[Co-exposure of carbon black and cadmium induces autophagy and inflammation in human bronchial epithelial cells via PERK pathway].

Objective: To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells. Methods: In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 µg/ml Cd, 100 µg/ml CBNPs, 100 µg/ml CBNPs+2 µg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results: There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined (P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group (P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression (P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group (P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group (P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene (P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 (P<0.05), but the interaction between the two chemicals had no statistical significance (P>0.05) . Conclusion: CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.

20(S)-protopanaxadiol induces apoptosis in human umbilical vein endothelial cells by activating the PERK-eIF2alpha-ATF4 signaling pathway.

20(S)-protopanaxadiol (PPD)-type ginsenosides are generally believed to be the most pharmacologically active components of Panax ginseng. These compounds induce apoptotic cell death in various cancer cells, which suggests that they have anti-cancer activity. Anti-angiogenesis is a promising therapeutic approach for controlling angiogenesis-related diseases such as malignant tumors, age-related macular degeneration, and atherosclerosis. Studies showed that 20(S)-PPD at low concentrations induces endothelial cell growth, but in our present study, we found 20(S)-PPD at high concentrations inhibited cell growth and mediated apoptosis in human umbilical vein endothelial cells (HUVECs). The mechanism by which high concentrations of 20(S)-PPD mediate endothelial cell apoptosis remains elusive. The present current study investigated how 20(S)-PPD induces apoptosis in HUVECs for the first time. We found that caspase-9 and its downstream caspase, caspase-3, were cleaved into their active forms after 20(S)-PPD treatment. Treatment with 20(S)-PPD decreased the level of Bcl-2 expression but did not change the level of Bax expression. 20(S)-PPD induced endoplasmic reticulum stress in HUVECs and stimulated UPR signaling, initiated by protein kinase R-like endoplasmic reticulum kinase (PERK) activation. Total protein expression and ATF4 nuclear import were increased, and CEBP-homologous protein (CHOP) expression increased after treatment with 20(S)-PPD. Furthermore, siRNA-mediated knockdown of PERK or ATF4 inhibited the induction of CHOP expression and 20(s)-PPD-induced apoptosis. Collectively, our findings show that 20(S)-PPD inhibits HUVEC growth by inducing apoptosis and that ATF4 expression activated by the PERK-eIF2α signaling pathway is essential for this process. These findings suggest that high concentrations of 20(S)-PPD could be used to treat angiogenesis-related diseases.

Protein kinase R-like endoplasmatic reticulum kinase is a mediator of stretch in ventilator-induced lung injury.

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.

Protein-rich foods, sea foods, and gut microbiota amplify immune responses in chronic diseases and cancers - Targeting PERK as a novel therapeutic strategy for chronic inflammatory diseases, neurodegenerative disorders, and cancer.

The endoplasmic reticulum (ER) is a cellular organelle that is physiologically responsible for protein folding, calcium homeostasis, and lipid biosynthesis. Pathological stimuli such as oxidative stress, ischemia, disruptions in calcium homeostasis, and increased production of normal and/or folding-defective proteins all contribute to the accumulation of misfolded proteins in the ER, causing ER stress. The adaptive response to ER stress is the activation of unfolded protein response (UPR), which affect a wide variety of cellular functions to maintain ER homeostasis or lead to apoptosis. Three different ER transmembrane sensors, including PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme-1 (IRE1), are responsible for initiating UPR. The UPR involves a variety of signal transduction pathways that reduce unfolded protein accumulation by boosting ER-resident chaperones, limiting protein translation, and accelerating unfolded protein degradation. ER is now acknowledged as a critical organelle in sensing dangers and determining cell life and death. On the other hand, UPR plays a critical role in the development and progression of several diseases such as cardiovascular diseases (CVD), metabolic disorders, chronic kidney diseases, neurological disorders, and cancer. Here, we critically analyze the most current knowledge of the master regulatory roles of ER stress particularly the PERK pathway as a conditional danger receptor, an organelle crosstalk regulator, and a regulator of protein translation. We highlighted that PERK is not only ER stress regulator by sensing UPR and ER stress but also a frontier sensor and direct senses for gut microbiota-generated metabolites. Our work also further highlighted the function of PERK as a central hub that leads to metabolic reprogramming and epigenetic modification which further enhanced inflammatory response and promoted trained immunity. Moreover, we highlighted the contribution of ER stress and PERK in the pathogenesis of several diseases such as cancer, CVD, kidney diseases, and neurodegenerative disorders. Finally, we discuss the therapeutic target of ER stress and PERK for cancer treatment and the potential novel therapeutic targets for CVD, metabolic disorders, and neurodegenerative disorders. Inhibition of ER stress, by the development of small molecules that target the PERK and UPR, represents a promising therapeutic strategy.

Chloromethylisothiazolinone induces ER stress-induced stress granule formation in human 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.

[Effect of acupuncture on protein kinase R-like endoplasmic reticulum kinase/eukaryotic translation initiation factor 2α signaling pathway in hippocampus of rats with post-traumatic stress disorder].

OBJECTIVE: To observe the effect of acupuncture on the protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α (eIF2α) signaling pathway in the hippocampus of rats with post-traumatic stress disorder (PTSD), so as to explore the underlying mechanism of acupuncture in treating PTSD. METHODS: Twenty-eight SD rats were randomly divided into normal, model, acupuncture and sertraline groups, with 7 rats in each group. The PTSD model was established by single prolonged stress method. The next day after modeling, acupuncture was applied to "Baihui" (GV20) and "Dazhui" (GV14) of rats in the acupuncture group for 10 min, once a day for 7 days. Sertraline (10 mg/kg) was given by gavage to rats of the sertraline group daily for 7 days. The behavioral changes of rats were detected by elevated cross maze experiment and new object recognition experiment. The expression levels of PERK,phosphorylated(p)-PERK, eIF2α, p-eIF2α and activating transcription factor 4 (ATF4) proteins in hippocampus were detected by Western blot. The ultrastructure of hippocampal neurons was observed by transmission electron microscopy. RESULTS: Compared with the normal group, the percentage of times and retention time of entering the open arm of the elevated cross maze experiment, and new object recognition index were significantly decreased (P<0.01); the expression levels of p-PERK, p-eIF2α and ATF4 proteins in hippocampus were significantly increased (P<0.05) of rats in the model group. Compared with the model group, the percentage of times and retention time of entering the open arm, and new object recognition index were significantly increased (P<0.05，P<0.01), the expression levels of p-PERK, p-eIF2α and ATF4 proteins in hippocampus were significantly decreased (P<0.05, P<0.01) of rats in the acupuncture and sertraline groups; the expression level of eIF2α protein was significantly decreased (P<0.05) in the sertraline group. Hippocampal neurons in the model group were damaged, the rough endoplasmic reticulum showed severe dilation, the mitochondrial cristae showed reduction or mild cavitation; compared with the model group, hippocampal neurons structural damage and the rough endoplasmic reticulum dilation were alleviated， and only some of the mitochondrial cristae decreased in the acupuncture and sertraline groups. CONCLUSION: Acupuncture can alleviate the anxiety behavior as well as the recognition and memory ability of PTSD rats, and its mechanism may be related to the inhibition of hippocampus PERK/eIF2α signaling pathway and the reduction of hippocampal neuron damage caused by endoplasmic reticulum stress.

Trimethylamine N-oxide promotes hyperoxaluria-induced calcium oxalate deposition and kidney injury by activating autophagy.

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.

miR-340-5p affects oral squamous cell carcinoma (OSCC) cells proliferation and invasion by targeting endoplasmic reticulum stress proteins.

Lip and oral cancer is the 12th most common malignancy and oral squamous cell carcinoma (OSCC) represents about 90% of all oral malignant tumors, with an annual mortality rate exceeding 50%. Recent studies have concluded that endoplasmic reticulum stress may have a close link to tumor genesis, progression, and prognosis. As an epigenetic regulatory factor, miRNA exerts a substantial effect on tumor development. This study found that transcription factor 6 (ATF6) and Protein kinase R-like endoplasmic reticulum kinase (PERK) were abnormally increased within OSCC tissue samples and oral cancer cell lines. The biological functions of ATF6 and PERK within CAL-27 and SCC-9 oral cancer cell lines were investigated. In vitro experiments revealed that silencing ATF6 and PERK suppressed the ability of cells to proliferate and to invade and mitigated cell endoplasmic reticulum (ER) stress. As predicted by bioinformatics analyses and experiments, miR-340-5p could simultaneously bind to ATF6 and PERK 3' untranslated region (UTR) and inhibit ATF6 and PERK expression. miR-340-3p overexpression inhibited while down-regulation of miR-340-5p boosted the invading and proliferating ability of oral cancer cells, and miR-340-3p also affects ER stress. When co-transfected in oral cancer cells, dynamic effects of miR-340-5p and its targets PERK and ATF6 on cell phenotypes in vitro and in vivo were investigated. PERK or ATF6 overexpression dramatically attenuated phenotypes of miR-340-5p up-regulation. Altogether, miR-340-5p targets the endoplasmic reticulum stress proteins PERK and ATF6 to affect OSCC cell proliferation and invasion.

NEFA Promotes Autophagosome Formation through Modulating PERK Signaling Pathway in Bovine Hepatocytes.

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.

Elucidation of the mechanism of NEFA-induced PERK-eIF2α signaling pathway regulation of lipid metabolism in bovine hepatocytes.

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.

IRE1-mTOR-PERK Axis Coordinates Autophagy and ER Stress-Apoptosis Induced by P2X7-Mediated Ca2+ Influx in Osteoarthritis.

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.

Genotoxicity and mutagenicity assessment of a standardized extract (P2Et) obtained from Caesalpinia spinosa.

The standardized P2Et extract obtained from Caesalpinia spinosa has shown antioxidant, and direct antitumor activity, but also activation of specific immune response through the induction of tumor immunogenic cell death in breast and melanoma cancer models. The present work evaluated the mutagenicity and genotoxicity profile of P2Et to continue the development of the P2Et. Genotoxicity was evaluated by OECD 1997 a guideline and mutagenicity by OECD 2016. At P2Et's doses of 500, 1000, and 2000 mg/kg body weight in mice (Mus musculus), the difference between the number of micronuclei in PCE of the groups were not statistically significant (17 (negative control), 15 (500 mg/kg), 15 (1000 mg/kg), 19 (2000 mg/kg) and 271 (positive control). Similarly, P2Et did not induce gene mutations by base pair changes or frameshifts in the genome of Salmonella Typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 at the tested range of concentrations up to 5000 µg/plate in the absence and presence of metabolic activation. Therefore, the P2Et was considered as non- mutagenic and non-genotoxic at the conditions of the tests.

A simplified aortic ring assay: A useful ex vivo method to assess biochemical and functional parameters of angiogenesis.

We present a simplified method for conducting aortic ring assays which yields robust sprouting and high reproducibility targeted towards matrix biologists studying angiogenesis and extracellular matrix signaling. Main adjustments from previously established protocols include embedding aortic rings between two layers of 3D type I collagen matrix and supplementing with vascular endothelial media. We also introduce a concise and effective staining protocol for obtaining high-resolution images of intracellular and extracellular matrix proteins along with a more accurate protocol to quantify angiogenesis. Importantly, we present a novel method to perform biochemical analyses of vessel sprouting without contamination from the aortic ring itself. Overall, our refined method enables detection of low abundance and phosphorylated proteins and provides a straightforward ex vivo angiogenic assay that can be easily reproduced by those in the matrix biology field.

Porcine Reproductive and Respiratory Syndrome Virus Infection Induces Stress Granule Formation Depending on Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) in MARC-145 Cells.

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-κB 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.

Tumor necrosis factor α accelerates Hep-2 cells proliferation by suppressing TRPP2 expression.

TRPP2, a Ca2+-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 Ca2+ concentration measurements that TNF-α treatment suppressed both TRPP2 expression and ATP-induced Ca2+ release in a laryngeal cancer cell line (Hep-2). Knockdown of TRPP2 by a specific siRNA significantly decreased ATP-induced Ca2+ release and abolished the effect of TNF-α on the ATP-induced Ca2+ 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 Ca2+ 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.