Activating Transcription Factor 6 Pathways Research Articles

Modulation of the unfolded protein response by white spot syndrome virus via wsv406 targeting BiP to facilitate viral replication

Outbreaks of diseases are often linked to environmental stress, which can lead to endoplasmic reticulum (ER) stress and subsequently trigger the unfolded protein response (UPR). The replication of the white spot syndrome virus (WSSV), the most serious pathogen in shrimp aquaculture, has been shown to rely on the UPR signaling pathway, although the detailed mechanisms remain poorly understood. In this study, we discovered that WSSV enhances its replication by hijacking the UPR pathway via the viral protein wsv406. Our analysis revealed a significant upregulation of wsv406 in the hemocytes and gills of infected shrimp. Mass spectrometry analysis identified that wsv406 interacts specifically with the immunoglobulin heavy-chain-binding protein (BiP) in shrimp Litopenaeus vannamei. Further examination revealed that wsv406 binds to multiple domains of LvBiP, inhibiting its ATPase activity without disrupting its binding to UPR stress receptors. Silencing either wsv406 or LvBiP resulted in a reduction in WSSV replication and improved shrimp survival rates. Further, wsv406 activation of the PRKR-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α) and activating transcription factor 6 (ATF6) pathways was demonstrated by a decrease in the phosphorylation of eIF2α and the nuclear translocation of ATF6 when wsv406 was silenced during WSSV infection. This activation facilitated the transcription of WSSV genes, promoting viral replication. In summary, these findings reveal that wsv406 manipulates the host UPR by targeting LvBiP, thereby enhancing WSSV replication through the PERK-eIF2α and ATF6 pathways. These insights into the interaction between WSSV and host cellular machinery offer potential targets for developing therapeutic interventions to control WSSV outbreaks in shrimp aquaculture.

Activating Transcription Factor 6 Mediates Inflammation in Experimental Varicocele-Induced Epididymal Epithelial Cells.

Varicocele is a dilatation of the internal spermatic vein and it is generally recognized as one cause of male infertility. This study aimed to analyze the roles of activating transcription factor 6 (ATF-6) in experimental varicocele-induced epididymal epithelial cells. Experimental left varicocele was established in rats through partial left renal vein ligation. At 8weeks after surgery, the left epididymal damage was observed using H&E and TUNEL staining. The expressions of neutral α-glucosidase (NAG), ATF-6, tumor necrosis factor (TNF)-α, and phospho-nuclear factor (p-NF)-κB p65 (S536) in the left epididymis were measured by immunohistochemistry. ATF-6 silence in rat epididymal epithelial cells was established by ATF-6 siRNA transfection. The cells were treated with hypoxia for 24h, and cell viability was measured by CCK-8, levels of NAG, TNF-α, and interleukin (IL)-8 in cells were measured by ELISA, levels of p-NF-κB p65 (S536)/NF-κB p65 protein in cells were measured by Western blotting. The results showed that the experimental left varicocele induced hypertrophy and apoptosis of epididymal epithelial cells (p<0.05), and decreased the expressions of NAG in the epididymal epithelial cells compared with the sham-operated control rats (p<0.01). Meanwhile, the expressions of ATF-6, TNF-α, and p-NF-κB p65 (S536) were increased in the epididymal epithelial cells after the experimental left varicocele compared with the sham-operated control rats (p<0.05). In the hypoxia-treated cells, ATF-6 silence increased the cell viability and decreased the levels of TNF-α, IL-8, and p-NF-κB p65 (S536) compared with the control cells (p<0.05). The ATF-6 pathway was activated in a rat's left varicocele-induced epididymal damage. Inhibition of the ATF-6 pathway might be a possible novel therapeutic approach for left varicocele-induced epididymal damage.

Impaired stemness in aging periodontal ligament stem cells is mediated by the progerin/endoplasmic reticulum stress/p53 axis

IntroductionDecreased periodontal ligament stem cells (PDLSCs) stemness is a key factor in age-related alveolar bone loss. Endoplasmic reticulum (ER) stress is closely related to age-related diseases and the mesenchymal stem cell (MSC) stemness. However, the role of ER stress in regulating the stemness of senescent PDLSCs and its potential mechanism remain unclear. ObjectivesTo investigate the detailed effect and mechanism of ER stress on impaired stemness in old periodontal ligament stem cells (OPDLSCs). MethodsThe level of ER stress of Young PDLSCs (YPDLSCs) and OPDLSCs were detected, and ER stress was regulated to observe its effect on PDLSCs stemness. The expression levels of ER stress sensors (protein kinase R-like ER kinase (PERK), activating transcription factor 6 (ATF6), inositol requiring enzyme 1 (IRE1)) were upregulated in YPDLSCs and downregulated in OPDLSCs by transfection experiments to verify the detailed unfolded protein response (UPR) pathway. Mechanismly, the regulatory effect of UPR pathway on p53/p21 pathway was explored. Further study was performed to investigated the important role of progerin accumulation during aging process on ER stress, UPR and p53/p21 pathway. ResultsDecreased stemness and ER stress activation were found in OPDLSCs. ER stress activation resulted in decreased stemness of YPDLSCs, while ER stress inhibition rescued compromised stemness of OPDLSCs. Mechanismly, ATF6 pathway regulated the OPDLSC stemness via the p53/p21 signaling as confirmed by transfection assay. Further study showed that progerin was accumulated in PDLSCs and progerin overexpression could resulted in ER stress activation, activating the ATF6/p53/p21 axis, leading to decreased stemness of aging PDLSCs. ConclusionsProgerin accumulation during the aging process can lead to ER stress activation, which can suppress OPDLSC stemness via the ATF6/p53/p21 axis.

Porcine epidemic diarrhea virus E protein induces unfolded protein response through activating both PERK and ATF6 rather than IRE1 signaling pathway.

Porcine epidemic diarrhea virus (PEDV) small envelope protein (E) plays important roles in virus budding, assembly, and release. Our previous study found that PEDV E protein localizes in the endoplasmic reticulum (ER) to trigger the unfolded protein response (UPR). However, how UPR is directly regulated by PEDV E protein remains elusive. Thus, in this study, we investigated the expression of ER chaperone glucose-regulated protein 78 (GRP78) and activations of the three main UPR signaling pathways to elucidate the underlying mechanisms of UPR triggered by PEDV E protein. The results showed that over-expression of PEDV E protein increased expression of GRP78 and induced stronger phosphorylation of both protein kinase RNA-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), as well as caused the significant degradation of activating transcription factor 6 (ATF6), in both dose- and time-dependent manners. However, PEDV E protein did not induce UPR through the inositol-requiring enzyme 1 (IRE1) signaling pathway, as revealed by the splicing of XBP1 remaining unaffected and unchanged when PEDV E protein was overexpressed. Taken together, these results demonstrate that PEDV E protein induces UPR through activation of both PERK and ATF6 pathways rather than IRE1 signaling. This study not only provides mechanistic details of UPR induced by the PEDV E protein, but also provides insights into these new biologic functions to help us better understand the interactions between PEDV and host cells.

African swine fever virus modulates the endoplasmic reticulum stress-ATF6-calcium axis to facilitate viral replication

ABSTRACT African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. Endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with unfolded protein response (UPR), which represents a response to ER stress, are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.

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.

Tyrosine Kinase Inhibitor Lenvatinib Causes Cardiotoxicity by Inducing Endoplasmic Reticulum Stress and Apoptosis through Activating ATF6, IRE1α and PERK Signaling Pathways.

Lenvatinib is a tyrosine kinase inhibitor that can improve progression-free survival in patients with thyroid cancer and hepatocellular carcinoma. However, it is limited by adverse cardiovascular events, including hypertension and cardiac dysfunction. Activation of endoplasmic reticulum stress is involved in cardiomyocyte apoptosis. This study aimed to confirm whether the cardiotoxicity of lenvatinib is associated with endoplasmic reticulum stress by targeting the activating transcription factor 6 (ATF6), inositol- requiring enzyme 1α (IRE1α) and protein kinase RNA-like ER kinase (PERK) signaling pathways. Male C57/BL6 mice were intragastric administration with 30 mg/kg/day lenvatinib. Electrocardiography (ECG) and echocardiography were used to detect arrhythmias and cardiac function. Neonatal rat cardiomyocytes were treated with lenvatinib for 48h. Cell counting kit (CCK8), 2´,7´-dichlorodihydrofluoresceine diacetate (H2DCFHDA), Hoechst 33258 and dihydrorhodamine 123 were respectively used for evaluating cell viability, the level of reactive oxygen species (ROS), nuclear morphological changes and mitochondrial membrane potential (MMP) level. Lenvatinib remarkably decreased the posterior wall thickness of left ventricle during diastole and systole but caused little decrease to the left ventricular ejection fraction (LVEF, %). Furthermore, lenvatinib greatly prolonged the corrected QT interval (QTc) and altered the morphology of cardiomyocytes. No dramatic difference in fibrosis was found in mouse cardiac slices. Lenvatinib upregulates apoptosis-related protein expression. In addition, lenvatinib increased ERS-related protein expression (GRP78, CHOP, and ATF6) and enhanced PERK phosphorylation. In neonatal rat cardiac myocytes, lenvatinib markedly decreased the viability of cardiomyocytes and induced apoptosis. Furthermore, ROS production increased and MMP decreased. Similar to the mice experiment, lenvatinib caused upregulation of apoptosis-related and ERS-related proteins and increased the phosphorylation levels of PERK and IRE1α. Lenvatinib-induced cardiotoxicity is associated with ERS-induced apoptosis by targeting the ATF6, IRE1α, and PERK signaling pathways.

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.

AA147 ameliorates post-cardiac arrest cerebral ischemia/reperfusion injury through the co-regulation of the ATF6 and Nrf2 signaling pathways.

Ischemia/reperfusion caused by cardiac arrest (CA) disturbs endoplasmic reticulum (ER) homeostasis and redox balance in neurons. AA147, originally developed as a pharmacologic activator of the activating transcription factor 6 (ATF6), can protect multiple tissues from ischemia/reperfusion injury (IRI) by decreasing reactive oxygen species (ROS) and restoring ER function. However, it is unclear whether pharmacologic treatment of AA147 could ameliorate post-CA cerebral IRI and whether it is associated with proteostasis regulation and anti-oxidative stress mechanism. In the present study, mice were subjected to 9 min-CA surgery followed by cardiopulmonary resuscitation (CPR). AA147 or vehicle was administered 1 day before the operation and 15min after the return of spontaneous circulation. We found that AA147 restored neurological function and reduced dead neurons in mice suffering from CA. Moreover, AA147 inhibited CA/CPR-caused neuronal apoptosis and ER stress, indicated by reduced TUNEL-positive neurons, surged expression of Bcl-2/Bax, and down expression of cleaved caspase-3, caspase-12, C/EBP homologous protein (CHOP). The expression of ATF6 and its regulated gene glucose-regulated protein 78 (GRP78) increased significantly after the administration of AA147, suggesting the activation of the ATF6 pathway. In addition, AA147 also alleviated the upsurge of the ROS generation and MDA levels as well as increased SOD activity, accompanied by enhancement of the nuclear factor E2-related factor 2 (Nrf2) and its modulated heme-oxygenase-1 (HO-1) expressions. Cotreatment of AA147 with inhibitors of the ATF6 or Nrf2 significantly suppressed AA147-dependent reductions in ROS scavenging and neuronal death after CA/CPR. The results suggested that AA147 could confer neuroprotection against post-CA cerebral IRI through inhibition of oxidative stress along with ER stress-associated apoptosis, which is attributed to the coregulation of both ATF6 and Nrf2 signaling pathways activity. Our findings support the potential for AA147 as a therapeutic approach to improve post-CA brain injury.

Lutein activates downstream signaling pathways of unfolded protein response in hyperglycemic ARPE-19 cells

We have earlier demonstrated that lutein effectively prevents hyperglycemia generated sustained oxidative stress in ARPE-19 cells by activating Nrf2 (nuclear factor erythroid 2–related factor 2) signaling. Since evidence portrays an intricate connection between ER (endoplasmic reticulum) stress and hyperglycemia-mediated oxidative stress, we aimed to explore the protective mechanism of lutein on hyperglycemia-induced ER stress in ARPE-19 cells. To determine the effect of lutein, we probed three major downstream branches of unfolded protein response (UPR) signaling pathways using western blot, immunofluorescent and RT-PCR techniques. The data showed a reduction (38%) in protein expression of an imperative ER chaperon, BiP (binding immunoglobulin protein), in glucose-treated ARPE-19 cells. At the same time, lutein pretreatment blocked this glucose-mediated effect, leading to a significant increase in BiP expression. Lutein promoted the phosphorylation of IRE1 (inositol requiring enzyme 1) and subsequent splicing of XBP1 (X-box binding protein 1), leading to enhanced nuclear translocation. Likewise, lutein activated the expression and translocation of transcription factors, ATF6 (activating transcription factor 6) and ATF4 (activating transcription factor 4) suppressed by hyperglycemia. Lutein also increased CHOP (C/EBP-homologous protein) levels in ARPE-19 cultured under high glucose conditions. The mRNA expression study showed that lutein pretreatment upregulates downstream UPR genes HRD1 (ERAD-associated E3 ubiquitin-protein ligase HRD1), p58IPK (protein kinase inhibitor p58) compared to high glucose treatment alone. From our study, it is clear that lutein show protection against hyperglycemia-mediated ER stress in ARPE-19 cells by activating IRE1-XBP1, ATF6, and ATF4 pathways and their downstream activators. Thus, lutein may have the pharmacological potential for protection against widespread disease conditions of ER stress.

Metabolically Activated Proteostasis Regulators Protect against Glutamate Toxicity by Activating NRF2.

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.

Myeloproliferative Neoplasm-Associated Type 2 Calreticulin Mutations Differentially Activate and Depend on the ATF6 Pathway of the UPR

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). 80% of CALR mutations are classified as either type 1 or type 2, exemplified by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5), respectively. Despite their shared mutant C-termini and mutual ability to bind and activate MPL, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. Type 1 mutations are primarily associated with an MF phenotype and a higher risk of fibrotic transformation from ET, while type 2 mutations are more common in ET. Molecularly, type 2 mutations retain many of the calcium binding sites present in the wild type protein, type 1 mutant proteins lose these residues. As a result, we have found that type 1-expressing cells demonstrate increased chronically depleted ER calcium levels, which leads to activation of and dependency on the IRE1α-XBP1 pathway of the UPR. Type 2 mutant CALR-expressing cells do not demonstrate activation of this pathway, leading us to explore whether type 2 CALR mutations may differentially activate and depend on another UPR branch altogether.RNA sequencing studies revealed differential up-regulation of the activating transcription factor 6 (ATF6) pathway of the UPR in CALRins5- versus CALRdel52-expressing cells. This finding was validated by western blot as well as in an ATF6 reporter assay. To determine whether CALRins5 cells depend on ATF6 for survival, we knocked down ATF6 gene using shRNA and found that while CALRwt and CALRdel52 cells were unaffected, the proliferation of CALRins5 cells was significantly inhibited, suggesting that CALRins5 cells do indeed depend on ATF6 for proliferation and survival.To dissect the mechanism by which CALRins5 cells activate the ATF6 pathway, we measured the chaperone function of CALRins5 protein. Given that the two main functions of CALR in the ER are to bind and store calcium and act as a molecular chaperone, we were interested to see whether CALRins5, which we found retains calcium binding ability, may lose chaperone function. Furthermore, since the major transcriptional targets of ATF6 are chaperones, we hypothesized that CALRins5 may activate ATF6 upon loss of chaperone function, to help cope with this loss of function and continue to survive. We employed an in vitro chaperone assay to measure the chaperone function of CALRwt, CALRdel52, and CALRins5, and indeed, found that only CALR ins5 loses its ability to chaperone. We performed structural modeling studies to determine how this loss of function may occur, and found that the mutant C-terminus of the CALRins5 protein, but not the CALRdel52 protein, interacts with the chaperone domain of CALR, potentially leading to its inhibition. In line with this, we found up-regulation of ATF6 chaperone targets specifically in CALRins5 cells, suggesting that loss of chaperone function may underlie the differential activation of ATF6 by CALRins5.Taken together, we have identified a novel molecular dependency unique to type 2 CALR mutant cells, and determined the molecular mechanism underlying its activation. This work suggests that ATF6 may represent a promising therapeutic target for treatment of type 2 CALR+ MPN. DisclosuresNo relevant conflicts of interest to declare.

ATF6 is essential for human cone photoreceptor development

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

Activation of the ATF6 (Activating Transcription Factor 6) Signaling Pathway in Neurons Improves Outcome After Cardiac Arrest in Mice.

BackgroundIschemia/reperfusion injury impairs proteostasis, and triggers adaptive cellular responses, such as the unfolded protein response (UPR), which functions to restore endoplasmic reticulum homeostasis. After cardiac arrest (CA) and resuscitation, the UPR is activated in various organs including the brain. However, the role of the UPR in CA has remained largely unknown. Here we aimed to investigate effects of activation of the ATF6 (activating transcription factor 6) UPR branch in CA.Methods and ResultsConditional and inducible sATF6‐KI (short‐form ATF6 knock‐in) mice and a selective ATF6 pathway activator 147 were used. CA was induced in mice by KCl injection, followed by cardiopulmonary resuscitation. We first found that neurologic function was significantly improved, and neuronal damage was mitigated after the ATF6 pathway was activated in neurons of sATF6‐KI mice subjected to CA/cardiopulmonary resuscitation. Further RNA sequencing analysis indicated that such beneficial effects were likely attributable to increased expression of pro‐proteostatic genes regulated by ATF6. Especially, key components of the endoplasmic reticulum–associated degradation process, which clears potentially toxic unfolded/misfolded proteins in the endoplasmic reticulum, were upregulated in the sATF6‐KI brain. Accordingly, the CA‐induced increase in K48‐linked polyubiquitin in the brain was higher in sATF6‐KI mice relative to control mice. Finally, CA outcome, including the survival rate, was significantly improved in mice treated with compound 147.ConclusionsThis is the first experimental study to determine the role of the ATF6 UPR branch in CA outcome. Our data indicate that the ATF6 UPR branch is a prosurvival pathway and may be considered as a therapeutic target for CA.

The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon beta

The open reading frame 8 (orf8) is an accessory protein of SARS-CoV-2. It has 121 amino acids with two genotypes, orf8L and orf8S. In this study, we overexpressed the orf8L and orf8S of SARS-CoV-2 as well as the orf8b of SARS-CoV to investigate their roles in the regulation of endoplasmic reticulum (ER) stress and the inhibition of interferon beta (IFNß) production. We found that the two genotypes of SARS-CoV-2 orf8 are capable of inducing ER stress without significant difference by triggering the activating transcription factor 6 (ATF6) and inositol-requiring enzymes 1 (IRE1) branches of the ER stress pathway. However, the third branch of ER stress pathway, i.e. the protein kinase-like ER kinase (PERK), was unaffected by the overexpression of SARS-CoV-2 orf8L or orf8S. Moreover, both orf8L and orf8S of SARS-CoV-2 are capable of down regulating the production of IFNß and interferon-stimulated genes (ISG), ISG15 and ISG56 induced by polyinosinic-polycytidylic acid (poly (I:C)). Moreover, we also found decreased nuclear translocation of Interferon regulatory factor 3 (IRF3), after overexpressing orf8L and orf8S induced by poly (I:C). Our data demonstrated that SARS-CoV-2 orf8 protein could induce ER stress by activating the ATF6 and IRE1 pathways, but not the PERK pathway, and functions as an interferon antagonist to inhibit the production of IFNß. However, these functions appeared not to be affected by the genotypes of SARS-CoV-2 orf8L and orf8S.

MiR-185-5p ameliorates endoplasmic reticulum stress and renal fibrosis by downregulation of ATF6

AbstractEndoplasmic reticulum (ER) stress is considered an important factor in the formation of fibrosis. Therefore, modulation of ER stress may represent a promising therapeutic strategy in renal fibrosis. MiR-185-5p has been identified to be implicated in TGF-β1-induced renal fibrosis; however, it is largely unknown whether and how miR-185-5p regulates ER stress in renal fibrosis. In this study, we demonstrated that miR-185-5p directly bound to ATF6, an ER stress-related protein, and downregulated the expression thereof. We subsequently constructed an in vitro model of renal fibrosis using HK2 cells treated with TGF-β1, and found that miR-185-5p attenuated ER stress and dedifferentiation of tubular epithelia by suppression of ATF6. In addition, we constructed an in vivo mouse model using unilateral urethral obstruction (UUO). Our in vivo findings showed that miR-185-5p reduced the expression of ER stress-related proteins and inhibited epithelial dedifferentiation via downregulation of ATF6, thereby improving UUO-induced renal fibrosis. Overall, our findings revealed that miR-185-5p exerts beneficial effects in renal fibrosis. Thus, the miR-185-5p/ATF6 regulatory pathway may be a potential target for therapeutic intervention in renal fibrosis.This study reveals that miR-185-5p alleviates endoplasmic reticulum (ER) stress, prevents epithelial dedifferentiation and subsequently improves renal fibrosis by downregulating activating transcription factor 6 (ATF6). The miR-185-5p/ATF6 pathway is proposed as a novel regulatory mechanism for renal fibrosis and may provide a therapeutic strategy for renal fibrosis associated with ER stress.

Quercetin provides protection against the peripheral nerve damage caused by vincristine in rats by suppressing caspase 3, NF-κB, ATF-6 pathways and activating Nrf2, Akt pathways

In the present study, the protective effects of quercetin on peripheral neurotoxicity caused by vincristine, which is used effectively in the treatment of various types of cancers, were investigated by using different techniques. In the study, for 12 days, male Sprague Dawley rats were given 25 and 50 mg/kg doses of quercetin orally and were administered a 0.1 mg/kg dose of vincristine (a total cumulative dose of 1.2 mg/kg) intraperitoneally 30 min later. The protein levels of nuclear factor erythroid 2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase-1 (NQO1), glial fibrillary acidic protein (GFAP), and nuclear factor kappa B (NF-κB) were measured with ELISA; the immunopositivity of 8-hydroxy-2′-deoxyguanosine (8−OHdG) and caspase 3 were determined with immunohistochemistry; the mRNA transcript levels of double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1), activating transcription factor-6 (ATF-6), glucose-regulated protein 78 (GRP78), Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), caspase 3, protein kinase B1/2 (Akt-1/2), and forkhead box transcription factor, class O1 (FOXO1) were determined with RT-PCR. The reduction of Nrf2 levels and HO-1 and NQO1 activities in the sciatic nerve tissue, the increase in the levels of 8−OHdG, and the increase in the levels of GFAP and NF-κB caused by vincristine was observed to cause oxidative stress, oxidative DNA damage, neuronal cell damage, and inflammation, respectively. Additionally, vincristine was determined to cause ER stress and apoptosis by increasing PERK, IRE1, ATF-6, and GRP78 and caspase 3 and Bax expressions and by decreasing Bcl-2 expressions. Vincristine causing Akt inhibition also shows that it prevents neuronal survival. However, quercetin was determined to relieve oxidative stress, oxidative DNA damage, neuronal cell damage, inflammation, ER stress, and apoptosis caused by vincristine and enable Akt activation. These results show that in rats, quercetin may have a protective effect against peripheral neurotoxicity caused by vincristine.

Induction of the Unfolded Protein Response during Bovine Alphaherpesvirus 1 Infection.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes great economic losses in the cattle industry. Herpesvirus infection generally induces endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) in infected cells. However, it is not clear whether ER stress and UPR can be induced by BoHV-1 infection. Here, we found that ER stress induced by BoHV-1 infection could activate all three UPR sensors (the activating transcription factor 6 (ATF6), the inositol-requiring enzyme 1 (IRE1), and the protein kinase RNA-like ER kinase (PERK)) in MDBK cells. During BoHV-1 infection, the ATF6 pathway of UPR did not affect viral replication. However, both knockdown and specific chemical inhibition of PERK attenuated the BoHV-1 proliferation, and chemical inhibition of PERK significantly reduced the viral replication at the post-entry step of the BoHV-1 life cycle. Furthermore, knockdown of IRE1 inhibits BoHV-1 replication, indicating that the IRE1 pathway may promote viral replication. Further study revealed that BoHV-1 replication was enhanced by IRE1 RNase activity inhibition at the stage of virus post-entry in MDBK cells. Furthermore, IRE1 kinase activity inhibition and RNase activity enhancement decrease BoHV1 replication via affecting the virus post-entry step. Our study revealed that BoHV-1 infection activated all three UPR signaling pathways in MDBK cells, and BoHV-1-induced PERK and IRE1 pathways may promote viral replication. This study provides a new perspective for the interactions of BoHV-1 and UPR, which is helpful to further elucidate the mechanism of BoHV-1 pathogenesis.

TNFα selectively activates the IRE1α/XBP1 endoplasmic reticulum stress pathway in human airway smooth muscle cells.

Airway inflammation is a key aspect of diseases such as asthma. Proinflammatory cytokines such as TNFα mediate the inflammatory response. In various diseases, inflammation leads to endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins, which triggers homeostatic responses to restore normal cellular function. We hypothesized that TNFα triggers ER stress through an increase in reactive oxygen species generation in human airway smooth muscle (hASM) with a downstream effect on mitofusin 2 (Mfn2). In hASM cells isolated from lung specimens incidental to patient surgery, dose- and time-dependent effects of TNFα exposure were assessed. Exposure of hASM to tunicamycin was used as a positive control. Tempol (500 μM) was used as superoxide scavenger. Activation of three ER stress pathways were evaluated by Western blotting: 1) autophosphorylation of inositol-requiring enzyme1 (IRE1α) leading to splicing of X-box binding protein 1 (XBP1); 2) autophosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) leading to phosphorylation of eukaryotic initiation factor 2α; and 3) translocation and cleavage of activating transcription factor 6 (ATF6). We found that exposure of hASM cells to tunicamycin activated all three ER stress pathways. In contrast, TNFα selectively activated the IRE1α/XBP1 pathway in a dose- and time-dependent fashion. Our results indicate that TNFα does not activate the PERK and ATF6 pathways. Exposure of hASM cells to TNFα also decreased Mfn2 protein expression. Concurrent exposure to TNFα and tempol reversed the effect of TNFα on IRE1α phosphorylation and Mfn2 protein expression. Selective activation of the IRE1α/XBP1 pathway in hASM cells after exposure to TNFα may reflect a unique homeostatic role of this pathway in the inflammatory response of hASM cells.

ATF6 Aggravates Angiogenesis-Osteogenesis Coupling During Ankylosing Spondylitis by Mediating FGF2&amp;nbsp;Expression in Chondrocytes

Background: Although angiogenesis-osteogenesis coupling is important in ankylosing spondylitis (AS), therapeutic agents targeting the vasculature remain elusive. Here, we identified activating transcription factor 6 (ATF6) as an important regulator of angiogenesis. Methods: ATF6 and fibroblast growth factor 2 (FGF2) levels were quantified in SKG mice and AS patient cartilage by immunohistochemistry. Next, we isolated and cultured human chondrocytes and stimulated them with TNF-α, IFN-γ, and IL-17. ATF6 and FGF2 expression was measured by quantitative real-time PCR and western blotting. The angiogenic ability of chondrocytes was assessed with human umbilical vein endothelial cell tube formation and transwell migration assays. Theendoplasmic reticulum stress alleviator 4-phenyl butyric acid and the ATF6 inhibitor Ceapin-A7 were used to indicate the relationship between ATF6 and FGF2. We also performed luciferase reporter and chromatin immunoprecipitation assays to test whether ATF6 was directly responsible for FGF2 transcription, treated SKG mice with Ceapin-A7 to evaluate the role of ATF6 in AS and performed micro-CT and immunofluorescence analyses. Findings: ATF6 and FGF2 expression was elevated in AS cartilage following long-term stimulation with inflammatory factors. Among the three pathways involved in the unfolded protein response, the ATF6 pathway was activated mostly in chondrocytes stimulated long-term with proinflammatory cytokines. In chondrocytes, ATF6 knockdown decreased FGF2 expression and inhibited angiogenesis, whereas ATF6 overexpression had the opposite effects. Mechanistically, ATF6 interacted with the FGF2 promotor and promoted its transcription. Treatment with the ATF6 inhibitor Ceapin-A7 inhibited angiogenesis in vitro and angiogenesis-osteogenesis coupling in vivo. Interpretation: ATF6 may aggravate angiogenesis-osteogenesis coupling during AS by mediating FGF2 transcription in chondrocytes, implying that ATF6 represents a promising therapeutic target for AS. Funding Statement: This study was financially supported by grants from the National Natural Science Foundation of China (81971518), the National Natural Science Foundation of China (81871750), the Natural Science Foundation of Guangdong Province (2018A030313232), the Shenzhen Key Medical Discipline Construction Fund (ZDSYS20190902092851024) and the Health Welfare Fund Project of Futian District (FTWS2020078) Declaration of Interests: All authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethics Committee of The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, China. Written informed consent was obtained from all participants.