The kinase PERK and the transcription factor ATF4 play distinct and essential roles in autophagy resulting from tunicamycin-induced ER stress

Abstract

Endoplasmic reticulum (ER) stress is thought to activate autophagy via unfolded protein response (UPR)-mediated transcriptional up-regulation of autophagy machinery components and modulation of microtubule-associated protein 1 light chain 3 (LC3). The upstream UPR constituents pancreatic EIF2-α kinase (PERK) and inositol-requiring enzyme 1 (IRE1) have been reported to mediate these effects, suggesting that UPR may stimulate autophagy via PERK and IRE1. However, how the UPR and its components affect autophagic activity has not been thoroughly examined. By analyzing the flux of LC3 through the autophagic pathway, as well as the sequestration and degradation of autophagic cargo, we here conclusively show that the classical ER stressor tunicamycin (TM) enhances autophagic activity in mammalian cells. PERK and its downstream factor, activating transcription factor 4 (ATF4), were crucial for this induction, but surprisingly, IRE1 constitutively suppressed autophagic activity. TM-induced autophagy required autophagy-related 13 (ATG13), Unc-51–like autophagy-activating kinases 1/2 (ULK1/ULK2), and GABA type A receptor–associated proteins (GABARAPs), but interestingly, LC3 proteins appeared to be redundant. Strikingly, ATF4 was activated independently of PERK in both LNCaP and HeLa cells, and our further examination revealed that ATF4 and PERK regulated autophagy through separate mechanisms. Specifically, whereas ATF4 controlled transcription and was essential for autophagosome formation, PERK acted in a transcription-independent manner and was required at a post-sequestration step in the autophagic pathway. In conclusion, our results indicate that TM-induced UPR activates functional autophagy, and whereas IRE1 is a negative regulator, PERK and ATF4 are required at distinct steps in the autophagic pathway.

Causal roles of mitochondrial dynamics in longevity and healthy aging

“…Stress caused by the UPR orchestrates a series of cellular events that culminate in changes in both mitochondrial dynamics and lysosomal function. ER stress‐induced PERK activation results in increased lysosomal amino‐acid sensing and autophagy via the transcription factor ATF4 , while mitochondrial biogenesis is induced by the ATF6 target gene PGC‐1α . Together, these studies highlight that crosstalk between ER–mitochondria–lysosome axis and their malleable response to stress via altering dynamics and function is key to tackle cellular UPR stress .…”

Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B, and distinct contributions from unfolded protein response components

“…Autophagy and autophagy-related (ATG) proteins are frequently implicated in cell death regulation [28], but their role in ER stress-induced cell death is unclear. ER stress potently upregulates the expression of microtubuleassociated proteins 1A/1B light chain 3B (LC3B), as well as other ATGs [29][30][31][32][33]. This correlates with enhanced autophagic flux in cells treated with the ER stressor tunicamycin (Tm) [30], whereas Tg blocks general autophagy due to ER Ca 2+ depletion [11,33].…”

“…ER stress potently upregulates the expression of microtubuleassociated proteins 1A/1B light chain 3B (LC3B), as well as other ATGs [29][30][31][32][33]. This correlates with enhanced autophagic flux in cells treated with the ER stressor tunicamycin (Tm) [30], whereas Tg blocks general autophagy due to ER Ca 2+ depletion [11,33]. Variable and inconsistent results have been reported with respect to the effect of genetic depletion of key ATGs (Beclin1 or ATG5) on Tgand Tm-induced cell death [25,[34][35][36][37].…”

Hypoxia-induced SETX links replication stress with the unfolded protein response

“…To further investigate whether SETX is a UPR target, we exposed cells to a range of stresses including tunicamycin and thapsigargin, both of which are known to induce the UPR, and found that both led to a significant increase in SETX mRNA similar to the induction observed in hypoxia (<0.1% O 2 ) ( Figure 4D, S7D). Consistent with this, SETX was found to be upregulated in a RNA sequencing dataset upon tunicamycin addition although this was not specifically reported 44 . As expected, we also demonstrated that SETX expression was not induced with hypoxia mimetics desferrioxamine (DFO) and 2% O 2 , which lead to HIF stabilisation ( Figure 4D, S7E).…”