The Role Of Dual Specificity Phosphatase 5 In BCG Infection Induced Autophagy In RAW264.7 Cell Lines

Tuberculosis is a chronic pulmonary infectious disease caused by Mycobacterium tuberculosis(Mtb).Macrophages are the host cells of Mtb.Numbers of studies have indicated macrophages clear intracellular Mtb through autophagy,however,Mtb have developed strategies to escape from the clearance.Multiple pathways,including MAPK are involved in autophagy regulation.Dual specificity protein phosphatases(DUSPs)are the endogenous inhibitors of MAPKs and play a key role in MAPK regulation.Research showed,DUSP5,a member of the DUSPs,participated in regulating autophagy of many cells,few reports if it regulated autophagy of macrophages which induce by Mtb infection.To investigate the regulation and mechanism of DUSP5 in Mtb induced autophagy of macrophages,lentivirus transfection were applied to establish DUSP5 stably overexpressed/interfered RAW264.7 cell strains.Rapamycin and bafilomycin stimulated DUSP5 stably overexpressed/interfered RAW264.7 cell strains respectively to explore the regulation of DUSP5 in macrophage autophagy preliminarily.Then BCG and inhibitor PD98059 were used to stimulate DUSP5 stably overexpressed/interfered RAW264.7 cell strains to investigate the regulation and mechanism of DUSP5 in macrophages autophagy induced by BCG.The results are as following:1.We established DUSP5 stably overexpressed/interfered RAW264.7 cell strains by using lentivirus transfection which provided a good experimental materials for subsequent investigation of regulation of DUSP5 in macrophage autophagy.2.With the stimulation of rapamycin,the expression of autophagy-related protein LC3? was significantly decreased in DUSP5 overexpressed RAW264.7 cell strains,whereas it was reversed as DUSP5 was inhibited,indicating DUSP5 suppressed LC3? expression.3.LC3? expression and the number of autophagosomes of DUSP5 interfered RAW264.7 cell strains were increased as the cells were treated by bafilomycin A1,suggesting DUSP5 could inhibits the formation of autophagosomes and play negative regulatory role in macrophages autophagy.4.By the stimulation of BCG,over-expression of DUSP5 was significantly reduced the expression of autophagy-related protein Beclinl?Atg5?Atg7 and LC3? and the number of autophagosomes,while they were up-regulated in DUSP5 stably interfered RAW264.7 cell strains.This demonstrated that DUSP5 supressed the expression of RAW264.7 autophagy-related protein in BCG-infected macrophages,which may play a negative regulatory role in macrophages RAW264.7 autophagy induced by BCG infection.5.The expression of p-ERK was up-regulated after BCG stimulation,however,the expression of it was down-regulated in DUSP5 stably overexpressed RAW264.7 cell strains compared with cells transfected empty vector and this phenomenon was reversed as DUSP5 was interfered and it had on effect on the expression oftotal-ERK1/2.It indicated BCG could activate ERK1/2 signaling pathway as macrophages was infected,while DUSP5 inhibited the phosphorylation of ERK1/2.6.When PD98059 was used to inhibit ERK1/2 signaling pathway,the expression of autophagy-related protein Beclinl was significantly down-regulated both in DUSP5 stably overexpressed and interfered RAW264.7 cell strains,while had no significant influence on LC3?.This indicated the expression ofbeclinl was regulated by ERK1/2 signaling pathway.In summary,DUSP5 is involved in the regulation of M.tuberculosis infection-induced autophagy of macrophages.It inhibited the phosphorylation of ERK1/2 and participated in the process of protein expression which correlated with ERK1/2 signaling pathway activation.Among autophagy.related protein expression,this process mainly mediates the expression of Beclinl.Beclinl is the initial protein of autophagy and it participates in the formation of autophagosome membranes.It is speculated that DUSP5 is involved in the regulation of BCG-induced macrophages RAW264.7 autophagy may be through inhibition of ERK1/2 signaling pathway,inhibition of Beclinl expression,resulting in blocked autophagosome membrane formation,and then play a negative regulatory role in autophagy.