Studies on the cytoprotective role of autophagy in necrosis

Abstract

Programmed cell death (PCD) is an intrinsically regulated cellular suicide process that can be categorized into apoptosis and necrosis based on their distinct morphological characteristics. Autophagy refers to an evolutionarily conserved process that sequesters and targets bulk cellular constituents for lysosomal degradation. Autophagy has been found to be implicated in regulation of PCD under various cellular settings. At present, the role of autophagy on PCD is highly controversial. Although autophagy generally serves as a cell survival mechanism under stress conditions such as starvation, there are reports showing that autophagy executes caspase-independent cell death, known as autophagic cell death. However, in many cases the evidence supporting autophagy as a cell death mechanism is frequently circumstantial and appears inadequate. zVAD, a pan-caspase inhibitor, has been shown to induce robust necrosis in L929 cells, and such necrosis has been reported as autophagic cell death. However, the molecular mechanism underlying such cell death has not been fully elucidated. Therefore, the main objective of this study is to investigate the regulatory role of autophagy in necrosis and to elucidate the underlying molecular mechanisms using in vitro mammalian cell models. The following investigations have been conducted: (i) examining the role of autophagy in zVAD-induced necrosis by modulation of autophagy via either pharmacological or genetic approaches; (ii) studying the regulatory role of class I PI3K-Akt-mTOR signaling axis in modulation of autophagy and necrosis; and (iii) elucidating the molecular mechanism underlying zVAD-induced necrosis. In this study, we first demonstrated that autophagy played a cytoprotective role during zVAD-induced necrosis. Moreover, zVAD was able to suppress autophagy via suppression of lysosome function via inhibition of cathepsin enzyme activity. One surprising finding of this study was that growth factors such as insulin and IGF-1 and nutrients such as amino acids were able to enhance zVAD-induced necrosis via activation of the PI3K-Akt-mTOR pathway and subsequent suppression of autophagy. Moreover, the pro-death function of insulin/amino acids was also observed in other two necrosis models, including MNNG-induced necrosis in L929 cells and H2O2-induced necrosis in Bax/Bak double knockout cells, where autophagy acted as a pro-survival mechanism. Finally, we identified that zVAD-induced necrosis was RIP1- and RIP3-mediated necroptosis that depended on the autocrine production of TNFa. zVAD promoted the autocrine production of TNFa at the transcription level, which was required for induction of cell death. We also demonstrated that zVAD promoted TNFa production via the PKC-MAPKs-AP-1 pathway. Moreover, we presented evidence showing that defects in autophagy might promote zVAD-induced cell death by enhancing AP-1 activity. In conclusion, data from this study demonstrate that (i) autophagy plays a cell survival strategy in the three necrosis models tested in this study; (ii) growth factors and amino acids promote necrosis in these models via activation of the PI3K-Akt-mTOR pathway and subsequent suppression of autophagy; and (iii) zVAD-induced necroptosis depends on autocrine production of TNFa that is mediated via the PKC-MAPKs-AP-1 signaling pathway. Taken together, results from the above-described studies provide novel insights for a better understanding of the role of autophagy in necrosis.