Atg5-deficient Mouse Embryo Fibroblasts Resistant To ROS-induced Autophagy-independent Apoptosis And The Potential Mechanisms

[Objective]Autophagy is one of the highlights of biomedical research in the pase decade. Under starvation or other stress conditions,cell will self-digest those non-essential macromolecule substances,including intracellular long-lived proteins and damaged subcellular organelles,like mitochondria,to provide the material and energy for cell survival.And under physiological conditions,autophagy has a number of vital roles such as maintenance of the amino acid pool during starvation,pre-implantation development,prevention of neurodegeneration,anti-aging,tumor suppression, clearance of intracellular microbes,and regulation of innate and adaptive immunity.At present,the molecular mechanism controlling autophagy has just emergered to be understood.Autophagy-related genes(Atgs) have been identified as the essential machinery required for the different stages of autophagy,such as the formation or elongation of isolating membrane,the formation of autophagosome or autolysosome. So far,more than 30 Atgs have been identified in yeast and many of them have homologues in mammlians.Among them,Atg5 is one of the key Atgs and plays an essential role in the autophagic process.Apoptosis is probably the most important form of programmed cell death,which is a relative slower,energy dependent,gene-guide progress.It involved a series of biochemistry changes,such as gene expression,synthesis of functional proteins, activation of enzymes and the transmission of cell signals.It has been well established that apoptosis is closely associated with various diseases such as cancer,and physio-pathological processes,including tumorigenesis,immunity,and inflammation.Reaactive oxygen species(ROS) is a group of highly reactive molecules, including singlet oxygen,hydroxyl radicals,superoxide anion,nitric oxide and hydrogen peroxides,which have been shown to play an important role in apoptosis. ROS can oxidize a wide range of cell constituents,such as lipids,proteins and DNA, results in damaging cell structures and compromising the function.Up to now,the role of autophagy in apoptosis has been largely controversial.A number of cross talks between autophagy and apoptosis have been discovered, revealing a rather complicated nature of interaction of these two important cellular processes.For instance,there are contradictory reports whether cells with deficiency in autophagy are more resistant or sensitive to apoptotic stimuli.Thus,the main objective of this study is to systematically investigate the involvement of Atg5 in oxidative stress-mediated apoptotic cell death,using the wild-type(WT) and Atg5-/- mouse embryonic fibroblasts(MEFs) as the model system.[Methods]1.Cell culture and treatment:WT and Atg5-/- MEFs were maintained in Dulbecco's modified Eagle's medium,containing 10%fetal bovine serum and 1% penicillin-streptomycin in a 5%CO₂ atmosphere at 37℃.Both of these two MEFs were treated with SNP(1.0 mM) for 3,6,9,12h or treated with H₂O₂(0.25mM) for 3,6,9,12h.2.Detection of cell death/apoptosis1) PI-live cell staining coupled with flow cytometry:after treatments,flow cytometry PI staining was employed to investigate the cell viability of WT and Atg5-/-MEFs.2) Hoechst 33258 staining:Hoechst 33258 staning was used to examine the morphological changes after treatments for 12h using fluorescence microscope.3) Western blotting:western blotting was applied to detect the expressions of apoptosis-related proteins after treatments for different time points.3.Detection of autophagy1) Rapamycin(10nM) or Chloroquine(20μM) was pre-treated for 1h to induce or inhibitor the autophagy in WT MEFs,then followed by SNP(1.0 mM) for 12h.2) Western blotting detected the expressions of LC3-â…¡and p-S6K.4.SiRNA:SiRNA technique was used to achieve knockdown of mouse Atg5 or Atg7 in WT MEFs,then after 24h,treated with SNP(1.0 mM) for 12h,5.Transient transfection:transient transfection technique was used to transfect GFP (0.06 ng) or GFP-Atg5(0.2 ng) plasmid to Atg5-/- MEFs for 18h.[Results]1.Atg5-/- MEFs are more resistant to apoptosis induced by SNP or H₂O₂.When treated with SNP(1.0 mM) or H₂O₂(0.25 mM),WT MEFs showed typical apoptotic morphological features using Hoecchst 33258 staining examined under fluorescence microscope,while Atg5-/- MEFs were more resistant.The cell death was further quantified using the PI-live cell staining coupled with flow cytometry.It was found that when treated with SNP(1.0 mM) for 9h,more than 60%Atg5-/- MEFs were alive, but more than 80%WT MEFs died.Similarly,after treated with H₂O₂(0.25 mM) for 12h,the cell viability was over 80%in Atg5-/- MEFs,but less than 10%in WT MEFs. Consistently,there were evident cleavage of caspase-3,-8,-9 and PARP in WT MEFs, but not in Atg5-/- MEFs.2.The resistance to apoptosis of Atg5-/- MEFs is likeyl to be autophagy-independent. Firstly,the flow and western data showed that promotion of autophagy with Rapa(10 nM×1h) or suppression of autophagy with CDP(20μM×1h) had no effect on SNP-induced cell death in WT MEFs.Secondly,Atg5 or Atg7 knockdown in WT MEFs demonstrated different effect:knockdown of Atg5 offered partial protection against SNP-induced apoptosis,while knockdown of Atg7 had no evident effect.3.The resistance to apoptosis of AtgS-/- MEFs is due to the deficiency of Atg5 itself. When GFP-Atg5 was transiently over-expressed in Atg5-/- MEFs,over-expression of Atg5 alone,even without any other treatment,could induce apoptosis in AtgS-/- MEFs, suggesting that Atg5 is a pro-apoptotic factor,in addition to its role in autophagy.4.Involvement of the JNK-p53 signaling pathway in oxidative stress-mediated apoptosis.In order to understand the molecular mechanism underlying the resistance to oxidative stress-mediated resistance in AtgS-/- MEFs,we examined the role of the JNK-p53 pathway.Aider treated with SNP for 3h,the level of p-JNK was increased markedly in WT MEFs,but not in Atg5-/- MEFs.Meanwhile,the phosphorylation levels of p53 at Ser 15 and Ser 20 were much more evident in WT-MEFs than that in Atg5-/- MEFs.Moreover,knockdown of JNK was able to reduce p-JNK,p-P53 and PARP cleavage,suggesting the critical role of the JNK-p53 pathway in cell death in the WT-MEFs,but not in the Atg5-/- MEFs.[Conclusions]1.Compared to the WT MEFs,Atg5-/- MEFs were more resistant to SNP or H₂O₂ induced-apoptosis.2.The resistance was not related to autophagy,but the role of Atg5 itself,and more likely through the JNK-p53 signaling pathway.