Activating transcription factor 6 alleviates secondary brain injury by increasing cystathionine γ-lyase expression in a rat model of intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) comprises primary and secondary injuries, the latter of which induces increased inflammation and apoptosis and is more severe. 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 cryoprotection. Hence, we predict that ATF6 will have a protective effect on brain tissue after ICH. METHOD: The ICH rat model was generated through autologous blood injection into the right basal ganglia, the expression of ATF6 after ICH was determined by WB and IF. The expression of ATF6 was effectively controlled by means of intervention, and a series of measures was used to detect cell death, neuroinflammation, brain edema, blood-brain barrier and other indicators after ICH. Finally, the effects on long-term neural function of rats were measured by behavioral means. RESULT: ATF6 was significantly increased in the ICH-induced brain tissues. Further, ATF6 was found to modulate the expression of cystathionine γ-lyase (CTH) after ICH. Upregulation of ATF6 attenuated neuronal apoptosis and inflammation in ICH rats, along with mitigation of ICH-induced brain edema, blood-brain barrier deterioration, and cognitive behavior defects. Conversely, ATF6 genetic knockdown induced effects counter to those aforementioned. CONCLUSIONS: This study thereby emphasizes the crucial role of ATF6 in secondary brain injury in response to ICH, indicating that ATF6 upregulation may potentially ameliorate ICH-induced secondary brain injury. Consequently, ATF6 could serve as a promising therapeutic target to alleviate clinical ICH-induced secondary brain injuries.

Effect of Calcium Supplementation and TMEM16A Inhibition on Endoplasmic Reticulum Stress Induced by Dental Fluorosis in Mice.

BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.

Identification of activating transcription factor 6 (ATF6) as a novel prognostic biomarker and potential target in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is originating from oral mucosal epithelial cells. Autophagy plays a crucial role in cancer treatment by promoting cellular self-degradation and eliminating damaged components, thereby enhancing therapeutic efficacy. In this study, we aim to identify a novel autophagy-related biomarker to improve OSCC therapy. METHODS: We firstly utilized Cox and Lasso analyses to identify that ATF6 is associated with OSCC prognosis, and validated the results by Kaplan-Meier survival analysis. We further identified the downstream pathways and related genes by enrichment analysis and WGCNA analysis. Subsequently, we used short interfering RNA to investigate the effects of ATF6 knockdown on proliferation, migration, apoptosis, and autophagy in SCC-9 and SCC-15 cells through cell viability assay, transwell assay, EdU incorporation assay, flow cytometry analysis, western blot analysis and immunofluorescence analysis, etc. RESULTS: Bioinformatics analyses showed that ATF6 overexpression was associated with prognosis and detrimental to survival. In vitro studies verified that ATF6 knockdown reduced OSCC cell proliferation and migration. Mechanistically, ATF6 knockdown could promote cellular autophagy and apoptosis. CONCLUSION: We propose that ATF6 holds potential as a prognostic biomarker linked to autophagy in OSCC. This study provides valuable clues for further exploration of targeted therapy against OSCC.

Inhibition of HDAC6 with CAY10603 alleviates acute and chronic kidney injury by suppressing the ATF6 branch of UPR.

BACKGROUND: Histone deacetylase 6 (HDAC6) inhibitor CAY10603 has been identified as a potential therapeutic agent for the treatment of diabetic kidney disease (DKD). The objective of this study was to investigate the therapeutic effects of CAY10603 in mice with acute kidney injury (AKI) and chronic kidney diseases (CKD). METHODS: Renal immunohistology was performed to assess the expression levels of HDAC6 in both human and mouse kidney samples. C57BL/6J mice were intraperitoneal injected with lipopolysaccharide (LPS) to induce AKI; CD-1 mice were fed with adenine diet to induce adenine-nephropathy as CKD model. Serum creatinine, blood urea nitrogen and uric acid were measured to reflect renal function; renal histology was applied to assess kidney damage. Western blot and immunohistology were used to analyze the unfolded protein response (UPR) level. RESULTS: HDAC6 was significantly upregulated in renal tubular epithelial cells (RTECs) of both AKI and CKD patients as well as mice. In the murine models of AKI induced by LPS and adenine-induced nephropathy, CAY10603 exhibited notable protective effects, including improvement in biochemical indices and pathological changes. In vivo and in vitro studies revealed that CAY10603 effectively suppressed the activation of activating transcription factor 6 (ATF6) branch of UPR triggered by thapsigargin (Tg), a commonly employed endoplasmic reticulum (ER) stressor. Consistent with these findings, CAY10603 also displayed substantial inhibition of ATF6 activation in RTECs from both murine models of LPS-induced AKI and adenine-induced nephropathy. CONCLUSIONS: Collectively, these results suggest that CAY10603 holds promise as a potential therapeutic agent for both acute and chronic kidney injury.

Downregulating miRNA-199a-5p exacerbates fluorouracil-induced cardiotoxicity by activating the ATF6 signaling pathway.

BACKGROUND: Fluorouracil (5-FU) might produce serious cardiac toxic reactions. miRNA-199a-5p is a miRNA primarily expressed in myocardial cells and has a protective effect on vascular endothelium. Under hypoxia stress, the expression level of miRNA-199a-5p was significantly downregulated and is closely related to cardiovascular events such as coronary heart disease, heart failure, and hypertension. We explored whether 5-FU activates the endoplasmic reticulum stress ATF6 pathway by regulating the expression of miRNA-199a-5p in cardiac toxicity. METHODS: This project established a model of primary cardiomyocytes derived from neonatal rats and treated them with 5-FU in vitro. The expression of miRNA-199a-5p and its regulation were explored in vitro and in vivo. RESULTS: 5-FU decreases the expression of miRNA-199a-5p in cardiomyocytes, activates the endoplasmic reticulum stress ATF6 pathway, and increases the expression of GRP78 and ATF6, affecting the function of cardiomyocytes, and induces cardiac toxicity. The rescue assay further confirmed that miRNA-199a-5p supplementation can reduce the cardiotoxicity caused by 5-FU, and its protective effect on cardiomyocytes depends on the downregulation of the endoplasmic reticulum ATF6 signaling pathway. CONCLUSIONS: 5-FU can down-regulate expression of miRNA-199a-5p, then activate the endoplasmic reticulum stress ATF6 pathway, increase the expression of GRP78 and ATF6, affect the function of cardiomyocytes, and induce cardiac toxicity.

Ntsr1 contributes to pulmonary hypertension by enhancing endoplasmic reticulum stress via JAK2-STAT3-Thbs1 signaling.

Pulmonary hypertension (PH) is a severe clinical syndrome with pulmonary vascular remodeling and poor long-term prognosis. Neurotensin receptor 1 (Ntsr1), serve as one of the G protein-coupled receptors (GPCRs), implicates in various biological processes, but the potential effects of Ntsr1 in PH development are unclear. The Sugen/Hypoxia (SuHx) or monocrotaline (MCT) induced rat PH model was used in our study and the PH rats showed aggravated pulmonary artery remodeling and increased right ventricular systolic pressure (RVSP). Our results revealed that Ntsr1 induced endoplasmic reticulum (ER) stress response via ATF6 activation contributed to the development of PH. Moreover, RNA-sequencing (RNA-seq) and phosphoproteomics were performed and the Ntsr1-JAK2-STAT3-thrombospondin 1 (Thbs1)-ATF6 signaling was distinguished as the key pathway. In vitro, pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition showed enhanced proliferation and migration properties, which could be inhibited by Ntsr1 knockdown, JAK2 inhibitor (Fedratinib) treatment, STAT3 inhibitior (Stattic) treatment, Thbs1 knockdown or ATF6 knockdown. In addition, adeno-associated virus 1 (AAV1) were used to knockdown the expression of Ntsr1, Thbs1 or ATF6 in rats and reversed the phenotype of PH. In summary, our results reveal that Ntsr1-JAK2-STAT3-Thbs1 pathway can induce enhanced ER stress via ATF6 activation and increased PASMC proliferation and migration capacities, which can be mechanism of the pulmonary artery remodeling and PH. Targeting Ntsr1 might be a novel therapeutic strategy to ameliorate PH.

Mechanism of Qili Qiangxin Capsule for Heart Failure Based on miR133a-Endoplasmic Reticulum Stress.

OBJECTIVE: To investigate the pharmacological mechanism of Qili Qiangxin Capsule (QLQX) improvement of heart failure (HF) based on miR133a-endoplasmic reticulum stress (ERS) pathway. METHODS: A left coronary artery ligation-induced HF after myocardial infarction model was used in this study. Rats were randomly assigned to the sham group, the model group, the QLQX group [0.32 g/(kg·d)], and the captopril group [2.25 mg/(kg·d)], 15 rats per group, followed by 4 weeks of medication. Cardiac function such as left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), the maximal rate of increase of left ventricular pressure (+dp/dt max), and the maximal rate of decrease of left ventricular pressure (-dp/dt max) were monitored by echocardiography and hemodynamics. Hematoxylin and eosin (HE) and Masson stainings were used to visualize pathological changes in myocardial tissue. The mRNA expression of miR133a, glucose-regulated protein78 (GRP78), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), X-box binding protein1 (XBP1), C/EBP homologous protein (CHOP) and Caspase 12 were detected by RT-PCR. The protein expression of GRP78, p-IRE1/IRE1 ratio, cleaved-ATF6, XBP1-s (the spliced form of XBP1), CHOP and Caspase 12 were detected by Western blot. TdT-mediated dUTP nick-end labeling (TUNEL) staining was used to detect the rate of apoptosis. RESULTS: QLQX significantly improved cardiac function as evidenced by increased EF, FS, LVSP, +dp/dt max, -dp/dt max, and decreased LVEDP (P<0.05, P<0.01). HE staining showed that QLQX ameliorated cardiac pathologic damage to some extent. Masson staining indicated that QLQX significantly reduced collagen volume fraction in myocardial tissue (P<0.01). Results from RT-PCR and Western blot showed that QLQX significantly increased the expression of miR133a and inhibited the mRNA expressions of GRP78, IRE1, ATF6 and XBP1, as well as decreased the protein expressions of GRP78, cleaved-ATF6 and XBP1-s and decreased p-IRE1/IRE1 ratio (P<0.05, P<0.01). Further studies showed that QLQX significantly reduced the expression of CHOP and Caspase12, resulting in a significant reduction in apoptosis rate (P<0.05, P<0.01). CONCLUSION: The pharmacological mechanism of QLQX in improving HF is partly attributed to its regulatory effect on the miR133a-IRE1/XBP1 pathway.

Partial limitation of cellular functions and compensatory modulation of unfolded protein response pathways caused by double-knockout of ATF6α and ATF6ß.

Mammalian cells have three types of endoplasmic reticulum (ER) stress-sensing molecules: ATF6, IRE1, and PERK. Among these, ATF6 is unique in that it is processed in an ER-stress-specific manner and functions as a transcription factor for the activation of anti-ER stress genes (such as BiP). ATF6 is known to have two homologues, ATF6α and ATF6ß, and a greater understanding of their functions has been achieved through analyses using cultured cells. Physiological functions are also gradually being investigated in mice lacking ATF6α or ATF6ß. However, little is known about the effects on mouse organisms of the deletion of both the ATF6α and ATF6ß genes, since such double-knockout (DKO) mice suffer embryonic lethality at an early developmental stage. In this study, we generated and analyzed ATF6 DKO mice in which embryonic lethality was evaded by using Cre/loxP technology. Pancreatic ß cell-specific ATF6 DKO mice were born normally and lived without dysregulation of blood-glucose levels but had a reduced tolerance to glucose. Islets isolated from ATF6 DKO mice also showed low production and secretion of insulin and mild enhancement of IRE1 and PERK activity. We further examined the developmental abnormalities of systemic ATF6 DKO mice. The phenotypes of ATF6α-/-; ATF6ß-/- mice were similar to those previously reported, but ATF6α+/-; ATF6ß-/- and ATF6α-/-; ATF6ß+/- mice showed embryonic lethality at middle developmental stages, unlike those reported. Analysis of embryonic fibroblasts derived from these mice revealed that ATF6α and ATF6ß have a gene-dose-dependent functional redundancy and display distinct differences in their ability to induce BiP expression. (250 words).

Curcumin alleviates atrazine-induced cardiotoxicity by inhibiting endoplasmic reticulum stress-mediated apoptosis in mice through ATF6/Chop/Bcl-2 signaling pathway.

Atrazine (ATR), a water-soluble herbicide commonly used to control broad-leaf and monocotyledonous weeds, presents a significant risk to environmental soil and water quality. Exposure to ATR adversely affects human and animal health, frequently resulting in cardiac impairment. Curcumin (Cur), an acidic polyphenol derivative from plants acclaimed for its pronounced anti-inflammatory and antioxidant properties, has garnered interest as a potential therapeutic agent. However, whether it has the potential to ameliorate ATR-induced cardiac toxicity via modulation of endoplasmic reticulum stress (ERS) and apoptosis pathways in mice remains unclear. Our results showed that Cur supplementation attenuates ATR-induced cardiotoxicity, evidenced by decrease in creatine kinase and lactate dehydrogenase, key biochemical markers of myocardial injury, which have a more significant protecting effect in high-dose ATR induced injury. Histopathological and electron microscopy examinations further solidified these findings, demonstrating an amelioration in organellar damage, particularly in endoplasmic reticulum swelling and subsequent mitochondrial impairment. Additionally, ATR exposure augments ERS and triggers apoptotic pathways, as indicated by the upregulation of ERS-related gene expression (ATF6, CHOP, IRE1, GRP78) and pro-apoptotic markers (BAX, BAK1, Caspase3, Caspase. Intriguingly, Cur counteracts this detrimental response, significantly reducing ERS and pro-apoptotic signals at both transcriptional and translational levels. Collectively, our findings illuminate Cur's cardioprotective effect against ATR-induced injury, primarily through its anti-ERS and anti-apoptotic activities, underscoring Cur's potential as a therapeutic for ATR-induced cardiotoxicity.

Arrestin beta-2 deficiency exacerbates periodontal inflammation by mediating activating transcription factor 6 activation and abnormal remodelling of the extracellular matrix.

AIM: To investigate the specific role of arrestin beta-2 (ARRB2) in the progression of periodontitis and the underlying mechanisms. MATERIALS AND METHODS: Single-cell RNA sequencing data were used to analyse gene expression in periodontal tissues from healthy controls and patients with periodontitis. Real-time quantitative polymerase chain reaction, Western blotting and immunohistochemical staining were performed to detect the expression of ARRB2. Furthermore, a ligature-induced periodontitis model was created. Using radiographic and histological methods, RNA sequencing and luciferase assay, the role of ARRB2 in periodontitis and the underlying mechanisms were explored. Finally, the therapeutic effect of melatonin, an inhibitor of activating transcription factor 6 (ATF6), on periodontitis in mice was assessed in both in vivo and in vitro experiments. RESULTS: ARRB2 expression was up-regulated in inflammatory periodontal tissue. In the ligature-induced mouse model, Arrb2 knockout exacerbated alveolar bone loss (ABL) and extracellular matrix (ECM) degradation. ARRB2 exerted a negative regulatory effect on ATF6, an essential targeted gene. Melatonin ameliorated ABL and an imbalance in ECM remodelling in Arrb2-deficient periodontitis mice. CONCLUSIONS: ARRB2 mediates ECM remodelling via inhibition of the ATF6 signalling pathway, which ultimately exerts a protective effect on periodontal tissues.

A new insight into fluoride induces cardiotoxicity in chickens: Involving the regulation of PERK/IRE1/ATF6 pathway and heat shock proteins.

Fluorosis poses a significant threat to human and animal health and is an urgent public safety concern in various countries. Subchronic exposure to fluoride has the potential to result in pathological damage to the heart, but its potential mechanism requires further investigation. This study investigated the effects of long-term exposure to sodium fluoride (0, 500, 1000, and 2000 mg/kg) on the hearts of chickens were investigated. The results showed that an elevated exposure dose of sodium fluoride led to congested cardiac tissue and disrupted myofiber organisation. Sodium fluoride exposure activated the ERS pathways of PERK, IRE1, and ATF6, increasing HSP60 and HSP70 and decreasing HSP90. The NF-κB pathway and the activation of TNF-α and iNOS elicited an inflammatory response. BAX, cytc, and cleaved-caspase3 were increased, triggering apoptosis and leading to cardiac injury. The abnormal expression of HSP90 and HSP70 affected the stability and function of RIPK1, RIPK3, and MLKL, which are crucial necroptosis markers. HSPs inhibited TNF-α-mediated necroptosis and apoptosis of the death receptor pathway. Sodium fluoride resulted in heart injury in chickens because of the ERS and variations in HSPs, inducing inflammation and apoptosis. Cardiac-adapted HSPs impeded the activation of necroptosis. This paper may provide a reference for examining the potential cardiotoxic effects of sodium fluoride.

Activating transcription factor (ATF) 6 upregulates cystathionine ß synthetase (CBS) expression and hydrogen sulfide (H2S) synthesis to ameliorate liver metabolic damage.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum stress responsive gene. We previously reported that conditional knockout of hepatic ATF6 exacerbated liver metabolic damage by repressing autophagy through mTOR pathway. However, the mechanism by which ATF6 influence liver metabolism has not been well established. Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays an important role in regulating inflammation, and suppress nonalcoholic fatty liver in mice. Based on the previous study, we assumed that ATF6 may regulate H2S production to participate in liver metabolism. In order to clarify the mechanism by which ATF6 regulates H2S synthesis to ameliorate liver steatosis and inflammatory environment, we conducted the present study. We used the liver specific ATF6 knockout mice and fed on high-fat-diet, and found that H2S level was significantly downregulated in hepatic ATF6 knockout mice. Restoring H2S by the administration of slow H2S releasing agent GYY4137 ameliorated the hepatic steatosis and glucose tolerance. ATF6 directly binds to the promoter of cystathionine ß synthetase (CBS), an important enzyme in H2S synthesis. Thus, ATF6 could upregulate H2S production through CBS. Sulfhydrated Sirtuin-1 (SIRT1) was downregulated in ATF6 knockout mice. The expression of pro-inflammatory factor IL-17A was upregulated and anti-inflammatory factor IL-10 was downregulated in ATF6 knockout mice. Our results suggest that ATF6 can transcriptionally enhance CBS expression as well as H2S synthesis. ATF6 increases SIRT1 sulfhydration and ameliorates lipogenesis and inflammation in the fatty liver. Therefore, ATF6 could be a novel therapeutic strategy for high-fat diet induced fatty liver metabolic abnormalities.

Repaglinide Induces ATF6 Processing and Neuroprotection in Transgenic SOD1G93A Mice.

The interaction of the activating transcription factor 6 (ATF6), a key effector of the unfolded protein response (UPR) in the endoplasmic reticulum, with the neuronal calcium sensor Downstream Regulatory Element Antagonist Modulator (DREAM) is a potential therapeutic target in neurodegeneration. Modulation of the ATF6-DREAM interaction with repaglinide (RP) induced neuroprotection in a model of Huntington's disease. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with no cure, characterized by the progressive loss of motoneurons resulting in muscle denervation, atrophy, paralysis, and death. The aim of this work was to investigate the potential therapeutic significance of DREAM as a target for intervention in ALS. We found that the expression of the DREAM protein was reduced in the spinal cord of SOD1G93A mice compared to wild-type littermates. RP treatment improved motor strength and reduced the expression of the ALS progression marker collagen type XIXα1 (Col19α1 mRNA) in the quadriceps muscle in SOD1G93A mice. Moreover, treated SOD1G93A mice showed reduced motoneuron loss and glial activation and increased ATF6 processing in the spinal cord. These results indicate that the modulation of the DREAM-ATF6 interaction ameliorates ALS symptoms in SOD1G93A mice.

Seneca Valley Virus Degrades STING via PERK and ATF6-Mediated Reticulophagy.

Seneca Valley Virus (SVV), a member of the Picornaviridae family, is an emerging porcine virus that can cause vesicular disease in pigs. However, the immune evasion mechanism of SVV remains unclear, as does its interaction with other pathways. STING (Stimulator of interferon genes) is typically recognized as a critical factor in innate immune responses to DNA virus infection, but its role during SVV infection remains poorly understood. In the present study, we observed that STING was degraded in SVV-infected PK-15 cells, and SVV replication in the cells was affected when STING was knockdown or overexpressed. The STING degradation observed was blocked when the SVV-induced autophagy was inhibited by using autophagy inhibitors (Chloroquine, Bafilomycin A1) or knockdown of autophagy related gene 5 (ATG5), suggesting that SVV-induced autophagy is responsible for STING degradation. Furthermore, the STING degradation was inhibited when reticulophagy regulator 1 (FAM134B), a reticulophagy related receptor, was knocked down, indicating that SVV infection induces STING degradation via reticulophagy. Further study showed that in eukaryotic translation initiation factor 2 alpha kinase 3 (PERK)/activating transcription factor 6 (ATF6) deficient cells, SVV infection failed to induce reticulophagy-medaited STING degradation, indicating that SVV infection caused STING degradation via PERK/ATF6-mediated reticulophagy. Notably, blocking reticulophagy effectively hindered SVV replication. Overall, our study suggested that SVV infection resulted in STING degradation via PERK and ATF6-mediated reticulophagy, which may be an immune escape strategy of SVV. This finding improves the understanding of the intricate interplay between viruses and their hosts and provides a novel strategy for the development of novel antiviral drugs.

KCNH2A561V Heterozygous Mutation Inhibits KCNH2 Protein Expression via The Activation of UPR Mediated by ATF6.

The potassium channel protein KCNH2 is encoded by KCNH2 gene, and there are more than 300 mutations of KCNH2. Unfolded protein response (UPR) is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER). The present study aimed to explore the UPR process and the role of activating transcription factor 6 (ATF6) in the abnormal expression of potassium voltage-gated channel subfamily H member 2 (KCNH2)A561V. The wild-type (wt) KCNH2 and A561V mutant KCNH2 was constructed with his-tag. The 293 cells were used and divided into KCNH2wt+KCNH2A561V, KCNH2wt and KCNH2A561V groups. The expression levels of ATF6 and KCNH2 in different groups were detected by Western blotting, reverse transcription-quantitative PCR, immunofluorescence and immuno-coprecipitation assays. The protein types and abundance of immuno-coprecipitation samples were analyzed by mass spectrometry. The proteomic analysis of the mass spectrometry results was carried out by using the reactome database and GO (Gene Ontology) tool. The mRNA expression levels of KCNH2 and ATF6 in the KCNH2wt+KCNH2A561V group were higher compared with the KCNH2A561V group. However, the full-length protein expression of ATF6 was inhibited, indicating that ATF6 was highly activated and a substantial number of ATF6 was sheared in KCNH2wt+KCNH2A561V group compared with control group. Furthermore, A561V-KCNH2 mutation leading to the accumulation of the immature form of KCNH2 (135 kDa bands) in ER, resulting in the reduction of the ratio of 155 kDa/135 kDa. In addition, the abundance of UPR-related proteins in the KCNH2A561V group was higher compared with the KCNH2wt+KCNH2A561V group. The 'cysteine biosynthetic activity' of GO:0019344 process and the 'positive regulation of cytoplasmic translation activity' of GO:2000767 process in the KCNH2A561V group were higher compared with the KCNH2wt+KCNH2A561V group. Hence, co-expression of wild-type and A561V mutant KCNH2 in 293 cells activated the UPR process, which led to the inhibition of protein translation and synthesis, in turn inhibiting the expression of KCNH2. These results provided a theoretical basis for clinical treatment of Long QT syndrome.

Identification and functional characterization of activating transcription factor 6 (ATF6) from the mud crab (Scylla paramamosain) in response to hydrogen peroxide and bacterial challenge.

Activating transcription factor 6 (ATF6) is critical for regulation of unfolded protein response (UPR), which is involved in the endoplasmic reticulum (ER) proteostasis maintenance and cellular redox regulation. In the present study, a ATF6 gene from the mud crab (designated as Sp-ATF6) has been cloned and identified. The open reading frame of Sp-ATF6 was 1917 bp, encoding a protein of 638 amino acids. The deduced amino acid sequences of Sp-ATF6 contained a typical basic leucine zipper (BZIP domain). Sp-ATF6 was widely expressed in all tested tissues, with the highest expression levels in the hemocytes and the lowest in the muscle. Subcellular localization showed that Sp-ATF6 was expressed in both nucleus and cytoplasm of S2 cells. The expression level of Sp-ATF6 was induced by hydrogen peroxide and V. parahaemolyticus challenge, indicating that the ATF6 pathway was activated in response to ER stress. In order to know more about the regulation mechanism of the Sp-ATF6, RNA interference experiment was investigated. Knocking down Sp-ATF6 in vivo can decrease the expression of antioxidant-related genes (CAT and SOD) and heat shock proteins (HSP90 and HSP70) after V. parahaemolyticus infection. All these results suggested that Sp-ATF6 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

Downregulation of PDCD4 through STAT3/ATF6/autophagy mediates MIF-induced PASMCs proliferation/migration and vascular remodeling.

To address the molecular mechanisms underlying macrophage migration inhibitory factor (MIF) induced pulmonary artery smooth muscle cells (PASMCs) proliferation, migration and vascular remodeling in pulmonary hypertension (PH), primary cultured rat PASMCs and monocrotaline (MCT)-induced rats with PH were applied in the present study. The results showed that MIF increased signal transducer and activator of transcription 3 (STAT3) phosphorylation, and then stimulated activating transcription factor 6 (ATF6) activation, subsequently triggered autophagy activation, which further led to programmed cell death factor 4 (PDCD4) lysosomal degradation, and eventually promoted PASMCs proliferation/migration. In lung tissues of MCT rats, MIF protein expression was elevated, phosphorylation of STAT3 and activation of ATF6 were increased, activation of autophagy was evident, and reduction of PDCD4 was observed. Intervention with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP), ATF6 blocker melatonin or autophagy inhibitor chloroquine, confirmed the in vitro interaction among MIF, STAT3, ATF6, autophagy and PDCD4 in MCT induced rats with PH. Targeting MIF/STAT3/ATF6/autophagy/PDCD4 axis effectively prevented the development of PH by suppressing PASMCs proliferation and vascular remodeling. In conclusions, we demonstrate that MIF activates the STAT3/ATF6/autophagy cascade and then degrades PDCD4 leading to PASMCs proliferation/migration and pulmonary vascular remodeling, suggesting that intervention this axis might have potential value in management of PH.

Protocadherin 20 maintains intestinal barrier function to protect against Crohn's disease by targeting ATF6.

BACKGROUND: Intestinal barrier dysfunction plays a central role in the pathological onset of Crohn's disease. We identify the cadherin superfamily member protocadherin 20 (PCDH20) as a crucial factor in Crohn's disease. Here we describe the function of PCDH20 and its mechanisms in gut homeostasis, barrier integrity, and Crohn's disease development. RESULTS: PCDH20 mRNA and protein expression is significantly downregulated in the colonic epithelium of Crohn's disease patients and mice with induced colitis compared with controls. In mice, intestinal-specific Pcdh20 knockout causes defects in enterocyte proliferation and differentiation, while causing morphological abnormalities. Specifically, the deletion disrupts barrier integrity by unzipping adherens junctions via ß-catenin regulation and p120-catenin phosphorylation, thus aggravating colitis in DSS- and TNBS-induced colitis mouse models. Furthermore, we identify activating transcription factor 6 (ATF6), a key chaperone of endoplasmic reticulum stress, as a functional downstream effector of PCDH20. By administering a selective ATF6 activator, the impairment of intestinal barrier integrity and dysregulation of CHOP/ß-catenin/p-p120-catenin pathway was reversed in Pcdh20-ablated mice with colitis and PCDH20-deficient colonic cell lines. CONCLUSIONS: PCDH20 is an essential factor in maintaining intestinal epithelial homeostasis and barrier integrity. Specifically, PCDH20 helps to protect against colitis by tightening adherens junctions through the ATF6/CHOP/ß-catenin/p-p120-catenin axis.

ATF6 aggravates apoptosis in early porcine embryonic development by regulating organelle homeostasis under high-temperature conditions.

Activating transcription factor 6 (ATF6), one of the three sensor proteins in the endoplasmic reticulum (ER), is an important regulator of ER stress-induced apoptosis. ATF6 resides in the ER and, upon activation, is translocated to the Golgi apparatus, where it is cleaved by site-1 protease (S1P) to generate an amino-terminal cytoplasmic fragment. Although recent studies have made progress in elucidating the regulatory mechanisms of ATF6, its function during early porcine embryonic development under high-temperature (HT) stress remains unclear. In this study, zygotes were divided into four groups: control, HT, HT+ATF6 knockdown, and HT+PF (S1P inhibitor). Results showed that HT exposure induced ER stress, which increased ATF6 protein expression and led to a decrease in the blastocyst rate. Next, ATF6 expression was knocked down in HT embryos under microinjection of ATF6 double-stranded RNA (dsRNA). Results revealed that ATF6 knockdown (ATF6-KD) attenuated the increased expression of CHOP, an ER stress marker, and Ca 2+ release induced by HT. In addition, ATF6-KD alleviated homeostasis dysregulation among organelles caused by HT-induced ER stress, and further reduced Golgi apparatus and mitochondrial dysfunction in HT embryos. AIFM2 is an important downstream effector of ATF6. Results showed that ATF6-KD reduced the occurrence of AIFM2-mediated embryonic apoptosis at HT. Taken together, our findings suggest that ATF6 is a crucial mediator of apoptosis during early porcine embryonic development, resulting from HT-induced ER stress and disruption of organelle homeostasis.

Targeting the ATF6-Mediated ER Stress Response and Autophagy Blocks Integrin-Driven Prostate Cancer Progression.

Prostate cancer progression to the lethal metastatic castration-resistant phenotype (mCRPC) is driven by αv integrins and is associated with Golgi disorganization and activation of the ATF6 branch of unfolded protein response (UPR). Overexpression of integrins requires N-acetylglucosaminyltransferase-V (MGAT5)-mediated glycosylation and subsequent cluster formation with Galectin-3 (Gal-3). However, the mechanism underlying this altered glycosylation is missing. For the first time, using HALO analysis of IHC, we found a strong association of integrin αv and Gal-3 at the plasma membrane (PM) in primary prostate cancer and mCRPC samples. We discovered that MGAT5 activation is caused by Golgi fragmentation and mislocalization of its competitor, N-acetylglucosaminyltransferase-III, MGAT3, from Golgi to the endoplasmic reticulum (ER). This was validated in an ethanol-induced model of ER stress, where alcohol treatment in androgen-refractory PC-3 and DU145 cells or alcohol consumption in patient with prostate cancer samples aggravates Golgi scattering, activates MGAT5, and enhances integrin expression at PM. This explains known link between alcohol consumption and prostate cancer mortality. ATF6 depletion significantly blocks UPR and reduces the number of Golgi fragments in both PC-3 and DU145 cells. Inhibition of autophagy by hydroxychloroquine (HCQ) restores compact Golgi, rescues MGAT3 intra-Golgi localization, blocks glycan modification via MGAT5, and abrogates delivery of Gal-3 to the cell surface. Importantly, the loss of Gal-3 leads to reduced integrins at PM and their accelerated internalization. ATF6 depletion and HCQ treatment synergistically decrease integrin αv and Gal-3 expression and temper orthotopic tumor growth and metastasis. IMPLICATIONS: Combined ablation of ATF6 and autophagy can serve as new mCRPC therapeutic.