CCCP‐induced mitochondrial dysfunction – characterization and analysis of integrated stress response to cellular signaling and homeostasis

Mitochondrial dysfunction mediated by CCCP (carbonyl cyanide m‐chlorophenyl hydrazone), an inhibitor of mitochondrial oxidative phosphorylation, evokes the integrated stress response (ISR), which is analyzed here by eIF2α phosphorylation and expression profiles of ATF4 and CHOP proteins. Our findings suggest that the CCCP‐induced ISR pathway is mediated by activation of HRI kinase, but not by GCN2, PERK, or PKR. Also, CCCP activates AMPK, a cellular energy sensor, and AKT, a regulator implicated in cell survival, and suppresses phosphorylation of mTORC1 substrates eIF4E‐BP1 and S6K. CCCP also downregulates translation and promotes autophagy, leading to noncaspase‐mediated cell death in HepG2 cells. All these events are neutralized by NAC, an anti‐ROS, suggesting that CCCP‐induced mitochondrial dysfunction promotes oxidative stress. ISRIB, an inhibitor of the ISR pathway, mitigates CCCP‐induced expression of ATF4 and CHOP, activation of AKT, and autophagy, similar to NAC. However, it fails to reverse CCCP‐induced AMPK activation, suggesting that CCCP‐induced autophagy is dependent on ISR and independent of AMPK activation. ISRIB restores partly, inhibition in eIF4E‐BP1 phosphorylation, promotes eIF2α phosphorylation, albeit slowly, and mitigates suppression of translation accordingly, in CCCP‐treated cells. These findings are consistent with the idea that CCCP‐induced oxidative stress leading to eIF2α phosphorylation and ATF4 expression, which is known to stimulate genes involved in autophagy, play a pro‐survival role together with AKT activation and regulate mTOR‐mediated eIF4E‐BP1 phosphorylation. ISRIB inhibits CCCP‐induced expression of ATF4 and CHOP, autophagy, and AKT activation but not AMPK, slows down eIF2α phosphorylation, and partially restores eIF4E‐BP1 phosphorylation and translation, accordingly. NAC neutralizes all CCCP‐induced signals. ISR, but not AMPK, mediates autophagy. Inhibition of CCCP‐induced AKT activation by ISRIB, and its initial decline in CCCP‐treated cells suggest that ISRIB may be activating PPP or changing the proteome that can generate antioxidants.