The Steroid Hormone 20-hydroxyecdysone Promotes Switching From Autophagy To Apoptosis By Increasing Intracellular Calcium Levels

Background and scientific questionsThe programmed cell death mainly consists of autophagy and poptosis.Autophagy is a process that cells degrade their own orangells and proteins to produce energy for survive when confronted with stress or nutrients shortage.Apoptosis is an orderly,autonomously death process regulated by cells' gene or their production.To date,there are two views about the function of autophagy.One insists that autophagy is a survival process,such as in many mammal cells fibroblast,T cells and so on.Another is autophigic cell death,such as the degradation of Drosophila melanogaster midgut.So,whether autophagy maintains survival or leads to cell death,the relationship between autophagy and apoptosis and the regulative mechanism of autophagy transformation to apoptosis are the scientific questions to be elucidated.The midgut is seperated into imaginal disk,larval midgut and the larval midgut is degraded by PCD including autophagy and apoptosis from larvae to pupae in insects.So,insect midgut is a good model to study autophagy,apoptosis and the relationship between autophagy and apoptosis.Autophagic cell death but apoptotic cell death occurs in diptera D.melanogaster midgut.However,autophagy precedes apoptosis in Lepidoptera Bombyx mori midgut and the mechanism of autophagy transformation to apoptosis is unclear.So,the mechanism of PCD is different in different insects.Insect PCD is dependent on molting hormone(20-Hydroxyecdysone,20E).In B.mori midgut,20E promotes PCD by upregulating autophagic related genes and apoptotic related genes.In B.mori silk gland,20E can induce the increase of intracellular Ca2+,lastly activate Caspase-3 leading to PCD.Calcium change and homeostasis in cells are important for a number of cellular functions.Cytosolic Ca2+ is kept at low levels,whereas extracellular Ca2+ is maintained at much higher levels.In a few cell types,increasing Ca2+ can trigger autophagy,whereas increasing Ca2+ in others can activate Calpain to cleave ATG5 to N-terminal ATG5(NtATG5),thereby inhibiting autophagy but promoting apoptosis.So,we speculated 20E-induced the increase of Ca2+ played an important role in the autophagy transformation to apoptosis.To verify our hypothesis and solve the scientific questions in autophagy area,we took the Helicoverpa armigera migut as research model and cooperation with the platform of H.armigera cell line(HaEpi)to investigate autophagic cell survival or death,connection between autophagy and apoptosis and the mechanism of autophagy turning to apoptosis.ResultsIn lepidopteran insect Helicoverpa armigera midgut,autophagy related genes(ATGs)and apoptosis related genes(Caspases)are highly expressed during the metamorphosis,also answerred to 20E induction in H.armigera epidermal cell line(HaEpi).Most ATGs express earlier than Caspases.20E induced microtubule-associated protein light chain 3-phosphatidylethanolamine(LC3-II,also named ATG8)formation,which demonstreated 20E induces autophagy.However,transfection with the bifluorescence reporter plasmid of pIEx-RFP-GFP-LC3-His to further detect autophagy,which showed 20E inductive autophagy can't be continuously,that is to say,autophagy was declining at the late stage under 20E induction.However,apoptosis occurred at the late stage under 20E induction.So,20E induces autophagy lastly turning to apoptosis.Autophagy and apoptosis coexsisted,sequentially occurred in the midgut of the lepidopteran insect H.armigera during midgut programmed cell death(PCD),as indicated by LC3-II formation and cleavage of autophagy-related gene5(ATG5)to N-terminal ATG5(NtATG5)and Caspase-3 to active-caspase-3.Lower concentrations of steroid hormone 20E induced LC3-? formation and autophagy.However,higher concentrations of 20E induced cleavage of ATG5 to NtATG5 and Caspase-3 to active-caspase-3 and led to a switch from autophagy to apoptosis in 24 h in HaEpi cells.Blocking autophagy by knockdown of ATG5 or with the autophagy inhibitor 3-Methyladenine resulted in inhibition of 20E-induced autophagy and apoptosis.However,blocking apoptosis by using the apoptosis inhibitor Ac-DEVD-CHO did not prevent 20E-induced autophagy,suggesting that autophagy controlled and determined apoptosis.Higher concentrations of 20E induced higher levels of intracellular Ca2+,promoting ATG5 cleavage into NtATG5 and Caspase-3 activation,and mediated the switch from autophagy to apoptosis.Blocking 20E-mediated increase of cellular Ca2+decreased levels of NtATG5 and active-caspase-3,and repressed the switch from autophagy to apoptosis,thus promoting cell survival.In human breast cells,20E also promoted autophagy;however,when addition of high concentration Ca2+,autophagy transformed to apoptosis.These results reveal that a higher concentration of intracellular Ca2+ induces a switch from autophagic cell survival to apoptotic cell death,and that 20E may induce the increase of Ca2+ levels and promotes the switch from autophagy to apoptosis.Conclusions1.In lepidopteran insect H.armigera midgut PCD,autophagy results in survival and apoptosis leads to cell death.Inhibition of apoptosis,cells maintains autophagy and keeps cells survival.This view is dffrrent from the autophagic cell death in D.melanogaster midgut.It may be the regulative mechanism is distinct in different spieces.2.Autophagy precedes apoptosis in midgut and 20E promotes the switch from autophagy to apoptosis.Autophagy is the premise of apoptosis and apoptosis terminates autophagy.It demonstrates that the relationship beween autophagy and apoptosis is not merely an antagonism.3.20E promotes switching from autophagic cell survival to apoptotic cell death by increasing intracellular calcium levels.Higher concentration of 20E induces higher level of Ca2+ which cleaves ATG5 to NtATG5 and then activitates the Caspase-3 to apoptosis.Blocking Ca2+ channels inhibits autophagy transformation to apoptosis.Autophagy is unable to switch to apoptosis without Ca2+.This result is further conformed in human breast cells.It determined that Ca2+ is the key factor in the transformation of autophagy to apoptosis.Scientific significancesThis article explains that 20E promotes the switching from autophagy to apoptosis by increasing the calcium,which is the mechanism of midgut PCD.Autophagy leads to cells survival and apoptosis results in cell death.Ca2+ determines the switching from autophagy to apoptosis.It provides the new target for insect disease prevention and new explainations for the function of autophagy,the mechanism about cell survival or cell death.