Activating Transcription Factor Research Articles

CTGF regulated by ATF6 inhibits vascular endothelial inflammation and reduces hepatic ischemia–reperfusion injury

Vascular endothelial inflammation is crucial in hepatic ischemia–reperfusion injury (IRI). Our previous research has shown that connective tissue growth factor (CTGF), secreted by endothelial cells, protects against acute liver injury, but its upstream mechanism is unclear. We aimed to clarify the protective role of CTGF in endothelial cell inflammation during IRI and reveal the regulation between endoplasmic reticulum stress-induced activating transcription factor 6 (ATF6) and CTGF. Hypoxia/reoxygenation in endothelial cells, hepatic IRI in mice and clinical specimens were used to examine the relationships between CTGF and inflammatory factors and determine how ATF6 regulates CTGF and reduces damage. We found that activating ATF6 promoted CTGF expression and reduced liver damage in hepatic IRI. In vitro, activated ATF6 upregulated CTGF and downregulated inflammation, while ATF6 inhibition had the opposite effect. Dual-luciferase assays and chromatin immunoprecipitation confirmed that activated ATF6 binds to the CTGF promoter, enhancing its expression. Activated ATF6 increases CTGF and reduces extracellular regulated protein kinase 1/2 (ERK1/2) phosphorylation, decreasing inflammatory factors. Conversely, inhibiting ATF6 decreases CTGF and increases the phosphorylation of ERK1/2, increasing inflammatory factor levels. ERK1/2 inhibition reverses this effect. Clinical samples have shown that CTGF increases after IRI, inversely correlating with inflammatory cytokines. Therefore, ATF6 activation during liver IRI enhances CTGF expression and reduces endothelial inflammation via ERK1/2 inhibition, providing a novel target for diagnosing and treating liver IRI.

Polydatin ameliorates early brain injury after subarachnoid hemorrhage through up-regulating SIRT1 to suppress endoplasmic reticulum stress.

This study aims to investigate the inhibitory effect of Polydatin (PD) on endoplasmic reticulum (ER) stress following subarachnoid hemorrhage (SAH) and to elucidate the underlying mechanisms. A standard intravascular puncture model was established to mimic SAH in mice. Neurological functions were assessed using neurological scoring, Grip test, and Morris water maze. Brain edema and Evans blue extravasation were measured to evaluate blood-brain barrier permeability. Western blot and quantitative real-time polymerase chain reaction (PCR) analyses were performed to examine protein and mRNA expressions related to ER stress. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to detect cell apoptosis, and transmission electron microscopy was used to observe the ultrastructure of the endoplasmic reticulum. The results indicated that PD significantly reduced brain edema and Evans blue extravasation after SAH, improving neurological function. Compared to the SAH group, the expression levels of ER stress-related proteins including glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), were significantly lower in the PD-treated group. Moreover, PD significantly enhances the protein expression of Sirtuin 1 (SIRT1). Validation with sh-SIRT1 confirmed the critical role of SIRT1 in ER stress, with PD's inhibitory effect on ER stress being dependent on SIRT1 expression. Additionally, PD attenuated ER stress-mediated neuronal apoptosis and SAH-induced ferroptosis through upregulation of SIRT1. PD alleviates ER stress following SAH by upregulating SIRT1 expression, thereby mitigating early brain injury. The protective effects of PD are mediated through SIRT1, which inhibits ER stress and reduces neuronal apoptosis and ferroptosis.

MLLT6/ATF2 Axis Restrains Breast Cancer Progression by Driving DDIT3/4 Expression.

Epigenetic deregulation is strongly associated with tumor progression. The identification of natural tumor suppressors to overcome cancer metastasis is urgent for cancer therapy. We investigate whether myeloid/lymphoid or mixed-lineage leukemia translocated (MLLT) family members contribute to breast cancer progression and found that high MLLT6 expression predicted a better prognosis and that gradually decreased MLLT6 expression was accompanied by breast cancer malignancy. MLLT6 was downregulated by hypoxia-induced enrichment of DNMT1 at the MLLT6 promoter. The results of in vitro functional experiments indicated that MLLT6 depletion promoted colony formation and cell migration, probably by hampering apoptosis. RNA profiling revealed that the apoptotic pathway was downregulated following stable knockdown of MLLT6. DNA damage-inducible transcript 3/4 (DDIT3/4) were among the top 10 downregulated genes and may have expression patterns similar to that of MLLT6. Restoring DDIT3/4 expression in cells with MLLT6 depletion blocked colony formation and cell migration and attenuated the successful colonization of breast cancer cells in vivo. We also determined that the transcription factor activating transcription factor 2 is a binding partner of MLLT6 and participates in the MLLT6/ATF2 axis, which was reinforced by inhibition of AKT signaling, in turn inducing DDIT3/4 expression by establishing an active chromatin structure at the DDIT3/4 gene promoters. As MLLT6 promotes breast cancer cell apoptosis by inducing DDIT3/4 expression during metastasis, it could be a novel tumor suppressor. Implications: Control of MLLT6 expression via inhibition of PI3K/AKT kinase activity is a potential therapeutic approach for the management of metastatic breast cancer.

Visual analysis on ferroptosis and its cross-talk to coronavirus disease 2019 (COVID-19)

BackgroundFerroptosis is a new type of programmed cell death. Although ferroptosis has been studied in various aspects, there has been no visual analysis of ferroptosis in coronavirus disease 2019 (COVID-19) to date. It is still a global health concern of the COVID-19 pandemic worldwide, three years after its outbreak. Yet the emergence of the mutant strain Omicron has caused a fourth wave of infections in many countries. The pathogenesis of COVID-19 is still undergoing extensive exploration, which holds paramount importance in mitigating future epidemics. MethodsFor this study, CiteSpace 6.2 R4 software was used for bibliometric and visual atlas analysis of ferroptosis-related research, and the Genecards database was used to mine ferroptosis and COVID-19-related genes. ResultsWe found increasing studies about ferroptosis. China and the United States have demonstrated robust scientific innovation over recent years, with extensive collaboration between their institutions and authors. Ferroptosis and COVID-19 were seen to have 13 shared genes, which may be new targets for the treatment of COVID-19 in the future. Most of the shared genes are enriched in tumor necrosis factor (TNF) pathways. The majority of those genes are up-regulated under the cellular response to oxidative stress. Genes including Tumour necrosis factor (TNF), RELA proto-oncogene (RELA), Activating transcription factor 4 (ATF4), Cytochrome b-245 beta chain (CYBB), Jun proto-oncogene (JUN), Mitogen-activated protein kinase 1 (MAPK1) and Heme oxygenase 1 (HMOX1), maybe a breakthrough for ferroptosis and COVID‐19. Whilst previous research has shown there to be a relationship between ferroptosis and COVID‐19, the specific role of ferroptosis remained unclear. Our study aimed to analyze the research status of ferroptosis and its relationship with COVID-19, to provide a useful reference for further prevention and treatment of COVID-19. Overall, uncovering the role of ferroptosis in SARS-CoV-2 infection is important for the development of new treatment strategies for COVID-19.

Novel therapeutic approach for psoriasis: Upregulating FcRn to inhibit ferroptosis and alleviate lesional skin

Psoriasis, a chronic inflammatory skin disease, is characterized by complex immune dysregulation and oxidative stress responses. The neonatal Fc receptor (FcRn) plays a crucial role in the development of autoimmune diseases. Analysis of clinical psoriasis samples demonstrated a negative correlation between FcRn expression in skin lesions and disease severity. However, the role of FcRn in this process remains unclear. This study aimed to investigate the involvement of FcRn in the pathogenesis and progression of psoriasis. In an imiquimod (IMQ)-induced psoriasis-like mouse model, FcRn expression was significantly decreased in the lesional skin, and transcriptome sequencing of the skin revealed activation of the ferroptosis pathway in psoriasis. This led to the hypothesis that FcRn could potentially regulate ferroptosis via the signal transducer and activating transcription factor 3 (STAT3)/solute carrier family 7 member 11 (SLC7A11) axis. Further experiments showed exacerbated psoriasis-like lesional skin and ferroptosis in FcRn-knockout mice, whereas intervention with the ferroptosis inhibitor Fer-1 or STAT3 inhibitor Stattic alleviated these symptoms. Critical binding sites for the transcription factor STAT3 were identified in the SLC7A11 promoter region at positions −1185 and −564 using the luciferase reporter assays and chromatin immunoprecipitation. The administration of 1,4-naphthoquinone (NQ), an FcRn agonist, effectively alleviated psoriasis-like skin lesions by inhibiting ferroptosis. This study highlights the molecular mechanisms of action of FcRn in psoriasis and provides an experimental basis for the development of novel therapeutic strategies targeting FcRn.

Anabolic deficits and divergent unfolded protein response underlie skeletal and cardiac muscle growth impairments in the Yoshida hepatoma tumor model of cancer cachexia.

Cancer cachexia manifests as whole body wasting, however, the precise mechanisms governing the alterations in skeletal muscle and cardiac anabolism have yet to be fully elucidated. In this study, we explored changes in anabolic processes in both skeletal and cardiac muscles in the Yoshida AH-130 ascites hepatoma model of cancer cachexia. AH-130 tumor-bearing rats experienced significant losses in body weight, skeletal muscle, and heart mass. Skeletal and cardiac muscle loss was associated with decreased ribosomal (r)RNA, and hypophosphorylation of the eukaryotic factor 4E binding protein 1. Endoplasmic reticulum stress was evident by higher activating transcription factor mRNA in skeletal muscle and growth arrest and DNA damage-inducible protein (GADD)34 mRNA in both skeletal and cardiac muscles. Tumors provoked an increase in tissue expression of interferon-γ in the heart, while an increase in interleukin-1β mRNA was apparent in both skeletal and cardiac muscles. We conclude that compromised skeletal muscle and heart mass in the Yoshida AH-130 ascites hepatoma model involves a marked reduction translational capacity and efficiency. Furthermore, our observations suggest that endoplasmic reticulum stress and tissue production of pro-inflammatory factors may play a role in the development of skeletal and cardiac muscle wasting.

Astaxanthin inhibits apoptosis in a cell model of tauopathy by attenuating endoplasmic reticulum stress and unfolded protein response

The accumulation of misfolded proteins is a common pathological characteristic shared by many neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. The disruption of proteostasis triggers endoplasmic reticulum (ER) stress, during which the unfolded protein response (UPR) is initiated by the activation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). These three branches of UPR signals act in concert to reduce the levels of abnormal proteins and restore ER homeostasis. However, the overactivation of UPR impairs cell function and induces apoptosis, which has been implicated in neurodegeneration. Astaxanthin is a xanthophyll carotenoid which has been shown to have neuroprotective effects in both cell and animal models; however, its effects on ER stress and UPR induced by disrupted proteostasis remain unclear. In this study, the effects of astaxanthin on ER stress and cytotoxicity were investigated in N2a cells stably expressing the pro-aggregant tau repeat domain carrying FTDP-17 mutation ΔK280 (Tau4RDΔK280). The results demonstrated that astaxanthin significantly inhibited Tau4RDΔK280-induced loss of cell viability and apoptosis, attenuating Tau4RDΔK280-induced caspase-3 activation and decrease of Bcl-2. Further studies revealed that astaxanthin treatment alleviated Tau4RDΔK280-induced ER stress and suppressed the activation of PERK, IRE1 and ATF6 signaling pathways. These findings suggested that astaxanthin might inhibit Tau4RDΔK280-induced cytotoxicity by attenuating UPR and ER stress. In addition, astaxanthin treatment resulted in a great reduction in the production of intracellular reactive oxygen species and a significant decrease in calcium influx induced by Tau4RDΔK280, which also contributed to the protective effects of astaxanthin against Tau4RDΔK280-induced cytotoxicity.

Palmitic acid-induced cell death: impact of endoplasmic reticulum and oxidative stress, mitigated by L-citrulline.

Palmitic acid (PA), the most abundant saturated free fatty acids, induces apoptosis in bovine mammary epithelial cells. It is suggested that oxidative stress and endoplasmic reticulum (ER) stress are key mechanisms underlying PA-induced cell death. This study aimed to investigate the interaction between ER stress and oxidative stress during PA-induced cell death in MAC-T cells. Additionally, we examined whether L-citrulline can protect against PA-induced damage of MAC-T cells. MAC-T cells were treated with 4-phenyl butyric acid (4-PBA) or N-acetyl-L-cysteine (NAC) to inhibit PA-induced ER stress and oxidative stress, respectively. MAC-T cells were pretreated with or without L-citrulline for 48 h followed by PA treatment. Cell viability was measured with MTT assays. Intracellular reactive oxygen species (ROS) levels in MAC-T cells were assessed using 5-(and-6)-chloromethyl- 2`,7`-dichlorodihydrofluorescein diacetate acetyl ester dye. Real-time qPCR was used to explore the regulation of genes associated with oxidative stress, and ER stress genes. Western blotting analysis was also carried out. 4-PBA significantly reduced PA-induced mRNA expressions of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and intracellular ROS levels. Furthermore, NAC dramatically reduced PA-induced ROS levels and the mRNA expressions of NRF2, ATF4, and CHOP. L-citrulline pretreatment effectively rescued cell viability decreased by PA. Moreover, L-citrulline pretreatment significantly downregulated the PA-induced upregulation of GRP78, ATF4, and CHOP mRNA expression, and protein expression of p-PERK and cleaved caspase-3. PA increased intracellular ROS levels and NRF2 mRNA expression, whereas L-citrulline pretreatment remarkably reduced these levels. Both ER and oxidative stresses interact during PA-induced cell death in MAC-T cells, and L-citrulline could attenuate this cell death by inhibiting ER and oxidative stresses. Therefore, L-citrulline may be a promising supplement for protecting against PA-induced cell death in bovine MECs during the lactation period of dairy cows.

Spermidine mitigates ferroptosis in free fatty acid-induced AML-12 cells through the ATF4/SLC7A11/GCLM/GPX4 pathway

Non-alcoholic fatty liver disease (NAFLD) is a prominent cause of chronic liver disease worldwide. Spermidine (SPD), a naturally occurring polyamine, has shown potential in alleviating the accumulation of hepatic lipids and reducing NAFLD symptoms in overweight mice. Nonetheless, the specific mechanisms through which SPD exerts its effects remain largely unknown. This study seeks to explore the protective effects of SPD on NAFLD and to clarify the underlying mechanisms. An in vitro model of NAFLD was established by inducing steatosis in AML-12 cells through the use of free fatty acids (FFAs). Our experimental results demonstrate that SPD significantly reduces NAFLD development induced by FFAs. This reduction is primarily achieved through the inhibition of cellular ferroptosis, as evidenced by decreased levels of Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS). Additionally, SPD was found to enhance cellular activity and ameliorate mitochondrial dysfunction and oxidative stress caused by FFA exposure.Further mechanistic studies have revealed that SPD upregulates the expression of solute transporter family 7a member 11 (SLC7A11), glutamate-cysteine ligase modifier subunit (GCLM), and glutathione peroxidase (GPX4). This upregulation is mediated by the activation of activating transcription factor 4 (ATF4). Knockdown experiments of ATF4 confirmed that its inhibition reverses the upregulation of SLC7A11, GCLM, and GPX4, thereby negating the protective effects of SPD. In conclusion, our findings suggest that SPD mitigates NAFLD by modulating the ATF4/SLC7A11/GCLM/GPX4 signaling pathway, resulting in the suppression of ferroptosis and the improvement of cellular health. These insights provide a novel molecular mechanism and identify potential therapeutic targets for the treatment of NAFLD.

The autophagy-targeting compound V46 enhances antimicrobial responses to Mycobacteroides abscessus by activating transcription factor EB

Mycobacteroides abscessus (Mabc) is a rapidly growing nontuberculous mycobacterium that poses a considerable challenge as a multidrug-resistant pathogen causing chronic human infection. Effective therapeutics that enhance protective immune responses to Mabc are urgently needed. This study introduces trans-3,5,4′-trimethoxystilbene (V46), a novel resveratrol analogue with autophagy-activating properties and antimicrobial activity against Mabc infection, including multidrug-resistant strains. Among the resveratrol analogues tested, V46 significantly inhibited the growth of both rough and smooth Mabc strains, including multidrug-resistant strains, in macrophages and in the lungs of mice infected with Mabc. Additionally, V46 substantially reduced Mabc-induced levels of pro-inflammatory cytokines and chemokines in both macrophages and during in vivo infection. Mechanistic analysis showed that V46 suppressed the activation of the protein kinase B/Akt-mammalian target of rapamycin signaling pathway and enhanced adenosine monophosphate-activated protein kinase signaling in Mabc-infected cells. Notably, V46 activated autophagy and the nuclear translocation of transcription factor EB, which is crucial for antimicrobial host defenses against Mabc. Furthermore, V46 upregulated genes associated with autophagy and lysosomal biogenesis in Mabc-infected bone marrow-derived macrophages. The combination of V46 and rifabutin exerted a synergistic antimicrobial effect. These findings identify V46 as a candidate host-directed therapeutic for Mabc infection that activates autophagy and lysosomal function via transcription factor EB.

Dietary xylo-oligosaccharides alleviates LPS-induced intestinal injury via endoplasmic reticulum-mitochondrial system pathway in piglets.

The beneficial effects of xylo-oligosaccharides (XOS) on the intestine have been widely reported, including anti-inflammation, antioxidant, maintenance of intestinal epithelial barrier, and treatment of intestinal injury. However, the specific mechanism of XOS in mitigating intestinal injury in weaned piglets remains unclear. Therefore, this study aimed to explore the specific mechanism of XOS in mitigating intestinal injury. The study is a complete randomized design with 24 weaned piglets in a 2 × 2 factorial arrangement that includes diet treatments (basal diet vs 0.02% XOS) and immunological challenge [saline vs lipopolysaccharide (LPS)]. All piglets were fed a basal diet or a XOS diet for 21 days. On day 22, all piglets received an injection of LPS or saline. In this study, dietary XOS increased jejunal villus height, reduced crypt depth and oxidative stress, and enhanced the gene and protein expression of Claudin-1, Occludin, and zonula occludens 1 (P < 0.05). The piglets fed the XOS diet had lower serum Diamine oxidase activity and D-lactic acid content (P < 0.05). In addition, dietary XOS regulates endoplasmic reticulum (ER)-mitochondria system function and the expression of key molecules, including mitochondrial dynamics dysfunction [mitofusin (Mfn)-1, optic atrophy 1, fission 1, and dynamin-related protein 1], ER stress [activating transcription factor 4 (ATF4), ATF6, C/EBP homologous protein, eukaryotic initiation factor 2α, glucose-regulated protein (GRP) 78, GRP94 and protein kinase R-like ER kinase] and the mitochondria-associated ER membranes (MAM) disorders (Mfn2, GRP75 and voltage-dlependent anion channel 1) (P < 0.05). Therefore, the findings to indicate that dietary XOS is effective against LPS-induced jejunal injury may be attributed to its ability to alleviate mitochondrial dynamics dysfunction, ER stress, and MAM disorders.

Epigallocatechin gallate induces apoptosis in multiple myeloma cells through endoplasmic reticulum stress induction and cytoskeletal disruption

Multiple myeloma (MM) is an incurable plasma cell malignancy that has prompted investigations into new potential therapeutic avenues. Epigallocatechin-3-gallate (EGCG), a major component of green tea, confers antioxidant, anti-inflammatory, and anti-tumor properties. Previous studies have shown that EGCG inhibits proliferation and induces apoptosis of multiple myeloma cells, however its underlying molecular mechanisms are largely unknown. In this study, we accordingly sought to examine the therapeutic effects and underlying mechanisms of EGCG on MM.Initially, using CCK8 (Cell Counting Kit-8) assays and Annexin V-FITC/PI staining, we demonstrated that EGCG dose-dependently reduced cell viability and induced apoptosis in the MM cell lines MM.1S and RPMI 8226. Subsequently, mRNA sequencing of EGCG-treated MM.1S cells revealed a significant upregulation of genes associated with endoplasmic reticulum stress (ERS), including P-eIF2α (phosphorylation-eukaryotic translation initiation factor 2 alpha), ATF4 (activating transcription factor 4), CHOP (C/EBP homologous protein, DDIT3), and PUMA (p53 upregulated modulator of apoptosis, BBC3), which were confirmed at the protein level by western blotting. Furthermore, treatment with the eIF2α inhibitor ISRIB reduced the rates of EGCG-induced apoptosis and promoted increases in the protein expression of all four ER stress-related molecules in MM cells. Additionally, mRNA-seq data revealed a downregulation of α-Tubulin 1b (TUBA1B) expression in EGCG-treated MM cells, which was confirmed by western blotting and immunofluorescence analyses. Moreover, we utilized a mouse model to show that EGCG inhibited myeloma tumor growth, which was inhibited by ISRIB.In summary, the findings of this novel study indicated that EGCG promotes apoptosis of MM cells, both via activation of the ER stress pathway and disruption of cytoskeletal integrity. These findings highlight the multi-faceted anti-tumor effects of EGCG and its potential clinical application in MM treatment.

The rs2341471-G/G genotype of activating transcription factor 6 (ATF6) is the risk factor of type 2 diabetes in subjects with obesity or overweight.

Numerous studies have demonstrated that the onset of type 2 diabetes (T2D) is linked to the reduction in ß-cell mass caused by apoptosis, a process initiated by endoplasmic reticulum (ER) stress. The aim of this study was to investigate the associations between single nucleotide polymorphisms (SNPs) in the ATF6 gene (activating transcription factor 6), a key sensor of ER stress, and T2D susceptibility. The study involved 3229 unrelated individuals, including 1569 patients with T2D and 1660 healthy controls from Central Russia. Four functionally significant intronic SNPs, namely rs931778, rs90559, rs2341471, and rs7517862, were genotyped using the MassARRAY-4 system. The rs2341471-G/G genotype of ATF6 was found to be associated with an increased risk of T2D (OR = 1.61, 95% CI 1.37-1.90, PFDR < 0.0001). However, a BMI-stratified analysis showed that this genotype and haplotypes CGGA and TAGA are associated with T2D risk exclusively in subjects with obesity or overweight (PFDR < 0.05). Despite these patients being found to have higher consumption of high-carbohydrate and high-calorie diets compared to normal-weight individuals (P < 0.0001), the influence of the rs7517862 polymorphism on T2D risk was observed independently of these dietary habits. Functional SNP annotation revealed the following: (1) the rs2341471-G allele is associated with increased ATF6 expression; (2) the SNP is located in a region exhibiting enhancer activity epigenetically regulated in pancreatic islets; (3) the rs2341471-G was predicted to create binding sites for 18 activating transcription factors that are part of gene-regulatory networks controlling glucose metabolism and maintaining proteostasis. The present study revealed, for the first time, a strong association between the rs2341471-G/G ATF6 genotype and an increased risk of type 2 diabetes in people with obesity or overweight, regardless of known dietary risk factors. Further research is needed to support the potential of silencing the ATF6 gene as a means for the treatment and prevention of type 2 diabetes.

Safflower Yellow Injection Alleviates Myocardial Ischemia/Reperfusion Injury by Reducing Oxidative and Endoplasmic Reticulum Stress.

Safflower yellow is an extract of the famous Chinese medicine Carthamus tinctorious L, and safflower yellow injection (SYI) is widely used clinically to treat angina pectoris. However, there are few studies on the anti-myocardial ischemia/reperfusion (I/R) injury effect of SYI, and its mechanisms are unclear. In the present study, we aimed to investigate the protective effect of SYI on myocardial I/R injury and explore its underlying mechanisms. Male Sprague Dawley rats were randomly divided into a control group, sham group, model group, and SYI group (20 mg/kg, femoral vein injection 1 h before modeling). The left anterior descending coronary artery was ligated to establish a myocardial I/R model. H9c2 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) after incubation with 80 μg/mL SYI for 24 h. In vivo, TsTC, HE, and TUNEL staining were performed to evaluate myocardial injury and apoptosis. A kit was used to detect superoxide dismutase (SOD) and malondialdehyde (MDA) to assess oxidative stress. In vitro, flow cytometry was used to detect the reactive oxygen species (ROS) content and apoptosis rate. Protein levels were determined via Western blotting. Pretreatment with SYI significantly reduced infarct size and pathological damage in rat hearts and suppressed cardiomyocyte apoptosis in vivo and in vitro. In addition, SYI inhibited oxidative stress by increasing SOD activity and decreasing MDA content and ROS production. Myocardial I/R and OGD/R activate endoplasmic reticulum (ER) stress, as evidenced by increased expression of activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), cysteinyl aspartate-specific proteinase caspase-12, and C/EBP-homologous protein (CHOP), which were all inhibited by SYI. SYI ameliorated myocardial I/R injury by attenuating apoptosis, oxidative damage, and ER stress, which revealed new mechanistic insights into its application.

Pharmacologic activation of activating transcription factor 6 contributes to neuronal survival after spinal cord injury in mice.

The impact of primary and secondary injuries of spinal cord injury (SCI) results in the demise of numerous neurons, and there is still no efficacious pharmacological intervention for it. Recently, studies have shown that endoplasmic reticulum stress (ERS) plays a pivotal role in recovery of neurological function after spinal cord injury. As a process to cope with intracellular accumulation of misfolded and unfolded proteins which triggers ERS, the unfolded protein response (UPR) plays an important role in maintaining protein homeostasis. And, a recently disclosed small molecule AA147, which selectively activates activating transcription factor 6 (ATF6), has shown promising pharmacological effects in several disease models. Thus, it seems feasible to protect the neurons after spinal cord injury by modulating UPR. In this study, primary neurons were isolated from E17-19 C57BL/6J mouse embryos and we observed that AA147 effectively promoted the survival of neurons and alleviated neuronal apoptosis after oxygen-glucose deprivation/reoxygenation (OGD/R) invitro. This was evident through a decrease in the proportion of PI-positive and TUNEL-positive cells, an increase in BCL-2 expression, and a decrease in the expression of BAX and C-caspase3. In in-vivo experiments, these findings were corroborated by TUNEL staining and immunohistochemistry. It was also found that AA147 enhanced three arms of the unfolded protein response with reduced CHOP expression. Besides, AA147 mitigated the accumulation of ROS in neurons probably by upregulating catalase expression. Furthermore, spinal cord injury models of C57BL/6J mice were established and behavioral experiments revealed that AA147 facilitated the recovery of motor function following SCI. Thus, pharmacologic activation of ATF6 represents a promise therapeutic approach to ameliorate the prognosis of SCI.

Varicella Zoster Virus disrupts MAIT cell polyfunctional effector responses.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that respond to riboflavin biosynthesis and cytokines through TCR-dependent and -independent pathways, respectively. MAIT cell activation plays an immunoprotective role against several pathogens, however the functional capacity of MAIT cells following direct infection or exposure to infectious agents remains poorly defined. We investigated the impact of Varicella Zoster Virus (VZV) on blood-derived MAIT cells and report virus-mediated impairment of activation, cytokine production, and altered transcription factor expression by VZV infected (antigen+) and VZV exposed (antigen-) MAIT cells in response to TCR-dependent and -independent stimulation. Furthermore, we reveal that suppression of VZV exposed (antigen-) MAIT cells is not mediated by a soluble factor from neighbouring VZV infected (antigen+) MAIT cells. Finally, we demonstrate that VZV impairs the cytolytic potential of MAIT cells in response to riboflavin synthesising bacteria. In summary, we report a virus-mediated immune-evasion strategy that disarms MAIT cell responses.

Abstract Mo004: The Protective Effects of Empagliflozin in Carfilzomib-induced Cardiotoxicity in Mice: Unveiling the Crosstalk between Oxidative Stress, Inflammation, Endoplasmic Reticulum Stress, and Autophagy Axes.

Introduction: Multiple myeloma ranks as the second most prevalent hematologic cancer in the US. Survival rates have significantly improved owing to advances in chemotherapy treatment. Carfilzomib, an irreversible proteasome inhibitor, has been used globally for the treatment of multiple myeloma. Unfortunately, its clinical use has been linked to cardiovascular complications. Empagliflozin is a sodium glucose co-transporter-2 inhibitor with potential cardioprotective activities, for which it is approved for heart failure management. Aims: The current study determined the beneficial effects of Empagliflozin against Carfilzomib-induced cardiotoxicity in C57BL/6 mice and the possible underlying mechanisms. Methods: Thirty-two mice were randomly divided into four groups and treated for two days as follows: Control (vehicle), Empagliflozin (10 mg/kg, oral), Carfilzomib (8 mg/kg, i.p.), and Carfilzomib + Empagliflozin groups. Results: The impact of Carfilzomib and Empagliflozin treatment on markers of cardiotoxicity and heart tissue architecture was assessed. Empagliflozin corrected histological alterations, CK-MB activity, and troponin I level induced by Carfilzomib. Additionally, Empagliflozin mitigated Carfilzomib-induced oxidative deficits via its effect on catalase activity, reduced glutathione level, and superoxide dismutase activity, restoring the balance of redox circuit. Moreover, Empagliflozin averted Carfilzomib-induced inflammatory response as evidenced by reducing nuclear factor-κB (p65), tumor necrosis factor-α, and interleukin-1β levels. Mechanistically, a substantial amelioration in the activating transcription factor 6-mediated endoplasmic reticulum stress was demonstrated with Empagliflozin treatment owing to its effect on glucose regulated protein-78, activating transcription factor-6, and C/EBP homologous protein. Intriguingly, the induction of autophagy by Carfilzomib was notably amplified following Empagliflozin administration as evidenced by higher mRNA expression of LC3B and beclin-1, and lower expression of p62. Accordingly, the apoptotic marker cleaved caspase-3 expression was markedly reduced following Empagliflozin treatment. Importantly, Empagliflozin did not alter the cytotoxic effect of Carfilzomib on human U266B1 myeloma cell line. Conclusion: This research suggests Empagliflozin could offer cardioprotection against Carfilzomib-induced cardiotoxicity in multiple myeloma patients.

Activating transcription factor 3 mediates apoptosis and cell cycle arrest in TP53-mutated anaplastic thyroid cancer cells

BackgroundIt is believed that loss of p53 function plays a crucial role in the progression of well to poorly differentiated thyroid cancers including anaplastic thyroid carcinoma (ATC). Given the poor prognosis of ATC due to its strong therapeutic resistance, there is a need to establish new therapeutic targets to extend the survival of ATC patients. Activating transcription factor 3 (ATF3) can inhibit the oncogenic activity of mutant p53 and, as a result, contribute to tumor suppression in several TP53-mutated cancers. Herein, we demonstrate that the ectopic overexpression of ATF3 leads to the suppression of oncogenic mutant p53 activity in chemo-resistant 8305 C thyroid cancer cells harboring R273C p53 gene mutation.MethodsThe biological behavior of 8305 C cells was assessed pre- and post-transfection with pCMV6-ATF3 plasmid using MTT assay, fluorescent microscopy, cell cycle, and annexin V/PI flow cytometric analysis. The effect of ectopic ATF3 overexpression on the cellular level of p53 was examined by western blotting assay. The mRNA expression levels of TP53, TAp63, ΔNp63, and SHARP1 were evaluated in ectopic ATF3-expressing cells compared to controls.ResultsThe overexpression of ATF3 in 8305 C thyroid cancer cells significantly decreased cell viability and induced apoptosis and cell cycle arrest in vitro. The immunoblotting of p53 protein revealed that ATF3 overexpression significantly increased the level of mutant p53 in 8305C cells compared to mock-transfected control cells. Additionally, elevated mRNA levels of TAp63 and SHARP1 and a decreased mRNA level of ΔNp63 were observed in PCMV6-AC-ATF3-transfected 8305 C cells with significant differences compared to the mock and untreated cells.ConclusionIn light of our findings, it is evident that therapeutic strategies aimed at increasing ATF3 expression or enhancing the interaction between ATF3 and mutant p53 can be a promising approach for the treatment of p53-mutated metastatic thyroid cancer.

The stress-responsive gene ATF3 drives fibroblast activation and collagen production through transcriptionally activating TGF-β receptor Ⅱ in skin wound healing.

Skin wound is an emerging health challenge on account of the high-frequency trauma, surgery and chronic refractory ulcer. Further study on the disease biology will help to develop new effective approaches for wound healing. Here, we identified a wound-stress responsive gene, activating transcription factor 3 (ATF3), and then investigated its biological action and mechanism in wound healing. In the full-thickness skin wound model, ATF3 was found to promote fibroblast activation and collagen production, resulted in accelerated wound healing. Mechanically, ATF3 transcriptionally activated TGF-β receptor Ⅱ via directly binding to its specific promoter motif, followed by the enhanced TGF-β/Smad pathway in fibroblasts. Moreover, the increased ATF3 upon skin injury was partly resulted from hypoxia stimulation with Hif-1α dependent manner. Altogether, this work gives novel insights into the biology and mechanism of stress-responsive gene ATF3 in wound healing, and provides a potential therapeutic target for treatment.

Differential effects of montelukast and zafirlukast on MDA‑MB‑231 triple‑negative breast cancer cells: Cell cycle regulation, apoptosis, autophagy, DNA damage and endoplasmic reticulum stress.

Montelukast and zafirlukast, cysteinyl leukotriene receptor antagonists (LTRAs), trigger apoptosis and inhibit cell proliferation of triple‑negative breast cancer MDA‑MB‑231 cells. By contrast, only zafirlukast induces G0/G1 cell cycle arrest. The present study compared the effects of these drugs on proteins regulating cell proliferation, apoptosis, autophagy, and endoplasmic reticulum (ER) and oxidative stress using reverse transcription‑quantitative PCR, western blotting and flow cytometry. The expression of proliferating markers, Ki‑67 and proliferating cell nuclear antigen, was decreased by both drugs. Zafirlukast, but not montelukast, decreased the expression of cyclin D1 and CDK4, disrupting progression from G1 to S phase. Zafirlukast also increased the expression of p27, a cell cycle inhibitor. Both drugs decreased the expression of anti‑apoptotic protein Bcl‑2 and ERK1/2 phosphorylation, and increased levels of the autophagy marker LC3‑II and DNA damage markers, including cleaved PARP‑1, phosphorylated (p)‑ATM and p‑histone H2AX. The number of caspase 3/7‑positive cells was greater in montelukast‑treated cells compared with zafirlukast‑treated cells. Montelukast induced higher levels of the ER stress marker CHOP compared with zafirlukast. Montelukast activated PERK, activating transcription factor 6 (ATF6) and inositol‑requiring enzyme type 1 (IRE1) pathways, while zafirlukast only stimulated ATF6 and IRE1 pathways. GSK2606414, a PERK inhibitor, decreased apoptosis mediated by montelukast, but did not affect zafirlukast‑induced cell death. The knockdown of CHOP by small interfering RNA reduced apoptosis triggered by montelukast and zafirlukast. In conclusion, the effects on cell cycle regulator proteins may contribute to cell cycle arrest caused by zafirlukast. The greater apoptotic effects of montelukast may be caused by the higher levels of activated caspase enzymes and the activation of three pathways of ER stress: PERK, ATF6, and IRE1.