Effect Of Calpain-2 On CCPG1-mediated ER-phagy By Regulating Atg5 And Its Role In Hepatocyte Apoptosis

Objective:To explore whether calcium neutral protease 2(calpain-2)affects cell cycle progression gene 1(CCPG1)-mediated ER-phagy by regulating autophagy related protein 5(Atg5)during the process of dithiothreitol(DTT)-induced endoplasmic reticulum stress(ERS).Methods:BRL-3A cells were treated with 2.0mmol/L DTT for 0,6,12,and 24 h to induce ERS.The effect of DTT on the proliferation of BRL-3A cells was detected by real time cellular dynamic analysis(RTCA).Cell apoptosis and cell cycle were detected by flow cytometry.The m RNA expression of glucose regulated protein 78(GRP78),CCPG1,focal adhesion kinase family interacting protein of 200(FIP200),microtubule-associated protein 1 light chain 3(LC3),Atg5 and calpain-2 was detected by real-time PCR.The proteins expression of GRP78,CCPG1,FIP200,LC3,Atg5,autophagy related protein 12(Atg12),calpain-2 was detected by Western blot.Immunoprecipitation was used to investigate the interaction between CCPG1 and LC3,CCPG1 and FIP200,calpain-2 and Atg5.Additionally,in order to clarify the role of CCPG1 in ERS,siRNA technology was used to knock down the expression of CCPG1 in BRL-3A cells,and the effect of CCPG1 gene silencing on ERS-induced apoptosis of hepatocytes was observed.Moreover,in order to clarify the regulation mechanism of calpain-2 on endoplasmic reticulophagy,BRL-3A cells were pretreated with 10 μmol/L calpain specific inhibitor Z-LLY FMK for 1 h,followed by DTT treatment for 24 h,the protein expression of calpain-2 and Atg5 after Z-LLY-FMK pretreatment and the hepatocyte apoptosis were detected.Results:The proliferation of BRL-3A cells was significantly inhibited after DTT treatment.Flow cytometry showed that cell proliferation of BRL-3A cells was blocked in G1 phase after DTT treatment.The apoptosis of BRL-3A cells after DTT treatment was significantly increased compared with control group.After treated with DTT for 6,12,24 h,the m RNA expression levels of GRP78,CCPG1,FIP200,LC3,Atg5 and calpain-2 in BRL-3A cells were significantly up-regulated compared with those in control group.The protein levels of GRP78,CCPG1,FIP200,Atg12,Atg7,calpain-2 and the ratio of LC3 II /LC3 I were also significantly increased after DTT treatment,while the protein expression level of Atg5 was significantly decreased after DTT treatment.Protein immunoprecipitation showed that CCPG1 antibody could precipitate FIP200 and LC3 protein from cell lysates,which confirmed the interaction between CCPG1 and FIP200,CCPG1 and LC3.The calpain-2 antibody could precipitate Atg5 protein from the cell lysates,and the Atg5 antibody also could precipitate calpain-2 protein from the cell lysates,confirming the interaction between calpain-2 and Atg5.After down-regulation of CCPG1 gene expression in BRL-3A cells by small interfering RNA technology,The m RNA level and protein expression level of CCPG1 siRNA group were significantly decreased compared with the control group,and the apoptosis level of hepatocytes in CCPG1 siRNA group was significantly increased compared with the control group.After calpain specific inhibitor Z-LLY-FMK pretreatment,the apoptosis of hepatocytes in the Z-LLY-FMK pretreatment group was significantly lower than that in the DTT group.Meanwhile,the expression level of Atg5 protein in the Z-LLY-FMK pretreatment group was significantly higher than that in the DTT group.Conclusion:CCPG1-mediated ER-phagy plays a protective role in DTT-induced endoplasmic reticulum stress,while calpain-2 may inhibit the process of ER-phagy and promote hepatocyte apoptosis by regulating Atg5.