Curcumin affects apoptosis of colorectal cancer cells through ATF6-mediated endoplasmic reticulum stress.

Colorectal cancer (CRC) is the main cause of cancer-associated death. Herein, we treated SW620 and HT-29 CRC cells with different curcumin concentrations, followed by treatment with the half maximal inhibitory concentration (IC50) curcumin/endoplasmic reticulum stress (ERS) inhibitor 4-phenyl butyric acid (4-PBA)/activating transcription factor 6 (ATF6) interference plasmid (si-ATF6). We detected cell proliferation/apoptosis, ATF6 cellular localization/nuclear translocation, ion concentration, ATF6 protein/apoptotic protein (Bax/Bcl-2/Cleaved Caspase-3) levels, and ERS-related proteins (glucose-regulated protein 78 [Grp78]/C/EBP homologous protein [CHOP]). We discovered inhibited cell proliferation/growth, enhanced cell apoptosis/(Bax/Bcl-2) ratio/Cleaved Caspase-3 levels/Ca2+ concentration in the cytoplasm/ERS-related protein (Grp78/CHOP) levels, and activated ERS following treatment with IC50 curcumin. 4-PBA partially reversed the inhibitory effect of curcumin on SW620 cells by restraining ERS. Curcumin stimulated ATF6 expression and its nuclear translocation to activate ERS. ATF6 silencing partly annulled the inhibitory effect of curcumin on SW620 cells. Our study explored the molecular mechanism of curcumin affecting CRC cell apoptosis through ATF6.

Hugan Qingzhi tablets attenuates endoplasmic reticulum stress in nonalcoholic fatty liver disease rats by regulating PERK and ATF6 pathways.

BACKGROUND: Endoplasmic reticulum (ER) stress, promoting lipid metabolism disorders and steatohepatitis, contributes significantly to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Hugan Qingzhi tablets (HQT) has a definite effect in the clinical treatment of NAFLD patients, but its mechanism is still unclear. This study aims to investigate the effects of HQT on ER stress in the liver tissues of NAFLD rats and explore the underlying mechanism. METHODS: The NAFLD rat model was managed with high-fat diet (HFD) for 12weeks. HQT was administrated in a daily basis to the HFD groups. Biochemical markers, pro-inflammatory cytokines, liver histology were assayed to evaluate HQT effects in HFD-induced NAFLD rats. Furthermore, the expression of ER stress-related signal molecules including glucose regulating protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), p-PERK, eukaryotic translation initiation factor 2α (EIF2α), p-EIF2α, activating transcription factor 4 (ATF4), acetyl-coenzyme A-carboxylase (ACC), activating transcription factor (ATF6), and nuclear factor-kappa B-p65 (NF-κB-p65) were detected by western blot and/or qRT-PCR. RESULTS: The histopathological characteristics and biochemical data indicated that HQT exhibited protective effects on HFD-induced NAFLD rats. Furthermore, it caused significant reduction in the expression of ERS markers, such as GRP78, PERK, p-PERK, and ATF6, and subsequently downregulated the expression of EIF2α, p-EIF2α ATF4, ACC, and NF-κB-p65. CONCLUSIONS: The results suggested that HQT has protective effect against hepatic steatosis and inflammation in NAFLD rats by attenuating ER stress, and the potential mechanism is through inhibition of PERK and ATF6 pathways.

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.

Repaglinide Elicits a Neuroprotective Effect in Rotenone-Induced Parkinson's Disease in Rats: Emphasis on Targeting the DREAM-ER Stress BiP/ATF6/CHOP Trajectory and Activation of Mitophagy.

Repaglinide, a meglitinide insulinotropic antidiabetic, was unraveled as a promising therapeutic agent for Huntington's disease by targeting the neuronal calcium sensor downstream regulatory element antagonist modulator (DREAM). However, its mechanistic profile in Parkinson's disease (PD) especially its impact on endoplasmic reticulum (ER) stress, mitophagy, and their interconnections is poorly elucidated. This study is the first to examine the neuroprotective potential of repaglinide in rotenone-induced PD in rats by exploring its effects on DREAM, BiP/ATF6/CHOP ER stress pathway, apoptosis, mitophagy/autophagy, oxidative stress, astrogliosis/microgliosis, and neuroinflammation. Male Wistar rats were randomly assigned to four groups: groups 1 and 2 received the vehicle or repaglinide (0.5 mg/kg/day p.o). Groups 3 and 4 received rotenone (1.5 mg/kg/48 h s.c) for 21 days; meanwhile, group 4 additionally received repaglinide (0.5 mg/kg/day p.o) for 15 days starting from day 11. Interestingly, repaglinide lessened striatal ER stress and apoptosis as evidenced by reduced BiP/ATF6/CHOP and caspase-3 levels; however, it augmented striatal DREAM mRNA expression. Repaglinide triggered the expression of the mitophagy marker PINK1 and the autophagy protein beclin1 and alleviated striatal oxidative stress through escalating catalase activity. In addition, repaglinide halted astrocyte/microglial activation and neuroinflammation in the striatum as expressed by reducing glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor protein 1 (Iba1) immunostaining and decreasing interleukin (IL)-6 and IL-1ß levels. Repaglinide restored striatum morphological alterations, intact neuron count, and neurobehavioral motor performance in rats examined by an open field, grip strength, and footprint gait analysis. Conclusively, repaglinide modulates the DREAM-ER stress BiP/ATF6/CHOP cascade, increases mitophagy/autophagy, inhibits apoptosis, and lessens oxidative stress, astrocyte/microglial activation, and neuroinflammation in PD.

[Research progress of the unfolded protein response-activating transcription factor 6 pathway in ischemia-reperfusion injury post cardiac arrest].

Ischemia/reperfusion (I/R) caused by cardiac arrest (CA) and subsequent cardiopulmonary resuscitation (CPR) was the primary cause of post-cardiac arrest syndrome (PCAS), including post-cardiac arrest myocardial dysfunction and post-cardiac arrest brain injury. Disturbance of endoplasmic reticulum proteostasis, so-called endoplasmic reticulum stress (ERS) was one of the pathological changes induced by I/R injury. The unfolded protein response (UPR) was an adaptive response triggered by ERS in cells. Modulating the UPR arms to alleviate ERS to promote cell survival was promising for attenuating I/R injury. Activating the activating transcription factor6 (ATF6) signaling pathway, one of the arms of the UPR, confers protection against I/R injury in multiple tissues by restoring endoplasmic reticulum proteostasis and reducing oxygen free radicals. This article reviewed the structural characteristics and biological function of ATF6 and focused on its essential role in cardiac and cerebral I/R injury as well as potential therapeutic targets, hoping to provide new ideas for the effective treatment of PCAS.

Activating transcription factor 6 protects against endothelial barrier dysfunction.

BACKGROUND: Endothelial hyperpermeability is associated with sepsis and acute respiratory distress syndrome (ARDS). The identification of molecular pathways involved in barrier dysfunction; may reveal promising therapeutic targets to combat ARDS. Unfolded protein response (UPR) is a highly conserved molecular pathway, which ameliorates endoplasmic reticulum stress. The present work focuses on the effects of ATF6, which is a UPR sensor, in lipopolysaccharides (LPS)-induced endothelial hyperpermeability. METHODS: The in vitro effects of AA147 and Ceapin-A7 in LPS-induced endothelial barrier dysfunction were investigated in bovine pulmonary artery endothelial cells (BPAEC). Small interfering (si) RNA was utilized to "silence" ATF6, and electric cell-substrate impedance sensing (ECIS) measured transendothelial resistance. Fluorescein isothiocyanate (FITC)-dextran assay was utilized to assess paracellular permeability. Protein expression levels were evaluated with Western blotting, and cell viability with MTT assay. RESULTS: We demonstrated that AA147 prevents LPS-induced barrier disruption by counteracting Cofilin and myosin light chain 2 (MLC2) activation, as well as VE-Cadherin phosphorylation. Moreover, this ATF6 inducer opposed LPS-triggered decrease in transendothelial resistance (TEER), as well as LPS-induced paracellular hyperpermeability. On the other hand, ATF6 suppression due to Ceapin-A7 or small interfering RNA exerted the opposite effects, and potentiated LPS-induced endothelial barrier disruption. Moderate concentrations of both ATF6 modulators did not affect cell viability. CONCLUSIONS: ATF6 activation protects against endothelial barrier function, suggesting that this UPR sensor may serve as a therapeutic target for sepsis and ARDS.

Luteolin restricts ASFV replication by regulating the NF-κB/STAT3/ATF6 signaling pathway.

African swine fever (ASF) is a devastating infectious disease that causes significant economic losses to the pig industry worldwide. Luteolin is abundant in onion leaves, carrots, broccoli, and apple skin and exerts various biological activities, including anti-cancer and anti-virus effects. Our aim was to demonstrate the mechanism of action and potent antiviral activity of luteolin against ASF virus (ASFV) in porcine alveolar macrophages. We performed cell viability, hemadsorption, indirect immunofluorescence, western blotting, and quantitative real-time polymerase chain reaction assays to investigate the effect of luteolin on ASFV. Notably, luteolin restricted ASFV replication in a dose-dependent manner. The anti-ASFV activity of luteolin was maintained for 24-72 h. Subsequent experiments revealed that luteolin could block multiple stages of the ASFV replication cycle, including those at 6-9 h and 12-15 h after infection, instead of directly interacting with ASFV. Moreover, ASFV infection stimulated the expression of phosphorylated nuclear factor (NF)-κB, interleukin (IL)- 6, and phosphorylated signal transducer and activator of transcription 3 (STAT3). However, luteolin downregulated ASFV-induced NF-κB, IL-6, and STAT3 expression. Importantly, NF-κB agonist CU-T12-9 weakened the inhibitory effects of luteolin on NF-κB and STAT3. Moreover, CU-T12-9 partially restored the inhibitory effect of luteolin on ASFV. Similarly, luteolin reduced ASFV-induced activating transcription factor 6 (ATF6) expression, and CU-T12-9 weakened the inhibitory effect of luteolin on ATF6. Our findings suggested that luteolin inhibited ASFV replication by regulating the NF-κB/STAT3/ATF6 signaling pathway and might provide a rationale for anti-ASFV drug development.

Flurochloridone Induced Cell Apoptosis via ER Stress and eIF2α-ATF4/ATF6-CHOP-Bim/Bax Signaling Pathways in Mouse TM4 Sertoli Cells.

Flurochloridone (FLC), as a novel herbicide, has been widely used in many countries since 1980s. Current studies have shown that FLC has toxic effects on male reproduction and its target organ is testis, while the underlying mechanism is still unknown. Mouse testis Sertoli cell line TM4 cells were used as an in vitro model and treated with FLC at different doses (40, 80, 160 µM) for different times (6, 12, 24 h). Cell viability, cytotoxicity and apoptotic cells were detected by CCK-8 assay, LDH leakage assay and flow cytometry. The protein levels of GRP78, phosphorylated-eIF2α, ATF4, ATF6, CHOP, Bim and Bax were observed by Western Blot and Immunofluorescence staining. FLC inhibited cell viability and induced cytotoxicity in dose-dependent way in TM4 cells. The percentage of apoptotic cells were 6.2% ± 0.6%, 7.3% ± 0.3%, 9.8% ± 0.4%, 13.2% ± 0.2%, respectively. The expression levels of ER stress and UPR related proteins were activated over dose. Meanwhile, the pro-apoptotic proteins (Bim and Bax) were also up-regulated in dose-dependent. After pretreated with ISRIB, the inhibitor of eIF2α phosphorylation, the elevated expression of GRP78, phosphorylated-eIF2α, ATF4, ATF6, CHOP and Bim was down to normal level accordingly. In conclusion, FLC induced apoptosis in TM4 cells mediated by UPR signaling pathways.

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP.

OBJECTIVE: The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1 (TSP1) in human renal tubular epithelial cells (HK-2 cells). METHODS: HK-2 cells were exposed to high concentrations of glucose (HG). The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis. The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry. Western blotting was used to detect the apoptosis- and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA. RESULTS: HG treatment induced high cell apoptosis, abundantly expressed TSP1 level and restrained viability in HK-2 cells. Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions. Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression. 4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers, such as PERK, ATF4, ATF6, p-eIF2α, IRE1, CHOP and XBP1, suggesting that the administration of 4-PBA was successful. CONCLUSION: Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis. TSP1 restrained cell proliferation, and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

MiR-195-5p represses inflammation, apoptosis, oxidative stress, and endoplasmic reticulum stress in sepsis-induced myocardial injury by targeting activating transcription factor 6.

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

Activation of the ATF6 branch of the unfolded protein response in neurons improves stroke outcome.

Impaired function of the endoplasmic reticulum (ER stress) is a hallmark of many human diseases including stroke. To restore ER function in stressed cells, the unfolded protein response (UPR) is induced, which activates 3 ER stress sensor proteins including activating transcription factor 6 (ATF6). ATF6 is then cleaved by proteases to form the short-form ATF6 (sATF6), a transcription factor. To determine the extent to which activation of the ATF6 UPR branch defines the fate and function of neurons after stroke, we generated a conditional and tamoxifen-inducible sATF6 knock-in mouse. To express sATF6 in forebrain neurons, we crossed our sATF6 knock-in mouse line with Emx1-Cre mice to generate ATF6-KI mice. After the ATF6 branch was activated in ATF6-KI mice with tamoxifen, mice were subjected to transient middle cerebral artery occlusion. Forced activation of the ATF6 UPR branch reduced infarct volume and improved functional outcome at 24 h after stroke. Increased autophagic activity at early reperfusion time after stroke may contribute to the ATF6-mediated neuroprotection. We concluded that the ATF6 UPR branch is crucial to ischemic stroke outcome. Therefore, boosting UPR pro-survival pathways may be a promising therapeutic strategy for stroke.

Role of the host cell's unfolded protein response in arenavirus infection.

Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.