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ABSTRACTChloromethylisothiazolinone (CMIT), a humidifier disinfectant, is known to be toxic to the respiratory system. While the toxic effect of CMIT on the lungs has been widely investigated, its effect on the skin is well unknown. In this study, we examined stress granule (SG) formation to investigate the cytotoxic effects of CMIT on human keratinocytes. We assessed the viability of the cells following CMIT exposure and performed immunofluorescence microscopy and immunoblot analyses to determine SG formation and downstream pathways. The IC50 values in human keratinocyte HaCaT cells after CMIT exposure for 1 and 24 h were 11 and 8 μg/mL, respectively, showing no significant difference. As determined using immunofluorescence microscopy, SG formation was effectively induced after CMIT exposure. Moreover, the phosphorylation of eukaryotic initiation factor-2α (eIF2α), a translation initiation factor, and protein kinase R-like endoplasmic reticulum (ER) kinase, which plays a role in the ER stress-mediated eIF2α phosphorylation, was confirmed by CMIT exposure. These results suggest that exposure to CMIT can have detrimental effects on the skin, even briefly, by inducing SG formation through ER stress in keratinocytes.

Chloromethylisothiazolinone (CMIT), a humidifier disinfectant, is known to be toxic to the respiratory system. While the toxic effect of CMIT on the lungs has been widely investigated, its effect on the skin is well unknown. In this study, we examined stress granule (SG) formation to investigate the cytotoxic effects of CMIT on human keratinocytes. We assessed the viability of the cells following CMIT exposure and performed immunofluorescence microscopy and immunoblot analyses to determine SG formation and downstream pathways. The IC50 values in human keratinocyte HaCaT cells after CMIT exposure for 1 and 24 h were 11 and 8 μg/mL, respectively, showing no significant difference. As determined using immunofluorescence microscopy, SG formation was effectively induced after CMIT exposure. Moreover, the phosphorylation of eukaryotic initiation factor-2α (eIF2α), a translation initiation factor, and protein kinase R-like endoplasmic reticulum (ER) kinase, which plays a role in the ER stress-mediated eIF2α phosphorylation, was confirmed by CMIT exposure. These results suggest that exposure to CMIT can have detrimental effects on the skin, even briefly, by inducing SG formation through ER stress in keratinocytes. [ABSTRACT FROM AUTHOR]

Calcium oxalate (CaOx) is the most common component of kidney stones. Oxidative stress, inflammation and autophagy-induced cell death are the major causes of CaOx crystal deposition and CaOx crystal deposition can further lead to kidney injury. Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, plays an important role in the pathogenesis of many diseases, such as atherosclerosis, diabetes and chronic kidney disease, but the effect of TMAO on hyperoxaluria-induced CaOx crystal deposition and kidney injury remains unknown. We hypothesize that TMAO aggravates CaOx crystal deposition via promoting CaOx-mediated cell death. C57Bl/6 mice were given high-oxalate diet as a model of hyperoxaluria. TMAO was provided via drinking water. Serum TMAO levels increased 15 days after CaOx treatment (6.30 ± 0.17 μmol/L vs. 34.65 ± 8.95 μmol/L). High-oxalate diet induced inflammation, CaOx deposition and kidney injury, which TMAO aggravated. In accordance, TMAO intensified high-oxalate diet induced oxidative stress, autophagy and apoptosis. Moreover, TMAO enhanced CaOx crystal adhesion to HK-2 cells and reduced cell viability (from 88.9 ± 1.6% to 75.0 ± 2.7%). Protein kinase R-like endoplasmic reticulum kinase (PERK) may mediate these TMAO effects, as TMAO promoted PERK phosphorylation. Consistently, PERK knockdown alleviated TMAO-evoked CaOx-autophagy, apoptosis and oxidative stress in HK-2 cells. In conclusion, TMAO can aggravate hyperoxaluria-induced kidney injury by triggering the PERK/ROS pathway, which enhances autophagy, apoptosis and inflammation, and facilitates CaOx crystal deposition in renal tubular cells. [Display omitted] • High-oxalate diet increases plasma TMAO levels via upregulation of CntA in mice. • TMAO increases the deposition of calcium oxalate crystals in kidney and promotes hyperoxaluria-induced kidney injury. • TMAO enhances calcium oxalate crystals-induced oxidative stress ,inflammation, autophagy and apoptosis via PERK. • TMAO is a potential target for treating hyperoxaluria. [ABSTRACT FROM AUTHOR]

Moderate-intensity exercise can help delay the development of osteoarthritis (OA). Previous studies have shown that the purinergic receptor P2X ligand gated ion channel 7 (P2X7) is involved in OA development and progression. To investigate the effect of exercise on P2X7 activation and downstream signaling in OA, we used the anterior cruciate ligament transection (ACLT)-induced OA rat model and primary chondrocyte culture system. Our in vivo experiments confirmed that treadmill exercise increased P2X7 expression and that this effect was more pronounced at the later time points. Furthermore, P2X7 activation induced endoplasmic reticulum (ER) stress and increased the expression levels of ER stress markers, such as 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6). At the early time points, IRE1 and PERK were activated, and mTOR was inhibited. At the later time points, mTOR was activated, mediating PERK to promote ER stress-apoptosis, whereas IRE1 and autophagy were inhibited. To confirm our observations in vitro, we treated primary chondrocytes with the P2X7 agonist benzoylbenzoyl-ATP (Bz-ATP). Our results confirmed that P2X7-mediated Ca2+ influx activated IRE1-mediated autophagic flux and induced PERK-mediated ER stress-apoptosis. To further investigate the role of P2X7 in OA, we injected mTOR antagonist rapamycin or P2X7 antagonist A740003 into the knee joints of ACLT rats. Our results demonstrated that mTOR inhibition induced autophagy, decreased apoptosis, and reduced cartilage loss. However, injection of mTOR agonist MHY1485 or Bz-ATP had the opposite effect. In summary, our results indicated that during the early stages of moderate-intensity exercise, P2X7 was activated and autophagic flux was increased, delaying OA development. At the later stages, P2X7 became over-activated, and the number of apoptotic cells increased, promoting OA development. We propose that the IRE1-mTOR-PERK signaling axis was involved in the regulation of autophagy inhibition and the induction of apoptosis. Our findings provide novel insights into the positive and preventative effects of exercise on OA, suggesting that the intensity and duration of exercise play a critical role. We also demonstrated that on a molecular level, P2X7 and its downstream pathways could be potential therapeutic targets for OA.

Abstract Background Acute respiratory distress syndrome (ARDS) is a severe form of lung injury characterized by damage to the epithelial barrier with subsequent pulmonary edema and hypoxic respiratory failure. ARDS is a significant medical problem in intensive care units with associated high care costs. There are many potential causes of ARDS; however, alveolar injury associated with mechanical ventilation, termed ventilator-induced lung injury (VILI), remains a well-recognized contributor. It is thus critical to understand the mechanism of VILI. Based on our published preliminary data, we hypothesized that the endoplasmic reticulum (ER) stress response molecule Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) plays a role in transmitting mechanosensory signals the alveolar epithelium. Methods ER stress signal responses to mechanical stretch were studied in ex-vivo ventilated pig lungs. To explore the effect of PERK inhibition on VILI, we ventilated live rats and compared lung injury parameters to non-ventilated controls. The effect of stretch-induced epithelial ER Ca2+ signaling on PERK was studied in stretched alveolar epithelial monolayers. To confirm the activation of PERK in human disease, ER stress signaling was compared between ARDS and non-ARDS lungs. Results Our studies revealed increased PERK-specific ER stress signaling in response to overstretch. PERK inhibition resulted in dose-dependent improvement of alveolar inflammation and permeability. Our data indicate that stretch-induced epithelial ER Ca2+ release is an activator of PERK. Experiments with human lung tissue confirmed PERK activation by ARDS. Conclusion Our study provides evidences that PERK is a mediator stretch signals in the alveolar epithelium.

During the perinatal period, the abnormally high plasma non-esterified fatty acids (NEFA) concentration caused by the negative energy balance (NEB) can impose a significant metabolic stress on the liver of dairy cows. Endoplasmic reticulum (ER) stress is an important adaptive response that can serve to maintain cell homeostasis in the event of stress. The protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is the most rapidly activated cascade when ER stress occurs in cells and has an important impact on the regulation of hepatic lipid metabolism and autophagy modulation. However, it is unknown whether NEFA can affect autophagy through modulating the PERK pathway, under NEB conditions. In this study, we provide evidence that NEFA treatment markedly increased lipid accumulation, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the expression of glucose-regulated protein 78 (Grp78), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). More importantly, NEFA treatment can cause a substantial increase in the protein levels of autophagy-related gene 7 (ATG7), Beclin-1 (BECN1), sequestosome-1 (p62), and microtubule-associated protein 1 light chain 3 (LC3)-II, and in the number of autophagosomes in primary bovine hepatocytes. The addition of GSK2656157 (PERK phosphorylation inhibitor) can significantly inhibit the effect of NEFA on autophagy and can further increase lipid accumulation. Overall, our results indicate that NEFA could promote autophagy via the PERK pathway in bovine hepatocytes. These findings provide novel evidence about the potential role of the PERK signaling pathway in maintaining bovine hepatocyte homeostasis.

TRPP2, a Ca-permeable non-selective cation channel, has been shown to negatively regulate cell cycle, but the mechanism underlying this regulation is unknown. Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine extensively involved in immune system regulation, cell proliferation and cell survival. However, the effects and mechanisms for the role of TNF-α in laryngeal cancer remain unclear. Here, we demonstrated using western blot analyses and intracellular Ca concentration measurements that TNF-α treatment suppressed both TRPP2 expression and ATP-induced Ca release in a laryngeal cancer cell line (Hep-2). Knockdown of TRPP2 by a specific siRNA significantly decreased ATP-induced Ca release and abolished the effect of TNF-α on the ATP-induced Ca release. TNF-α treatment also enhanced Hep-2 cell proliferation and growth, as determined using cell counting and flow cytometry cell cycle assays. Moreover, TNF-α treatment down-regulated phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK) and phosphorylated eukaryotic translation initiation factor (p-eIF2α) expression levels, without affecting PERK and eIF2α expression levels in Hep-2 cells. We concluded that suppressing TRPP2 expression and TRPP2-mediated Ca signaling may be one mechanism underlying TNF-α-enhanced Hep-2 cell proliferation. These results offer new insights into the mechanisms of TNF-α-mediated laryngeal cancer cell proliferation, and provide evidences showing a potential role of TNF-α in the development of laryngeal cancer. [ABSTRACT FROM AUTHOR]

GSK2606414 is a novel, highly selective inhibitor of protein kinase R-like endoplasmic reticulum kinase (PERK). GSK2606414 and its analogues have recently been demonstrated to delay tumor growth and prevent neurodegeneration. The present study investigated the effects of GSK2606414 on proliferation, apoptosis, and the expression of activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein homologous protein (CHOP) and vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (RPE) cells under endoplasmic reticulum (ER) stress. ARPE-19 human RPE cells were treated with 0.01-50 µM GSK2606414, and ER stress was induced by thapsigargin (TG) treatment. Cell proliferation was assessed using the Cell Counting kit-8 cell viability assay. Apoptosis was detected by Annexin-V/propidium iodide double staining using flow cytometry. Western blot analysis was used to measure eukaryotic initiation factor 2a (eIF2a) phosphorylation levels. ATF4, CHOP and VEGF mRNA expression levels were assessed using reverse transcription-quantitative polymerase chain reaction. GSK2606414 treatment inhibited RPE cell proliferation in a dose-dependent manner, however it did not induce apoptosis. In addition, GSK2606414 treatment inhibited eIF2a phosphorylation and reduced CHOP and VEGF mRNA expression levels in RPE cells under TG-induced ER stress. To the best of our knowledge, the present study is the first to demonstrate that GSK2606414 has a potential antiproliferative effect in RPE cells in vitro. This effect appeared to be achieved via inhibition of the PERK/ATF4/CHOP signaling pathway and suppression of VEGF expression levels. [ABSTRACT FROM AUTHOR]

Stress granules (SGs) are sites of mRNA storage that are formed in response to various conditions of stress, including viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. In this study, we found that infection of PRRSV strain WUH3 (genotype 2 PRRSV) induced stable formation of robust SGs in MARC-145 cells, as demonstrated by the recruitment of marker proteins of SGs, including TIA1, G3BP1, and eIF3η. Treatment with specific inhibitors or siRNAs against the stress kinases that are involved in SG formation revealed that PRRSV induced SG formation through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Impairment of SG assembly by concomitant knockdown of the SG marker proteins (TIA1, G3BP1, and TIAR) did not affect PRRSV growth, while significantly enhanced PRRSV-induced NF-kB subunit p65 phosphorylation and inflammatory cytokine production. Taken together, our results demonstrate that PRRSV induces SG formation via a PERK-dependent pathway and that SGs are involved in the signaling pathway of the PRRSV-induced inflammatory response in MARC-145 cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Negative energy balance can lead to the mobilization of large amounts of body fat. A large amount of non-esterified fatty acids (NEFA) produced by lipolysis not only can be used for energy generation by β-oxidation in the liver but can also act as a potential regulator of lipid metabolism. The present study provides evidence that NEFA can activate hepatocyte autophagy through the protein kinase R-like endoplasmic reticulum kinase (PERK) signaling pathway. Autophagy has been reported to contribute to lipid metabolism through promoting the breakdown of intracellular lipids. These findings enable a better understanding of the redistribution and utilization of energy during the perinatal period of dairy cows. During the perinatal period, the abnormally high plasma non-esterified fatty acids (NEFA) concentration caused by the negative energy balance (NEB) can impose a significant metabolic stress on the liver of dairy cows. Endoplasmic reticulum (ER) stress is an important adaptive response that can serve to maintain cell homeostasis in the event of stress. The protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is the most rapidly activated cascade when ER stress occurs in cells and has an important impact on the regulation of hepatic lipid metabolism and autophagy modulation. However, it is unknown whether NEFA can affect autophagy through modulating the PERK pathway, under NEB conditions. In this study, we provide evidence that NEFA treatment markedly increased lipid accumulation, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the expression of glucose-regulated protein 78 (Grp78), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). More importantly, NEFA treatment can cause a substantial increase in the protein levels of autophagy-related gene 7 (ATG7), Beclin-1 (BECN1), sequestosome-1 (p62), and microtubule-associated protein 1 light chain 3 (LC3)-II, and in the number of autophagosomes in primary bovine hepatocytes. The addition of GSK2656157 (PERK phosphorylation inhibitor) can significantly inhibit the effect of NEFA on autophagy and can further increase lipid accumulation. Overall, our results indicate that NEFA could promote autophagy via the PERK pathway in bovine hepatocytes. These findings provide novel evidence about the potential role of the PERK signaling pathway in maintaining bovine hepatocyte homeostasis. [ABSTRACT FROM AUTHOR]

• The activation of the PERK signaling pathway can effectively alleviate the accumulation of TG in hepatocytes induced by NEFA. • The NEFA-mediated PERK signaling pathway can promote lipid de novo synthesis, stimulate lipolysis, and inhibit lipid transport. • The PERK signaling pathway activation has been deemed necessary for the stability of lipid metabolism in the liver of dairy cows. During the periparturient transition period, negative energy balance (NEB) characterized by high concentrations of non-esterified fatty acids (NEFA) may cause fatty liver and ketosis in dairy cows. Previous studies have shown that the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the endoplasmic reticulum stress (ERS) response plays an important role in lipid metabolism in hepatocytes. This study, therefore, investigated the role of the PERK-branch in NEFA-induced fatty liver. Different concentrations of NEFA or GSK2656157 (a novel catalytic inhibitor of PERK) were used to treat hepatocytes isolated from calves. The NEFA treatment significantly increased the triacylglycerol (TG) content, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the abundance of glucose-regulated protein 78 (Grp78), C/EBP homologous protein (CHOP), sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FASN), peroxisome proliferator-activated receptor-α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), apolipoprotein B (APOB), and the low-density lipoprotein receptor (LDLR). Compared with the 1.2 mM NEFA group, inhibition of PERK activity further increased the TG content in hepatocytes, the very-low-density lipoprotein (VLDL) content in the supernatant and the protein abundance of APOB while reducing the expression and nuclear levels of SREBP-1c and PPARα, as well as the expression of CPT1A and CPT2. In conclusion, the results showed that the NEFA-induced PERK-eIF2α signaling pathway promotes lipid synthesis, lipid oxidation, but inhibits the assembly and secretion of VLDL. Therefore, during the transition period, the activation of the PERK-eIF2α signaling pathway in the liver of dairy cows could defeat the acid-induced lipotoxicity and provide energy to alleviate NEB. [ABSTRACT FROM AUTHOR]

Background: Acute respiratory distress syndrome (ARDS) is a severe form of lung injury characterized by damage to the epithelial barrier with subsequent pulmonary edema and hypoxic respiratory failure. ARDS is a significant medical problem in intensive care units with associated high care costs. There are many potential causes of ARDS; however, alveolar injury associated with mechanical ventilation, termed ventilator-induced lung injury (VILI), remains a well-recognized contributor. It is thus critical to understand the mechanism of VILI. Based on our published preliminary data, we hypothesized that the endoplasmic reticulum (ER) stress response molecule Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) plays a role in transmitting mechanosensory signals the alveolar epithelium.Methods: ER stress signal responses to mechanical stretch were studied in ex-vivo ventilated pig lungs. To explore the effect of PERK inhibition on VILI, we ventilated live rats and compared lung injury parameters to non-ventilated controls. The effect of stretch-induced epithelial ER Ca2+ signaling on PERK was studied in stretched alveolar epithelial monolayers. To confirm the activation of PERK in human disease, ER stress signaling was compared between ARDS and non-ARDS lungs.Results: Our studies revealed increased PERK-specific ER stress signaling in response to overstretch. PERK inhibition resulted in dose-dependent improvement of alveolar inflammation and permeability. Our data indicate that stretch-induced epithelial ER Ca2+ release is an activator of PERK. Experiments with human lung tissue confirmed PERK activation by ARDS.Conclusion: Our study provides evidences that PERK is a mediator stretch signals in the alveolar epithelium. [ABSTRACT FROM AUTHOR]