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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.

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.

High-density lipoprotein (HDL) has beneficial effects in coronary artery disease. Latz and colleagues show that HDL's benefits stem at least in part by activating an anti-inflammatory program dependent on the transcription factor ATF3. High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo . Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.

The histone deacetylases (HDACs) are a superfamily of chromatin-modifying enzymes that silence transcription through the modification of histones. Among them, HDAC3 is unique in that interaction with nuclear receptor corepressors 1 and 2 (NCoR1/2) is required to engage its catalytic activity 1 – 3 . However, global loss of HDAC3 also results in the repression of transcription, the mechanism of which is currently unclear 4 – 8 . Here we report that, during the activation of macrophages by lipopolysaccharides, HDAC3 is recruited to activating transcription factor 2 (ATF2)-bound sites without NCoR1/2 and activates the expression of inflammatory genes through a non-canonical mechanism. By contrast, the deacetylase activity of HDAC3 is selectively engaged at ATF3-bound sites that suppress Toll-like receptor signalling. Loss of HDAC3 in macrophages safeguards mice from lethal exposure to lipopolysaccharides, but this protection is not conferred upon genetic or pharmacological abolition of the catalytic activity of HDAC3. Our findings show that HDAC3 is a dichotomous transcriptional activator and repressor, with a non-canonical deacetylase-independent function that is vital for the innate immune system. During the activation of mouse macrophages by lipopolysaccharides, histone deacetylase 3 controls inflammatory responses by both repressing and activating gene transcription depending on its differential association with transcription factors.

Skeletal muscle stem cells (also called satellite cells, SCs) are important for maintaining muscle tissue homeostasis and damage-induced regeneration. However, it remains poorly understood how SCs enter cell cycle to become activated upon injury. Here we report that AP-1 family member ATF3 (Activating Transcription Factor 3) prevents SC premature activation. Atf3 is rapidly and transiently induced in SCs upon activation. Short-term deletion of Atf3 in SCs accelerates acute injury-induced regeneration, however, its long-term deletion exhausts the SC pool and thus impairs muscle regeneration. The Atf3 loss also provokes SC activation during voluntary exercise and enhances the activation during endurance exercise. Mechanistically, ATF3 directly activates the transcription of Histone 2B genes, whose reduction accelerates nucleosome displacement and gene transcription required for SC activation. Finally, the ATF3-dependent H2B expression also prevents genome instability and replicative senescence in SCs. Therefore, this study has revealed a previously unknown mechanism for preserving the SC population by actively suppressing precocious activation, in which ATF3 is a key regulator. Muscle regeneration relies on activation and expansion of skeletal muscle stem cells. Here, authors show that ATF3 induction prevents precocious activation of skeletal muscle stem cells by binding and promoting the transcription of Histone2B.

Non-obese diabetes (NOD) mice are an established, spontaneous model of type 1 diabetes in which diabetes develops through insulitis. Using next-generation sequencing, coupled with pathway analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from 4 to 12 weeks of age. The resulting dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6 and 8 weeks that increased to a regulatory plateau between 10 and 12 weeks. The inflammatory signature is identified by the activation of central immunogenic factors such as Infg, Il1b , and Tnfa , and activation of canonical inflammatory signaling. Analysis of the regulatory landscape revealed the transcription factor Atf3 as a potential novel modulator of inflammatory signaling in the NOD islets. Furthermore, the Hedgehog signaling pathway correlated with Atf3 regulation, suggesting that the two play a role in regulating islet inflammation; however, further studies are needed to establish the nature of this connection.

Cells rapidly and extensively remodel their transcriptome in response to stress to restore homeostasis, but the underlying mechanisms are not fully understood. Here, we characterize the dynamic changes in transcriptome, epigenetics, and 3D genome organization during the integrated stress response (ISR). ISR induction triggers widespread transcriptional changes within 6 h, coinciding with increased binding of ATF4, a key transcriptional effector. Notably, ATF4 binds to hundreds of genes even under non-stress conditions, priming them for stronger activation upon stress. The transcriptional changes at ATF4-bound sites during ISR do not rely on increased H3K27 acetylation, chromatin accessibility, or rewired enhancer-promoter looping. Instead, ATF4-mediated gene activation is linked to the redistribution of CEBPγ from non-ATF4 sites to a subset of ATF4-bound regions, likely by forming an ATF4/CEBPγ heterodimer. CEBPγ preferentially targets the sites pre-occupied by ATF4, as well as genomic regions exhibiting a unique higher-order chromatin structure signature. Thus, the transcriptional responses during ISR are largely pre-wired by intrinsic chromatin properties. These findings provide critical insights into transcriptional remodeling during ISR with broader implications for other stress responses. Cells reprogram gene expression to adapt to stress, but the mechanisms remain unclear. Here, the authors show that transcriptional responses during the integrated stress response are pre-wired by ATF4 binding and chromatin features, enabling rapid stress adaptation.

Proteostasis collapse, the diminished ability to maintain protein homeostasis, has been established as a hallmark of nematode aging. However, whether proteostasis collapse occurs in humans has remained unclear. Here, we demonstrate that proteostasis decline is intrinsic to human senescence. Using transcriptome-wide characterization of gene expression, splicing, and translation, we found a significant deterioration in the transcriptional activation of the heat shock response in stressed senescent cells. Furthermore, phosphorylated HSF1 nuclear localization and distribution were impaired in senescence. Interestingly, alternative splicing regulation was also dampened. Surprisingly, we found a decoupling between different unfolded protein response (UPR) branches in stressed senescent cells. While young cells initiated UPR-related translational and transcriptional regulatory responses, senescent cells showed enhanced translational regulation and endoplasmic reticulum (ER) stress sensing; however, they were unable to trigger UPR-related transcriptional responses. This was accompanied by diminished ATF6 nuclear localization in stressed senescent cells. Finally, we found that proteasome function was impaired following heat stress in senescent cells, and did not recover upon return to normal temperature. Together, our data unraveled a deterioration in the ability to mount dynamic stress transcriptional programs upon human senescence with broad implications on proteostasis control and connected proteostasis decline to human aging.

The amino acid antiporter system Xc− is important for the synthesis of glutathione (GSH) that functions to prevent lipid peroxidation and protect cells from nonapoptotic, iron-dependent death (i.e., ferroptosis). While the activity of system Xc− often positively correlates with the expression level of its light chain encoded by SLC7A11, inhibition of system Xc− activity by small molecules (e.g., erastin) causes a decrease in the intracellular GSH level, leading to ferroptotic cell death. How system Xc− is regulated during ferroptosis remains largely unknown. Here we report that activating transcription factor 3 (ATF3), a common stress sensor, can promote ferroptosis induced by erastin. ATF3 suppressed system Xc−, depleted intracellular GSH, and thereby promoted lipid peroxidation induced by erastin. ATF3 achieved this activity through binding to the SLC7A11 promoter and repressing SLC7A11 expression in a p53-independent manner. These findings thus add ATF3 to a short list of proteins that can regulate system Xc− and promote ferroptosis repressed by this antiporter.