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Background and Purpose: Traumatic brain injury (TBI) destroys white matter, and this destruction is aggravated by secondary neuroinflammatory reactions. Although white matter injury (WMI) is strongly correlated with poor neurological function, understanding of white matter integrity maintenance is limited, and no available therapies can effectively protect white matter. One candidate approach that may fulfill this goal is cannabinoid receptor 2 (CB2) agonist treatment. Here, we confirmed that a selective CB2 agonist, JWH133, protected white matter after TBI., Methods: The motor evoked potentials (MEPs), open field test, and Morris water maze test were used to assess neurobehavioral outcomes. Brain tissue loss, WM damage, Endoplasmic reticulum stress (ER stress), microglia responses were evaluated after TBI. The functional integrity of WM was measured by diffusion tensor imaging (DTI) and transmission electron microscopy (TEM). Primary microglia and oligodendrocyte cocultures were used for additional mechanistic studies., Results: JWH133 increased myelin basic protein (MBP) and neurofilament heavy chain (NF200) levels and anatomic preservation of myelinated axons revealed by DTI and TEM. JWH133 also increased the numbers of oligodendrocyte precursor cells and mature oligodendrocytes. Furthermore, JWH133 drove microglial polarization toward the protective M2 phenotype and modulated the redistribution of microglia in the striatum. Further investigation of the underlying mechanism revealed that JWH133 downregulated phosphorylation of the protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK) signaling pathway and its downstream signals eukaryotic translation initiation factor 2 α (eIF2α), activating transcription factor 4 (ATF4) and Growth arrest and DNA damage-inducible protein (GADD34); this downregulation was followed by p-Protein kinase B(p-Akt) upregulation. In primary cocultures of microglia and oligodendrocytes, JWH133 decreased phosphorylated PERK expression in microglia stimulated with tunicamycin and facilitated oligodendrocyte survival. These data reveal that JWH133 ultimately alleviates WMI and improves neurological behavior following TBI. However, these effects were prevented by SR144528, a selective CB2 antagonist., Conclusions: This work illustrates the PERK-mediated interaction between microglia and oligodendrocytes. In addition, the results are consistent with recent findings that microglial polarization switching accelerates WMI, highlighting a previously unexplored role for CB2 agonists. Thus, CB2 agonists are potential therapeutic agents for TBI and other neurological conditions involving white matter destruction., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Endoplasmic reticulum (ER) stress has been reported to play a role in acute lung injury (ALI), yet the in-depth mechanism remains elusive. This study aims to investigate the effect of ER stress-induced autophagy of alveolar macrophage (AM) on acute lung injury (ALI) and airway inflammation using mouse models. ALI models were induced by intranasal instillation of lipopolysaccharide (LPS). The lung weight/body weight (LW/BW) ratio and excised lung gas volume (ELGV) in each group were measured. Mouse bronchoalveolar lavage fluid (BALF) was collected for cell sorting and protein concentration determination. Expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in lung tissues and BALF was also detected. Mouse AMs were isolated to observe the autophagy. Expression of GRP78, PERK, LC3I, LC3II and Beclin1 was further determined. The results indicated that tunicamycin (TM) elevated GRP78 and PERK expression of AMs in ALI mice in a dose-dependent manner. Low dosage of TM abated LC3I expression, increased LC3II and Beclin1 expression, triggered ER stress and AM autophagy, and alleviated pathological changes of AMs in ALI mice. Also, in ALI mice, low dosage of TM attenuated goblet cell proliferation of tracheal wall, and declined LW/BW ratio, ELGV, total cells and neutrophils, protein concentrations in BALF, and IL-6 and TNF-α expression in lung tissues and BALF. Collectively, this study suggests that a low dosage of TM-induced ER stress can enhance the autophagy of AM in ALI mice models, thus attenuating the progression of ALI and airway inflammation.

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a multimeric protein complex that mediates maturation of the cytokines IL-1β and IL-18 as well as release of the proinflammatory protein high-mobility group box 1 (HMGB1) and contributes to several inflammatory diseases, including sepsis, gout, and type 2 diabetes. In this context, the well-studied active complement fragment C5a and its receptor C5aR1 or C5aR2 orchestrate the inflammatory responses in many diseases. Although a C5a-C5aR interaction in NLRP3-associated diseases has been suggested, little is known about the details of C5a-C5aR cross-talk with the NLRP3 inflammasome in macrophages. In this study, using mice and murine macrophages and cytokines, immunoblotting, siRNA, and quantitative real-time PCR assays, we demonstrate that C5aR2 deficiency restricts activation of the NLRP3 inflammasome and release of HMGB1 both in vitro and in vivo Mechanistically, we found that C5aR2 promotes NLRP3 activation by amplifying dsRNA-dependent PKR expression, which is an important NLRP3-activating factor. We also observed that elevation of PKR expression because of the C5a-C5aR2 interaction depends on the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase pathway and type I IFN signaling. In conclusion, these findings reveal that C5aR2 contributes to NLRP3 inflammasome activation and HMGB1 release from macrophages., (© 2019 Yu et al.)

Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, infects epithelial surfaces and establishes latency in the central nervous system, where astrocytes are a major immune cell type. Here, we report changes that occur in the expression of pathogen recognition receptors, such as Toll-like receptors, DNA and RNA sensors, interferons, and interferon-stimulated genes, when astrocytes are infected with HSV-1 strain F. We observed upregulation of Toll-like receptors 2, 6 and 9, MDA5, and DAI along with an increase in the expression of type I interferons and interferon-stimulated genes such as IFIT1, IFIT3 and RNase L. These genes encode proteins that mediate the antiviral immune response.

Cerebral autosomal dominant arteriolopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is the most common form of hereditary small vessel disease (SVD) of the brain. Neuronal apoptosis has been demonstrated in the cortex of patients. Whether it is associated with an activation of the pro-apoptotic protein PKR pathway is unknown. Similarly, activation of autophagy in CADASIL has never been explored. Immunostaining of four CADASIL brains previously analyzed for cortical neuronal apoptosis and five control brains for PKR (phosphoPKR) and autophagy (ATG5, LC3II) activation markers. Significant nuclear pPKR staining was observed in CADASIL neurons comparatively to controls (p = 0.001). No difference was observed between patients and controls with autophagy markers. We demonstrated the activation of PKR pathway in CADASIL. This was not associated with a detectable modulation of autophagy. These results open a new field to explore in order to better understand the mechanisms underlying cortical neurons apoptosis.

IRF9 is a key factor in the JAK-STAT pathway. Under the stimulation of type I IFN, IRF9 interacts with STAT1 and STAT2 to form the IFN-I-stimulated gene factor 3 (ISGF3) which activates the transcription of ISG. However, many studies also showed that the dimmer IRF9/STAT2 rather than the tripolymer IRF9/STAT1/STAT2 acts as the ISGF3 in cells in response to IFN signals. In the present study, the full-length cDNA sequence of IRF9 (termed CiIRF9, KT601055) and STAT2 (term CiSTAT2, KT781914) from grass carp were cloned and identified. A low level of constitutive expression of CiIRF9 was detected by RT-PCR in grass carp tissues, but it was significantly up-regulated by LPS and poly I:C stimulation. In vitro, a high-affinity interaction between CiIRF9 and the promoter of CiIFN or CiPKR was demonstrated by gel mobility shift assay. In vivo, the promoter activities of CiIFN and CiPKR were not only increased by transient transfection of CiIRF9, but also prominently increased by co-transfection of CiIRF9 and CiSTAT2. Moreover, the interaction of CiIRF9 and CiSTAT2 was further investigated by in vivo and in vitro protein interaction assays. Recombinant CiIRF9 and CiSTAT2, both tagged with FLAG (or HA), were expressed in HEK 293T cells by transient transfection experiment. Co-immunoprecipitation assays showed that CiIRF9 can interact with CiSTAT2 in vivo. Soluble GST-ST2-936 (containing the N-terminal and coiled-coil domain of CiSTAT2) was expressed and purified from E. coli. A GST pull-down assay suggested that GST-tagged ST2-936 efficiently bound to FLAG-tagged IRF9. The data indicated that interaction of IRF9 and STAT2 synergistically up-regulated the transcriptional level of IFN and ISG genes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The unfolded protein response (UPR) regulates cell fate following exposure of cells to endoplasmic reticulum stresses. PERK, a UPR protein kinase, regulates protein synthesis and while linked with cell survival, exhibits activities associated with both tumor progression and tumor suppression. For example, while cells lacking PERK are sensitive to UPR-dependent cell death, acute activation of PERK triggers both apoptosis and cell cycle arrest, which would be expected to contribute tumor suppressive activity. We have evaluated these activities in the BRAF-dependent melanoma and provide evidence revealing a complex role for PERK in melanoma where a 50% reduction is permissive for BrafV600E-dependent transformation, while complete inhibition is tumor suppressive. Consistently, PERK mutants identified in human melanoma are hypomorphic with dominant inhibitory function. Strikingly, we demonstrate that small molecule PERK inhibitors exhibit single agent efficacy against BrafV600E-dependent tumors highlighting the clinical value of targeting PERK., Competing Interests: KAS and MCGP are employed by Eli Lilly.

Colorectal cancer is a common type of tumor among both men and women worldwide. Conventional remedies such as chemotherapies pose the risk of side‑effects, and in many cases cancer cells develop chemoresistance to these treatments. Non‑thermal gas plasma (NTGP) was recently identified as a potential tool for cancer treatment. In this study, we investigated the potential use of NTGP to control SNUC5 human colon carcinoma cells. We hypothesized that NTGP would generate reactive oxygen species (ROS) in these cells, resulting in induction of endoplasmic reticulum (ER) stress. ROS generation, expression of ER stress‑related proteins and mitochondrial calcium levels were analyzed. Our results confirmed that plasma‑generated ROS induce apoptosis in SNUC5 cells. Furthermore, we found that plasma exposure resulted in mitochondrial calcium accumulation and expression of unfolded protein response (UPR) proteins such as glucose‑related protein 78 (GRP78), protein kinase R (PKR)‑like ER kinase (PERK), and inositol‑requiring enzyme 1 (IRE1). Elevated expression of spliced X‑box binding protein 1 (XBP1) and CCAAT/enhancer‑binding protein homologous protein (CHOP) further confirmed that ROS generated by NTGP induces apoptosis through the ER stress signaling pathway.

We have previously demonstrated that radiation induced cell death in PKR (-/-) deficient mouse embryo fibroblasts (MEFs) but not in PKR (+/+) wild type MEFs. Our study indicated that PKR can also be involved in survival pathways following radiation therapy through activation of the AKT survival pathways in these MEFs is mediated in part through PKR. The role of PKR on radiation sensitivity in cancer cells has not been evaluated. In this study, we demonstrated that radiation treatment causes nuclear translocation of PKR in human lung cancer cells. The transduction of lung cancer cells with a dominant negative adenoviral PKR vector blocks nuclear translocation of PKR and leads to the reversal of radiation resistance. Plasmid transduction of lung cancer cells with nuclear targeted wild type PKR vectors also increased radiation resistance. This effect is selectively abrogated by plasmid transduction of dominant negative PKR vectors which restore radiation sensitivity. These findings suggest a novel role for PKR in lung cancer cells as a mediator of radiation resistance possibly through translocation of the protein product to the nucleus., Competing Interests: The authors declare no conflicts of interest.

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. We previously showed that the inhibition of placental growth factor (PlGF) exerts antitumour effects and induces vessel normalisation, possibly reducing hypoxia. However, the exact mechanism underlying these effects remains unclear. Because hypoxia and endoplasmic reticulum stress, which activates the unfolded protein response (UPR), have been implicated in HCC progression, we assessed the interactions between PlGF and these microenvironmental stresses., Methods: PlGF knockout mice and validated monoclonal anti-PlGF antibodies were used in a diethylnitrosamine-induced mouse model for HCC. We examined the interactions among hypoxia, UPR activation and PlGF induction in HCC cells., Results: Both the genetic and pharmacological inhibitions of PlGF reduced the chaperone levels and the activation of the PKR-like endoplasmic reticulum kinase (PERK) pathway of the UPR in diethylnitrosamine-induced HCC. Furthermore, we identified that tumour hypoxia was attenuated, as shown by reduced pimonidazole binding. Interestingly, hypoxic exposure markedly activated the PERK pathway in HCC cells in vitro, suggesting that PlGF inhibition may diminish PERK activation by improving oxygen delivery. We also found that PlGF expression is upregulated by different chemical UPR inducers via activation of the inositol-requiring enzyme 1 pathway in HCC cells., Conclusions: PlGF inhibition attenuates PERK activation, likely by tempering hypoxia in HCC via vessel normalisation. The UPR, in turn, is able to regulate PlGF expression, suggesting the existence of a feedback mechanism for hypoxia-mediated UPR that promotes the expression of the angiogenic factor PlGF. These findings have important implications for our understanding of the effect of therapies normalising tumour vasculature.

We have previously shown that the replication of avian reovirus (ARV) in chicken embryo fibroblasts (CEF) is more resistant to the antiviral action of interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study we examined the capacity of these three viruses to induce the expression of IFN when infecting avian cells. Efficient expression of both type-α and type-β IFNs, as well as of the double-stranded RNA (dsRNA)-activated protein kinase (PKR), takes place in ARV-infected CEF, but not in cells infected with VSV or VV. PKR expression is not directly induced by ARV infection, but by the IFN secreted by ARV-infected cells. IFN induction in ARV-infected cells requires viral uncoating, but not viral gene expression, a situation similar to that reported for apoptosis induction by ARV-infected cells. However, our results demonstrate that IFN induction by ARV-infected CEF occurs by a caspase-independent mechanism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR., (© 2015 John Wiley & Sons Ltd.)

The pathogenesis of rheumatoid arthritis (RA) is characterized by inflammation. We aimed to examine the roles of double-stranded RNA-activated protein kinase (PKR) and high-mobility group box chromosomal protein 1 (HMGB1) in a rat model of RA. Male SD rats were divided into three groups: control, RA model, and intervention (RA model plus treatment). The model of RA was made by injecting Freund's adjuvant into the posterior right limb of the rat and the intervention group received a PKR-specific inhibitor C16 after RA modeling. The degree of limb swelling was measured following RA modeling and intervention. In addition, plasma levels of HMGB1 were determined using ELISA. The mRNA and protein levels of PKR and HMGB1 were detected in rat synovium using quantitative PCR and western blot, respectively. The degree of limb swelling in the RA model was increased compared to control, while it was decreased in the intervention model compared to the RA model. Plasma HMGB1 levels in the model group were significantly higher compared to the control group but were lower in the intervention group compared to the model group. PKR and HMGB1 protein and mRNA levels in the rat synovium were elevated in the model group and markedly reduced in the intervention group. Increased levels of PKR and HMGB1 in synovium or blood appear to be involved in the occurrence and development of RA in a rat model. Selective inhibition of PKR improves the symptoms of RA, perhaps by inhibiting the release of HMGB1.

Resveratrol, a natural polyphenolic compound found in foods and beverages, has attracted increasing attention in recent years because of its potent chemopreventive and anti-tumor effects. In this study, the effects of resveratrol on the expression of P-glycoprotein/multi-drug resistance protein 1 (P-gp/MDR1), and the underlying molecular mechanisms, were investigated in oxaliplatin (L-OHP)-resistant colorectal cancer cells (HCT116/L-OHP). Resveratrol downregulated MDR1 protein and mRNA expression levels and reduced MDR1 promoter activity. It also enhanced the intracellular accumulation of rhodamine 123, suggesting that resveratrol can reverse multi-drug resistance by downregulating MDR1 expression and reducing drug efflux. Resveratrol treatment also reduced nuclear factor-κB (NF-κB) activity, reduced phosphorylation levels of IκBα, and reduced nuclear translocation of the NF-κB subunit p65. Moreover, downregulation of MDR1 expression and promoter activity was mediated by resveratrol-induced AMP-activated protein kinase (AMPK) phosphorylation. The inhibitory effects of resveratrol on MDR1 expression and cAMP-responsive element-binding protein (CREB) phosphorylation were reversed by AMPKα siRNA transfection. We found that the transcriptional activity of cAMP-responsive element (CRE) was inhibited by resveratrol. These results demonstrated that the inhibitory effects of resveratrol on MDR1 expression in HCT116/L-OHP cells were closely associated with the inhibition of NF-κB signaling and CREB activation in an AMPK-dependent manner.

Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, and PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to karyopherin alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An imbalance in pro- and anti-inflammation is an important mechanism of steroid resistance in UC (ulcerative colitis), and miRNAs may participate in this process. The present study aimed to explore whether miRNAs play a role in the steroid resistance of UC by regulating gene expression of the inflammation signal pathway. SS (steroid-sensitive) patients, SR (steroid-resistant) patients and healthy individuals were recruited. In vivo miRNA profiles of serum samples showed that miR-195 was decreased significantly in the SR group compared with the SS group (P<0.05). This result was confirmed by qPCR (quantitative real-time PCR) and miRNA ISH (in situ hybridization) in serum and colon tissue samples. Online software was used to identify Smad7 mRNA as a potential target of miR-195. The direct interaction of miR-195 and Smad7 mRNA was investigated using a biotinylated miR-195 pull-down assay. Overexpression of a miR-195 precursor lowered cellular levels of Smad7 protein; conversely, antagonism of miR-195 enhanced Smad7 translation without disturbing Smad7 mRNA levels. A luciferase reporter assay revealed a repressive effect of miR-195 via a single Smad7 3'-UTR target site, and point mutation of this site prevented miR-195-induced repression of Smad7 translation. Furthermore, increased levels of miR-195 led to a decrease in c-Jun and p65 expression. In contrast, transfection with anti-miR-195 led to increased levels of c-Jun and p65 protein. The decrease in miR-195 led to an increase in Smad7 expression and corresponding up-regulation of p65 and the AP-1 (activator protein 1) pathway, which might explain the mechanism of steroid resistance in UC patients., (© 2015 Authors; published by Portland Press Limited.)

Serum amyloid A (SAA) proteins are known to be surrogate markers of sepsis, but their pathogenic roles remain poorly elucidated. Here we provide evidence to support a possible role of SAA as a pathogenic mediator of lethal sepsis. In a subset of septic patients for which serum high mobility group box 1 (HMGB1) levels paralleled the clinical scores, some anti-HMGB1 antibodies detected a 12-kDa protein belonging to the SAA family. In contrast to the most abundant SAA1, human SAA induced double-stranded RNA-activated protein kinase R (PKR) expression and HMGB1 release in the wild-type, but not toll-like receptor 4/receptor for advanced glycation end products (TLR4/RAGE)-deficient, macrophages. Pharmacological inhibition of PKR phosphorylation blocked SAA-induced HMGB1 release, suggesting an important role of PKR in SAA-induced HMGB1 release. In animal models of lethal endotoxemia and sepsis, recombinant SAA exacerbated endotoxemic lethality, whereas SAA-neutralizing immunoglobulins G (IgGs) significantly improved animal survival. Collectively, these findings have suggested SAA as an important mediator of inflammatory diseases. Highlights of this study include: human SAA is possibly only expressed in a subset of septic patients; SAA induces HMGB1 release via TLR4 and RAGE receptors; SAA supplementation worsens the outcome of lethal endotoxemia; whereas SAA-neutralizing antibodies confer protection against lethal endotoxemia and sepsis.

Background: Brain cell death is a major pathological consequence of alcohol neurotoxicity. However, the molecular cascades in alcohol-induced brain tissue injury are unclear., Methods: Using Western blot and double immunofluorescence, we examined the expression of interferon (IFN)-induced protein kinase R (PKR), phosphorylated-PKR (p-PKR), and IFN gamma (IFNγ) in the prefrontal cortex (PFC) of postmortem brains from subjects with alcohol use disorders (AUD)., Results: The protein levels of PKR, p-PKR, and IFNγ were significantly increased in subjects with AUD compared with control subjects without AUD, and a younger age of onset of AUD was significantly correlated with higher protein levels of p-PKR. In addition, elevated PKR- and p-PKR-IR were observed in both neurons and astrocytes in the PFC of subjects with AUD compared to subjects without AUD., Conclusions: The activation of the IFNγ-PKR pathway in PFC of humans is associated with chronic excessive ethanol use with an age of onset dependent manner, and activation of this pathway may play a pivotal role in AUD-related brain tissue injury. This study provides insight into neurodegenerative key factors related to AUD and identifies potential targets for the treatment of alcohol-induced neurotoxicity., (Copyright © 2015 by the Research Society on Alcoholism.)

Objectives: Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in severe pre-eclampsia (SPE) and is characterized by the activation of three signaling pathways: PKR-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring 1 (Ire1). This study was designed to investigate the role of ER stress in the pathogenesis of SPE., Materials and Methods: Placental tissues were collected from 32 women with normal pregnancies and two cohorts of women with early (n = 32) or late onset (n = 32) SPE. The expression of glucose-regulated protein 78 (GRP78), PERK, eukaryotic initiation factor 2 subunit a (eIF2α), activating transcription factor 6 (ATF4), ATF6, Ire1, CHOP (ClEBP homologus protein), and caspase 12 mRNA and protein in the placentas was analyzed using real-time reverse transcription-polymerase chain reaction and Western blotting, respectively., Results: The levels of GRP78, PERK, eIF2α, CHOP, ATF6, and caspase 12 mRNA and protein expression were significantly higher in the placentas of women with early and late SPE than in the control women, whereas there were no differences in ATF6 and Ire1 mRNA and protein., Conclusion: ER stress-induced apoptosis was important in the development of SPE, especially in early onset SPE and was probably due to the activation of the PERK signaling pathway., (Copyright © 2014. Published by Elsevier B.V.)

Unlabelled: Pathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2α and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense., Importance: Protein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported here reveal that the hantavirus nucleocapsid protein counteracts the PKR antiviral response by inhibiting PKR dimerization, which is required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus-infected cells prevents the PKR-induced host translational shutdown to ensure the continuous synthesis of viral proteins required for efficient virus replication., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Elevated intraocular pressure (IOP) is considered as the major risk factor for the loss of retinal ganglion cells (RGCs) and their axons in glaucoma. Lithium chloride (LiCl) inhibits glycogen synthase kinase-3 beta (GSK-3β) and attends PERK-induced endoplasmic reticulum stress (ERs) transition. PERK is a type I transmembrane protein located in the endoplasmic reticulum. PERK pathway activation takes place in ERs early inhibiting protein synthnesis to protect cell and promote cell survival. Here, we firstly evaluate that LiCl reduced IOP when administered intraperitoneally. After 6 weeks, IOP dropped by around 21.9% in LiCl treated rats. Then we investigated the effects of LiCI on PERK-mediated signaling pathways. LiCl treatment activated PERK and inhibited the expression of ROCK-1 and ROCK-2 in a rat model of glaucoma. Collectively, these results suggest that LiCl reduced the IOP through the phosphorylation of PERK by the regulation of PERK/ROCK signaling in glaucoma rat model.

Histone deacetylase 4 (HDAC4) is involved in the regulation of many fundamental cell processes such as proliferation, differentiation, and survival via the modification of their substrates or protein-protein interactions. In this study, we found that HDAC4 could be upregulated under ER stress. There exists a direct interaction between HDAC4 and activating transcription factor 4 (ATF4). In vitro, overexpression of HDAC4 caused the retention of ATF4 in cytoplasm and inhibition of ATF4 transcriptional activity. ER stress could promote cell apoptosis through the upregulation of ATF4 levels and its target genes such as CHOP and TRB3. This effect was exacerbated by downregulation of HDAC4 levels. These results demonstrated that HDAC4 played an important role in the regulation of ER stress-induced apoptosis through interacting with ATF4 and inhibiting its transcriptional activity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Herpes simplex virus type 1 (HSV-1) causes a lytic infection in epithelial cells before being captured and moved via retrograde axonal transport to the nuclei of the sensory neurons of the trigeminal ganglion or dorsal root, where it establishes a latent infection. HSV-1 infection induces an antiviral response through the production of Beta Interferon (IFN-β) in infected trigeminal ganglia. The aim of this work was to characterize the response induced by IFN-β in neuron-enriched trigeminal ganglia primary cultures infected with HSV-1. An antiviral effect of IFN-β in these cultures was observed, including reduced viral production and increased cell survival. In contrast, viral infection significantly decreased both double stranded RNA dependent protein kinase (Pkr) transcription and Jak-1 and Stat-1 phosphorylation, suggesting a possible HSV-1 immune evasion mechanism in trigeminal cells. Additionally, HSV-1 infection upregulated Suppressor of Cytokine Signaling-3 (Socs3) mRNA; upregulation of socs3 was inhibited in IFN-β treated cultures. HSV-1 infection increased the number of Socs3 positive cells and modified the intracellular distribution of Socs3 protein, in infected cells. This neuron-enriched trigeminal ganglia culture model could be used to elucidate the HSV-1 viral cycle in sensory neurons and to study cellular antiviral responses and possible viral evasion mechanisms that underlie the choice between viral replication and latency., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purpose: Molecular mechanism of ossification of the posterior longitudinal ligament (OPLL) remains unclear. This study was to investigate different expressions of PERK between the spinal ligament fibroblasts from OPLL patients and non-OPLL patients, and demonstrate knockdown of PERK protein expression by RNA interference inhibiting expression of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) in the cells from OPLL patients., Methods: Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of OCN, ALP and COL I was detected in the cells from OPLL and non-OPLL patients by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of PERK was detected by Western blotting. And then, after 72 h, when RNA interference against PERK was performed on the cells from OPLL patients, expression of the osteoblast-specific genes was compared again between the transfection group and non-transfection group., Results: Spinal ligament fibroblasts were observed 7-10 days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of OCN, ALP and COL I and protein expression of PERK in the cells from OPLL patients were significantly greater than those from non-OPLL patients. In addition, knockdown of PERK protein expression inhibited the mRNA expressions of OCN, ALP and COL I remarkably in the transfection group compared with the non-transfection group, at 72 h after RNA interference targeting PERK was performed on the cells from OPLL patients., Conclusions: The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and PERK-mediated ER stress might be involved in development of OPLL.

Although the potential pathogenesis of nonalcoholic fatty liver disease (NAFLD) is unclear, increasing evidence indicates that endoplasmic reticulum (ER) stress may link free fatty acids to NAFLD. Since we previously reported that hepatic stimulator substance (HSS) could protect the liver from steatosis, this study is aimed to investigate whether HSS protection could be related with its inhibition on ER stress. The HSS gene was stably transfected into BEL-7402 hepatoma cells and effectively expressed in ER. The palmitic acid (PA)-induced heptocyte lipotoxicity was reproduced in the HSS-transfected cells, and HSS alleviation of the ER stress and apoptosis were subsequently examined. The results showed that PA treatment led to a heavy accumulation of fatty acids within the cells and a remarkable increase in reactive oxygen species (ROS). However, in the HSS-expressing cells, production of ROS was inhibited and ER stress-related marker glucose-regulated protein 78 (GRP-78), sterol regulatory element-binding protein (SREBP), anti-phospho-PRK-1ike ER kinase (p-PERK), anti-phospho-eukaryotic initiation factor 2α (p-eIF2α), and anti-C/EBP homologous protein (CHOP) were downregulated compared with the wild-type or mutant HSS-transfected cells. Furthermore, PA treatment severely impaired the activity of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), leading to imbalanced calcium homeostasis during ER stress, which could be rescued in the HSS-trasfected cells. The protection provided by HSS to the SERCA is identical to that observed with N-acetyl-l-cysteine (NAC) and sodium dimercaptopropane sulfonate (Na-DMPS), which are two typical free radical scavengers. As a consequence, the rate of ER stress-mediated apoptosis in the HSS-expressing cells was significantly reduced. In conclusion, the protective effect of HSS against ER stress may be associated with the removal of ROS to restore the activity of the SERCA.

Human interleukin-24 (IL-24) has been found recently to play a tumor-suppressor role in a variety of tumors, including gliomas. However, the exact mechanism of glioma tumor suppression by IL-24 remains unclear. We collected by surgery 30 gliomas at different grades and evaluated IL-24 and double-stranded RNA-activated protein kinase (PKR) expression using fluorescence quantitative real-time PCR and immunohistochemical techniques. Two human glioma cell lines, U87 and U251, were transfected with Ad5F35-IL24 via recombinant adenovirus-mediated gene transfer and apoptosis, as well as PKR and eIF-2α expression analyzed. The results showed that IL-24 and PKR expression decreased with increasing tumor grade. Compared with cells of the control groups, Ad5F35-IL24-infected U87 and U251 cells exhibited a significantly increased apoptosis and elevated PKR, eIF-2α, p-PKR, and p-eIF-2α levels, while the expression of Bcl-2 was decreased. Finally, IL-24 also sensitized apoptosis of glioma cells to temozolomide (TMZ). This study indicates that IL-24 upregulates expression and activation of PKR, further increasing expression and activation of eIF-2α, and decreasing Bcl-2 to promote apoptosis. IL-24 also increases chemosensitivity of glioma cells to TMZ.

The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-β, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-β, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of β-actin, glycerophosphate dehydrogenase, and β2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.

Introduction: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined., Methods: A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion., Results: Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids., Conclusions: Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α) to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1), resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV) infection in human fibroblasts (HF) induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA), resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1) protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP complexes for SREBP1 processing.

Aims/hypothesis: Immunohistochemical staining reveals that the enteroviral capsid protein VP1 is present at higher frequency in the insulin-containing islets of patients with recent-onset type 1 diabetes than in controls. This is consistent with epidemiological evidence suggesting that enteroviral infection may contribute to the autoimmune response in type 1 diabetes. However, immunostaining of VP1 is not definitive since the antibody widely used to detect the protein (Clone 5D8/1) might also cross-react with additional proteins under some conditions. Therefore, we sought to verify that VP1 immunopositivity correlates with additional markers of viral infection., Methods: Antigen immunoreactivity was examined in formalin-fixed, paraffin-embedded, pancreases from two different collections of type 1 diabetes and control cases: a historical collection from the UK and the nPOD (network of Pancreatic Organ donors with Diabetes) cohort from the USA., Results: VP1 immunoreactivity was present in ~20% of insulin-containing islets of both cohorts under stringent conditions but was absent from insulin-deficient islets. The presence of VP1 was restricted to beta cells but only a minority of these contained the antigen. The innate viral sensor, protein kinase R (PKR) was upregulated selectively in beta cells that were immunopositive for VP1. The anti-apoptotic protein myeloid cell leukaemia sequence-1 (Mcl-1) was abundant in beta cells that were immunonegative for VP1 but Mcl-1 was depleted in cells containing VP1., Conclusions/interpretation: The presence of immunoreactive VP1 within beta cells in type 1 diabetes is associated with a cellular phenotype consistent with the activation of antiviral response pathways and enhanced sensitivity to apoptosis. However, definitive studies confirming whether viral infections are causal to beta cell loss in human diabetes are still awaited.

Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic reticulum (ER) that is unusually susceptible to perturbations in protein synthesis. This biology is believed to account for the exquisite sensitivity of multiple myeloma (MM) to the proteasomal inhibitor bortezomib (BTZ). Despite remarkable response rates to BTZ in MM, BTZ carries the potential for serious side-effects and development of resistance. We, therefore, sought to identify therapeutic combinations that effectively disrupt proteostasis in order to provide new potential treatments for MM. We found that sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, inhibits TNFα-induced Iκβ proteasomal degradation in a manner similar to BTZ. Like BTZ, sulforaphane synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent with clinical activity in MM. ATO and sulforaphane co-treatment augmented apoptotic induction as demonstrated by cleavage of caspase-3, -4 and PARP. The enhanced apoptotic response was dependent upon production of reactive oxygen species (ROS) as demonstrated by glutathione depletion and partial inhibition of the apoptotic cascade after pretreatment with the radical scavenger N-acetyl-cysteine (NAC). Combination treatment resulted in enhanced ER stress signaling and activation of the unfolded protein response (UPR), indicative of perturbation of proteostasis. Specifically, combination treatment caused elevated expression of the molecular chaperone HSP90 (heat shock protein 90) along with increased PERK (protein kinase RNA-like endoplasmic reticulum kinase) and eIF2α phosphorylation and XBP1 (X-box binding protein 1) splicing, key indicators of UPR activation. Moreover, increased splicing of XBP1 was apparent upon combination treatment compared to treatment with either agent alone. Sulforaphane in combination with ATO effectively disrupts protein homeostasis through ROS generation and induction of ER stress to culminate in inhibition of protein secretion and apoptotic induction in MM. Our results suggest that sulforaphane deserves further investigation in combination with ATO in the treatment of MM.

Background: Pregnant women are considered to have a high risk for influenza virus infection, although little is known about the biological reasons for this risk. Antiviral immunity is critical during influenza virus infection, and understanding the changes that occur during pregnancy and the effect of vaccination is essential for improving health outcomes for mother and baby., Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 26 healthy, nonpregnant women and 28 healthy pregnant women and cultured with 2009 pandemic influenza A virus subtype H1N1 (H1N1/09). Protein concentrations of interferon α (IFN-α), IFN-λ, and IFN-γ were measured from culture supernatant. Messenger RNA expression of protein kinase R (PKR) and Toll-like receptors 3, 7, and 9 was also measured from cell lysates., Results: PBMCs from pregnant women produced significantly less IFN-α (median level, 114.06 pg/mL [range, 51.48-394.9]) and IFN-λ (median level, 30.65 pg/mL [range, 0-260]), compared with PBMCs from nonpregnant women (median level, 800.38 pg/mL [range, 259-1458] and 479.87 pg/mL [257.1-1113], respectively; P < .01). PKR expression was also significantly reduced in PBMCs from pregnant women (P < .05). Vaccination significantly improved innate and adaptive immunity in pregnancy (P < .01)., Conclusion: PBMCs from nonvaccinated pregnant women have attenuated antiviral immunity following H1N1/09 stimulation, but vaccination improves this response. These novel findings help improve understanding of the increased susceptibility and disease severity to influenza virus infection during pregnancy and the importance of influenza vaccination.

Objective: Inflammatory bowel diseases (IBDs) feature multiple cellular stress responses, including endoplasmic reticulum (ER) unfolded protein responses (UPRs). UPRs represent autoregulatory pathways that adjust organelle capacity to cellular demand. A similar mechanism, mitochondrial UPR (mtUPR), has been described for mitochondria. ER UPR in intestinal epithelial cells (IECs) contributes to the development of intestinal inflammation, and since mitochondrial alterations and dysfunction are implicated in the pathogenesis of IBDs, the authors characterised mtUPR in the context of intestinal inflammation., Methods: Truncated ornithine transcarbamylase was used to selectively induce mtUPR in a murine IEC line. Dextran sodium sulphate (DSS) was administered to PKR (double-stranded-RNA-activated protein kinase) knockout mice to induce IEC stress in vivo and to test for their susceptibility to DSS-induced colitis. Expression levels of the mitochondrial chaperone chaperonin 60 (CPN60) and PKR were quantified in IECs from patients with IBDs and from murine models of colitis using immunohistochemistry and Western blot analysis., Results: Selective mtUPR induction by truncated ornithine transcarbamylase transfection triggered the phosphorylation of eukaryotic translation initiation factor (eIF) 2α and cJun through the recruitment of PKR. Using pharmacological inhibitors and small inhibitory RNA, the authors identified mtUPR-induced eIF2α phosphorylation and transcription factor activation (cJun/AP1) as being dependent on the activities of the mitochondrial protease ClpP and the cytoplasmic kinase PKR. Pkr(-/-) mice failed to induce CPN60 in IECs upon DSS treatment at early time points and subsequently showed an almost complete resistance to DSS-induced colitis. Under inflammatory conditions, primary IECs from patients with IBDs and two murine models of colitis exhibited a strong induction of the mtUPR marker protein CPN60 associated with enhanced expression of PKR., Conclusion: PKR integrates mtUPR into the disease-relevant ER UPR via eIF2α phosphorylation and AP1 activation. Induction of mtUPR and PKR was observed in IECs from murine models and patients with IBDs. The authors' results indicate that PKR might link mitochondrial stress to intestinal inflammation.

MAPK (mitogen-activated protein kinase) pathway is considered a control regulator in various malignant tumors but its role in esophageal carcinomas remains elusive. In our study, we examined the possible prognostic significance of MAPK pathway in human esophageal cancer. We searched for mutations in exons 18-21 of EGFR gene, codons 12 and 13 of K-RAS gene and exon 15 of B-RAF gene by high resolution melting analysis (HRMA) and pyrosequencing in 44 esophageal carcinomas. Immunohistochemistry was performed in 29 cases in order to evaluate expression levels of pERK (extracellular-signal regulated kinase). In one laser microdissected squamous cell carcinoma, a somatic K-RAS mutation at codon 12 was detected, whereas none of the cases displayed mutations in EGFR and B-RAF genes. Elevated nuclear as well as cytoplasmic pERK expression (100% and 62% of cases respectively) was observed independently of EGFR and B-RAF mutational status. Increasing pERK nuclear and cytoplasmic expression as well as the intensity of nuclear staining was found to be significantly correlated with tumor grade in univariate and multivariate statistical analysis. Our findings depict the presence of activated ERK despite the low frequency of upstream alterations, implicating ERK activation in the acquisition of a more aggressive phenotype in esophageal cancer., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The aim of the study was to determine the ability of several chemically different antiviral substances to induce the expression of interferon α(IFNα), PKR, OAS1a and RNAse L genes in the rat liver. The investigated substances included Amizon, Altabor and Proteflasid, which are already used in practical medicine, and 3',7-dimethylquercetin extracted from Proteflasid, the mixture of synthesized trimethyl- and tetramethylquercetin and Sialospecific lectin from persimmon, which are at the stage of preclinical trial and experimental research respectively. The content of corresponding mRNAs in total RNA was detected with the help of reverse transcription and polymerase chain reaction in real time. The results have shown that all investigated substances induce the expression of genes α, PKR, OAS RNAse L in specific manner. The combination of 3',7-dimethylquercetin + lectin from persimmon had the highest stimulating effect exceeding the effect of each component of the mixture and the influence of Heberon (recombinant IFNα2b) and PolyI-polyC as the standard inducers of IFNα and its target genes. The ability of all substances to specifically induce the expression of IFNa and its target genes, the absence of correlation between the levels of IFNα and its target genes expression as well as between target genes themselves indicate that the mechanism of antiviral activity of the investigated substances is connected not only with up-regulation of IFNα and potential IFNα mediated effects.

IFN-α is a widely used treatment for hepatitis B virus (HBV) infection, and IFN resistance caused by viral and/or host factors is currently a challenging clinical problem. A better understanding of the molecular mechanisms underlying IFN immunotherapy in the treatment of viral infection would be very beneficial clinically and is of immense clinical importance. Calreticulin (CRT) is an endoplasmic reticulum luminal calcium-binding chaperone that is involved in the regulation of calcium homoeostasis, the folding of newly synthesized proteins, and many other cellular functions. However, little is known about the role of CRT in HBV infection. In this study, we observed high levels of CRT expression in the sera and PBMCs of patients with HBV relative to those of healthy individuals. HBV upregulated the expression of CRT at the transcriptional level. Further investigation showed that HBV-induced CRT enhanced HBV replication by antagonizing the IFN pathway. CRT suppressed the production of endogenous IFN-α by reducing the nuclear translocation of IFN regulatory factor-7 but not IFN regulatory factor-3. Furthermore, CRT also suppressed the antiviral activity of IFN-α by inhibiting the phosphorylation of STAT1 and decreasing the expression of two IFN-α downstream effectors, protein kinase R and 2',5'-oligoadenylate synthetase. Our results offer new insights into the pathogenesis of HBV infection and may provide potential targets for anti-HBV therapy.

We have previously shown that voluntary wheel running activity in mice is associated with an increase in the Endoplasmic Reticulum (ER) Unfolded Protein stress response in multiple regions of the brain. Mice that are given access to running wheels show large variations in individual running activity. In contrast, when food is placed on the lid of their cages, rather than within the cage, all mice must undertake significant physical activity in order to gain access to their food. Hence we investigated the effects of food location on food intake and growth of C57BL/6 mice and on the activity of the ER stress system in the brain as reflected in the expression of two marker genes, Xbp1 and Atf6. Mice that had food in cups within their cages and allowed access to running wheels showed the anticipated changes in food intake, body weight and ER stress in the hippocampus compared to mice with no access to running wheels. Locating the food on the lid had no effect on food intake but reduced weight gain significantly. Likewise, locating food on the lid increased the expression of both Xbp1 and Atf6 in the hippocampi in the absence of any running wheel activity. Voluntary wheel running activity was reduced in mice whose food was located on the cage lid and this running actually reduced the expression of the two marker ER stress genes. We conclude that the usual practice of providing food for mice on their cage lids provides a significant level of physical activity that alters the metabolic status and increases ER stress. As such, this may not be the optimal model for the majority of mouse studies that are reported in the literature and it may significantly alter the interpretation of the effect of wheel running activity on ER stress. The differential effects of food location on hippocampal Bdnf gene expression also suggest that BDNF does not directly regulate UPR activity but may be coordinately regulated in response to running activity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: To investigate the myxovirus-resistance protein A (MxA) and double-stranded RNA-activated protein kinase (PKR) genetic response to interferon (IFN) therapy in hepatitis C virus (HCV) genotype 4-infected patients. Moreover, we studied the association between suppressor of cytokine signaling 3 (SOCS3) gene expression and therapy resistance in genotype 4. Finally, we investigated the novel link between p53 and IFN-stimulated genes (ISGs) in humans., Methods: Gene expression analyses were performed in peripheral blood using TaqMan real-time PCR. Virologic response was assessed with a branched-DNA assay. Genotyping was confirmed., Results: Early virologic responders (EVRs, n = 23) but not non-EVRs (n = 7) showed strong upregulation of PKR at week 12 of therapy compared to baseline. Both EVRs and non-EVRs showed MxA upregulation at week 12 compared to baseline. Baseline SOCS3 expression did not distinguish EVRs from non-EVRs in genotype 4. An association was found between p53 and MxA and PKR gene expression., Conclusion: Measurement of MxA and PKR transcriptional induction during treatment may distinguish EVRs from non-EVRs in genotype 4. SOCS3 gene does not seem to be implicated in therapy resistance in genotype 4. An association between p53 and ISGs expression was shown for the first time in HCV-infected patients, further supporting the contribution of p53 to host antiviral response., (Copyright © 2011 S. Karger AG, Basel.)

Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection.

Chronic spinal cord injury (SCI) results in an accelerated trajectory of several cardiovascular disease (CVD) risk factors and related aging characteristics, however the molecular mechanisms that are activated have not been explored. Adipokines and leptin signaling are known to play a critical role in neuro-endocrine regulation of energy metabolism, and are now implicated in central inflammatory processes associated with CVD. Here, we examine hypothalamic adipokine gene expression and leptin signaling in response to chronic spinal cord injury and with advanced age. We demonstrate significant changes in fasting-induced adipose factor (FIAF), resistin (Rstn), long-form leptin receptor (LepRb) and suppressor of cytokine-3 (SOCS3) gene expression following chronic SCI and with advanced age. LepRb and Jak2/stat3 signaling is significantly decreased and the leptin signaling inhibitor SOCS3 is significantly elevated with chronic SCI and advanced age. In addition, we investigate endoplasmic reticulum (ER) stress and activation of the uncoupled protein response (UPR) as a biological hallmark of leptin resistance. We observe the activation of the ER stress/UPR proteins IRE1, PERK, and eIF2alpha, demonstrating leptin resistance in chronic SCI and with advanced age. These findings provide evidence for adipokine-mediated inflammatory responses and leptin resistance as contributing to neuro-endocrine dysfunction and CVD risk following SCI and with advanced age. Understanding the underlying mechanisms contributing to SCI and age related CVD may provide insight that will help direct specific therapeutic interventions.

Toxoplasmosis is a significant opportunistic infection caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular pathogen that relies on host cell nutrients for parasite proliferation. Toxoplasma parasites divide until they rupture the host cell, at which point the extracellular parasites must survive until they find a new host cell. Recent studies have indicated that phosphorylation of Toxoplasma eukaryotic translation initiation factor 2-alpha (TgIF2α) plays a key role in promoting parasite viability during times of extracellular stress. Here we report the cloning and characterization of a TgIF2α kinase designated TgIF2K-D that is related to GCN2, a eukaryotic initiation factor 2α (eIF2α) kinase known to respond to nutrient starvation in other organisms. TgIF2K-D is present in the cytosol of both intra- and extracellular Toxoplasma parasites and facilitates translational control through TgIF2α phosphorylation in extracellular parasites. We generated a TgIF2K-D knockout parasite and demonstrated that loss of this eIF2α kinase leads to a significant fitness defect that stems from an inability of the parasite to adequately adapt to the environment outside host cells. This phenotype is consistent with that reported for our nonphosphorylatable TgIF2α mutant (S71A substitution), establishing that TgIF2K-D is the primary eIF2α kinase responsible for promoting extracellular viability of Toxoplasma. These studies suggest that eIF2α phosphorylation and translational control are an important mechanism by which vulnerable extracellular parasites protect themselves while searching for a new host cell. Additionally, TgIF2α is phosphorylated when intracellular parasites are deprived of nutrients, but this can occur independently of TgIF2K-D, indicating that this activity can be mediated by a different TgIF2K.

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.

The cellular mechanisms by which articular cartilage responds to load are poorly understood, but such responses may involve regulation at the level of protein translation rather than synthesis of mRNA. We investigated the role of translational control in cyclically (0.5 Hz, 0.1 Hz and 0.05 Hz) and statically loaded porcine articular cartilage explants. Messenger RNA was extracted for real time polymerase chain reaction (RT-PCR) and newly synthesised proteins were measured by their incorporation of radiolabelled 35S[methionine/cysteine] or 35SO4. Some medium from loaded and unloaded explants was immunoblotted for type II collagen, CTGF and TIMP3. The pathways that control protein translation were investigated by immunoblotting explant lysates for PKR, PERK (PKR like endoplasmic reticulum kinase), eIF2a (eukaryotic initiation factor 2a), eEFs (eukaryotic elongation factors), and AMP-dependent kinase. Explants were also loaded in the presence of inhibitors of PKR, the fibroblast growth factor (FGF) receptor and PI3 kinase. Cyclic loading caused complete global translational arrest as evidenced by a total suppression of new protein synthesis whilst maintaining mRNA levels. Translational arrest did not occur following static loading and was partly dependent upon the load frequency. There was a rebound increase in protein synthesis when labelling was performed after load had been withdrawn. Phosphorylation of PKR occurred in explants following cyclic load and inhibition of PKR modestly reversed suppression of newly synthesised proteins suggesting that PKR, at least in part, was responsible for loading induced translational arrest. These results show that translational control provides a rapid and potentially important mechanism for controlling the synthetic responses of articular chondrocytes in response to different types of mechanical load.

The purpose of this study was to investigate the effect of NT-4 on the endoplasmic reticulum (ER) stress-related apoptosis of retinal neurons of isolated retinas. The retinas were isolated from normal and diabetic rats, and the normal retinas were exposed to high glucose (HG). Our results showed that the number of TUNEL-positive, and PERK- and CHOP-positive cells was significantly higher in diabetic and HG exposed retinas than in normal retinas. In diabetic and HG exposed retinas supplemented with NT-4, the number of TUNEL-positive, and PERK- and CHOP-positive cells was significantly lower than in retinas without NT-4. The neuroprotective effect of NT-4 on retinas cultured under diabetic stress was correlated with the suppression in the expression of PERK and CHOP, ER stress-related factors., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

As indicated in the Global Initiative for Asthma guidelines, short-acting β2-adrenoreceptor agonists (SABAs) are important relievers in asthma exacerbation. Interferon γ-inducible protein (IP)-10/CXCL 10 is a T-helper type 1 (Th1) cell-related chemokine which is important in the recruitment of Th1 cells involved in host immune defense against intracellular pathogens such as viral infection. Regulated on activation, normal T expressed and secreted (RANTES)/CCL 5 is a chemokine which plays a role in attractant of eosinophils, mast cells, and basophils toward the site of allergic inflammation. Bronchial epithelial cells are first-line barriers against pathogen invasion. However, whether SABAs have regulatory effects on the expression of IP-10 and RANTES in bronchial epithelial cells is unknown. BEAS-2B cells, the human bronchial epithelial cell lines, were pretreated with procaterol (one of the SABAs) or dibutyryl-cAMP (a cyclic AMP analog) at different doses for 1 h and then stimulated with poly I:C (10 μg/mL). Supernatants were collected 12 and 24 h after poly I:C stimulation to determine the concentrations of IP-10 and RANTES by ELISA. In some cases, the cells were pretreated with selective β2-adrenoreceptor antagonist, ICI-118551, 30 min before procaterol treatment. To investigate the intracellular signaling, the cells were pretreated with mitogen-activated protein kinase (MAPK) inhibitors and a NF-κB inhibitor 30 min before procaterol treatment. Western blot was also used to explore the intracellular signaling. Procaterol significantly suppressed poly I:C-induced IP-10 and RANTES in BEAS-2B cells in a dose-dependent manner. ICI-118551, a selective β2-adrenoreceptor antagonist, could significantly reverse the suppressive effects. Dibutyryl-cAMP could confer the similar effects of procaterol on poly I:C-induced IP-10 and RANTES expression. Data of Western blot revealed that poly I:C-induced p-ERK, p-JNK, and pp38 expression, but not pp65, were suppressed by procaterol. SABAs could suppress poly I:C-induced IP-10 and RANTES expression in bronchial epithelial cells, at least in part, via β2-adrenoreceptor-cAMP and MAPK-ERK, JNK, and p38 pathways.

The replication of viral nucleic acids triggers cellular antiviral responses. The double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays a key role in this antiviral response. We have recently reported that JFH-1 HCV replication in Huh-7 cells triggers PKR activation. Here we show that the HCV-induced PKR activation is further stimulated by the mitogen- and stress-activated protein kinase 2 (MSK2), a member of the 90kDa ribosomal S6 kinase (RSK) family that has emerged as an important downstream effector of ERK and p38 MAPK signaling pathways. We show that MSK2 binds PKR and stimulates PKR phosphorylation, whereas the closely related MSK1 and RSK2 have no effect. Our data further indicate that MSK2 functions as an adaptor in mediating PKR activation, apparently independent of its catalytic activity. These results suggest that, in addition to viral dsRNA, stress signaling contributes to the regulation of cellular antiviral response., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The pathogenic mechanism of dengue virus infection is related to the host responses within target cells, and therefore we assessed intracellular changes in stress proteins following dengue virus infection. This study provides evidence that Hsp70 helps in viral multiplication by suppressing the type 1 interferon response. Dengue virus infection in human monocytic THP-1 cells led to overexpression of Hsp70, which also acts as a chaperone. The functional role of Hsp70 in dengue virus multiplication was identified by downregulating the Hsp70 gene with its specific siRNA duplexes, which led to a decrease in viral RNA copy numbers in cellular supernatants and intracellular viral load. It also resulted in an increased IFN-α level, which mediates its antiviral effect through double-stranded RNA-induced protein kinase-PKR. Collectively these results suggest that an increased level of Hsp70 expression in dengue-virus-infected THP-1 cells assists in viral replication by escaping the antiviral effect of type 1 interferon.

Purpose: The role of RNA-dependent protein kinase (PKR) in antiviral defense mechanisms and in cellular differentiation, growth, and apoptosis is well known, but the role of PKR in human lung cancer remains poorly understood. To explore the role of PKR in human lung cancer, we evaluated the expression of PKR in tissue microarray (TMA) specimens from both non-small cell lung cancer (NSCLC) and normal human bronchial epithelium tissue., Experimental Design: TMA samples (TMA-1) from 231 lung cancers were stained with PKR antibody and validated on TMA-2 from 224 lung cancers. Immunohistochemical expression score was quantified by three pathologists independently. Survival probability was computed by the Kaplan-Meier method., Results: The NSCLC cells showed lower levels of PKR expression than normal bronchial epithelium cells did. We also found a significant association between lower levels of PKR expression and lymph node metastasis. We found that loss of PKR expression is correlated with a more aggressive behavior, and that a high PKR expression predicts a subgroup of patients with a favorable outcome. Univariate and multivariate Cox proportional hazards regression models showed that a lower level of PKR expression was significantly associated with shorter survival in NSCLC patients. We further validated and confirmed PKR to be a powerful prognostic factor in TMA-2 lung cancer (hazard ratio, 0.22; P < 0.0001)., Conclusions: Our findings first indicate that PKR expression is an independent prognostic variable in NSCLC patients., (©2010 AACR.)

Influenza virus (Flu) infection and secondary complications are a leading cause of morbidity and mortality worldwide. The increasing number of annual Flu cases, coupled with the recent Flu pandemic, has amplified concerns about the impact of Flu on human and animal health. Similar to humans, Flu is problematic in pigs, not only as a primary pathogen but as an agent in polymicrobial pneumonia. Bordetella species play a role in mixed infections and often colonize the respiratory tract without overt clinical signs. Pigs serve as a valuable animal model for several respiratory pathogens, including Bordetella (Bb) and Flu. To investigate Flu/Bb coinfection pathogenesis, a study was completed in which pigs were inoculated with Flu-only, Bb-only or both agents (Flu/Bb). Results indicate that Flu clearance is not altered by Bb infection, but Flu does enhance Bb colonization. Pulmonary lesions in the Flu/Bb group were more severe when compared to Flu-only or Bb-only groups and Bb did not cause significant lesions unless pigs were coinfected with Flu. The type I interferon response was elevated in coinfected pigs, but increased expression of antiviral genes Mx and PKR did not appear to enhance Flu clearance in coinfected pigs, as viral clearance was similar between Flu/Bb and Flu-only groups. IL-1beta and IL-8 were elevated in lungs of coinfected pigs, correlating to the days enhanced lesions were observed. Overall, Flu infection increased Bb colonization and enhanced production of proinflammatory mediators that likely contribute to exacerbated pulmonary lesions., (Published by Elsevier India Pvt Ltd.)