The Interaction Of Atg4B And Bcl-2 Plays An Important Role In Cdinduced Crosstalk Between Apoptosis And Autophagy Through Disassociation Of Bcl-2-Beclin1 In A549 Cells

Cadmium(Cd)is a non-essential element and toxic to human body.Cd enters into human bodies through inhalation or skin absorption.Because of its long half-life and low rate of excretion,Cd gradually accumulated into various tissues and organs and caused organ injury,such as pulmonary edema,hepatic injury and even promoted cancer development.A large number of studies reported that low level of Cd-induced apoptosis and autophagy.Apoptosis and autophagy are not mutually independent pathways.A growing number of studies showed the cooperative relationship between apoptosis and autophagy.Several commonly known inducers of autophagy have been shown to activate apoptosis,such as Atg12,which can also induce apoptosis through associates with antiapoptotic Bcl-2 family members.Similarly,activation of apoptosis can also mediate autophagy.Data from several reports suggested that the members of the Bcl-2 family regulate autophagy in addition to their classical function in controlling the pathways of caspase activation.Atg4 B is one of the autophagy-related(Atg)genes,numerous studies have demonstrated the correlation between Atg4 B and autophagy.In our previous study,we found that Cd-induced reactive oxygen species(ROS)formation provoked Atg4 B up-regulation,caused cell cycle changes and led to cell proliferation in MRC-5 cells.There is no report demonstrated the role of Atg4 B in Cd-induced apoptosis and the mechanism of Cd-induced apoptosis and autophagy is not clear.So,in this study,we aimed to know the function of Atg4 B in apoptosis and autophagy in Cdtreated A549 cells.Objective: To demonstrate the effects of low-dose Cd on apoptosis and autophagy,investigated the involvement of Atg4 B in Cd-induced apoptosis and autophagy.Methods: In this study,A549 cells were treated as in vitro models.Western blot was used to observe the expression of apoptosis and autophagy-related proteins in A549 cells after Cd treated 0,12,24,36 and 48 hrs.The effects of Cd on apoptosis and autophagy were further examined by direct observation of the nuclei fragmentation and condensation and autophagolysosomes(AVOs)formation using Hoechst and Acridine orange(AO)assay respectively.AO and Western blot were used to observe the number of AVOs and the expression of autophagy-related proteins to examined the effect of ZVAD-FMK on Cd-induced autophagy in A549 cells.To verify the effect of autophagy on Cd-induced apoptosis,3MA,the autophagy inhibitor was employed.Hoechst and Western blot were used to observe the number of nuclei fragmentation and the expression of apoptosis-related proteins in A549 cells.JC-1 was used to detect the effect of Cd and 3MA on mitochondrial membrane potential(MMP)in A549 cells.To investigate the function of Atg4 B in Cd-induced apoptosis,A549 cells were transfected by siRNA and plasmids.To investigate whether the depolarization of MMP plays a role in Cd-induced Atg4 B expression,CCCP,a mitochondrial function inhibitor was used.Cells were used for Western blot to observe the expression of Atg4 B.To determine the role of Atg4 B on depolarization of MMP,cells were investigated using JC-1.To investigate the function of Bcl-2 in Cd-induced autophagy,A549 cells were transfected by siRNA and plasmids.To verify the regulative mechanism underlying the crosstalk between apoptosis and autophagy induced by Cd,the interaction of Atg4 B and Bcl-2 was investigated.Plasmids Atg4 B and Bcl-2 were transfected separately or transfected together in A549 cells.The expressions of apoptosis and autophagy-related proteins were further investigated by Western blot analysis.To confirm a direct relationship between Atg4 B,Bcl-2 and Beclin1,Co-immunoprecipitation(COIP)assay was carried out.Results: After Cd treatment for 0,12,24,36 and 48 hrs,apoptosis-related proteins,cleaved Caspase9(CL-CASP9),cleaved Caspase3(CL-CASP3)and cytosol cytochrome c were increased and Bcl-2 was down-regulated significantly during 12 to 24 hrs(P<0.05 and P<0.01).The Cd-treated cells showed nuclear shrinkage,chromatin condensation,or fragmentation when treated for 12-24 hrs(P<0.01).These results suggested that Cd-induced apoptosis at 12 and 24 hrs.Meanwhile,a significant increase in LC3-II protein levels and a decrease in P62 protein levels were observed after Cd treatment for 36 and 48 hrs(P<0.01).The number of AVOs(positive red puncta)in Cdtreated cells significantly increased after 24 hrs in A549 cells(P<0.01).These results suggested that Cd-induced autophagy at 36 and 48 hrs.Cd-induced LC3-II and Beclin1 protein expression was reduced,and P62 was upregulated significantly in Z-VAD-FMK pretreatment group compared with that of only Cd-treated group(P<0.05 and P<0.01).Also,the percentage of AVOs induced by Cd was significantly decreased after pretreatment with Z-VAD-FMK compared with only Cd-treated cells(P<0.01).These data suggested that apoptosis induced by Cd treatment played an important role in triggering Cd-induced autophagy.On the other hand,pretreatment with 3MA raised Cdinduced CL-CASP9,CL-CASP3 and cytosol cytochrome c significantly after incubation with Cd for 48 hrs compared to the only Cd-treated A549 cells(P<0.01).Hoechst staining also illustrated that there was a significant increase in the number of apoptotic cells in 3MA pretreated groups compared to only Cd-treated cells(P<0.01).These results demonstrated that autophagy induced by Cd inhibited Cd-induced apoptosis and might have a protective effect on Cd-induced apoptosis.To further confirm the protective effect of autophagy in Cd-induced apoptosis,Cd-induced depolarization of MMP was detected by JC-1.Cd treatment reduced MMP activity significantly around 12 to 24 hrs and then significantly rescued during 36-48 hrs(P<0.01).However,after 3MA pretreatment,the rescue of MMP was reduced significantly compared with only Cd-treated cells(P<0.01).This result indicated that exposure to Cd caused a depolarization of MMP and Cd-induced autophagy might impose a protective effect on Cd-induced apoptosis via the ability of rescuing the damage to MMP induced by Cd.A549 cells were transfected with Atg4 B siRNA to knock down the expression of Atg4 B.There was a significant decrease of CL-CASP9 and CL-CASP3 protein level in Cd-treated Atg4B-siRNA A549 cells compared with the only Cd-treated cells(P<0.05 and P<0.01).Bcl-2 was increased in Cd-treated Atg4 BsiRNA A549 cells compared with the only Cd-treated cells(P<0.05).In addition,to obtain overexpression of Atg4 B,plasmid Atg4 B and empty vector were transfected in A549 cells.It was observed that both the expression of CL-CASP9 and CL-CASP3 increased in the plasmid Atg4 B transfected A549 cells compared with the empty vector cells(P<0.01),whereas the level of Bcl-2 protein had a evident decrease in plasmid Atg4 B transfected A549 cells.The role of Atg4 B in Cd-induced apoptosis was further examined by TUNEL assay and Hoechst 33342 staining.The number of TUNEL positive cells increased significantly in plasmid Atg4 B group compared with empty vector group(P<0.05).Hoechst 33342 staining also showed that the number of pyknotic nuclei in plasmid Atg4 B group was significantly increased compared with empty vector group(P<0.01).To investigate whether the depolarization of MMP plays a role in Cdinduced Atg4 B expression,CCCP,a mitochondrial function inhibitor was used.After A549 cells were treated with different concentrations(0.1 and 0.5μM)of CCCP for 24 hrs,the expression of Atg4 B was significantly increased at the concentration of 0.5μM compared with that of the control cells(P<0.01).To determine the role of Atg4 B on depolarization of MMP,A549 cells were transfected with Atg4 B siRNA or plasmid Atg4 B.The MMP did not change significantly in both Atg4 B siRNA and plasmid Atg4 B transfected A549 cells compared to control cells,indicated that the depolarization of MMP had a strong effect on the expression of Atg4 B protein and Atg4 B had no effect on MMP.The role of Bcl-2 in Cd-induced autophagy was investigated using Bcl-2 siRNA and plasmid transfection to knock down or overexpress Bcl-2.Bcl-2 knockdown significantly increased the relative amount of LC3-II and Beclin1,and decreased P62 protein expression induced by Cd treatment compared to only Cd-treated cells(P<0.05 and P<0.01).Whereas Bcl-2 overexpression by Bcl-2 plasmid transfection in A549 cells decreased the relative amount of LC3-II and Beclin1,and increased P62 protein expression significantly compared with the empty vector cells(P<0.05).These results indicated that Bcl-2 played an important role in Cd-induced autophagy.To verify the regulative mechanism underlying the crosstalk between apoptosis and autophagy induced by Cd,the interaction of Atg4 B and Bcl-2 was investigated.Plasmids Atg4 B and Bcl-2 were transfected separately or transfected together in A549 cells.Overexpression of Bcl-2 reduced Atg4B-transfection-induced LC3-II,Beclin1,CL-CASP9 and CL-CASP3(P<0.05 and P<0.01).This result demonstrated that Bcl-2 plays an important role in Atg4B-induced autophagy and apoptosis.To further illustrate the regulative relationship between Atg4 B and Bcl-2,CoIP experiment was performed.IP of Bcl-2 with specific anti-Bcl-2 pulled down Atg4 B more from Cd-treated A549 cells than from control cells.At the same time,treatment with Cd restrained the Bcl-2-Beclin1 interaction,as indicated by the reduction of the specific pull-down bands.Conclusion: These results demonstrated that Atg4 B directly interacted with Bcl-2 causing the dissociation between Bcl-2 and Beclin1 and initiated the crosstalk of apoptosis and autophagy in Cd-treated A549 cells.