Cancer SLC6A6-mediated taurine uptake transactivates immune checkpoint genes and induces exhaustion in CD8+ T cells.

Taurine is used to bolster immunity, but its effects on antitumor immunity are unclear. Here, we report that cancer-related taurine consumption causes T cell exhaustion and tumor progression. The taurine transporter SLC6A6 is correlated with aggressiveness and poor outcomes in multiple cancers. SLC6A6-mediated taurine uptake promotes the malignant behaviors of tumor cells but also increases the survival and effector function of CD8+ T cells. Tumor cells outcompete CD8+ T cells for taurine by overexpressing SLC6A6, which induces T cell death and malfunction, thereby fueling tumor progression. Mechanistically, taurine deficiency in CD8+ T cells increases ER stress, promoting ATF4 transcription in a PERK-JAK1-STAT3 signaling-dependent manner. Increased ATF4 transactivates multiple immune checkpoint genes and induces T cell exhaustion. In gastric cancer, we identify a chemotherapy-induced SP1-SLC6A6 regulatory axis. Our findings suggest that tumoral-SLC6A6-mediated taurine deficiency promotes immune evasion and that taurine supplementation reinvigorates exhausted CD8+ T cells and increases the efficacy of cancer therapies.

Effects of electroacupuncture of different intensities and durations on PERK/ATF4/CHOP signaling pathway in liver of non-alcoholic fatty liver disease rats. / ä¸åŒå¼ºåº¦åŠæ—¶ç¨‹ç”µé’ˆå¹²é¢„å¯¹éžé ’ç²¾æ€§è„‚è‚ªè‚ç— å¤§é¼ è‚è„PERK/ATF4/CHOP通路的影响.

OBJECTIVES: To analyze the effects of electroacupuncture (EA) at "Fenglong" (ST40) and "Zusanli" (ST36) of different intensities and durations on rats with non-alcoholic fatty liver disease (NAFLD) based on the protein kinase R-like endoplasmic reticulum kinase (PERK)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) signaling pathway, so as to explore its mechanism underlying improvement of NAFLD. METHODS: SD rats were randomly divided into normal diet group, high-fat model group, sham EA group, strong stimulation EA (SEA) group, and weak stimulation EA (WEA) group, with 15 rats in each group. Each group was further divided into 2, 3, and 4-week subgroups. NAFLD rat model was established by feeding a high-fat diet. After successful modeling, rats in the SEA and WEA groups received EA at bilateral ST40 and ST36 with dense and sparse waves (4 Hz/20 Hz) at current intensities of 4 mA (SEA group) and 2 mA (WEA group), lasting for 20 minutes, once a day, 5 days a week with 2 days of rest. The sham EA group only had the EA apparatus connected without electricity. Different duration subgroups were intervened for 2, 3, and 4 weeks. After the intervention, the contents of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rats were detected by an automatic biochemical analyzer；liver morphological changes were observed by Oil Red O staining；real-time fluorescence quantitative PCR and Western blot were used to detect the expression of PERK, ATF4, and CHOP mRNAs and proteins in the rat liver tissue. RESULTS: In the high-fat model group, there was a significant accumulation of red lipid droplets in the liver cells, which was reduced significantly in the SEA group at the 4th week. Compared with the normal diet group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and proteins in the liver tissue were elevated (P<0.01) in the high-fat model group . Compared with the high-fat model group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, CHOP mRNAs and proteins in the liver tissue were decreased (P<0.01, P<0.05) in the SEA and WEA groups. Compared with the sham EA group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs were decreased (P<0.01, P<0.05) in the SEA and WEA groups, the expression of PERK, ATF4, and CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA group at the 2nd, 3rd, and 4th week, the expression of PERK and CHOP proteins at the 2nd, 3rd, 4th week and ATF4 protein at 2nd week in the liver tissue were decreased (P<0.01, P<0.05) in the WEA group. Compared with the SEA group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and proteins in the liver tissue were elevated (P<0.05, P<0.01) in the WEA group. Compared with the 2-week time point within the groups, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and PERK proteins in the liver tissue were decreased (P<0.01, P<0.05) in the SEA and WEA groups at 3rd and 4th week, the expression of ATF4 proteins in the liver tissue was decreased (P<0.01) in the SEA group at 3rd and 4th week, and the expression of CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA group at 4th week and in the WEA group at 3rd and 4th week. Compared with the 3-week time point within the groups, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs were significantly decreased (P<0.05, P<0.01) in the SEA and WEA groups at 4th week, the expression of PERK and CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA and WEA groups at 4th week, and the expression of ATF4 protein in the liver tissue was decreased (P<0.05) in the SEA group at 4th week. CONCLUSIONS: EA at ST40 and ST36 can significantly improve liver function in NAFLD rats, and its mechanism of action may involve inhibiting PERK expression thereby targeting the downstream ATF4/CHOP signaling pathway to suppress endoplasmic reticulum stress, exerting a liver protective effect；the optimal effect was observed with EA intensity of 4 mA for 4 weeks.

Withaferin A, a natural thioredoxin reductase 1 (TrxR1) inhibitor, synergistically enhances the antitumor efficacy of sorafenib through ROS-mediated ER stress and DNA damage in hepatocellular carcinoma cells.

BACKGROUND: Sorafenib (Sora), a multi-target tyrosine kinase inhibitor, is widely recognized as a standard chemotherapy treatment for advanced hepatocellular carcinoma (HCC). However, drug resistance mechanisms hinder its anticancer efficacy. Derived from Withania somnifera, Withaferin A (WA) exhibits remarkable anti-tumor properties as a natural bioactive compound. This study aimed to examine the mechanisms that underlie the impacts of Sora and WA co-treatment on HCC. METHODS: Cell proliferation was evaluated through colony formation and MTT assays. Flow cytometry was employed to determine cellular apoptosis and reactive oxygen species (ROS) levels. The evaluation of apoptosis-related protein levels, DNA damage, and endoplasmic reticulum stress was conducte utilizing IHC staining and western blotting. Moreover, the caspase inhibitor Z-VAD-FMK, ATF4 siRNA, ROS scavenger N-acetyl cysteine (NAC), and TrxR1 shRNA were used to elucidate the underlying signaling pathways. To validate the antitumor effects of Sora/WA co-treatment, in vivo experiments were ultimately executed using Huh7 xenografts. RESULTS: Sora/WA co-treatment demonstrated significant synergistic antitumor impacts both in vivo and in vitro. Mechanistically, the enhanced antitumor impact of Sora by WA was achieved through the inhibition of TrxR1 activity, resulting in ROS accumulation. Moreover, ROS generation induced the activation of DNA damage and endoplasmic reticulum (ER) stress pathways, eventually triggering cellular apoptosis. Pre-treatment with the antioxidant NAC significantly inhibited ROS generation, ER stress, DNA damage, and apoptosis induced by Sora/WA co-treatment. Additionally, the inhibition of ATF4 by small interfering RNA (siRNA) attenuated Sora/WA co-treatment-induced apoptosis. In vivo, Sora/WA co-treatment significantly suppressed tumor growth in HCC xenograft models and decreased TrxR1 activity in tumor tissues. CONCLUSION: Our study suggests that WA synergistically enhances the antitumor effect of Sora, offering promising implications for evolving treatment approaches for HCC.

Loss of the mitochondrial protein Abcb10 results in altered arginine metabolism in MEL and K562 cells and nutrient stress signaling through ATF4.

Abcb10 is a mitochondrial membrane protein involved in hemoglobinization of red cells. Abcb10 topology and ATPase domain localization suggest it exports a substrate, likely biliverdin, out of mitochondria that is necessary for hemoglobinization. In this study, we generated Abcb10 deletion cell lines in both mouse murine erythroleukemia and human erythroid precursor human myelogenous leukemia (K562) cells to better understand the consequences of Abcb10 loss. Loss of Abcb10 resulted in an inability to hemoglobinize upon differentiation in both K562 and mouse murine erythroleukemia cells with reduced heme and intermediate porphyrins and decreased levels of aminolevulinic acid synthase 2 activity. Metabolomic and transcriptional analyses revealed that Abcb10 loss gave rise to decreased cellular arginine levels, increased transcripts for cationic and neutral amino acid transporters with reduced levels of the citrulline to arginine converting enzymes argininosuccinate synthetase and argininosuccinate lyase. The reduced arginine levels in Abcb10-null cells gave rise to decreased proliferative capacity. Arginine supplementation improved both Abcb10-null proliferation and hemoglobinization upon differentiation. Abcb10-null cells showed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha, increased expression of nutrient sensing transcription factor ATF4 and downstream targets DNA damage inducible transcript 3 (Chop), ChaC glutathione specific gamma-glutamylcyclotransferase 1 (Chac1), and arginyl-tRNA synthetase 1 (Rars). These results suggest that when the Abcb10 substrate is trapped in the mitochondria, the nutrient sensing machinery is turned on remodeling transcription to block protein synthesis necessary for proliferation and hemoglobin biosynthesis in erythroid models.

Intestinal activating transcription factor 4 regulates stress-related behavioral alterations via paraventricular thalamus in male mice.

Chronic stress induces depression- and anxiety-related behaviors, which are common mental disorders accompanied not only by dysfunction of the brain but also of the intestine. Activating transcription factor 4 (ATF4) is a stress-induced gene, and we previously show that it is important for gut functions; however, the contribution of the intestinal ATF4 to stress-related behaviors is not known. Here, we show that chronic stress inhibits the expression of ATF4 in gut epithelial cells. ATF4 overexpression in the colon relieves stress-related behavioral alterations in male mice, as measured by open-field test, elevated plus-maze test, and tail suspension test, whereas intestine-specific ATF4 knockout induces stress-related behavioral alterations in male mice. Furthermore, glutamatergic neurons are inhibited in the paraventricular thalamus (PVT) of two strains of intestinal ATF4-deficient mice, and selective activation of these neurons alleviates stress-related behavioral alterations in intestinal ATF4-deficient mice. The highly expressed gut-secreted peptide trefoil factor 3 (TFF3) is chosen from RNA-Seq data from ATF4 deletion mice and demonstrated decreased in gut epithelial cells, which is directly regulated by ATF4. Injection of TFF3 reverses stress-related behaviors in ATF4 knockout mice, and the beneficial effects of TFF3 are blocked by inhibiting PVT glutamatergic neurons using DREADDs. In summary, this study demonstrates the function of ATF4 in the gut-brain regulation of stress-related behavioral alterations, via TFF3 modulating PVT neural activity. This research provides evidence of gut signals regulating stress-related behavioral alterations and identifies possible drug targets for the treatment of stress-related behavioral disorders.

Toosendanin induced hepatotoxicity via triggering PERK-eIF2α-ATF4 mediated ferroptosis.

Toosendanin (TSN) is the main active compound of Melia toosendan Sieb et Zucc with various bioactivities. In this study, we investigated the role of ferroptosis in TSN-induced hepatotoxicity. The characteristic indicators of ferroptosis were detected including reactive oxygen species (ROS), lipid-ROS, glutathione (GSH), ferrous ion and the expression of glutathione peroxidase 4 (GPX4), which showed that TSN caused ferroptosis in hepatocytes. The results of qPCR analysis and western blotting assay showed that TSN-induced activation of protein kinase R-like endoplasmic reticulum kinase (PERK)- eukaryotic initiation factor 2 α subunit (eIF2α)- activation transcription factor 4 (ATF4) signaling pathway resulted in increasing activation transcription factor 3 (ATF3) expression, which upregulated the expression of transferrin receptor 1 (TFRC). Furthermore, TFRC mediated iron accumulation leading to ferroptosis in hepatocytes. To clarify whether TSN triggered ferroptosis in vivo, male Balb/c mice were treated with the different doses of TSN. The results of hematoxylin-eosin (H&E) staining, 4-hydroxynonenal (4-HNE) staining, malondialdehyde (MDA) content and the protein expression of GPX4 showed that ferroptosis contributed to TSN-induced hepatotoxicity. Iron homeostasis relative protein and PERK- eIF2α- ATF4 signaling pathway also involved in hepatotoxicity of TSN in vivo.

Lathyrol promotes ER stress-induced apoptosis and proliferation inhibition in lung cancer cells by targeting SERCA2.

Lathyrol is a natural product isolated from the traditional Chinese medicine Semen Euphorbiae with unknown anti-tumor effects. We found that lathyrol had significant inhibitory effect on lung cancer cells by inducing apoptosis and inhibiting proliferation. Subsequently, we demonstrated for the first time that endoplasmic reticulum (ER) stress is a key anti-tumor mechanism of lathyrol. Furthermore, we found that lathyrol can induce ER stress in lung cancer cells by upregulating the protein expression levels of GRP78, PERK, p-eIF2α, CHOP, and ATF4, and the inhibitory effect of lathyrol on lung cancer cells was significantly reversed when cells were pretreated with ER stress inhibitor. In addition, we found that inhibition of SERCA2 resulted in depletion of the ER Ca2+ pool followed by a sustained increase in cytoplasmic Ca2+ levels, eventually leading to ER stress induced tumor cell apoptosis and proliferation inhibition. Lathyrol targeted SERCA2 to cause a significant upregulation of Ca2+ levels, and the inhibitory effect of lathyrol on lung cancer cells was significantly reversed after pretreatment with SERCA2 agonist. Taken together, our data suggest that lathyrol exerts its anti-tumor effect primarily by targeting SERCA2. Our findings highlight the potential for lathyrol as a new candidate drug for the treatment of lung cancer.

Wogonin, a Compound in Scutellaria baicalensis, Activates ATF4-FGF21 Signaling in Mouse Hepatocyte AML12 Cells.

Fibroblast growth factor 21 (FGF21), which is mainly synthesized and secreted by the liver, plays a crucial role in systemic glucose and lipid metabolism, ameliorating metabolic diseases. In this study, we screened the WAKANYAKU library derived from medicinal herbs to identify compounds that can activate Fgf21 expression in mouse hepatocyte AML12 cells. We identified Scutellaria baicalensis root extract and one of its components, wogonin, as an activator of Fgf21 expression. Wogonin also enhanced the expression of activating transcription factor 4 (ATF4) by a mechanism other than ER stress. Knockdown of ATF4 by siRNA suppressed wogonin-induced Fgf21 expression, highlighting its essential role in wogonin's mode of action. Thus, our results indicate that wogonin would be a strong candidate for a therapeutic to improve metabolic diseases by enhancing hepatic FGF21 production.

Resistance exercise enhances long-term mTORC1 sensitivity to leucine.

OBJECTIVE: Exercise enhances the sensitivity of mammalian target of rapamycin complex 1 (mTORC1) to amino acids, in particular leucine. How long this enhanced sensitivity lasts, and which mechanisms control enhanced leucine-mediated mTORC1 activation following exercise is currently unknown. METHODS: C57BL/6J mice were exercised for one night in a resistance-braked running wheel after a 12-day acclimatization period. Mice were gavaged with a submaximal dose of l-leucine or saline acutely or 48 h after exercise cessation, following 3 h food withdrawal. Muscles were excised 30 min after leucine administration. To study the contribution of mTORC1, we repeated those experiments but blocked mTORC1 activation using rapamycin immediately before the overnight running bout and one hour before the first dose of leucine. mTORC1 signaling, muscle protein synthesis and amino acid sensing machinery were assessed using immunoblot and qPCR. Leucine uptake was measured using L-[14C(U)]-leucine tracer labeling. RESULTS: When compared to sedentary conditions, leucine supplementation more potently activated mTORC1 and protein synthesis in acutely exercised muscle. This effect was observed in m. soleus but not in m. tibialis anterior nor m. plantaris. The synergistic effect in m. soleus was long-lasting as key downstream markers of mTORC1 as well as protein synthesis remained higher when leucine was administered 48 h after exercise. We found that exercise enhanced the expression of amino acid transporters and promoted uptake of leucine into the muscle, leading to higher free intramuscular leucine levels. This coincided with increased expression of activating transcription factor 4 (ATF4), a main transcriptional regulator of amino acid uptake and metabolism, and downstream activation of amino acid genes as well as leucyl-tRNA synthetase (LARS), a putative leucine sensor. Finally, blocking mTORC1 using rapamycin did not reduce expression and activation of ATF4, suggesting that the latter does not act downstream of mTORC1. Rather, we found a robust increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, suggesting that the integrated stress response pathway, rather than exercise-induced mTORC1 activation, drives long-term ATF4 expression in skeletal muscle after exercise. CONCLUSIONS: The enhanced sensitivity of mTORC1 to leucine is maintained at least 48 h after exercise. This shows that the anabolic window of opportunity for protein ingestion is not restricted to the first hours immediately following exercise. Increased mTORC1 sensitivity to leucine coincided with enhanced leucine influx into muscle and higher expression of genes involved in leucine sensing and amino acid metabolism. Also, exercise induced an increase in ATF4 protein expression. Altogether, these data suggest that muscular contractions switch on a coordinated program to enhance amino acid uptake as well as intramuscular sensing of key amino acids involved in mTORC1 activation and the stimulation of muscle protein synthesis.

Tong-Xie-Yao-Fang alleviates diarrhea-predominant irritable bowel syndrome in rats via the GCN2/PERK-eIF2α-ATF4 signaling pathway.

BACKGROUND: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common functional gastrointestinal disease. Tong-Xie-Yao-Fang (TXYF), the traditional Chinese herbal medicine prescription, is a classic and effective prescription for the treatment of IBS-D, but its mechanism of action is not fully clarified. OBJECTIVE: To evaluate the efficacy of TXYF in the treatment of IBS-D and to explore its potential mechanism of action. METHODS: Changes in the serum levels of 50 free amino acids were targeted for detection by high-performance liquid chromatography (HPLC), and the expression of glucose-regulated protein 78 (GRP78), general control nonderepressible 2 (GCN2), and endoplasmic reticulum-resident kinase (PERK) was detected by immunohistochemistry examinations in healthy volunteers and IBS-D patients. The IBS-D rat was constructed by the three-factor superposition method of neonatal maternal separation, 2,4,6-trinitrobenzene sulfonic acid enema, and chronic unpredictable stress stimulation. The treatment effect of TXYF on IBS-D rats was observed by recording the body weight, grasp force, fecal water content (FWC), and abdominal withdrawal reflex (AWR) of rats before and after treatment. The effects of GCN2/PERK-eukaryotic initiation factor-2 (eIF2α) -activating transcription Factor 4 (ATF4) pathway proteins and gene expression were analyzed by western blotting, reverse transcription-polymerase chain reaction (RT-qPCR), and immunohistochemistry evaluations. RESULTS: Compared with healthy volunteers, IBS-D patients exhibited lower levels of cysteine, γ-aminoacetic acid (GABA), homoproline, and lysine, and immunohistochemistry showed strong activation of GRP78, a marker of endoplasmic reticulum stress. Differential expression of GCN2 and PERK proteins was detected in IBS-D patients and rat colons. In the IBS-D rats, TXYF improved the body weight and grasp force, reduced the FWC, and improved the AWR score. TXYF increased the levels of p-GCN2 and GCN2 and reduced the levels of GRP78, p-PERK, PERK, p-eIF2α, and eIF2α, thereby affecting the expression of the apoptosis-related transcription factors ATF4, CHOP, Caspase-3, and Bcl-2. CONCLUSION: Our study showed that TXYF improved IBS-D by inhibiting apoptosis. The anti-apoptosis effects were potentially mediated by regulating the GCN2/PERK-eIF2a-ATF4 signaling pathway.

Activation of the eIF2α-ATF4 Pathway by Chronic Paracetamol Treatment Is Prevented by Dietary Supplementation with Cysteine.

Chronic treatment with acetaminophen (APAP) induces cysteine (Cys) and glutathione (GSH) deficiency which leads to adverse metabolic effects including muscle atrophy. Mammalian cells respond to essential amino acid deprivation through the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Phosphorylated eIF2α leads to the recruitment of activating transcription factor 4 (ATF4) to specific CCAAT/enhancer-binding protein-ATF response element (CARE) located in the promoters of target genes. Our purpose was to study the activation of the eIF2α-ATF4 pathway in response to APAP-induced Cys deficiency, as well as the potential contribution of the eIF2α kinase GCN2 and the effect of dietary supplementation with Cys. Our results showed that chronic treatment with APAP activated both GCN2 and PERK eIF2α kinases and downstream target genes in the liver. Activation of the eIF2α-ATF4 pathway in skeletal muscle was accompanied by muscle atrophy even in the absence of GCN2. The dietary supplementation with cysteine reversed APAP-induced decreases in plasma-free Cys, liver GSH, muscle mass, and muscle GSH. Our new findings demonstrate that dietary Cys supplementation also reversed the APAP-induced activation of GCN2 and PERK and downstream ATF4-target genes in the liver.

Protective effect of zinc supplementation on tricalcium phosphate particles-induced inflammatory osteolysis in mice.

Zinc (Zn), an essential trace element, can stimulate bone formation and inhibit osteoclastic bone resorption, which controls the growth and maintenance of bone. However, the effect of Zn supplementation on tricalcium phosphate (TCP) wear particles-induced osteolysis remains unknown. Here, we doped Zn into TCP particles (ZnTCP), and explore the protective effects of Zn on TCP particles-induced osteolysis in vivo. TCP particles and ZnTCP particles were embedded under the periosteum around the middle suture of the mouse calvaria. After 2 weeks, blood, the periosteal tissue, and the calvaria were collected to determine serum levels of Zn and osteocalcin, pro-inflammatory cytokines, bone biochemical markers, osteoclastogenesis and bone resorption area, and to explain its mechanism. Data revealed that Zn significantly prevented TCP particles-induced osteoclastogenesis and bone loss, and increased bone turnover. The Zn supplement remarkably suppressed the release of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6. Immunoblotting demonstrated that Zn alleviated expression levels of ER stress-related proteins such as glucose-regulated protein 78 (GRP78), PKR-like ER kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α (p-eIF2α), activating transcription factor 4 (ATF4), inositol-requiring enzyme 1α (IRE1-α) and transcription factor X-box binding protein spliced (XBP1s), leading to decreasing the ratios of p-PERK/PERK and p-eIF2α/eIF2α. Taken together, Zn supplementation strongly prevents TCP particles-induced periprosthetic osteolysis via inhibition of the ER stress pathway, and it may be a novel therapeutic approach for the treatment of aseptic prosthesis loosening.

Serine biosynthesis as a novel therapeutic target for dilated cardiomyopathy.

AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.

Endoplasmic reticulum stress contributes to autophagy and apoptosis in cantharidin-induced nephrotoxicity.

Mylabris, as a natural product of traditional Chinese medicine (TCM), exhibiting typical antitumor activity, and cantharidin (CTD) is the major bioactive component. However, drug-induced nephrotoxicity (DIN) extremely limited its clinical application. In this study, we proved that activation of the endoplasmic reticulum (ER) stress-dependent PERK/CHOP pathway exerts a toxic role in rats and HK-2 cells through inducing autophagy and apoptosis. Results showed that CTD could cause renal function damage, cytotoxicity, and apoptosis. The ER dilatation and autolysosomes were observed after CTD treatment. Furthermore, the distribution of LC3, ATF4, and CHOP proteins was observed in the nucleus and cytoplasm. In addition, the mRNA levels of ER stress-regulated genes (PERK, eIF2α, CHOP, and ATF4) were increased, and the expression levels of GRP78, ATF4, CHOP, LC3, Beclin-1, Atg3, Atg7, Caspase 3, and Bax/Bcl-2 proteins were increased both in vitro and in vivo. Consistently, this upregulation could be inhibited by an ER stress inhibitor 4-Phenylbutyric acid (4-PBA), indicating that ER stress is partly responsible for activation of autophagy and apoptosis in CTD-induced DIN. In conclusion, CTD could induce DIN by triggering ER stress, further activating autophagy and apoptosis both in vivo and in vitro.

Neonatal Hyperoxia Activates Activating Transcription Factor 4 to Stimulate Folate Metabolism and Alveolar Epithelial Type 2 Cell Proliferation.

Oxygen supplementation in preterm infants disrupts alveolar epithelial type 2 (AT2) cell proliferation through poorly understood mechanisms. Here, newborn mice are used to understand how hyperoxia stimulates an early aberrant wave of AT2 cell proliferation that occurs between Postnatal Days (PNDs) 0 and 4. RNA-sequencing analysis of AT2 cells isolated from PND4 mice revealed hyperoxia stimulates expression of mitochondrial-specific methylenetetrahydrofolate dehydrogenase 2 and other genes involved in mitochondrial one-carbon coupled folate metabolism and serine synthesis. The same genes are induced when AT2 cells normally proliferate on PND7 and when they proliferate in response to the mitogen fibroblast growth factor 7. However, hyperoxia selectively stimulated their expression via the stress-responsive activating transcription factor 4 (ATF4). Administration of the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia suppressed ATF4 and thus early AT2 cell proliferation, but it had no effect on normative AT2 cell proliferation seen on PND7. Because ATF4 and methylenetetrahydrofolate dehydrogenase are detected in hyperplastic AT2 cells of preterm infant humans and baboons with bronchopulmonary dysplasia, dampening mitochondrial oxidative stress and ATF4 activation may provide new opportunities for controlling excess AT2 cell proliferation in neonatal lung disease.

Bushen Jieyu Tiaochong Formula reduces apoptosis of granulosa cells via the PERK-ATF4-CHOP signaling pathway in a rat model of polycystic ovary syndrome with chronic stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder that is also an important cause of infertility. Adverse psychological stress can aggravate the occurrence and development of PCOS. Bushen Jieyu Tiaochong Formula (BJTF), a prescription of Traditional Chinese Medicine (TCM), has been used in the treatment of PCOS and shown to be effective in reducing negative emotion. However, the therapeutic mechanism has yet to be clearly elucidated. In the current study, we investigated the potential mechanism of action of BJTF. AIM OF THE STUDY: To investigate the role of PERK-ATF4-CHOP signaling in the molecular mechanisms that mediate the effects of BJTF in a rat model of PCOS, with chronic stress induced by letrozole and a chronic unpredictable mild stress (CUMS) paradigm. MATERIALS AND METHODS: In addition to the normal control group, the PCOS combined with CUMS model rats were randomly assigned to a model group, a Diane-35 (ethinylestradiol 35 µg/cyproterone acetate 2 mg)-treated positive control group, or one of three BJTF-treated groups receiving a low, medium, or high dose. Behavioral testing, including the sucrose preference test and open field test, was conducted, and hematoxylin and eosin (H&E) staining was used to observe changes in the pathological morphology of ovarian tissue. Free testosterone (FT), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels in serum were quantified by enzyme-linked immunosorbent assays (ELISA). The hippocampal levels of norepinephrine (NE), 5-hydroxytryptamine/serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were measured using high-performance liquid chromatography-electrochemical detection (HPLC-ECD). Apoptotic granulosa cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to detect the expression of glucose-regulated protein 78 (GRP78) and CHOP in the ovarian tissues. The expression levels of GRP78, CHOP, PERK, and ATF4 in ovarian tissues were also measured by western blotting. RESULTS: Treatment with either BJTF or Diane-35 ameliorated the abnormal cystic dilatation of follicles in the model rats and reduced the serum levels of FT and LH, and the LH/FSH ratio. BJTF treatment also attenuated chronic psychological stress-like behavior and regulated the expression and metabolism of cerebral monoamine neurotransmitters. The efficacy of BJTF was greater than that of Diane-35, with the optimal effects observed at the medium dose. BJTF also lowered the apoptotic index of ovarian granulosa cells and downregulated the expression of GRP78, CHOP, and ATF4. Although the expression level of PERK was not significantly altered by BJTF, the mean PERK expression level was the lowest in the medium-dose BJTF group. CONCLUSIONS: Administration of BJTF has the therapeutic potential to promote the homeostasis of the reproductive endocrine environment and to restore follicular development and ovulation, possibly through the inhibition of the PERK-ATF4-CHOP signaling pathway, leading to downregulation of GRP78 expression to further delay ovarian granule cell apoptosis mediated by endoplasmic reticulum stress (ERS). Moreover, BJTF could improve behavioral performance by regulating cerebral monoamine neurotransmitters in this rat model. These findings provide a new perspective for treating PCOS related to psychological stress using TCM.

Tissue-Specific Gamma-Flicker Light Noninvasively Ameliorates Retinal Aging.

Aging is a risk factor for multiple retinal degeneration diseases. Entraining brain gamma oscillations with gamma-flicker light (γFL) has been confirmed to coordinate pathological changes in several Alzheimer's disease mouse models and aged mice. However, the direct effect of γFL on retinal aging remains unknown. We assessed retinal senescence-associated beta-galactosidase (ß-gal) and autofluorescence in 20-month-old mice and found reduced ß-gal-positive cells in the inner retina and diminished lipofuscin accumulation around retinal vessels after 6 days of γFL. In immunofluorescence, γFL was further demonstrated to ameliorate aging-related retinal changes, including a decline in microtubule-associated protein 1 light chain 3 beta expression, an increase in complement C3 activity, and an imbalance between the anti-oxidant factor catalase and pro-oxidant factor carboxymethyl lysine. Moreover, we found that γFL can increase the expression of activating transcription factor 4 (ATF4) in the inner retina, while revealing a decrease of ATF4 expression in the inner retina and positive expression in the outer segment of photoreceptor and RPE layer for aged mice. Western blotting was then used to confirm the immunofluorescence results. After mRNA sequencing (NCBI Sequence Read Archive database: PRJNA748184), we found several main mechanistic clues, including mitochondrial function and chaperone-mediated protein folding. Furthermore, we extended γFL to aged Apoe-/- mice and showed that 1-m γFL treatment even improved the structures of retinal-pigment-epithelium basal infolding and Bruch's membrane. Overall, γFL can orchestrate various pathological characteristics of retinal aging in mice and might be a noninvasive, convenient, and tissue-specific therapeutic strategy for retinal aging.

Activation of mTORC1 by Free Fatty Acids Suppresses LAMP2 and Autophagy Function via ER Stress in Alcohol-Related Liver Disease.

Alcohol-related liver disease (ALD) is characterized by accumulation of hepatic free fatty acids (FFAs) and liver injury. The present study aimed to investigate if mechanistic target of rapamycin complex 1 (mTORC1) plays a role in FFA-induced organelle dysfunction, thereby contributing to the development of ALD. Cell studies were conducted to define the causal role and underlying mechanism of FFA-activated mTORC1 signaling in hepatocellular cell injury. C57BL/6J wild-type mice were subjected to chronic alcohol feeding with or without rapamycin to inhibit mTORC1 activation. We revealed that palmitic acid (PA)-induced ER stress and suppression of LAMP2 and autophagy flux were mTORC1-dependent as rapamycin reversed such deleterious effects. C/EBP homologous protein (CHOP) was downstream of ATF4 which partially modulated LAMP2. Supplementation with rapamycin to alcohol-fed mice attenuated mTORC1 activation and ER stress, restored LAMP2 protein, and improved autophagy, leading to amelioration of alcohol-induced liver injury. Induction of mTORC1 signaling and CHOP were also detected in the liver of patients with severe alcoholic hepatitis. This study demonstrates that hepatic FFAs play a crucial role in the pathogenesis of ALD by activating mTORC1 signaling, thereby inducing ER stress and suppressing LAMP2-autophagy flux pathway, which represents an important mechanism of FFA-induced hepatocellular injury.

Puerarin Inhibits the PERK-eIF2[Formula: see text]-ATF4-CHOP Pathway through Inactivating JAK2/STAT3 Signal in Pancreatic beta-Cells.

Type 1 diabetes (T1D) is an autoimmune and inflammatory disease with excessive loss of pancreatic islet [Formula: see text]-cells. Accumulating evidence indicated that endoplasmic reticulum (ER) stress played a critical role in [Formula: see text]-cells loss, leading to T1D. Therefore, promoting the survival of pancreatic [Formula: see text]cells would be beneficial for patients with T1D. Puerarin is a natural isoflavone that has been demonstrated to be able to decrease blood glucose in patients with T1D. However, it remains unknown whether puerarin improves ER stress to prevent [Formula: see text]-cells from apoptosis. Here, we sought to investigate the role of puerarin in ER stress-associated apoptosis and explore its underlying mechanism in the mouse insulinoma cell line (MIN6). Flow cytometry and cell counting kit-8 (CCK8) experiments showed that puerarin caused a significant increase in the viability of MIN6 cells injured by H2O2. Furthermore, the protein kinase R-like ER kinase (PERK) signal pathway, a critical branch of ER stress response, was found to be involved in this process. Puerarin inhibited the phosphorylation of PERK, subsequently suppressed the phosphorylation of eukaryotic initiation factor 2[Formula: see text] (eIF2[Formula: see text], then decreased the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, ultimately attenuating ER stress to prevent MIN6 cells from apoptosis. In addition, puerarin inhibited the activation of Janus kinase 2 (JAK2)/signal transducer and activators of transcription 3 (STAT3), which suppressed the PERK signal cascade with decreased ATF4 and CHOP levels. Taken together, our results firstly demonstrated that puerarin could prevent MIN6 cells from apoptosis at least in part by inhibiting the PERK-eIF2[Formula: see text]-ATF4-CHOP axis under ER stress conditions, which might be mediated by inactivation of the JAK2/STAT3 signal pathway. Therefore, investigating the mechanism underlying the effects of puerarin might highlight the potential roles of puerarin developing into an antidiabetic drug.

Very-low-protein diets lead to reduced food intake and weight loss, linked to inhibition of hypothalamic mTOR signaling, in mice.

The protein leverage hypothesis predicts that low dietary protein should increase energy intake and cause adiposity. We designed 10 diets varying from 1% to 20% protein combined with either 60% or 20% fat. Contrasting the expectation, very low protein did not cause increased food intake. Although these mice had activated hunger signaling, they ate less food, resulting in decreased body weight and improved glucose tolerance but not increased frailty, even under 60% fat. Moreover, they did not show hyperphagia when returned to a 20% protein diet, which could be mimicked by treatment with rapamycin. Intracerebroventricular injection of AAV-S6K1 significantly blunted the decrease in both food intake and body weight in mice fed 1% protein, an effect not observed with inhibition of eIF2a, TRPML1, and Fgf21 signaling. Hence, the 1% protein diet induced decreased food intake and body weight via a mechanism partially dependent on hypothalamic mTOR signaling.