PKR Senses Nuclear and Mitochondrial Signals by Interacting with Endogenous Double-Stranded RNAs.

Protein kinase RNA-activated (PKR) induces immune response by sensing viral double-stranded RNAs (dsRNAs). However, growing evidence suggests that PKR can also be activated by endogenously expressed dsRNAs. Here, we capture these dsRNAs by formaldehyde-mediated crosslinking and immunoprecipitation sequencing and find that various noncoding RNAs interact with PKR. Surprisingly, the majority of the PKR-interacting RNA repertoire is occupied by mitochondrial RNAs (mtRNAs). MtRNAs can form intermolecular dsRNAs owing to bidirectional transcription of the mitochondrial genome and regulate PKR and eIF2α phosphorylation to control cell signaling and translation. Moreover, PKR activation by mtRNAs is counteracted by PKR phosphatases, disruption of which causes apoptosis from PKR overactivation even in uninfected cells. Our work unveils dynamic regulation of PKR even without infection and establishes PKR as a sensor for nuclear and mitochondrial signaling cues in regulating cellular metabolism.

PERK signaling activation restores nucleus pulposus degeneration by activating autophagy under hypoxia environment.

OBJECTIVES: Intervertebral disc (IVD) degeneration is an important disease with no efficient biological therapy identified. Autophagy, a wildly known therapeutic target for human disease, has been demonstrated to be activated under hypoxia, with underlying mechanism remains elusive. Thus, this study aims to specify the role of autophagy in IVD degeneration, the regulating mechanism of hypoxia-inducing autophagy, and the therapeutic value of autophagy for IVD degeneration. METHODS: RNA-seq was used to screen the primary pathway affected in NP cells under hypoxia, the specific link between hypoxia and autophagy were investigated using ChIP-seq and dual luciferase reporter assay. Conditional ATG7 knockout mice (ATG7-/-) were constructed for assessing the effect of autophagy on IVD degeneration, and puncture induced mice model of IVD degeneration were used for intradiscal injection to evaluate the therapeutic value of autophagy. RESULTS: We demonstrated that hypoxia induces autophagy by transcriptional activation of autophagic gene LC3B and ATG7, which is controlled by PERK signaling. Then, we observed that inhibiting autophagy or PERK signaling leads to impaired NP cell viability and function, furthermore, using ATG7 knockout (ATG7-/-) mice, we identified the protective role of autophagy in IVD. Furthermore, we found that intradiscal injection of PERK signaling agonist, CCT020312, significantly restores the degeneration level of needle punctured mice IVD. CONCLUSION: We showed that the activation of PERK signaling upon hypoxia serves as a vital mechanism to induce autophagy and identified the therapeutic value of PERK signaling agonist for IVD degeneration treatment.

Sterile activation of invariant natural killer T cells by ER-stressed antigen-presenting cells.

Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.

The Integrated UPR and ERAD in Oligodendrocytes Maintain Myelin Thickness in Adults by Regulating Myelin Protein Translation.

Myelin proteins, which are produced in the endoplasmic reticulum (ER), are essential and necessary for maintaining myelin structure. The integrated unfold protein response (UPR) and ER-associated degradation (ERAD) are the primary ER quality control mechanism. The adaptor protein Sel1L (Suppressor/Enhancer of Lin-12-like) controls the stability of the E3 ubiquitin ligase Hrd1 (hydroxymethylglutaryl reductase degradation protein 1), and is necessary for the ERAD activity of the Sel1L-Hrd1 complex. Herein, we showed that Sel1L deficiency specifically in oligodendrocytes caused ERAD impairment, the UPR activation, and attenuation of myelin protein biosynthesis; and resulted in late-onset, progressive myelin thinning in the CNS of adult mice (both male and female). The pancreatic ER kinase (PERK) branch of the UPR functions as the master regulator of protein translation in ER-stressed cells. Importantly, PERK inactivation reversed attenuation of myelin protein biosynthesis in oligodendrocytes and restored myelin thickness in the CNS of oligodendrocyte-specific Sel1L-deficient mice (both male and female). Conversely, blockage of proteolipid protein production exacerbated myelin thinning in the CNS of oligodendrocyte-specific Sel1L-deficient mice (both male and female). These findings suggest that impaired ERAD in oligodendrocytes reduces myelin thickness in the adult CNS through suppression of myelin protein translation by activating PERK.SIGNIFICANCE STATEMENT Myelin is an enormous extended plasma membrane of oligodendrocytes that wraps and insulates axons. Myelin structure, including thickness, was thought to be extraordinarily stable in adults. Myelin proteins, which are produced in the endoplasmic reticulum (ER), are essential and necessary for maintaining myelin structure. The integrated unfolded protein response (UPR) and ER-associated degradation (ERAD) are the primary mechanism that maintains ER protein homeostasis. Herein, we explored the role of the integrated UPR and ERAD in oligodendrocytes in regulating myelin protein production and maintaining myelin structure using mouse models. The results presented in this study imply that the integrated UPR and ERAD in oligodendrocytes maintain myelin thickness in adults by regulating myelin protein production.

RNase L Amplifies Interferon Signaling by Inducing Protein Kinase R-Mediated Antiviral Stress Granules.

Virus infection leads to activation of the interferon (IFN)-induced endoribonuclease RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRRs), like retinoic acid-inducible I (Rig-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins Rig-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a key stress granule protein, G3BP1, with only PKR and Rig-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production, but not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection, demonstrating the importance of avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective antiviral response.IMPORTANCE Double-stranded RNAs produced during viral infections serve as pathogen-associated molecular patterns (PAMPs) and bind pattern recognition receptors to stimulate IFN production. RNase L is an IFN-regulated endoribonuclease that is activated in virus-infected cells and cleaves single-stranded viral and cellular RNAs. The RNase L-cleaved dsRNAs signal to Rig-like helicases to amplify IFN production. This study identifies a novel role of antiviral stress granules induced by RNase L as an antiviral signaling hub to coordinate the RNA ligands with cognate receptors to mount an effective host response during viral infections.

Inhibition of the PERK/TXNIP/NLRP3 Axis by Baicalin Reduces NLRP3 Inflammasome-Mediated Pyroptosis in Macrophages Infected with Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a significant threat to global health as it induces granuloma and systemic inflammatory responses during active tuberculosis. Mtb can induce macrophage pyroptosis, leading to the release of IL-1ß and tissue damage, promoting its spread. Here, we established an in vitro Mtb-infected macrophage model to seek an effective antipyroptosis agent. Baicalin, isolated from Radix Scutellariae, was found to reduce pyroptosis in Mtb-infected macrophages. Baicalin could inhibit activation of the PERK/eIF2α pathway and thus downregulates TXNIP expression and subsequently reduces activation of the NLRP3 inflammasome, resulting in reduced pyroptosis in Mtb-infected macrophages. In conclusion, baicalin reduced pyroptosis by inhibiting the PERK/TXNIP/NLRP3 axis and might thus be a new adjuvant host-directed therapy (HDT) drug.

ß-Arrestin1-mediated decrease in endoplasmic reticulum stress impairs intestinal stem cell proliferation following radiation.

Gastrointestinal toxicity limits the clinical application of abdominal and pelvic radiotherapy and currently has no effective treatment. Intestinal leucine-rich-repeat-containing GPCR 5 (Lgr5)-positive stem cell depletion and loss of proliferative ability due to radiation may be the primary factors causing intestinal injury following radiation. Here, we report the critical role of ß-arrestin1 (ßarr1) in radiation-induced intestinal injury. Intestinal ßarr1 was highly expressed in radiation enteritis and in a radiation model. ßarr1 knockout (KO) or knockdown mice exhibited increased proliferation in intestinal Lgr5+ stem cell, crypt reproduction, and survival following radiation. Unexpectedly, the beneficial effects of ßarr1 deficiency on intestinal stem cells in response to radiation were compromised when the endoplasmic reticulum stress-related protein kinase RNA-like ER kinase (PERK)/eukaryotic initiation factor-2α (eIF2α) pathway was inhibited, and this result was further supported in vitro. Furthermore, we found that ßarr1 knockdown with small interfering RNA significantly enhanced intestinal Lgr5+ stem cell proliferation after radiation via directly targeting PERK. ßarr1 offers a promising target for mitigating radiation-induced intestinal injury.-Liu, Z., Jiang, J., He, Q., Liu, Z., Yang, Z., Xu, J., Huang, Z., Wu, B. ß-Arrestin1-mediated decrease in endoplasmic reticulum stress impairs intestinal stem cell proliferation following radiation.

Activation of the Endoplasmic Reticulum Stress Response Impacts the NOD1 Signaling Pathway.

Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition receptor (PRR) responsible for sensing bacterial peptidoglycan fragments. Stimulation of NOD1 leads to a robust innate immune response via activation of the major transcription factor NF-κB. In addition to peptidoglycan sensing, NOD1 and the closely related PRR NOD2 have been linked to inflammation by responding to the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR). Here we show that differential ER stress induction renders cells more susceptible to Salmonella enterica serovar Typhimurium infection in a NOD1-dependent manner, measured by increased NF-κB activation and cytokine expression. In HeLa57A cells stably transfected with an NF-κB::luciferase reporter, we show that cells undergoing ER stress induced by thapsigargin display a significant increase in NF-κB activation in response to NOD1 stimulation by C12-iE-DAP (acylated derivative of the iE-DAP dipeptide [gamma-d-glutamyl-meso-diaminopimelic acid]) and the S Typhimurium effector protein SopE. Tunicamycin-induced ER stress had no effect on NOD1-stimulated NF-κB activation. We further show that the mouse intestinal epithelial cell line MODE-K and RAW264.7 macrophages are more responsive to Salmonella infection when treated with thapsigargin but not with tunicamycin. These profound differences between thapsigargin- and tunicamycin-treated cells upon inflammation suggest that different components downstream of the UPR contribute to NOD1 activation. We found that the NOD1-induced inflammatory response is dependent on protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) activation in conjunction with stimulation of the inositol triphosphate receptor (IP3R). Together, these results suggest that differential UPR activation makes cells more responsive to bacterial infections in a NOD1-dependent manner.

ER-stress regulates macrophage polarization through pancreatic EIF-2alpha kinase.

During the process of NAFLD progression, ER-stress is activated in macrophages and induces the pro-inflammatory polarization of macrophage. As one of the three ER membrane resident proteins, pancreatic eIF-2alpha kinase (PERK) plays an important role in ER stress, but its participation in macrophage polarization is largely unknown. In this study, we found that the PA mediated ER-stress activation could induce M1-type polarization in macrophages, and this phenotype polarization could be inhibited by ER-stress inhibitor 4-PBA as well as GSK2656157, an inhibitor of PERK. Moreover, the knockdown of PERK altered the STAT1 and STAT6 pathways in macrophages, which then led to the M1-to-M2 phenotypic shift. In summary, we found that PERK could regulate the phenotypic polarization of macrophages. This finding may provide new insight into the suppression of pathological progression of fatty liver or liver ischemia reperfusion injury induced by M1-type macrophages.

ER Protein Quality Control and the Unfolded Protein Response in the Heart.

Cardiac myocytes are the cells responsible for the robust ability of the heart to pump blood throughout the circulatory system. Cardiac myocytes grow in response to a variety of physiological and pathological conditions; this growth challenges endoplasmic reticulum-protein quality control (ER-PQC), a major feature of which includes the unfolded protein response (UPR). ER-PQC and the UPR in cardiac myocytes growing under physiological conditions, including normal development, exercise, and pregnancy, are sufficient to support hypertrophic growth of each cardiac myocyte. However, the ER-PQC and UPR are insufficient to respond to the challenge of cardiac myocyte growth under pathological conditions, including myocardial infarction and heart failure. In part, this insufficiency is due to a continual decline in the expression levels of important adaptive UPR components as a function of age and during myocardial pathology. This chapter will discuss the physiological and pathological conditions unique to the heart that involves ER-PQC, and whether the UPR is adaptive or maladaptive under these circumstances.

Molecular mechanisms relating to amino acid regulation of protein synthesis.

Some amino acids (AA) act through several signalling pathways and mechanisms to mediate the control of gene expression at the translation level, and the regulation occurs, specifically, on the initiation and the signalling pathways for translation. The translation of mRNA to protein synthesis proceeds through the steps of initiation and elongation, and AA act as important feed-forward activators that are involved in many pathways, such as the sensing and the transportation of AA by cells, in these steps in many tissues of mammals. For the translation, phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a critical molecule that controls the translation initiation and its functions can be regulated by some AA. Another control point in the mRNA binding step in the translation initiation is at the regulation by mammalian target of rapamycin, which requires a change of phosphorylation status of ribosomal protein S6. In fact, the change of phosphorylation status of ribosomal protein S6 might be involved in global protein synthesis. The present review summarises recent work on the molecular mechanisms of the regulation of protein synthesis by AA and highlights new findings.

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.

Integrated Stress Response Mediates Epithelial Injury in Mechanical Ventilation.

Ventilator-induced lung injury (VILI) is a severe complication of mechanical ventilation that can lead to acute respiratory distress syndrome. VILI is characterized by damage to the epithelial barrier with subsequent pulmonary edema and profound hypoxia. Available lung-protective ventilator strategies offer only a modest benefit in preventing VILI because they cannot impede alveolar overdistension and concomitant epithelial barrier dysfunction in the inflamed lung regions. There are currently no effective biochemical therapies to mitigate injury to the alveolar epithelium. We hypothesize that alveolar stretch activates the integrated stress response (ISR) pathway and that the chemical inhibition of this pathway mitigates alveolar barrier disruption during stretch and mechanical ventilation. Using our established rat primary type I-like alveolar epithelial cell monolayer stretch model and in vivo rat mechanical ventilation that mimics the alveolar overdistension seen in acute respiratory distress syndrome, we studied epithelial responses to mechanical stress. Our studies revealed that the ISR signaling pathway is a key modulator of epithelial permeability. We show that prolonged epithelial stretch and injurious mechanical ventilation activate the ISR, leading to increased alveolar permeability, cell death, and proinflammatory signaling. Chemical inhibition of protein kinase RNA-like endoplasmic reticulum kinase, an upstream regulator of the pathway, resulted in decreased injury signaling and improved barrier function after prolonged cyclic stretch and injurious mechanical ventilation. Our results provide new evidence that therapeutic targeting of the ISR can mitigate VILI.

PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression.

PURPOSE: The aim of this study was to investigate the functions of dsRNA-activated protein kinase (PKR) in choroidal neovascularization (CNV) and related signaling pathways in the production of vascular endothelial growth factor (VEGF). METHODS: A chemical hypoxia model of in vitro RF/6A cells, a rhesus choroid-retinal endothelial cell line, was established by adding cobalt chloride (CoCl2) to the culture medium. PKR, phosphophosphatidylinositol 3-kinase (p-PI3K), phosphoprotein kinase B (p-Akt), and VEGF protein levels in RF/6A cells were detected with western blotting. PKR siRNA and the PI3K inhibitor LY294002 were used to evaluate the roles of the PKR and PI3K signaling pathways in VEGF expression with western blotting. In an ARPE-19 (RPE cell line) and RF/6A cell coculture system, proliferation, migration, and tube formation of RF/6A cells under hypoxic conditions were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Transwell, and Matrigel Transwell assays, respectively. In vivo CNV lesions were induced in C57BL/6J mice using laser photocoagulation. The mice were euthanized in a timely manner, and the eyecups were dissected from enucleated eyes. PKR, p-PI3K, p-Akt, and VEGF protein levels in tissues were detected with western blotting. To evaluate the leakage area, fundus fluorescein angiography and choroidal flat mount were performed on day 7 after intravitreal injection of an anti-PKR monoclonal antibody. RESULTS: The in vitro RF/6A cell chemical hypoxia model showed that PKR expression was upregulated in parallel with p-PI3K, p-Akt, and VEGF expression, peaking at 12 h. PKR siRNA downregulated PKR, p-PI3K, p-Akt, and VEGF expression. In addition, the PI3K inhibitor LY294002 greatly decreased the p-PI3K, p-Akt, and VEGF protein levels, but PKR expression was unaffected, indicating that Akt was a downstream molecule of PKR that upregulated VEGF expression. In the ARPE-19 (RPE cell line) and RF/6A cell coculture system, PKR siRNA reduced the migration and tube formation of the RF/6A cells. In vivo, PKR, p-PI3K, p-Akt, and VEGF expression increased and peaked at 7 days in the mouse CNV model induced by laser photocoagulation. Furthermore, on the RPE and choroid cryosections, PKR colocalized with CD31, suggesting that PKR was expressed by the vascular endothelium. The intravitreal injection of an anti-PKR monoclonal antibody decreased the progression and leakage area of CNV in mice. CONCLUSIONS: PKR promotes CNV formation via the PI3K/Akt signaling pathway in VEGF expression. Additionally, the anti-PKR monoclonal antibody significantly decreased CNV in a mouse model, showing the antibody may have therapeutic potential in human CNV.

Pseudomonas aeruginosa quorum-sensing molecule homoserine lactone modulates inflammatory signaling through PERK and eI-F2α.

Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (HSL-C12) as a quorum-sensing molecule to regulate bacterial gene expression. Because HSL-C12 is membrane permeant, multiple cell types in P. aeruginosa-infected airways may be exposed to HSL-C12, especially adjacent to biofilms where local (HSL-C12) may be high. Previous reports showed that HSL-C12 causes both pro- and anti-inflammatory effects. To characterize HSL-C12's pro- and anti-inflammatory effects in host cells, we measured protein synthesis, NF-κB activation, and KC (mouse IL-8) and IL-6 mRNA and protein secretion in wild-type mouse embryonic fibroblasts (MEF). To test the role of the endoplasmic reticulum stress inducer, PERK we compared these responses in PERK(-/-) and PERK-corrected PERK(-/-) MEF. During 4-h treatments of wild-type MEF, HSL-C12 potentially activated NF-κB p65 by preventing the resynthesis of IκB and increased transcription of KC and IL-6 genes (quantitative PCR). HSL-C12 also inhibited secretion of KC and/or IL-6 into the media (ELISA) both in control conditions and also during stimulation by TNF-α. HSL-C12 also activated PERK (as shown by increased phosphorylation of eI-F2α) and inhibited protein synthesis (as measured by incorporation of [(35)S]methionine by MEF). Comparisons of PERK(-/-) and PERK-corrected MEF showed that HSL-C12's effects were explained in part by activation of PERK→phosphorylation of eI-F2α→inhibition of protein synthesis→reduced IκBα production→activation of NF-κB→increased transcription of the KC gene but reduced translation and secretion of KC. HSL-C12 may be an important modulator of early (up to 4 h) inflammatory signaling in P. aeruginosa infections.

The unfolded protein response as a target for cancer therapy.

Various physiological and pathological conditions generate an accumulation of misfolded proteins in the endoplasmic reticulum (ER). This results in ER stress followed by a cellular response to cope with this stress and restore homeostasis: the unfolded protein response (UPR). Overall, the UPR leads to general translational arrest and the induction of specific factors to ensure cell survival or to mediate cell death if the stress is too severe. In multiple cancers, components of the UPR are overexpressed, indicating increased dependence on the UPR. In addition, the UPR can confer resistance to anti-cancer treatment. Therefore, modification of the UPR should be explored for its anti-cancer properties. This review discusses factors associated with the UPR that represent potential therapeutic targets.

Double-stranded RNA-dependent protein kinase deficiency protects the heart from systolic overload-induced congestive heart failure.

BACKGROUND: Double-stranded RNA-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α kinase that inhibits mRNA translation under stress conditions. PKR also mediates inflammatory and apoptotic signaling independently of translational regulation. Congestive heart failure is associated with cardiomyocyte hypertrophy, inflammation, and apoptosis, but the role of PKR in left ventricular hypertrophy and the development of congestive heart failure has not been examined. METHODS AND RESULTS: We observed increased myocardial PKR expression and translocation of PKR into the nucleus in humans and mice with congestive heart failure. To determine the impact of PKR on the development of congestive heart failure, PKR knockout and wild-type mice were exposed to pressure overload produced by transverse aortic constriction. Although heart size increased similarly in wild-type and PKR knockout mice after transverse aortic constriction, PKR knockout mice exhibited very little pulmonary congestion, well-preserved left ventricular ejection fraction and contractility, and significantly less myocardial fibrosis compared with wild-type mice. Bone marrow-derived cells from wild-type mice did not abolish the cardiac protective effect observed in PKR knockout mice, whereas bone marrow-derived cells from PKR knockout mice had no cardiac protective effect in wild-type mice. Mechanistically, PKR knockout attenuated transverse aortic constriction-induced tumor necrosis factor-α expression and leukocyte infiltration and lowered cardiac expression of proapoptotic factors (Bax and caspase-3), so that PKR knockout hearts were more resistant to transverse aortic constriction-induced cardiomyocyte apoptosis. PKR depletion in isolated cardiomyocytes also conferred protection against tumor necrosis factor-α- or lipopolysaccharide-induced apoptosis. CONCLUSION: PKR is a maladaptive factor upregulated in hemodynamic overload that contributes to myocardial inflammation, cardiomyocyte apoptosis, and the development of congestive heart failure.

Activation of NADPH oxidase 4 in the endoplasmic reticulum promotes cardiomyocyte autophagy and survival during energy stress through the protein kinase RNA-activated-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway.

RATIONALE: Autophagy is an essential survival mechanism during energy stress in the heart. Oxidative stress is activated by energy stress, but its role in mediating autophagy is poorly understood. NADPH oxidase (Nox) 4 is an enzyme that generates reactive oxygen species (ROS) at intracellular membranes. Whether Nox4 acts as a sensor of energy stress to mediate activation of autophagy is unknown. OBJECTIVE: We investigated whether Nox4 is involved in the regulation of autophagy and cell survival during energy stress in cardiomyocytes. METHODS AND RESULTS: Production of ROS in cardiomyocytes was increased during glucose deprivation (GD) in a Nox4-dependent manner. Protein levels and the ROS-producing activity of Nox4 were increased in the endoplasmic reticulum (ER), but not in mitochondria, in response to GD. Selective knockdown of Nox4, but not Nox2, or selective reduction of ROS in the ER with ER-targeted catalase, but not mitochondria-targeted perioxiredoxin 3, abrogated GD-induced autophagy. Nox4 promoted autophagy during GD through activation of the protein kinase RNA-activated-like ER kinase pathway by suppression of prolyl hydroxylase 4. The decrease in cell survival during GD in the presence of Nox4 knockdown was rescued by reactivation of autophagy by Atg7 overexpression, indicating that the effect of Nox4 on cell survival is critically mediated through regulation of autophagy. Nox4 was activated during fasting and prolonged ischemia in the mouse heart, where Nox4 is also required for autophagy activation and cardioprotection. CONCLUSIONS: Nox4 critically mediates autophagy in response to energy stress in cardiomyocytes by eliciting ROS in the ER and stimulating the protein kinase RNA-activated-like ER kinase signaling pathway.

Inflammation and Oxidative Stress: The Molecular Connectivity between Insulin Resistance, Obesity, and Alzheimer's Disease.

Type 2 diabetes (T2DM), Alzheimer's disease (AD), and insulin resistance are age-related conditions and increased prevalence is of public concern. Recent research has provided evidence that insulin resistance and impaired insulin signalling may be a contributory factor to the progression of diabetes, dementia, and other neurological disorders. Alzheimer's disease (AD) is the most common subtype of dementia. Reduced release (for T2DM) and decreased action of insulin are central to the development and progression of both T2DM and AD. A literature search was conducted to identify molecular commonalities between obesity, diabetes, and AD. Insulin resistance affects many tissues and organs, either through impaired insulin signalling or through aberrant changes in both glucose and lipid (cholesterol and triacylglycerol) metabolism and concentrations in the blood. Although epidemiological and biological evidence has highlighted an increased incidence of cognitive decline and AD in patients with T2DM, the common molecular basis of cell and tissue dysfunction is rapidly gaining recognition. As a cause or consequence, the chronic inflammatory response and oxidative stress associated with T2DM, amyloid-ß (Aß) protein accumulation, and mitochondrial dysfunction link T2DM and AD.

EPSTI1 Is Involved in IL-28A-Mediated Inhibition of HCV Infection.

It has been reported that IFN-λs inhibit HCV replication in vitro. But the mechanisms of how IL-28A conducts antiviral activity and the functions of IL-28A-induced ISGs (IFN-stimulated genes) are not fully understood. In this study, we found that IL-28A has the antiviral effect on HCV life cycle including viral replication, assembly, and release. IL-28A and IFN-α synergistically inhibit virus replication. EPSTI1 (epithelial-stromal interaction 1), one of IL-28A-induced ISGs, plays a vital role in IL-28A-mediated antiviral activity. Furthermore, forced expression of EPSTI1 effectively inhibits HCV replication in the absence of interferon treatment, and knockdown of EPSTI1 contributes to viral enhancement. EPSTI1 can activate PKR promoter and induce several PKR-dependent genes, including IFN-ß, IFIT1, OAS1, and RNase L, which is responsible for EPSTI1-mediated antiviral activity.