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Purpose We tested the hypothesis that polyphenol-rich extracts can reduce endoplasmic reticulum (ER) stress induced by a high-fat diet (HFD) in skeletal muscle of mice. Methods Mice were randomly assigned to four groups receiving during 20 weeks either a standard chow control (CTRL), or a HFD supplemented, or not, with pomegranate (HFD + P) or green tea (HFD + GT) extracts. After the nutritional intervention, mice were killed and gastrocnemius muscles were taken. Proteins and mRNA were measured by Western blot and RT-qPCR, respectively. Results Body weight gain and visceral fat were higher in HFD, HFD + P and HFD + GT than in CTRL. The markers of the unfolded protein response BiP, XBP1u, XBP1s and ATF4 were higher only in HFD. In HFD + P and HFD + GT, this increase was not observed except for CHOP, which was elevated in all HFD groups. HFD increased also markers of ubiquitin–proteasome pathway, autophagy and oxidative stress, which were kept low in HFD + P and HFD + GT groups. Conclusion Our data provide evidence for a protective effect of pomegranate and green tea extracts against ER stress, oxidative stress and protein degradation induced by HFD in skeletal muscle. They give arguments for a usefulness of these natural nutritional compounds to fight against cellular dysfunctions related to fat excess.

The phosphorylation of eIF2α is essential for the endoplasmic reticulum (ER) stress response, the formation of stress granules, as well as macroautophagy. Several successful anticancer chemotherapeutics have the property to induce immunogenic cell death (ICD), thereby causing anticancer immune responses. ICD is accompanied by the translocation of calreticulin (CALR) from the ER lumen to the plasma membrane, which facilitates the transfer of tumor-associated antigens to dendritic cells. Here we systematically investigated the capacity of anticancer chemotherapeutics to induce signs of ER stress. ICD inducers including anthracyclines and agents that provoke tetraploidization were highly efficient in enhancing the phosphorylation of eIF2α, yet failed to stimulate other signs of ER stress including the transcriptional activation of activating transcription factor 4 (ATF4), the alternative splicing of X-box binding protein 1 (XBP1s) mRNA and the proteolytic cleavage of activating transcription factor 6 (ATF6) both in vitro and in cancers established in mice. Systematic analyses of clinically used anticancer chemotherapeutics revealed that only eIF2α phosphorylation, but none of the other signs of ER stress, correlated with CALR exposure. eIF2α phosphorylation induced by mitoxantrone, a prototype ICD-inducing anthracyline, was mediated by eIF2α kinase-3 (EIF2AK3). Machine-learning approaches were used to determine the physicochemical properties of drugs that induce ICD, revealing that the sole ER stress response relevant to the algorithm is eIF2α phosphorylation with its downstream consequences CALR exposure, stress granule formation and autophagy induction. Importantly, this approach could reduce the complexity of compound libraries to identify ICD inducers based on their physicochemical and structural characteristics. In summary, it appears that eIF2α phosphorylation constitutes a pathognomonic characteristic of ICD.

ObjectiveMitochondrial dysfunction plays a dominant role in the pathogenesis of alcoholic liver disease (ALD); however, the underlying mechanisms remain to be fully understood. We previously found that hepatic activating transcription factor 4 (ATF4) activation was associated with mitochondrial dysfunction in ALD. This study aimed to investigate the function and mechanism of ATF4 in alcohol-induced hepatic mitochondrial dysfunction.DesignATF4 activation was detected in the livers of patients with severe alcoholic hepatitis (AH). The role of ATF4 and mitochondrial transcription factor A (TFAM) in alcohol-induced liver damage was determined in hepatocyte-specific ATF4 knockout mice and liver-specific TFAM overexpression mice, respectively.ResultsHepatic PERK-eIF2α-ATF4 ER stress signalling was upregulated in patients with AH. Hepatocyte-specific ablation of ATF4 in mice ameliorated alcohol-induced steatohepatitis. ATF4 ablation also attenuated alcohol-impaired mitochondrial biogenesis and respiratory function along with the restoration of TFAM. Cell studies confirmed that TFAM expression was negatively regulated by ATF4. TFAM silencing in hepatoma cells abrogated the protective effects of ATF4 knockdown on ethanol-mediated mitochondrial dysfunction and cell death. Moreover, hepatocyte-specific TFAM overexpression in mice attenuated alcohol-induced mitochondrial dysfunction and liver damage. Mechanistic studies revealed that ATF4 repressed the transcription activity of nuclear respiratory factor 1 (NRF1), a key regulator of TFAM, through binding to its promoter region. Clinical relevance among ATF4 activation, NRF1–TFAM pathway disruption and mitochondrial dysfunction was validated in the livers of patients with AH.ConclusionThis study demonstrates that hepatic ATF4 plays a pathological role in alcohol-induced mitochondrial dysfunction and liver injury by disrupting the NRF1–TFAM pathway.

Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER. In the presence of stress stimuli, the endoplasmic reticulum either adapts the protein synthesis or triggers an apoptotic response, but the mechanisms underlying this decision are unknown. Kaufman and colleagues show that the ER stress response factors ATF4 and CHOP increase protein synthesis, which in turn induces oxidative stress and increased ATP consumption, leading to cell death during chronic ER stress.

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF4 1 – 3 . However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1–DELE1–HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1–DELE1–HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction. A genome-wide CRISPR interference screen shows that a signalling pathway involving OMA1, DELE1 and the eIF2α kinase HRI relays mitochondrial stress to the cytosol to trigger the integrated stress response.

The thrombospondin (Thbs) family of secreted matricellular proteins are stress- and injury-induced mediators of cellular attachment dynamics and extracellular matrix protein production. Here we show that Thbs1, but not Thbs2, Thbs3 or Thbs4, induces lethal cardiac atrophy when overexpressed. Mechanistically, Thbs1 binds and activates the endoplasmic reticulum stress effector PERK, inducing its downstream transcription factor ATF4 and causing lethal autophagy-mediated cardiac atrophy. Antithetically, Thbs1 −/− mice develop greater cardiac hypertrophy with pressure overload stimulation and show reduced fasting-induced atrophy. Deletion of Thbs1 effectors/receptors, including ATF6α, CD36 or CD47 does not diminish Thbs1-dependent cardiac atrophy. However, deletion of the gene encoding PERK in Thbs1 transgenic mice blunts the induction of ATF4 and autophagy, and largely corrects the lethal cardiac atrophy. Finally, overexpression of PERK or ATF4 using AAV9 gene-transfer similarly promotes cardiac atrophy and lethality. Hence, we identified Thbs1-mediated PERK-eIF2α-ATF4-induced autophagy as a critical regulator of cardiomyocyte size in the stressed heart. Beneficial and detrimental effects have been ascribed to the different Thrombospondin (Thbs) proteins in the adult mammalian heart. Here, the authors show that Thbs1-mediated activation of PERK-eIF2α-ATF4-induced autophagy regulates adult cardiomyocyte size in the stressed heart.

In response to growth signals, mechanistic target of rapamycin complex 1 (mTORC1) stimulates anabolic processes underlying cell growth. We found that mTORC1 increases metabolic flux through the de novo purine synthesis pathway in various mouse and human cells, thereby influencing the nucleotide pool available for nucleic acid synthesis. mTORC1 had transcriptional effects on multiple enzymes contributing to purine synthesis, with expression of the mitochondrial tetrahydrofolate (mTHF) cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) being closely associated with mTORC1 signaling in both normal and cancer cells. MTHFD2 expression and purine synthesis were stimulated by activating transcription factor 4 (ATF4), which was activated by mTORC1 independent of its canonical induction downstream of eukaryotic initiation factor 2α elF2α phosphorylation. Thus, mTORC1 stimulates the mTHF cycle, which contributes one-carbon units to enhance production of purine nucleotides in response to growth signals.

Endoplasmic reticulum stress is an evolutionarily conserved cell stress response associated with numerous diseases, including cardiac hypertrophy and heart failure. The major endoplasmic reticulum stress signaling pathway causing cardiac hypertrophy involves endoplasmic reticulum stress sensor PERK (protein kinase-like kinase) and eIF2α-ATF4-CHOP signaling. Here, we describe a non-canonical, AGGF1-mediated regulatory system for endoplasmic reticulum stress signaling associated with increased p-eIF2α and ATF4 and decreased sXBP1 and CHOP. Specifically, we see a reduced AGGF1 level consistently associated with induction of endoplasmic reticulum stress signaling in mouse models and human patients with heart failure. Mechanistically, AGGF1 regulates endoplasmic reticulum stress signaling by inhibiting ERK1/2 activation, which reduces the level of transcriptional repressor ZEB1, leading to induced expression of miR-183-5p. miR-183-5p post-transcriptionally downregulates CHOP and inhibits endoplasmic reticulum stress-induced apoptosis. AGGF1 protein therapy and miR-183-5p regulate endoplasmic reticulum stress signaling and block endoplasmic reticulum stress-induced apoptosis, cardiac hypertrophy, and heart failure, providing an attractive paradigm for treatment of cardiac hypertrophy and heart failure. Endoplasmic reticulum (ER) stress promotes cardiac dysfunction. Here the authors uncover a pathway whereby AGGF1 blocks ER stress by inhibiting ERK1/2 activation and the transcriptional repressor ZEB1, leading to induction of miR-183-5p and down-regulation of CHOP, and show that AGGF1 can effectively treat cardiac hypertrophy and heart failure.

Activating transcription factor 4 (ATF4) is a critical mediator of metabolic and oxidative homeostasis and cell survival. ATF4 is elevated in response to diverse microenvironmental stresses, including starvation, ER stress damages and exposure to toxic factors. Here we show that ATF4 expression fosters the malignancy of primary brain tumors (WHO grade III and IV gliomas) and increases proliferation and tumor angiogenesis. Hence, ATF4 expression promotes cell migration and anchorage-independent cell growth, whereas siRNA-mediated knockdown of ATF4 attenuates these features of malignancy in human gliomas. Further experiments revealed that ATF4-dependent tumor promoting effects are mediated by transcriptional targeting the glutamate antiporter xCT/SCL7A11 (also known as system Xc - ). Thus, xCT is elevated as a consequence of ATF4 activation. We further found evidence that ATF4-induced proliferation can be attenuated by pharmacological or genetic xCT inhibition and ferroptosis inducers such as sorafenib, erastin and GPx4 inhibitor RSL3. Further, fostered xCT expression promotes cell survival and growth in ATF4 knockdown cells. Moreover, increased xCT levels ameliorate sorafenib and erastin-induced ferroptosis. Conversely, ATF4 knockdown renders cells susceptible for erastin, sorafenib and RSL3-induced ferroptosis. We further identified that ATF4 promotes tumor-mediated neuronal cell death which can be alleviated by xCT inhibition. Moreover, elevated ATF4 expression in gliomas promotes tumor angiogenesis. Noteworthy, ATF4-induced angiogenesis could be diminished by ferroptosis inducers erastin and by GPx4 inhibitor RSL3. Our data provide proof-of-principle evidence that ATF4 fosters proliferation and induces a toxic microenvironmental niche. Furthermore, ATF4 increases tumor angiogenesis and shapes the vascular architecture in a xCT-dependent manner. Thus, inhibition of ATF4 is a valid target for diminishing tumor growth and vasculature via sensitizing tumor cells for ferroptosis.

The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis. When building healthy tissue, the human body must carefully control the growth of new cells to prevent them from becoming cancerous. A core component of this regulation is the protein mTORC1, which responds to various growth-stimulating factors and nutrients, and activates the chemical reactions cells need to grow. Part of this process involves controlling ‘nutrient-sensing transcription factors’ – proteins that regulate the activity of specific genes based on the availability of different nutrients. One of these nutrient-sensing transcription factors, ATF4, has recently been shown to be involved in some of the processes triggered by mTORC1. The role this factor plays in how cells respond to stress – such as when specific nutrients are depleted, protein folding is disrupted or toxins are present – is well-studied. But how it reacts to the activation of mTORC1 is less clear. To bridge this gap, Torrence et al. studied mouse embryonic cells and human prostate cancer cells grown in the laboratory, to see whether mTORC1 influenced the behavior of ATF4 differently than cellular stress. Cells were treated either with insulin, which activates mTORC1, or an antibiotic that sparks the stress response. The cells were then analyzed using a molecular tool to see which genes were switched on by ATF4 following treatment. This revealed that less than 10% of the genes activated by ATF4 during cellular stress are also activated in response to mTORC1-driven growth. Many of the genes activated in both scenarios were involved in synthesizing and preparing the building blocks that make up proteins. This was consistent with the discovery that ATF4 helps mTORC1 stimulate growth by promoting protein synthesis. Torrence et al. also found that mTORC1’s regulation of ATF4 stimulated the synthesis of glutathione, the most abundant antioxidant in cells. The central role mTORC1 plays in controlling cell growth means it is important to understand how it works and how it can lead to uncontrolled growth in human diseases. mTORC1 is activated in many overgrowth syndromes and the majority of human cancers. These new findings could provide insight into how tumors coordinate their drive for growth while adapting to cellular stress, and reveal new drug targets for cancer treatment.