integrin ?4.Results1.Study on the molecular mechanism of sphingosylphosphorylcholine inhibited apoptosis while induced autophagy in vascular endothelial cells under the conditions of serum and FGF-2 deprivation1.1 Sphingosylphosphorylcholine induced autophagy and inhibited apoptosis in vascular endothelial cells by the deprivation of serum and FGF-2.We first used MTT to analyze the viability of HUVECs,and found that sphingosylphosphorylcholine(SPC)could inhibit the decreased cell viability induced by deprivation of serum and FGF-2.Hoechst 33258 staining showed that SPC inhibited cell apoptosis.As we know,there are close link between autophagy and apoptosis,we next examed the effect of SPC on autophagy.Using western blot and immunofluorescence microscopy,we analyzed the effect of SPC on autophagy and the results showed that SPC induce autophagy in HUVECs.1.2 Study on the molecular mechanism of sphingosylphosphorylcholine inducing autophagy in vascular endothelial cells.Our previous study showed that integrin ?4 was key factor involved in A549 lung cancer cell autophagy and cadmium-induced autophagy in HUVECs.In this study,we examed that during SPC induced autophagy,the level of integrin ?4,PC-PLC activity and the level of p53 were downregulated by SPC.Using the strategies of siRNA and overexpression of integrin ?4,we found that SPC promoted autophagy via integrin ?4;PC-PLC and p53 may be its downstream factors in autophagy of VECs deprived of serum and FGF-2.2.Using epoxy alkyl pyrazole derivatives ECPC as a tool,investigating integrin?4 nuclear translocation as well as the molecular mechanism in regulation of apoptosis in vascular endothelial cells2.1 Epoxy alkyl pyrazole derivatives ECPC promoted HUVEC apoptosis via integrin?4 in the absence of serum and FGF-2.After treatment with ECPC under conditions of serum and FGF-2 deprived,mostcells had detached from the substratum and become round.The viability of HUVECs was significantly decreased by SRB.Western blot showed that cleaved Poly(ADP-ribose)polymerase(PARP)was apparent in HUVECs stimulated with ECPC.In addition,TUNEL assay revealed that ECPC significantly promoted DNA fragmentation.Therefore,ECPC induced HUVEC apoptosis.Kncokdown the expression of integrin ?4 using its specific siRNA inhibited apoptosis by ECPC.We concluded that ECPC promoted HUVEC apoptosis via integrin ?4.2.2 Molecular mechanisms of ECPC inducing apoptosis via integrin ?4 in HUVEC.We investigated the phosphorylation of Y-1494 in integrin ?4 cytoplasmic domain and found that it increased with ECPC treatment.The results of immunofluorescence microscopy showed that integrin ?4 translocated from the cytoplasm to nucleus in response to ECPC in HUVECs;Moreover,integrin ?4 nuclear translocation promoted the expression of KITLG,IL-8,PTGS2 and ATF3,which were all involved in inflammation and apoptosis.Physiological concentration of FGF-2 is necessary for VEC survival via FGFR1;the much higher or lower levels would lead to cell apoptosis.FGF-2 pretreatment inhibited ECPC-induced integrin ?4 phosphorylation.With siRNA knockdown of FGFR1,ECPC could not trigger phosphorylation of Y-1494.In addition,the results of immunoprecipitation showed that the interaction between FGFR1 and integrin ?4 increased in response to ECPC stimulation,and FGFR1 could phosphorylate integrin P4 directly.Therefore,FGFR1 mediated phosphorylation of integrin ?4 induced by ECPC in the absence of serum and FGF-2.With inhibiting phosphorylation of integrin ?4 by FGF-2 pretreatment,ECPC-triggered nuclear translocation of integrin ?4 as well as subsequent upregulation of KITLG,IL-8,PTGS2 and ATF3 were also blocked.3.Using chiral pyrazole alkylene oxide derivative as a tool to study the molecular mechanism of integrin ?4 nuclear translocation3.1 Chiral pyrazole alkylene oxide derivative 31-S was identified to induce integrin ?4 nuclear translocation and apoptosis in endothelial cells.Based on the structure of ECPC,several chiral chemical small molecules was synthesized.In the absence of serum and FGF-2,HUVECs were treated with these chemical small molecules,through morphological observation,SRB,MMP,ROS detection,Hoechst 33258,Tunel staining as well as immunofluorescence to localization of integrin ?4,we identified 31-S that could induce integrin ?4 nuclear translocation and apoptosis in HUVECs.3.2 Using 31-S as the tool to investigate the molecular mechanism of integrin ?4 nuclear translocation HEK 293 cells were transfected with GFP-tagged full-length wide or mutated integrin p4 with tyrosine 1494 to phenylalanine,after 31-S treated,the level of mutated integrin ?4 in nuclear was declined;In addition,benzoxazine derivative ABO could inhibit integrin ?4 nuclear translocation induced by 31-S.Conclusions1.Sphingosylphosphorylcholine(SPC)inhibited apoptosis and induced autophagy by deprivation of serum and fibroblast growth factor 2;SPC promoted autophagy and inhibited apoptosis via downregulating integrin ?4;PC-PLC and p53 were the downstream factors of integrin ?4.2.Epoxy alkyl pyrazole derivatives ECPC promoted HUVEC apoptosis via integrin?4 in the absence of serum and FGF-2.3.In the absence of of serum and fibroblast growth factor 2,phosphorylation of Y-1494 in the integrin ?4 cytoplasmic domain via FGFR1 induced by ECPC contributed to the nuclear translocation of integrin ?4,which upregulated the expression of apoptosis and inflammation related genes:ATF3,PTGS2,KITLG and IL-8,resulting in apoptosis of VECs.FGF-2 inhibited phosphorylation of integrin ?4 and blocked integrin ?4 nuclear translocation,downregulating the expression of ATF3,PTGS2,KITLG and IL-8 and thus cell apoptosis in VECs.4.Using chiral pyrazole alkylene oxide derivative 31-S as a tool to study the nuclear translocation of integrin ?4,phosphorylation of integrin ?4 at Y-1494 is critical for integrin ?4 nuclear translocation induced by 31-S;ABO could inhibit integrin?4 nuclear translocation induced by 31-S.