Abstract
Autophagy is typically a prosurvival cellular process that promotes the turnover of long-lived proteins and damaged organelles, but it can also induce cell death. We have previously reported that the small molecule Z36 induces autophagy along with autophagic cell death in HeLa cells. In this study, we analyzed differential gene expression in Z36-treated HeLa cells and found that Z36-induced endoplasmic reticulum-specific autophagy (ER-phagy) results in ER stress and the unfolded protein response (UPR). This result is in contrast to the common notion that autophagy is generally activated in response to ER stress and the UPR. We demonstrate that Z36 up-regulates the expression levels of FAM134B, LC3, and Atg9, which together mediate excessive ER-phagy, characterized by forming increased numbers of autophagosomes with larger sizes. We noted that the excessive ER-phagy accelerates ER degradation and impairs ER homeostasis and thereby triggers ER stress and the UPR as well as ER-phagy-dependent cell death. Interestingly, overexpression of FAM134B alone in HeLa cells is sufficient to impair ER homeostasis and cause ER stress and cell death. These findings suggest a mechanism involving FAM134B activity for ER-phagy to promote cell death.
Highlights
Autophagy is typically a prosurvival cellular process that promotes the turnover of long-lived proteins and damaged organelles, but it can induce cell death
To better understand the mechanism for Z36 to induce autophagy and cell death, we first compared the morphology of autophagosomes induced by Z36 with those induced by rapamycin (Rapa), which results in canonical protective autophagy
The results showed that the phosphorylation of PERK, IRE1␣, and eIF2␣ and the level of CHOP are increased after Z36 treatment (Fig. 2A), confirming that ER stress is activated by Z36 treatment
Summary
Z36 up-regulates the expression of genes related to autophagy, ER stress, and the UPR. Using transmission EM (TEM), we found that there are many more autophagosomes in Z36-treated HeLa cells, compared with those of Rapa. In Z36treated cells, the estimated average number of autophagosomes per cell was 30, whereas there were 17 and 6 autophagosomes per cell in Rapa- or DMSO (as control)-treated cells, respectively (Fig. 1B). Z36 treatment led to a much larger size for autophagosomes, as the average maximal cross-sectional diameters of autophagosomes in Z36-, Rapa-, and DMSO-treated cells were 1.20, 0.74, and 0.70 m, respectively (Fig. 1, C and D). It is reported that Atg (homologue of LC3 in yeast) regulates the size of autophagosomes [26], whereas the level of Atg determines the number of autophagosomes [27, 28]. We have analyzed the expression of these two genes at both
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