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Activating transcription factors, ATFs, are a group of bZIP transcription factors that act as homodimers or heterodimers with a range of other bZIP factors. In general, ATFs respond to extracellular signals, indicating their important roles in maintaining homeostasis. The ATF family includes ATF1, ATF2, ATF3, ATF4, ATF5, ATF6, and ATF7. Consistent with the diversity of cellular processes reported to be regulated by ATFs, the functions of ATF are also diverse. ATFs play an important role in cell proliferation, apoptosis, differentiation and inflammation-related pathological processes. The expression and phosphorylation status of ATFs are also related to neurodegenerative diseases and polycystic kidney disease. Various miRNAs target ATF to regulate cancer proliferation, apoptosis, autophagy, sensitivity and resistance to radiotherapy and chemotherapy. Moreover, ATFs are necessary to maintain cell redox homeostasis. Therefore, deepening our understanding of the regulation and function of ATFs will provide insights into the basic regulatory mechanisms that influence how cells integrate extracellular and intracellular signals into genomic responses through transcription factors. Under pathological conditions, especially in cancer biology and response to treatment, the characterization of ATF dysfunction is important for understanding how to therapeutically utilize ATF2 or other pathways controlled by transcription factors. In this review, we will demonstrate how ATF1, ATF2, ATF3, ATF4, ATF5, ATF6, and ATF7 function in promoting or suppressing cancer development and identify their roles in tumour immunotherapy.

Endoplasmic reticulum (ER) stress has been associated with the dysfunction of intestinal barrier in humans and animals. We have previously shown that oral administration of glycine to suckling-piglets improves ER stress-related intestinal mucosal barrier impairment and jejunal epithelial apoptosis. However, the underlying mechanism remains unknown. In this study, the protective effect and the mechanism of glycine on apoptosis and dysfunction in intestinal barrier induced by brefeldin A (BFA), an ER stress inducer, was explored in porcine intestinal epithelial cells (IPEC-1). The results showed that BFA treatment led to enhanced apoptosis and upregulation of proteins involved in ER stress signaling, including inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6α (ATF6α), c-Jun N-terminal kinase (JNK), and C/EBP-homologous protein (CHOP). In addition, BFA induced a dysfunction in intestinal epithelial barrier, as evidenced by the increased paracellular permeability, decreased transepithelial electrical resistance (TEER), and reduced abundance of tight junction proteins (occludin, claudin-1, zonula occludens [ZO]-1, and ZO-2). These alterations triggered by BFA were significantly abolished by glycine treatment (P

Sorafenib and its derivative regorafenib are the first- and second-line targeted drugs for advanced HCC, respectively. Although both drugs improve overall survival, drug resistance remains the major barrier to their full efficacy. Thus, strategies to enhance sorafenib and regorafenib efficacy against HCC are solely needed. Interleukin-6 receptor alpha (IL-6Rα) is the receptor of IL-6, a multi-functional cytokine, which plays key roles in liver-regeneration, inflammation and development of hepatocellular carcinoma (HCC). Here we show the expression of IL-6Rα was induced in response to sorafenib. Depletion of IL-6Rα abolished IL-6 induced STAT3 phosphorylation at 705th tyrosine and tumor growth of HCC cells under sorafenib treatment. Mechanistically, activating transcription factor 3 (ATF3) was induced in response to sorafenib and subsequently bound to the promoter of IL-6Rα, leading to its transcriptional activation. Depletion of ATF3 or its upstream transcription factor, ATF4, attenuated IL-6Rα induction and IL-6 mediated sorafenib resistance. The ATF4-ATF3-IL-6Rα cascade is also activated by regorafenib. Furthermore, blockade of IL-6Rα with the FDA approved IL-6Rα antibody drug, Sarilumab, drastically attenuated both sorafenib and regorafenib resistance in patient-derived xenograft (PDX) tumors, where human IL-6 could be detected by a novel in situ hybridization technique, named RNAscope. Together, our data reveal that ATF3-mediated IL-6Rα up-regulation promotes both sorafenib and regorafenib resistance in HCC, and targeting IL-6Rα represents a novel therapeutic strategy to enhance sorafenib/regorafenib efficacy for advanced HCC treatment.

Myocardial injury (MI) is a common complication of sepsis. MicroRNAs (miRNAs) have been suggested as potential biomarkers of MI; however, their mechanisms in sepsis-induced MI remain unclear. A sepsis rat model was constructed by use of cecal ligation and puncture (CLP). The levels of miR-195-5p and activating transcription factor 6 (ATF6) expression were determined by quantitative reverse-transcription polymerase chain reaction, and cytokine levels were detected by ELISA. The levels of oxidative stress (OS)-related indicators and endoplasmic reticulum stress (ERS)-related proteins were examined, and the regulatory effect of miR-195-5p on ATF6 was determined by using the luciferase reporter assay. Our results showed that miR-195-5p expression was downregulated and ATF6 expression was upregulated in lipopolysaccharide-induced cardiomyocytes and mice with CLP-induced sepsis. We also found that miR-195-5p could markedly attenuate the inflammation, apoptosis, OS, and ERS associated with sepsis-induced MI. Additionally, we verified that miR-195-5p could relieve sepsis-induced MI by targeting ATF6. This study identified potential targets for treating MI after sepsis.

Background In bladder cancer, up to 70% of patients will relapse after resection within 5 years, in which the mechanism underlying the recurrence remains largely unclear. Methods Quantitative real-time PCR, western blot and immunohistochemistry were conducted. The assays of tumor sphere formation and tumor xenograft were further performed to assess the potential biological roles of ATF5 (activating transcription factor 5). Chromatin immunoprecipitation-qPCR and luciferase activity assays were carried out to explore the potential molecular mechanism. A two-tailed paired Student's t-test, χ2 test, Kaplan Meier and Cox regression analyses, and Spearman's rank correlation coefficients were used for statistical analyses. Results ATF5 is elevated in bladder urothelial cancer (BLCA) tissues, especially in recurrent BLCA, which confers a poor prognosis. Overexpressing ATF5 significantly enhanced, whereas silencing ATF5 inhibited, the capability of tumor sphere formation in bladder cancer cells. Mechanically, ATF5 could directly bind to and stimulate the promoter of DVL1 gene, resulting in activation of Wnt/β-catenin pathway. Conclusions This study provides a novel insight into a portion of the mechanism underlying high recurrence potential of BLCA, presenting ATF5 as a prognostic factor or potential therapeutic target for preventing recurrence in BLCA.

The extracellular accumulation of glutamate is a pathologic hallmark of numerous neurodegenerative diseases including ischemic stroke and Alzheimer's disease. At high extracellular concentrations, glutamate causes neuronal damage by promoting oxidative stress, which can lead to cellular death. This has led to significant interest in developing pharmacologic approaches to mitigate the oxidative toxicity caused by high levels of glutamate. Here, we show that the small molecule proteostasis regulator AA147 protects against glutamate-induced cell death in a neuronal-derived cell culture model. While originally developed as an activator of the activating transcription factor 6 (ATF6) arm of the unfolded protein response, this AA147-dependent protection against glutamate toxicity is primarily mediated through activation of the NRF2-regulated oxidative stress response. We demonstrate that AA147 activates NRF2 selectively in neuronal-derived cells through a mechanism involving metabolic activation to a reactive electrophile and covalent modification of KEAP1─a mechanism analogous to that involved in the AA147-dependent activation of ATF6. These results define the potential for AA147 to protect against glutamate-induced oxidative toxicity and highlight the potential for metabolically activated proteostasis regulators like AA147 to activate both protective ATF6 and NRF2 stress-responsive signaling pathways to mitigate oxidative damage associated with diverse neurologic diseases.

We investigated the mediating roles of activating transcription factor 3 (ATF3), an injury marker, or C-type lectin member 5A (CLEC5A), an inflammatory response molecule, in the induction of endoplasmic reticulum (ER) stress and neuroinflammation in diabetic peripheral neuropathy in ATF3 and CLEC5A genetic knockout (aft3−/− and clec5a−/−, respectively) mice. ATF3 was expressed intranuclearly and was upregulated in mice with diabetic peripheral neuropathy (DN) and clec5a−/− mice. The DN and clec5a−/− groups also exhibited neuropathic behavior, but not in the aft3−/− group. The upregulation profiles of cytoplasmic polyadenylation element-binding protein, a protein translation–regulating molecule, and the ER stress-related molecules of inositol-requiring enzyme 1α and phosphorylated eukaryotic initiation factor 2α in the DN and clec5a−/− groups were correlated with neuropathic behavior. Ultrastructural evidence confirmed ER stress induction and neuroinflammation, including microglial enlargement and proinflammatory cytokine release, in the DN and clec5a−/− mice. By contrast, the induction of ER stress and neuroinflammation did not occur in the aft3−/− mice. Furthermore, the mRNA of reactive oxygen species–removing enzymes such as superoxide dismutase, heme oxygenase-1, and catalase were downregulated in the DN and clec5a−/− groups but were not changed in the aft3−/− group. Taken together, the results indicate that intraneuronal ATF3, but not CLEC5A, mediates the induction of ER stress and neuroinflammation associated with diabetic neuropathy., Neuroinflammation and endoplasmic reticulum stress is a critical process that leads to diabetic peripheral neuropathy (DPN). Activating transcription factor 3 (ATF3) is a susceptible molecule, and loss-of-function of ATF3, but not C-type lectin member 5A (CLEC5A), prevents the development of neuroinflammation and ER stress. Functional blockade of ATF3 may be a potential treatment for diabetic peripheral neuropathy by inhibiting neuroinflammation and cellular stress-induced ER stress.

Activating transcription factor 3 (ATF3) has been shown to play an important role in HDL metabolism; yet, the role of hepatocytic ATF3 in the development of steatohepatitis remains elusive. Here we show that adenoassociated virus-mediated overexpression of human ATF3 in hepatocytes prevents diet-induced steatohepatitis in C57BL/6 mice and reverses steatohepatitis in db/db mice. Conversely, global or hepatocyte-specific loss of ATF3 aggravates diet-induced steatohepatitis. Mechanistically, hepatocytic ATF3 induces hepatic lipolysis and fatty acid oxidation and inhibits inflammation and apoptosis. We further show that hepatocyte nuclear factor 4α (HNF4α) is required for ATF3 to improve steatohepatitis. Thus, the current study indicates that ATF3 protects against steatohepatitis through, at least in part, hepatic HNF4α. Targeting hepatic ATF3 may be useful for treatment of steatohepatitis.

The pathogenesis of Parkinson's disease (PD) remains elusive, but mitochondrial dysfunction is believed to be one crucial step in its pathogenesis. The mitochondrial unfolded protein response (UPRmt) is an important mitochondrial quality control strategy that maintains mitochondrial function in response to disturbances of mitochondrial protein homeostasis. Activation of the UPRmt and the beneficial effect of rescuing mitochondrial proteostasis have been reported in several genetic models of PD. However, the pathogenic relevance of the UPRmt in idiopathic PD is unknown. The present study examined the link between the UPRmt and mitochondrial dysfunction in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells. Treatment with MPP + induced activation of the UPRmt, reflected by an increase in the expression of UPRmt-related chaperones, proteases, and transcription mediators. UPRmt activation that was induced by overexpressing mutant ornithine transcarbamylase significantly reduced the production of mitochondrial reactive oxygen species (ROS) and improved cell survival in SH-SY5Y cells following MPP+ treatment. Moreover, the overexpression of activating transcription factor 5 (mammalian UPRmt transcription factor) conferred protection against MPP+-induced ROS production and against cell death in SH-SY5Y cells. Overall, our results demonstrate the beneficial effect of UPRmt activation in MPP + -treated cells, shedding new light on the mechanism of mitochondrial dysfunction in the pathogenesis of PD.

Background and objective: Activating transcription factor 3 (ATF3) plays a crucial role in regulating tumor development depending on the cellular context or cancer cell type. However, the effect of ATF3 on stem cells-like features in colorectal cancer (CRC) has yet to be elaborated. Methods: In this study, we overexpressed ATF3 in SW620 CRC cells to investigate its effects on stem cells-like features. Results: Our results indicated that overexpressing ATF3 inhibited the proliferation, invasion, migration, and sphere formation capacity of SW620 CRC cells. ATF3 overexpression also decreased the size of tumorspheres and reduced expression of the cancer stem cell markers CD44 and CD133 in SW620 cells. Conclusion: In summary, our study revealed that ATF3 suppresses CRC growth and stem cells-like features. ATF3 is considered a potential target in CRC therapy.

Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho+/P23H) mice. Significantly increased rhodopsin protein levels were found in Atf6−/−Rho+/P23H retinas compared to Atf6+/−Rho+/P23H retinas at early ages (~ P12), while rhodopsin mRNA levels were not different. The IRE1 pathway of the UPR was hyper-activated in young Atf6−/−Rho+/P23H retinas, and photoreceptor layer thickness was unchanged at this early age in Rho+/P23H mice lacking Atf6. By contrast, older Atf6−/−Rho+/P23H mice developed significantly increased retinal degeneration in comparison to Atf6+/−Rho+/P23H mice in all retinal layers, accompanied by reduced rhodopsin protein levels. Our findings demonstrate that Atf6 is required for efficient clearance of rhodopsin protein in rod photoreceptors expressing P23H rhodopsin, and that loss of Atf6 ultimately accelerates retinal degeneration in P23H-KI mice.

The endoplasmic reticulum (ER) is an organelle that orchestrates the production and proper assembly of an extensive types of secretory and membrane proteins. Endoplasmic reticulum stress is conventionally related to prolonged disruption in the protein folding machinery resulting in the accumulation of unfolded proteins in the ER. This disruption is often manifested due to oxidative stress, Ca2+ leakage, iron imbalance, disease conditions which in turn hampers the cellular homeostasis and induces cellular apoptosis. A mild ER stress is often reverted back to normal. However, cells retaliate to acute ER stress by activating the unfolded protein response (UPR) which comprises three signaling pathways, Activating transcription factor 6 (ATF6), inositol requiring enzyme 1 alpha (IRE1α), and protein kinase RNA-activated-like ER kinase (PERK). The UPR response participates in both protective and pro-apoptotic responses and not much is known about the mechanistic aspects of the switch from pro-survival to pro-apoptosis. When ER stress outpaces UPR response then cell apoptosis prevails which often leads to the development of various diseases including cardiomyopathies. Therefore, it is important to identify molecules that modulate the UPR that may serve as promising tools towards effective treatment of cardiovascular diseases. In this review, we elucidated the latest advances in construing the contribution imparted by the three arms of UPR to combat the adverse environment in the ER to restore cellular homeostasis during cardiomyopathies. We also summarized the various therapeutic agents that plays crucial role in tilting the UPR response towards pro-survival.

It is known that β cell proliferation expands the β cell mass during development and under certain hyperglycemic conditions in the adult, a process that may be used for β cell regeneration in diabetes. Here, through a new high-throughput screen using a luminescence ubiquitination-based cell cycle indicator (LUCCI) in zebrafish, we identify HG-9-91-01 as a driver of proliferation and confirm this effect in mouse and human β cells. HG-9-91-01 is an inhibitor of salt-inducible kinases (SIKs), and overexpression of Sik1 specifically in β cells blocks the effect of HG-9-91-01 on β cell proliferation. Single-cell transcriptomic analyses of mouse β cells demonstrate that HG-9-91-01 induces a wave of activating transcription factor (ATF)6-dependent unfolded protein response (UPR) before cell cycle entry. Importantly, the UPR wave is not associated with an increase in insulin expression. Additional mechanistic studies indicate that HG-9-91-01 induces multiple signalling effectors downstream of SIK inhibition, including CRTC1, CRTC2, ATF6, IRE1 and mTOR, which integrate to collectively drive β cell proliferation. A high-throughput chemical screen identifies the salt-inducible kinase inhibitor HG-9-91-01 as a driver of β cell proliferation, acting through an ATF6-dependent unfolded protein response.

Background Esophageal cancer (ESCA) is one of the most common cancers worldwide and has a very poor prognosis. Hypoxia-inducible factor 1 (HIF1) signaling pathway plays a critical role in tumorigenesis and is therefore considered a potential therapeutic target in the treatment of many cancers. Activating transcription factor 5 (ATF5) facilitates the expression of various genes and has been extensively studied for its potential role in cancer treatment. Methods The expression level of ATF5 in clinic sample was detected by quantitative real time PCR and immunohistochemistry. ATF5 biological function was investigated by western blot, cell cycle analysis, cell viability assay, luciferase reporter assays, colony formation assay, transwell assay, wound healing assay, tube formation assay, and ELISA assay. CHIP and Re-CHIP assay, GST-pulldown, and RNA-sequencing were used to study the cross-talks between ATF5 and HIF1 complex. Mouse xenograft study was utilized to study the correlation of ATF5 and tumor growth in vivo. Student’s t-test or Chi-square test was used for statistical analysis. Results Here, we first found ATF5 was dramatically upregulated in ESCA cancer and related with poor survival time. Next, we found that the expression level of ATF5 had a positive relationship with the proliferation, migration, and invasion ability of ESCA cells. Besides, we innovatively found that ATF5 functions as a novel coactivator in HIF1 transcription complex by binding to HIF1α. Further, we demonstrated that silencing ATF5 phenocopies HIF1α knockdown in tumorigenic properties in vitro and inhibited ESCA tumor angiogenesis and proliferation in vivo. Conclusion Herein, we found ATF5 as a novel component of the HIF1 transcription complex. The findings of the present study may provide new insights into the development of a novel and more efficient therapeutic strategy against ESCA.

Activating transcription factor 5 (ATF5) is a stress-responsive transcription factor that belongs to the cAMP response element-binding protein (CREB)/ATF family, and is essential for the differentiation and survival of sensory neurons in murine olfactory organs. However, the study of associated proteins and target genes for ATF5 has been hampered due to the limited availability of immunoprecipitation-grade ATF5 antibodies. To overcome this issue, we generated hemagglutinin (HA)-tag knock-in mice for ATF5 using CRISPR/Cas9-mediated genome editing with one-step electroporation in oviducts (i-GONAD). ATF5-HA fusion proteins were detected in the nuclei of immature and some mature olfactory and vomeronasal sensory neurons in the main olfactory epithelium and vomeronasal organ, respectively, as endogenous ATF5 proteins were expressed, and some ATF5-HA proteins were found to be phosphorylated. Chromatin immunoprecipitation (ChIP) experiments revealed that ATF5-HA bound to the CCAAT/enhancer-binding protein (C/EBP)-ATF response element site in the promotor region of receptor transporting protein 1 (Rtp1), a chaperone gene responsible for proper olfactory receptor expression. These knock-in mice may be used to examine the expression, localization, and protein-protein/-DNA interactions of endogenous ATF5 and, ultimately, the function of ATF5 in vivo.

A key transcriptional activator, activating transcription factor 5 (ATF5), is aberrantly overexpressed in glioma and supports both poor prognosis and antiapototic potential. Unfortunately, data on ATF5 is largely based on its regulatory mechanism. Further investigation of the upstream regulatory factor for ATF5 transcription in glioma is required. Clinical data for patients with diagnosed glioma were obtained from The Cancer Genome Atlas (TCGA). Additionally, transcription factors potentially regulating the ATF5 promoter in glioma were screened with bioinformatics. A further experimental study was performed to investigate both the role of E74-like factor 1 (ELF1) and the binding of ELF1 and the ATF5 promoter in glioma. We show that ATF5 expression is upregulated in glioma tissues and associated with tumor malignancy and worse prognosis. As a putative upstream regulator, silencing ELF1 inhibits glioma cell growth and migration with ATF5 involvement. Moreover, ELF1 upregulation is also associated with poor prognosis in glioma. Importantly, the luciferase assay and chromatin immunoprecipitation (ChIP) reveal that the ATF5 gene promoter is essential for ELF1-dependent activation of ATF5 gene transcription. These results indicate that a high expression of ELF1 may be related to the malignant behavior of human glioma and ELF1 promotes glioma development mediated by transactivation of the ATF5 gene.

INTRODUCTION Nicotiflorin is the main characteristic component of Nymphaea candida, which is a natural product that reportedly ameliorates acute injury of the liver and cerebral cortex, but the effect of nicotiflorin on acute kidney injury (AKI) remains unknown. This study aimed to investigate the effects of nicotiflorin on ischaemia/reperfusion (I/R) AKI and the associated mechanisms. METHODS We performed both (a) in vivo experiments with C57BL/6 mice with bilateral renal pedicles clamped for 45 minutes and (b) in vitro experiments with human kidney epithelial cells (HK-2) exposed to hypoxia/reoxygenation to mimic I/R injury to study the role of nicotiflorin in AKI. RESULTS In vivo, nicotiflorin administration exerted protective effects on renal injury, as demonstrated by reductions in the levels of caspase3 and Bad (P

Cardiac hypertrophy can cause heart failure. However, the mechanisms underlying the progression of cardiac hypertrophy remain unclear. Emerging evidence suggests that circular RNAs (circRNAs) play a critical role in cardiac hypertrophy. However, the association between circ_nuclear factor I X (circNfix) and cardiac hypertrophy remain largely unknown. Therefore, the aim of the present study was to explore the role of circNfix in cardiac hypertrophy. In order to detect the function of circNfix in cardiac hypertrophy, cardiomyocytes were stimulated with angiotensin II (Ang II) to mimic the pathogenesis of the disease. In addition, pressure overload-induced cardiac hypertrophy in a mouse model was established using transverse aortic constriction (TAC) surgery. The mechanism via which circNfix regulated cardiac hypertrophy was investigated using RNA pull-down and luciferase reporter assays, and fluorescence in situ hybridization (FISH). circNfix was downregulated in Ang II-treated cardiomyocytes. Similarly, circNfix expression was markedly downregulated in mice following TAC surgery. In addition, circNfix overexpression significantly prevented the progression of cardiac hypertrophy in TAC-treated mice. Luciferase activity and RNA pull-down assays indicated that circNfix could indirectly target activating transcription factor 3 (ATF3) by binding with microRNA (miR)-145-5p in cardiomyocytes. miR-145-5p overexpression or ATF3 knockdown could reverse the effects of circNfix in Ang II-treated mouse cardiomyocytes. circNfix attenuated pressure overload-induced cardiac hypertrophy by regulating the miR-145-5p/ATF3 axis. Therefore, circNfix may serve as a molecular target for cardiac hypertrophy treatment., GRAPHICAL ABSTRACT

Background: Activating transcription factor 3 (ATF3) plays a significant role in cancer development and progression. We investigated the association between variants in expression quantitative trait loci (eQTLs) within ATF3 binding regions and the prognosis of non-small cell lung cancer (NSCLC) after surgery. Methods: A total of 772 patients with NSCLC who underwent curative surgery were enrolled. Using a public database (http://galaxyproject.org), we selected 104 single nucleotide polymorphisms (SNPs) in eQTLs in the ATF3 binding regions. The association of those SNPs with disease-free survival (DFS) was evaluated. Results: Among those SNPs, HAX1 rs11265425T>G was associated with significantly worse DFS (aHR = 1.30, 95% CI = 1.00–1.69, p = 0.05), and ME3 rs10400291C>A was associated with significantly better DFS (aHR = 0.66, 95% CI = 0.46–0.95, p = 0.03). Regarding HAX1 rs11265425T>G, the significant association remained only in adenocarcinoma, and the association was significant only in squamous cell carcinoma regarding ME3 rs10400291C>A. ChIP-qPCR assays showed that the two variants reside in active enhancers where H3K27Ac and ATF3 binding occurs. Promoter assays showed that rs11265425 G allele had significantly higher HAX1 promoter activity than T allele. HAX1 RNA expression was significantly higher in tumor than in normal lung, and higher in rs11265425 TG+GG genotypes than in TT genotype. Conversely, ME3 expression was significantly lower in tumor than in normal lung, and higher in rs10400291 AA genotype than in CC+CA genotypes. Conclusions: In conclusion, this study shows that the functional polymorphisms in ATF3 binding sites, HAX1 rs11265425T>G and ME3 rs10400291C>A are associated with the clinical outcomes of patients in surgically resected NSCLC.

Activating transcription factor (ATF)3 is known to have an anti-inflammatory function, yet the role of hepatic ATF3 in lipoprotein metabolism or atherosclerosis remains unknown. Here we show that overexpression of human ATF3 in hepatocytes reduces the development of atherosclerosis in Western-diet-fed Ldlr-/- or Apoe-/- mice, whereas hepatocyte-specific ablation of Atf3 has the opposite effect. We further show that hepatic ATF3 expression is inhibited by hydrocortisone. Mechanistically, hepatocyte ATF3 enhances high-density lipoprotein (HDL) uptake, inhibits intestinal fat and cholesterol absorption and promotes macrophage reverse cholesterol transport by inducing scavenger receptor group B type 1 (SR-BI) and repressing cholesterol 12α-hydroxylase (CYP8B1) in the liver through its interaction with p53 and hepatocyte nuclear factor 4α, respectively. Our data demonstrate that hepatocyte ATF3 is a key regulator of HDL and bile acid metabolism and atherosclerosis.

Vascular calcification, the ectopic deposition of calcium in blood vessels, develops in association with various metabolic diseases and atherosclerosis and is an independent predictor of morbidity and mortality associated with these diseases. Herein, we report that reduction of microRNA-27a-3p (miR-27a-3p) causes an increase in activating transcription factor 3 (ATF3), a novel osteogenic transcription factor, in vascular smooth muscle cells. Both microRNA (miRNA) and mRNA microarrays were performed with rat vascular smooth muscle cells, and reciprocally regulated pairs of miRNA and mRNA were selected after bioinformatics analysis. Inorganic phosphate significantly reduced the expression of miR-27a-3p in A10 cells. The transcript level was also reduced in vitamin D3-administered mouse aortas. miR-27a-3p mimic reduced calcium deposition, whereas miR-27a-3p inhibitor increased it. The Atf3 mRNA level was upregulated in a cellular vascular calcification model, and miR-27a-3p reduced the Atf3 mRNA and protein levels. Transfection with Atf3 could recover the miR-27a-3p-induced reduction of calcium deposition. Our results suggest that reduction of miR-27a-3p may contribute to the development of vascular calcification by de-repression of ATF3., Graphical Abstract, Vascular calcification causes stiffness of blood vessels, which causes secondary damage to the cardiovascular system. A reduction in the microRNA miR-27a-3p leads to an increase in the transcription factor ATF3, which induces calcium deposition. This miR-27a-3p/ATF signaling pathway may provide a novel therapeutic target for vascular calcification.

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

Growth hormone releasing hormone (GHRH) antagonists enhance endothelial barrier function and counteract the LPS-induced lung endothelial hyperpermeability, the cardinal feature of the acute respiratory distress syndrome (ARDS). The unfolded protein response (UPR) is a multifaceted molecular mechanism, strongly involved in tissue defense against injury. The current study introduces the induction of UPR by GHRH antagonists, since those peptides induced several UPR activation markers, including the inositol-requiring enzyme-1α (IRE1α), the protein kinase RNA-like ER kinase (PERK), and the activating transcription factor 6 (ATF6). On the other hand, the GHRH agonist MR-409 exerted the opposite effects. Furthermore, GHRH antagonists counteracted the kifunensine (UPR suppressor)-induced lung endothelial barrier dysfunction. Our observations suggest that UPR mediates, at least in part, the protective effects of GHRH antagonists in the lung microvasculature. To the best of our knowledge; this is the first study to provide experimental evidence in support of the hypothesis that UPR induction is a novel mechanism by which GHRH antagonists oppose severe human disease, including ARDS.

Increasing evidence in substantiating the roles of endoplasmic reticulum stress, oxidative stress, and inflammatory responses and their interplay is evident in various diseases. However, an in-depth mechanistic understanding of the crosstalk between the intracellular stress signaling pathways and inflammatory responses and their participation in disease progression has not yet been explored. Progress has been made in our understanding of the cross talk and integrated stress signaling network between endoplasmic reticulum stress and oxidative stress towards the pathogenesis of diabetic nephropathy. In this present study, we studied the crosstalk between the endoplasmic reticulum stress and oxidative stress by understanding the role of protein disulfide isomerase and endoplasmic reticulum oxidase 1α, a key player in redox protein folding in the endoplasmic reticulum. We had recruited a total of 90 subjects and divided into three groups (control (n = 30), type 2 diabetes mellitus (n = 30), and diabetic nephropathy (n = 30)). We found that endoplasmic reticulum stress markers, activating transcription factor 6, inositol-requiring enzyme 1α, protein kinase RNA-like endoplasmic reticulum kinase, C/EBP homologous protein, and glucose-regulated protein-78; oxidative stress markers, thioredoxin-interacting protein and cytochrome b-245 light chain; and the crosstalk markers, protein disulfide isomerase and endoplasmic reticulum oxidase-1α, were progressively elevated in type 2 diabetes mellitus and diabetic nephropathy subjects. The association between the crosstalk markers showed a positive correlation with endoplasmic reticulum stress and oxidative stress markers. Further, the interplay between endoplasmic reticulum stress and oxidative stress was investigated in vitro using a human leukemic monocytic cell line under a hyperglycemic environment and examined the expression of protein disulfide isomerase and endoplasmic reticulum oxidase-1α. DCFH-DA assay and flow cytometry were performed to detect the production of free radicals. Further, phosphorylation of eIF2α in high glucose–exposed cells was studied using western blot. In conclusion, our results shed light on the crosstalk between endoplasmic reticulum stress and oxidative stress and significantly contribute to the onset and progression of diabetic nephropathy and therefore represent the major therapeutic targets for alleviating micro- and macrovascular complications associated with this metabolic disturbance. [Figure: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12192-020-01176-z.

Graphical abstract The map illustrating mechanisms associated with lncRNA HIF1A-AS2-mediated inflammation in the atherosclerosis. LncRNA HIF1A-AS2 forms a complex with USF1, which is recruited into the ATF2 promoter to elevate ATF2 expression, thus promoting the development of atherosclerotic inflammation. Meanwhile, downregulation of lncRNA HIF1A-AS2 decreases the expression of ATF2 by reducing the binding of USF1 to the ATF2 promoter regions, thereby inhibiting atherosclerotic inflammation, as reflected by decreased inflammatory factors TNF-α, IL-1β and IL-6 in serum and protein levels of adhesion molecules VCAM-1, ICAM-1, and MCP-1, as well as increased cell viability and reduced apoptosis in ox-LDL-induced inflammation in ECs, SMCs, and HCAECs., Introduction In atherosclerotic lesions, extensive inflammation of the vessel wall contributes to plaque instability. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes in atherosclerosis. Objectives Here, we aim to identify the functional role and regulatory mechanisms of lncRNA hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) in atherosclerotic inflammation. Methods An atherosclerotic mouse model was induced in ApoE-/- mice by high fat diet (HFD). Endothelial cells (ECs), human aortic smooth muscle cells (SMCs) or human coronary artery endothelial cells (HCAECs) were exposed to ox-LDL to develop the in vitro model. The effects of lncRNA HIF1A-AS2 on inflammation were evaluated by determining levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and levels of adhesion molecules vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and macrophage cationic peptide 1 (MCP-1). Results It was established that lncRNA HIF1A-AS2 and ATF2 were highly expressed in atherosclerotic ApoE-/- mice. Downregulating lncRNA HIF1A-AS2 in ox-LDL-exposed ECs, SMCs and HCAECs inhibited inflammation by reducing levels of pro-inflammatory factors and adhesion molecules. LncRNA HIF1A-AS2 bound to the transcription factor USF1 to elevate ATF2 expression. USF1 overexpression counteracted the suppressive effect of lncRNA HIF1A-AS2 silencing on ox-LDL-induced inflammation. Knockdown of lncRNA HIF1A-AS2 or ATF2 could also attenuate inflammation in atherosclerotic mice. Collectively, the present study demonstrates that downregulation of lncRNA HIF1A-AS2 represses the binding of USF1 to the ATF2 promoter region and then inhibits ATF2 expression, thereby suppressing atherosclerotic inflammation. Conclusion This study suggests lncRNA HIF1A-AS2 as an promising therapeutic target for atherosclerosis.

X-box-binding protein 1 (XBP1) is a protein containing the basic leucine zipper structure. It belongs to the cAMP-response element binding protein (CREB)/activating transcription factor transcription factor family. As the main transcription factor, spliced XBP1 (XBP1s) participates in many physiological and pathological processes and plays an important role in embryonic development. Previous studies showed that XBP1-knockout mice died because of pancreatic exocrine function deficiency, indicating that XBP1 plays an important role in pancreatic development. However, the exact role of XBP1 in pancreatic development remains unclear. This study aimed to investigate the role of XBP1 in the pancreatic development of Xenopus laevis embryos. Whole-mount in situ hybridization and quantitative real-time PCR results revealed that the expression levels of pancreatic progenitor marker genes pdx1, p48, ngn3, and sox9 were downregulated in XBP1s morpholino oligonucleotide (MO)-injected embryos. The expression levels of pancreatic exocrine and endocrine marker genes insulin and amylase were also downregulated. Through the overexpression of XBP1s, the phenotype and gene expressions were opposite to those in XBP1s MO-injected embryos. Luciferase and chromatin immunoprecipitation assays showed that XBP1s could bind to the XBP1-binding site in the foxa2 promoter. These results revealed that XBP1 is required in the pancreatic development of Xenopus laevis and might function by regulating foxa2.

Background: Activation of the unfolded protein response (UPR) is closely associated with the pathogenesis of many metabolic diseases. There is increasing evidence for the interdependence of the UPR and metabolic signaling pathways. The AMP-activated protein kinase (AMPK) signaling pathway controls energy balance in eukaryotes. Objective: This study was to investigate the possible interaction between AMPK signaling and the UPR in muscle cells exposed to a saturated fatty acid, as well as the underlying mechanism. Methods: The UPR was induced in C2C12 myotubes by treatment with the saturated fatty acid palmitate. Compound C or knocking down AMPKα with short hairpin RNA (shRNA) was used to inhibit AMPK signaling pathway in palmitate-treated muscle cells. AMPK signaling was activated in myotubes using 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICAR) or ex229. Taurourdodeoxycholic acid (TUDCA) was pre-treated to C2C12 myotubes to inhibit UPR before adding palmitate. Real-time PCR and Western blotting were conducted to evaluate the expression of UPR markers and AMPK activation. Results: The UPR was induced in C2C12 myotubes by treatment with palmitate along with activation of AMPK signaling. Inhibiting the AMPK pathway with compound C or knocking down AMPKα with shRNA attenuated palmitate-induced UPR activation, while inhibiting the UPR with TUDCA alleviated palmitate-induced AMPK activation, suggesting a positive feedback loop between the UPR and AMPK. Additionally, pharmacologic activation of the AMPK pathway with either AICAR or ex229 was sufficient to induce significant upregulation of UPR markers. Conclusions: These results provide the first evidence for the involvement of AMPK signaling in the early activation of the UPR induced by saturated fatty acid in skeletal muscle, and suggest that physiologic or pharmacologic activation of the AMPK pathway (ie, by exercise or metformin, respectively) can promote muscle health and function and thus improve quality of life for individuals with metabolic disorders due to a high-fat diet or obesity.

Silent information regulator factor 2‑related enzyme 1 (Sirt1) is involved in the regulation of cell senescence, gene transcription, energy balance and oxidative stress. However, the effect of Sirt1 on atrial natriuretic factor (ANF) secretion, especially under hypoxic conditions is unclear. The present study aimed to investigate the effect of Sirt1, regulated by NADPH oxidase 4 (NOX4), on ANF secretion in isolated beating rat atria during hypoxia. ANF secretion was analyzed using radioimmunoassays and protein expression levels were determined by western blotting and immunofluorescence staining. Intra‑atrial pressure was recorded using a physiograph. Hypoxia significantly upregulated Sirt1 and nuclear factor erythroid‑2‑related factor 2 (Nrf2) protein expression levels, together with significantly increased ANF secretion. Hypoxia‑induced protein expression of Sirt1 was significantly blocked by a NOX4 inhibitor, GLX351322, and Nrf2 protein expression levels were significantly abolished using the Sirt1 inhibitor, EX527. Hypoxia also significantly elevated the protein expression levels of phosphorylated‑Akt and sequestosome 1 and significantly downregulated Kelch‑like ECH‑associated protein 1 protein expression levels. These effects were significantly blocked by EX527, preventing hypoxia‑induced Nrf2 expression. An Nrf2 inhibitor, ML385, significantly abolished the hypoxia‑induced upregulation of activating transcription factor (ATF)3, ATF4, T cell factor (TCF)3 and TCF4/lymphoid enhancer factor 1 (LEF1) protein expression levels, and significantly attenuated hypoxia‑induced ANF secretion. These results indicated that Sirt1 and Nrf2, regulated by NOX4, can potentially stimulate TCF3 and TCF4/LEF1 signaling via ATF3 and ATF4 activation, thereby potentially participating in the regulation of ANF secretion in beating rat atria during hypoxia. In conclusion, intervening with the Sirt1/Nrf2/ATF signaling pathway may be an effective strategy for resisting oxidative stress damage in the heart during hypoxia.

ATF3 (activating transcription factor 3) is a member of the mammalian activation transcription factor/cAMP-responsive element-binding (CREB) family. It plays a role in inflammation and innate immunity, and suggests that ATF3 is associated with atherosclerosis. In our study, we analyzed datasets of atherosclerosis from the NCBI-GEO (Gene Expression Omnibus) database and found that expression levels of ATF3 were lower in macrophages from ruptured atherosclerotic plaques than from stable atherosclerotic plaques. Expression levels of ATF3 correlated with the stability of atherosclerotic plaques. KEGG analysis of different expression genes (DEGs) between ruptured and stable atherosclerotic plaques was performed by Metascape database. The PI3K-AKT pathway may be a potential pathway of the formation of ruptured atherosclerotic plaques. High-fat diet-induced atherosclerosis apoE-/- mice were divided into two groups: a model group and an ATF3 overexpression (OE)-group. Tests on atherosclerotic plaques in the aortic root suggested that absence of ATF3 and increase of macrophages may be risk factors for the formation of ruptured atherosclerotic plaques. We found decreased areas of lesions in aortic roots and branches of aortic arch, as well as increased lesional content of macrophages as well as TUNEL-positive areas. Consistent with these results, we found reduced degradation and incidence of elastic plate cracks accompanied by suppressed MMPs expression and transduction pathway protein PI3K/AKT activation. These data suggest that ATF3 is a signaling molecule that mediates the progression and stability of atherosclerotic plaques. ATF3 could be a potential new biomarker for the prognosis of atherosclerosis and may be a therapeutic target to reduce atherosclerosis.

Spinal cord injury (SCI) is one of the most common devastating injuries, with little possibility of recovery in humans. However, zebrafish efficiently regenerate functional nervous system tissue after SCI. Therefore, the spinal cord transection model of adult zebrafish was applied to explore the role of ATF6 in neuro-recovery. Activating transcription factor 6 (ATF6) is a type-II transmembrane protein in the endoplasmic reticulum (ER). ATF6 target genes could improve ER homeostasis, which contributes to cytoprotection. Herein, we found that the ATF6 level increased at 12 h and 3 days post SCI, and returned to sham levels at 7 days post SCI. ATF6-expressing motor neurons were present in the central canal of the spinal cord and increased at 12 h post SCI. ATF6 morpholino treatment showed that inhibition of ATF6 delayed locomotor recovery and hindered neuron axon regrowth in SCI zebrafish. Furthermore, we investigated the role of both binding immunoglobulin protein (Bip) and C/EBP homologous transcription factor protein (CHOP), the two target genes of ATF6. We found that Bip expression significantly increased in the spinal cord at 7 days after SCI, which served as a pro-survival chaperone. Our results also showed that CHOP expression significantly decreased in the spinal cord at 7 days after SCI, which was identified as a protein involved in apoptosis. Taken together, our data demonstrate that ATF6 may contribute to the functional recovery after SCI in adult zebrafish, via up-regulation of Bip and down-regulation of CHOP to restore the homeostasis of ER.

Background Centromere protein F (CENPF) is a key component of the kinetochore complex involved in mitosis, cell differentiation and cellular response to stresses. However, the alteration of CENPF in response to endoplasmic reticulum (ER) stress has not been well described. In the present study, we investigate CENPF regulation in response to ER stress. Methods Quantitative real-time polymerase chain reaction and western blotting were used to determine CENPF expression under ER stress. Luciferase activity analysis was performed to investigate the promoter regions contributing to CENPF transcription in response to TG. Chromatin immunoprecipitation (ChIP) and ChIP Re-IP assays were used to determine if X-box binding protein 1 (XBP1) and/or activating transcription factor 6α (ATF6α) bind in the CENPF promoter region. Cell apoptosis and proliferation were analyzed using TUNEL, cell growth and clonogenic assays. Results CENPF expression is dramatically reduced under ER stress induced by thapsigargin (TG), brefeldin A (BFA), or tunicamycin (TM) and this downregulation of CENPF expression was dependent on XBP1 and ATF6α. Luciferase activity analysis of the truncated CENPF promoter indicates that regions from bases − 679 to − 488 and from − 241 to − 78 in the CENPF promoter were sensitive to TG treatment. Additionally, ChIP and ChIP Re-IP assays reveal that XBP1 and ATF6α were assembled on the same regions of CENPF promoter. Notably, we identify two XBP1 binding sequences at positions − 567 and − 192, to which XBP1 binding was enhanced by TG. Finally, CENPF overexpression inhibits cell apoptosis and promotes cell proliferation in response to ER stress. Conclusion In summary, these results demonstrate that ER stress plays a crucial role in CENPF expression, and XBP1 may up-regulate DNA-binding affinities after TG treatment to the promoter of CENPF. These findings may contribute to the understanding of the molecular mechanism of CENPF regulation.

Tandem transcription interference occurs when the act of transcription from an upstream promoter suppresses utilization of a co-oriented downstream promoter. Because eukaryal genomes are liberally interspersed with transcription units specifying long non-coding (lnc) RNAs, there are many opportunities for lncRNA synthesis to negatively affect a neighboring protein-coding gene. Here, I review two eukaryal systems in which lncRNA interference with mRNA expression underlies a regulated biological response to nutrient availability. Budding yeast SER3 is repressed under serine-replete conditions by transcription of an upstream SRG1 lncRNA that traverses the SER3 promoter and elicits occlusive nucleosome rearrangements. SER3 is de-repressed by serine withdrawal, which leads to shut-off of SRG1 synthesis. The fission yeast phosphate homeostasis (PHO) regulon comprises three phosphate acquisition genes – pho1, pho84, and tgp1 – that are repressed under phosphate-replete conditions by 5′ flanking lncRNAs prt, prt2, and nc-tgp1, respectively. lncRNA transcription across the PHO mRNA promoters displaces activating transcription factor Pho7. PHO mRNAs are transcribed during phosphate starvation when lncRNA synthesis abates. The PHO regulon is de-repressed in phosphate-replete cells by genetic manipulations that favor ‘precocious’ lncRNA 3′-processing/termination upstream of the mRNA promoters. PHO lncRNA termination is governed by the Pol2 CTD code and is subject to metabolite control by inositol pyrophosphates.

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, was used to protect liver function through antiapoptosis or reducing endoplasmic reticulum stress (ER stress). Previous studies showed that ER stress was modulated by herpes simplex virus types 1 (HSV-1) infection to facilitate viral replication. Here, we investigated the effect of TUDCA on HSV-1 infection of HEC-1-A cells and showed that both replication and multiplication of the virus were inhibited by TUDCA in a dose dependent manner. Unfolded protein response was induced to deliver stress signals from ER to nucleus. We found that TUDCA alleviated activating transcription factor 6 branch inhibition, partially enhanced protein kinase RNA-like ER kinase pathway activation, and repressed inositol-requiring protein 1α arm activation significantly in infected cells. The findings of this study suggest that TUDCA inhibits HSV-1 replication through ER stress pathway, which may provide a potential therapeutic strategy for HSV-1 infection.

Yanbian yellow cattle are one of the top five largest breeds of cattle in China. We had previously found that bta-miR-1271 is differentially expressed in the longissimus dorsi muscles of Yanbian yellow bulls and steers. However, whether bta-miR-1271 affects bovine fat formation is unclear. In this study, we used target gene prediction, dual-luciferase reporter assay, and transfection-mediated overexpression and inhibition of bta-miR-1271 in a culture of Yanbian yellow cattle preadipocytes to investigate the role of bta-miR-1271 in adipogenesis. We showed that bta-miR-1271 directly targets the 3'-untranslated region (3'-UTR) of the activating transcription factor 3 (ATF3) mRNA and downregulates its expression. Overexpression of bta-miR-1271 enforced by the miRNA mimics promoted triglyceride accumulation and significantly upregulated expression of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα) genes at both the protein and mRNA levels, as demonstrated by RT-qPCR and Western blot analyses. Conversely, inhibition of bta-miR-1271 expression produced the opposite effect. Our results show that bta-miR-1271 regulates differentiation of Yanbian yellow cattle preadipocytes by inhibiting ATF3 expression, which highlights the importance of microRNA-mediated regulation of adipogenesis. miR-1271 and its target gene(s) may provide a new research direction for investigating biological agents affecting intramuscular fat deposition in cattle.

An overwhelming endoplasmic reticulum stress (ERS) and the following unfolded protein response (UPR) can induce hepatic inflammation, fibrosis and hepatocellular carcinoma (HCC). Caudatin, one of the species of C‑21 steroidal glycosides mainly isolated from the roots of Cynanchum bungei Decne, exhibits potent anticancer activities in vivo. However, the effect of caudatin on HCC remains unclear. In the present study, a diethylnitrosamine (DEN)‑induced HCC model was established. Nodules and tumors in rat livers were monitored by T2‑/T1‑weighted‑magnetic resonance imaging (MRI) using a 1.5 T scanner. Caudatin reduced the number and size of nodules and alleviated the inflammatory foci in the liver. In addition, the hepatic pro‑inflammatory levels of interleukin (IL) 6, monocyte chemoattractant protein 1 and IL‑1β were decreased in caudatin‑treated rats. The DEN‑induced surge in malondialdehyde, aspartate aminotransferase, alanine transaminase and TBIL were alleviated following caudatin treatment. The expression of ERS chaperones glucose‑regulated protein, 94 kDa, glucose‑regulated protein, 78 kDa and protein disulfide‑isomerase A4 and the proliferation marker Ki‑67 in liver nodules were all downregulated by caudatin as demonstrated by immunohistochemistry, reverse transcription‑quantitative PCR and western blot analysis. Caudatin reduced the cytoprotective ERS sensor activating transcription factor 6‑mediated signal transduction and inhibited the PKR‑like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway. However, the effect of caudatin on inositol requiring enzyme 1 signaling was negligible. In conclusion, restoration of the dysregulated UPR program was involved in the antitumor efficacy of caudatin without inducing cumulative hepatotoxicity.

Heat shock factor 1 (HSF1) regulates cellular adaptation to challenges such as heat shock and oxidative and proteotoxic stresses. We have recently reported a previously unappreciated role for HSF1 in the regulation of energy metabolism in fat tissues; however, whether HSF1 is differentially expressed in adipose depots and how its levels are regulated in fat tissues remain unclear. Here, we show that HSF1 levels are higher in brown and subcutaneous fat tissues than in those in the visceral depot and that HSF1 is more abundant in differentiated, thermogenic adipocytes. Gene expression experiments indicated that HSF1 is transcriptionally regulated in fat by agents that modulate cAMP levels, by cold exposure, and by pharmacological stimulation of β-adrenergic signaling. An in silico promoter analysis helped identify a putative response element for activating transcription factor 3 (ATF3) at −258 to −250 base pairs from the HSF1 transcriptional start site, and electrophoretic mobility shift and ChIP assays confirmed ATF3 binding to this sequence. Furthermore, functional assays disclosed that ATF3 is necessary and sufficient for HSF1 regulation. Detailed gene expression analysis revealed that ATF3 is one of the most highly induced ATFs in thermogenic tissues of mice exposed to cold temperatures or treated with the β-adrenergic receptor agonist CL316,243 and that its expression is induced by modulators of cAMP levels in isolated adipocytes. To the best of our knowledge, our results show for the first time that HSF1 is transcriptionally controlled by ATF3 in response to classic stimuli that promote heat generation in thermogenic tissues.

Recent evidence suggests that lactic acid bacteria communicate with host cells via secretome components to influence immune responses but less is known about gut-pathogen secretomes, impact of lactic acid bacteria secretomes on host–pathogen interactions, and the mechanisms underlying these interactions. Genome-wide microarrays and cytokine profiling were used to interrogate the impact of the Lactobacillus rhamnosus R0011 secretome (LrS) on TNF-α and Salmonella enterica subsp. enterica serovar Typhimurium secretome (STS)–induced outcomes in human intestinal epithelial cells. The LrS attenuated both TNF-α– and STS-induced gene expression involved in NF-κB and MAPK activation, as well as expression of genes involved in other immune-related signaling pathways. Specifically, the LrS induced the expression of dual specificity phosphatase 1 (DUSP1), activating transcription factor 3 (ATF3), and tribbles pseudokinase 3 (TRIB3), negative regulators of innate immune signaling, in HT-29 intestinal epithelial cells challenged with TNF-α or STS. TNF-α– and STS-induced acetylation of H3 and H4 histones was attenuated by the LrS, as was the production of TNF-α– and STS-induced proinflammatory cytokines and chemokines. Interestingly, the LrS induced production of macrophage migration inhibitory factor (MIF), a cytokine involved in host–microbe interactions at the gut interface. We propose that the LrS attenuates proinflammatory mediator expression through increased transcription of negative regulators of innate immune activity and changes in global H3 and H4 histone acetylation. To our knowledge, these findings provide novel insights into the complex multifaceted mechanisms of action behind secretome-mediated interdomain communication at the gut-mucosal interface.

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

Endoplasmic reticulum (ER) stress triggers the unfolded protein response (UPR), which has been correlated with enhancedproduction of inflammatory cytokines. Given the important pathogenic roles of macrophages and inflammatory responses in theetiopathogenesis of Behcet’s disease (BD), this study aimed to assess the mRNA expression pattern of genes involved in theUPR pathway in macrophages from smoker and non-smoker BD patients. This case-control study was conducted between 2015and 2016 in Shariati Hospital, Tehran, Iran. Monocytes were enriched from obtained whole blood samples of 10 smokers and 10non-smoker BD patients as well as 10 healthy individuals. Using macrophage-colony stimulating factor (M-CSF), separatedmonocytes were differentiated into macrophages. After total RNA purification and cDNA synthesis, quantification analysis ofUPR genes, including activating transcription factor (ATF) 4, ATF6, X-box binding protein 1 (XBP1), binding immunoglobulinprotein (BIP), C/EBP homologous protein (CHOP), homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERP), and growth arrest and DNA damage-inducible protein (GADD34), was performed using SYBRgreen master mix and real-time PCR. Among the measured genes, HERP mRNA was overexpressed in macrophages from BDpatients in comparison with healthy macrophages. HERP and GADD34 genes were upregulated in smoker BD patients comparedwith non-smoker BD patients as well as healthy subjects. Cigarette smoke can induce UPR gene expression in BD patients. Thealtered UPR gene expression in BD macrophages may contribute to BD pathogenesis.

The homeostasis of immune cells during immune response is vital for hosts to defend against invaders. Activating transcription factor 6 (ATF6) is an important transcription factor in the unfolded protein response (UPR) to maintaining cellular homeostasis. In the present study, one ATF6 homologue was identified from Pacific oyster Crassostrea gigas (designated as CgATF6β). The full length cDNA of CgATF6β was of 2645 bp with a 1596 bp open reading frame (ORF) encoding a polypeptide of 531 amino acids. The deduced amino acid sequence of CgATF6β was predicted to contain a transmembrane region, a conserved basic leucine zipper (bZIP) domain, a site 1 protease cleavage site, a site 2 protease cleavage site, and a Golgi localization signal. CgATF6β mRNA was constitutively expressed in hemocytes, gill, mantle, gonad, hepatopancreas and labial palp, with a slightly higher expression level in muscle (2.45-fold of that in gill, p 0.05). After oysters were challenged with Vibrio splendidus, the mRNA expression levels of CgATF6β in hemocytes were significantly up-regulated at 3 h (2.68-fold of that in seawater group, p 0.01) and peaked at 12 h (3.14-fold of that in seawater group, p 0.01). The endogenic CgATF6β protein was mainly located in the cytoplasm of oyster hemocytes, and it was significantly transported into the nuclei of hemocytes at 1.5 h after the challenge with V. splendidus. After an injection with CgATF6β dsRNA, the mRNA expression of CgATF6β was knocked down to 0.26-fold of that in dsGFP group (p 0.01). In CgATF6β dsRNA-injected oysters, the mRNA expressions of glucose-regulated protein 78 (GRP78), calnexin (CNX) and anti-apoptotic B-cell lymphoma-2 (Bcl-2) in hemocytes were significantly decreased at 12 h after V. splendidus challenge, which were 0.65-fold (p 0.01), 0.54-fold (p 0.01) and 0.17-fold (p 0.01) of that in dsGFP-injected oysters, while the apoptotic rate of hemocytes was significantly up-regulated (1.97-fold of that in dsGFP group, p 0.05). Collectively, these results suggested that CgATF6β was involved in apoptosis inhibition of oyster hemocytes upon V. splendidus challenge by regulating the expression of CgGRP78, CgCNX and CgBcl-2.

Introduction Breast cancer is a common malignancy in females worldwide. In this study, we investigated the role of activating transcription factor 3 (ATF3) and ADP-ribosylation factor like-4 (ARL4) in human breast cancer, and the associated mechanisms. Materials and methods We measured ATF3 and ATL4C expressions in 15 paired breast cancer tissues using qRT-PCR, Western blotting and IHC. Cell growth, migration and invasion were tested in ATF3 or ARL4C overexpression breast cancer cells. TCGA database analysis was done to identify the correlation between ATF3 and ARL4C. We evaluated the binding of ATF3 to ARL4C promoter sequences and the effect of hypermethylation and demethylation of ATF3. A meta-analysis was done to investigate the relationship between the expression of ATF3 and/or ARL4C and the poor prognoses. Results Our results showed that ATF3 and ARL4C were decreased in breast cancer specimens at both mRNA and protein levels. Restoration of ATF3 or ARL4C reduced breast cancer tumorigenesis, evidenced by decreased cell growth, migration and invasion. The expression of ATF3 was positively correlated with ARL4C in breast cancer specimens, and ATF3 was shown to bind to the ARL4C promoter sequences. Furthermore, the expression of ATF3 was negatively regulated by hypermethylation, and demethylation of ATF3 stimulated ATF3 expression, which further promoted ARL4C transcription. Finally, a meta-analysis showed that patients with breast cancer with lower expression levels of ATF3 and/or ARL4C had worse prognoses. Conclusion Our results suggest that the ATF3/ARL4C axis may be a prospective biomarker for diagnosis and determination of prognosis, and a potential target for breast cancer treatment.

Epstein‐Barr virus (EBV) is associated with particular forms of gastric cancer (GC). We previously showed that EBV infection into gastric epithelial cells induced aberrant DNA hypermethylation in promoter regions and silencing of tumor suppressor genes. We here undertook integrated analyses of transcriptome and epigenome alteration during EBV infection in gastric cells, to investigate activation of enhancer regions and related transcription factors (TFs) that could contribute to tumorigenesis. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) sequencing (‐seq) data revealed 19 992 open chromatin regions in putative H3K4me1+ H3K4me3− enhancers in EBV‐infected MKN7 cells (MKN7_EB), with 10 260 regions showing increase of H3K27ac. Motif analysis showed candidate TFs, eg activating transcription factor 3 (ATF3), to possibly bind to these activated enhancers. ATF3 was considerably upregulated in MKN7_EB due to EBV factors including EBV‐determined nuclear antigen 1 (EBNA1), EBV‐encoded RNA 1, and latent membrane protein 2A. Expression of mutant EBNA1 decreased copy number of the EBV genome, resulting in relative downregulation of ATF3 expression. Epstein‐Barr virus was also infected into normal gastric epithelial cells, GES1, confirming upregulation of ATF3. Chromatin immunoprecipitation‐seq analysis on ATF3 binding sites and RNA‐seq analysis on ATF3 knocked‐down MKN7_EB revealed 96 genes targeted by ATF3‐activating enhancers, which are related with cancer hallmarks, eg evading growth suppressors. These 96 ATF3 target genes were significantly upregulated in MKN7_EB compared with MKN7 and significantly downregulated when ATF3 was knocked down in EBV‐positive GC cells SNU719 and NCC24. Knockdown of ATF3 in EBV‐infected MKN7, SNU719, and NCC24 cells all led to significant decrease of cellular growth through an increase of apoptotic cells. These indicate that enhancer activation though ATF3 might contribute to tumorigenesis of EBV‐positive GC., Through integrated analyses on alterations of transcriptome, histone modification, and open chromatin status during Epstein‐Barr virus (EBV) infection in gastric cells, we here identified activating transcription factor 3 (ATF3) as a critical transcriptional activator with involvement in enhancer activation. We undertook screening of putative transcription factors binding to activated enhancer regions and identified ATF3 as a transcription factor that is upregulated by EBV infection and induces aberrant enhancer activation. Effect of ATF3 expression on cellular proliferation was confirmed, suggesting a tumorigenic role of aberrant enhancer activation by ATF3 upregulation, which could help our understanding of the tumorigenic mechanisms in this particular subtype of gastric cancer.

Chemoresistance is one of the major reasons leading to ovarian cancer high mortality and poor survival. Studies have shown that the alteration of cellular autophagy is associated with cancer cell chemoresistance. Here, we investigated whether the ovarian cancer chemoresistance is associated with the autophagy induced by the inhibitor of DNA binding 1 (ID1). By using gene overexpression or silencing, luciferase assay and human specimens, we show that ID1 induces high autophagy and confers cancer cell chemoresistance. The mechanistic study demonstrates that ID1 first activates the NF-κB signaling through facilitating the nuclear translocation of NF-κB p65, which strengthens the expression and secretion of IL-6 from cancer cells to subsequently activate the signal transducer and activator of transcription 3 (STAT3) through the protein phosphorylation at Y705. We further identified that STAT3 functions to promote the transcription of the activating transcription factor 6 (ATF6), which induces endoplasmic reticulum stress to promote cellular autophagy, granting cancer cell resistance to both cisplatin and paclitaxel treatment. Moreover, we found a significant correlation between the expression of ID1 and ATF6 in 1104 high grade serous ovarian cancer tissues, and that patients with the high expression of ID1 or ATF6 were resistant to platinum treatment and had the poor overall survival and progression-free survival. Thus, we have uncovered a mechanism in which ID1 confers cancer cell chemoresistance largely through the STAT3/ATF6-induced autophagy. The involved molecules, including ID1, STAT3, and ATF6, may have a potential to be targeted in combination with chemotherapeutic agents to improve ovarian cancer survival.

Background and aims Although knowledge regarding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) has profoundly grown in recent decades, the internal restrictive mechanisms remain largely unknown. We have recently reported that the transcription repressor interferon regulatory factor-2 binding protein 2 (IRF2BP2) is enriched in cardiomyocytes and inhibits pathological cardiac hypertrophy in mice. Notably, IRF2BP2 is abundantly expressed in hepatocytes and dramatically down-regulated in steatotic livers, whereas the role of IRF2BP2 in NAFLD is unknown. Approach and results Herein, using gain-of-function and loss-of-function approaches in mice, we demonstrated that while hepatocyte-specific Irf2bp2 knockout exacerbated high-fat diet-induced hepatic steatosis, insulin resistance and inflammation, hepatic Irf2bp2 overexpression protected mice from these metabolic disorders. Moreover, the inhibitory role of IRF2BP2 on hepatosteatosis is conserved in a human hepatic cell line in vitro. Combinational analysis of digital gene expression and chromatin immunoprecipitation sequencing identified activating transcription factor 3 (ATF3) to be negatively regulated by IRF2BP2 in NAFLD. Chromatin immunoprecipitation and luciferase assay substantiated the fact that IRF2BP2 is a bona fide transcription repressor of ATF3 gene expression via binding to its promoter region. Functional studies revealed that ATF3 knockdown significantly relieved IRF2BP2 knockout-exaggerated hepatosteatosis in vitro. Conclusion IRF2BP2 is an integrative restrainer in controlling hepatic steatosis, insulin resistance, and inflammation in NAFLD through transcriptionally repressing ATF3 gene expression.

Amyloid plaques are the most important pathological hallmarks of Alzheimer's disease. The deposition of amyloid plaques will cause ER Stress. Activating Transcription Factor 6(ATF6) is a sensor of ER Stress. However, the role of ATF6 in Alzheimer's disease has not been reported yet.The levels of β-site APP-cleaving enzyme 1 (BACE1) and Aβ1-42 were detected by Western blot, ELISA and Thioflavin S staining. Y maze and Morris water maze tests were used to detect the learning and memory functions. Dual luciferase assay was used to test the promoter activity ofIn our study, we found that the expression of ATF6 was reduced in APPswe/PSNdE9 (APP/PS1) Alzheimer's disease model mice compared with wild type mice. Furthermore, in LN229 cell, we found that ATF6 reduced the expression of full length amyloid precursor protein (APP) in protein level. At the same time, the overexpression of ATF6 strikingly reduced the level of Aβ1-42. Interestingly, ATF6 also downregulated the promoter activity ofOur findings indicated that ATF6 rescued the amyloid pathology by downregulating BACE1. Therefore, we suggest that ATF6 could be a potential hub for targeting treatment of the Alzheimer's disease.