Role Of AMPK-FOXO3a-BECN1-dependent Autophagy In Cadmium Toxicity In Stem Cells

BackgroundCadmium(Cd)is a toxic contaminant of natural and occupational environments,arising mainly form food chain and industrial applications.It can cause nonoccupational-exposure and occupational-exposure in workplaces in mammals.Bone is a very important target of Cd.Exposure to this toxic metal may lead to bone damage,even at low doses because it accumulates and has a long biological half-life in humans.Mesenchymal stem cells(MSCs)are generally described as a nonhematopoietic subpopulation of cells with self-proliferation ability that can differentiate into osteoblasts.Certain studies have reported that Cd affects MSC viability which inhibits bone formation and affects the well-being of bone tissue.Autophagy is a highly conserved catabolic pathway that is essential for the maintenance of cellular homeostasis via the removal of damaged organelles and toxic macromolecules.However,it also leads to cell death possibly via the activation of apoptosis or as a result of the inability of cells to survive the nonspecific degradation of large amouts of cytoplasmic contents.The mammalian forkhead transcription factors of the O class(FOXOs)members typically display redundancies in biological functions.Numerous numbers of studies have suggested that FOXO3 a is likely involved in the induction of autophagy,including autophagosome formation and autophagy-related gene expression.And AMPK enhanced the transcriptional activity of FOXO3 a via direct phosphorylation of FOXO3 a.Therefore,the purpose of this study is to explore whether Cd induced MSCs death and elucidate the potential mechanism involved it.Methods1.To verify whether Cd induced autophagic cell death in MSCs,The MSCs were exposed continuously to Cd at different concentrations(3.5,7,and 14 ?M)for 24 h or with 14 ?M Cd for various periods(0,6,12,and 24 h).After treatment,CCK-8 assay was used to measure cell viability and trypan blue assay was used to detect cell death.We then performed image analyses to measure autophagosomes formation with or without chloroquine(CQ),an inhibitor of autophagy.Meanwhile,autophagy-related genes expressions were detected with Cd treatment in MSCs.Furthermore,Autophagy was then suppressed using siRNA against BECN1 and 3-MA for identification whether inhibition of autophagic process would reverse cadmium-induced autophagic cell death in MSCs.2.To further address the potential mechanism involved in the established model,the level of FOXO family members were measured after Cd treatment using Western blot,PCR and immunofluorescence technique.The upstream signaling AMPK was also detected in this model.Then gene silencing and AMPK inhibitor Compound C were used to evaluate whether AMPK-FOXO3 a signaling pathway was involved in Cd-induced autophagic cell death in MSCs.Results1.Cd reduced cell viability and increased cell death in a dose-and time-dependent manner with the increasing numbers of GFP-LC3-positive puncta compared with controls.Moreover,prior to exposure to Cd,CQ presence significantly increased the percentage of LC3 protein abundance as well as GFP-LC3 puncta in MSCs.mRNA levels of the autophagosome formation related genes,including BECN1,ULK1,ATG5,and ATG12,were elevated in a dose-and time-dependent manner in Cd-treated MSC,however,only BECN1 protein elevated.In particular,BECN1 gene silencing or 3-MA pretreatment blocked Cd-induced autophagic cell death in MSCs.2.Real-time PCR and Western blotting results revealed the upregulation of FOXO1 and FOXO3 a mRNA and protein levels after Cd treatment.And only a partial nuclear translocation of FOXO3 a was founded following Cd treatment in MSCs.Besides,luciferase reporter assays indicated that Cd treatment dramatically increased FOXO3 a but not FOXO1-induced transcription in MSCs.Moreover,Cd significantly elevated Ser588 phosphorylation of FOXO3 a and Thr172 phosphorylation of AMPK protein in a dosedependent manner in MSCs.AMPK gene silencing or AMPK inhibitor Compound C significantly suppressed Cd-induced Ser588 phosphorylation of FOXO3 a.Furthermore,knocking down FOXO3 a attenuated Cd-induced BECN1,LC3 expression level and autophagosome formation.And silencing of the FOXO3 a or FOXO1 gene displayed a certain protective effect in cell death against Cd in MSCs.ConclusionIn summary,our study provided evidence that Cd-induced autophagy in MSCs by increasing autophagy-related genes expression and autophagosome formation.Moreover,inhibition of autophagy by knockdown of BECN1 partially restored cell death.Notably,Cd increased FOXO3 a and FOXO1 m RNA,and AMPK was demonstrated to enhance FOXO3 a nuclear translocation and transcriptional activity by phosphorylating FOXO3 a at specific serine residues(Ser588)in Cd-treated MSCs.Specifically,FOXO3 a,but not FOXO1,gene silencing efficiently prevented autophagy-related genes expression and autophagosome formation,which attenuated the increased autophagy in Cd-exposed MSCs in vitro.