Search

It has been well documented that cells deficient in either TSC1 or TSC2 are highly sensitive to various cell death stimuli. In this study, we utilized the TSC2 (-/-) mouse embryonic fibroblasts (MEFs) to study the involvement of autophagy in the enhanced susceptibility of TSC2-null cells to cell death. We first confirmed that both TSC1-null and TSC2-null MEFs are more sensitive to apoptosis in response to amino acid starvation (EBSS) and hypoxia. Second, we found that both the basal and inducible autophagy in TSC2 (-/-) MEFs is impaired, mainly due to constitutive activation of mTORC1. Third, suppression of autophagy by chloroquine and Atg7 knockdown sensitizes TSC2 (+/+) cells, but not TSC2 (-/-) cells, to EBSS-induced cell death. Conversely, the inhibition of mTORC1 by raptor knockdown and rapamycin activates autophagy and subsequently rescues TSC2 (-/-) cells. Finally, in starved cells, nutrient supplementations (insulin-like growth factor-1 (IGF-1) and leucine) enhanced cell death in TSC2 (-/-) cells, but reduced cell death in TSC2 (+/+) cells. Taken together, these data indicate that constitutive activation of mTORC1 in TSC2 (-/-) cells leads to suppression of autophagy and enhanced susceptibility to stress-mediated cell death. Our findings thus provide new insights into the complex relationships among mTOR, autophagy and cell death, and support the possible autophagy-targeted intervention strategies for the treatment of TSC-related pathologies.

Our previous work has shown that autophagy plays a pro-survival function in two necrotic cell death models: zVAD-treated L929 cells as well as H(2)O(2)-treated Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (DKO MEF). This study aims to further explore the regulatory role of autophagy in necrosis by examining the functional role of the phosphoinositide-3 kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway. Our initial intriguing finding was that insulin is able to promote necrotic cell death induced by zVAD and MNNG in L929 cells or by H(2)O(2) in DKO MEF cells cultured in full-growth medium. The pro-necrosis function of insulin was further supported by the observations that insulin is capable of abolishing the protective effect of starvation on necrotic cell death induced by zVAD in L929 cells. Next, we demonstrated that insulin acts on the PI3K-Akt-mTOR pathway to promote necrosis as the suppression of the above pathway by either chemical inhibitors (LY294002 and rapamycin) or mTOR knockdown is able to mitigate the pro-death function of insulin. Finally, we provided evidence that the pro-death function of insulin is dependent on its inhibitory effect on autophagy, which serves as an important pro-survival function in necrosis. Taken together, here we provide compelling evidence to show that activation of the PI3K-Akt-mTOR signaling pathway can promote necrotic cell death via suppression of autophagy, at least in the necrosis models defined in our study in which autophagy serves as a pro-survival function. Data from this study not only further underscore the pro-survival function of autophagy in necrotic cell death, but also provide a novel insight into the intricate connections linking the PI3K-Akt-mTOR signaling pathway with cell death via modulation of autophagy.

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.