Cr(?)Induced Mitophagy Via The Interaction Of HMGA2 And BNIP3/PARK2 In A549 Cells

Chromium mainly exists in two forms: trivalent chromium(Cr(?))and hexavalent chromium(Cr(?)).Cr(?)is a proven carcinogen.A great deal of epidemiological studies have shown that lung cancer is associated with occupational exposure to Cr(?).Some researches reported that Cr(?)could induce autophagy.However,there was no report concerned with Cr(?)-induced mitophagy.Mitophagy is a selective engulfment of mitochondria,in which unwanted or damaged mitochondria are engulfed by autophagosomes.Growing evidence has suggested that the loss of mitochondrial membrane potential(MMP)is an important signal for mitophagy,and the PINK1-PARK2 pathway is regarded as a main pathway for mitophagy and has been well-characterized for mitophagy.Increasing number of studies imply that autophagy is activated under endoplasmic reticulum(ER)stress.HMGA2 participates in a variety of biological processes,it has emerged as a biomarker of tumors.In our previous study,we were the first to demonstrate that HMGA2 was involved in Cr(?)-induced autophagy in A549 cells.Meanwhile,Cr(?)-induced mitochondrial morphological changes,such as mitochondrial swelling and vacuolization were also observed when the autophagosomes were detected by transmission electron microscopy(TEM).Therefore,in this study,we investigated whether Cr(?)could induce mitophagy and the possible role of HMGA2 played in Cr(?)-induced mitophagy.Objective: To investigate the effects of low concentrations of Cr(?)on mitophagy,and the role of HMGA2 in Cr(?)-induced mitophagy.Methods: A549 and HEK 293 cells were selected as experimental models in vitro.The expressions of mitophagy-related proteins,HMGA2 and ER stress markers weredetected by Western Blot.The formation of double membrane autophagic vesicles(AVs)engulfing mitochondria and mitochondrial morphology were observed by TEM.The mitochondrial membrane potential(MMP)was observed by JC-1 staining.HMGA2 was down-regulated by HMGA2 siRNA.HMGA2 was up-regulated by plasmid HMGA2.Co-Immunoprecipitation assay was carried out to investigate the interaction of HMGA2 with PARK2,BNIP3.Results: The expression of mitophagy-related proteins(PINK1,PARK2,LC3 ?),ER stress markers(GRP78,p-PERK)and HMGA2 was induced in Cr(?)-treated A549cells(P<0.05 and P<0.01).And the transmission electron microscopy showed that the formation of double membrane autophagic vesicles(AVs)engulfing mitochondria was evident and mitochondrial damages(mitochondrial swelling,ridge fracture,vacuolar degeneration)were observed in Cr(?)-treated cells.Exposure to Cr(?)significantly reduced MMP activity during 6-12 h compared to that observed in control cells by JC-1staining.These results demonstrated that Cr(?)induced mitophagy,mitochondrial damage and ER stress in A549 cells.The expression of HMGA2,GRP78,p-PERK,PINK1,PARK2 and LC3 ? in A549 cells was significantly increased after treated with CCCP(0.1 ?M)for 24 hours(P<0.05),indicating that mitochondrial damage mediated Cr(?)-induced HMGA2,ER stress and mitophagy.Pretreatment with 4PBA(5 mM)for 4 h significantly decreased Cr(?)-induced HMGA2,PINK1,PARK2 and LC3 ? expression in A549 cells(P<0.05,P<0.01),indicating that ER stress mediated Cr(?)-induced mitophagy and HMGA2 expression.HMGA2 siRNA reduced Cr(?)-induced PINK1,PARK2 and LC3 ? expression significantly(P<0.05,P<0.01);Moreover,HMGA2 siRNA reduced CCCP-induced PINK1,PARK2 and LC3 ? expression significantly(P<0.05,P<0.01),indicating that HMGA2 played a key role in Cr(?)-induced mitophagy mediated by mitochondrial damage.In order to further prove the role of HMGA2,HMGA2 was overexpressed by transfected with plasmid HMGA2 in HEK 293 cells,Western Blot showed that the levels of PINK1,PARK2 and LC3 ? proteins were significantly increased.Co-IP assay indicated that the direct binding of HMGA2 to BNIP3 and to PARK2 underlay the mechanism of HMGA2 mediating Cr(?)-induced mitophagy.Conclusion: The binding of HMGA2 to BNIP3 and PARK2 under the crosstalk of mitochondrial dysfunction and endoplasmic reticulum stress might be the main mechanism of Cr(?)-induced mitophagy in A549 cells.