Phosphorylation Of Eukaryotic Initiation Factor 2α Research Articles

Activation of eukaryotic initiation factor-2α-kinases in okadaic acid-treated neurons

Phosphorylation of eukaryotic initiation factor-2α (eIF2α) is increased in Alzheimer's disease (AD) and this protein can be phosphorylated by several kinases, including double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), amino acids-regulated eIF2α kinase (GCN2) and heme-regulated eIF2α kinase (HRI). PKR and PERK especially are activated in the AD brain, and GCN2 is reported to increase presenilin-1 (PS1) activity. Okadaic acid (OA), a protein phosphatase-2A (PP2A) inhibitor, is known to increase tau phosphorylation, β-amyloid (Aβ) deposition and neuronal death, which are the pathological characteristics of AD. Here, we show that the phosphorylation of eIF2α is increased and its kinases, PKR, PERK and GCN2 are activated in rat neurons by OA. Activating transcription factor (ATF4) which induces apoptosis in response to eIF2α phosphorylation was increased and translocated to nuclei in OA-treated neurons. These results suggest that the successive events of activation of eIF2α kinases and eIF2α phosphorylation leading to ATF4 nuclear translocation may contribute to neuronal death. However, PKR inhibitors did not reduce eIF2α phosphorylation or neuronal toxicity despite inhibiting PKR activity. These results suggest that PKR might not be the most responsible kinase for eIF2α phosphorylation or cell death in PP2A-inhibited conditions such as AD.

OGFOD1, a Novel Modulator of Eukaryotic Translation Initiation Factor 2α Phosphorylation and the Cellular Response to Stress

Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2alpha and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2alpha, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2alpha and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2alpha and the eIF2alpha kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress.

Enhanced Signaling Downstream of Ribonucleic Acid-Dependent Protein Kinase-Like Kinase Potentiates Lipotoxic Endoplasmic Reticulum Stress in Human Islets

Free fatty acids cause pancreatic β-cell apoptosis and may contribute to β-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. Eukaryotic translation initiation factor 2α (eIF2α) phosphorylation is an adaptive response to ER stress, and reductions in eIF2α phosphorylation trigger β-cell failure. Salubrinal inhibits eIF2α dephosphorylation and has been proposed as a novel therapy for diabetes. The objective of the study was to examine whether salubrinal modulates human islet susceptibility to lipotoxicity. Human islets were treated with oleate or palmitate, alone or in combination with salubrinal, and examined for apoptosis, ultrastructure, and gene expression. Salubrinal enhanced signaling downstream of eIF2α and markedly induced the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein, but it did not induce the inositol requiring-1α or activating transcription factor 6 ER stress pathways. Salubrinal potentiated the deleterious effects of oleate and palmitate in human islets. This proapoptotic effect involved ER dilation and mitochondrial rounding and fragmentation. Excessive eIF2α phosphorylation is poorly tolerated by human islets and exacerbates fatty acid-induced apoptosis through ER and mitochondrial mechanisms. This should be taken into consideration when designing approaches to pharmacologically modulate the β-cell ER stress response in type 2 diabetes.

Apolipoprotein E-Deficient Lipoproteins Induce Foam Cell Formation by Activation of PERK-EIF-2α Signaling Cascade

Transformation of macrophages into foam cells by apolipoprotein (Apo) E-deficient, ApoB48-containing (E(-)/B48) lipoproteins has been shown to be associated with increased phosphorylation of eukaryotic initiation factor-2α (eIF-2α). The present report examined the causal relationship between eIF-2α phosphorylation and lipid accumulation in macrophages induced by E(-)/B48 lipoproteins. E(-)/B48 lipoproteins increased eIF-2α phosphorylation and cholesterol ester accumulation, while lipoprotein degradation decreased and lysosomal acid lipase and cathepsin B mRNA translation was inhibited in mouse peritoneal macrophages (MPMs). These responses were overcome by overexpression of a nonphosphorylatable eIF-2α mutant in MPMs. Incubation of MPMs with E(-)/B48 lipoproteins also increased the phosphorylation of RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK), but not other eIF-2α kinases. Overexpression of a nonphosphorylatable PERK mutant inhibited PERK and eIF-2α phosphorylation, and alleviated cholesterol ester accumulation induced by E(-)/B48 lipoproteins. PERK is an eIF-2α kinase activated by endoplasmic reticulum (ER) stress. Taken together, findings from this report suggest that induction of ER stress, i.e., activation of the PERK-eIF2α signaling cascade, is a mechanism by which E(-)/B48 lipoproteins down-regulate lysosomal hydrolase synthesis, inhibit lysosomal lipoprotein degradation, and increase intracellular lipoprotein and cholesterol ester accumulation, resulting in foam cell formation.

Baicalein protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis through inhibition of reactive oxygen species production and CHOP induction

Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi and possesses various effects, including cytoprotection and anti-inflammation. Because endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, we investigated the effects of baicalein on apoptotic death of HT22 mouse hippocampal neuronal cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were measured by flow cytometry. Expression level and phosphorylation status of ER stress-associated proteins and activation and cleavage of apoptosis-associated proteins were analyzed by Western blot. Baicalein reduced TG- and BFA-induced apoptosis of HT22 cells and activation and cleavage of apoptosis-associated proteins, such as caspase-12 and -3 and poly(ADP-ribose) polymerase. Baicalein also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78, the cleavage of X-box binding protein-1 and activating transcription factor 6α, and the phosphorylation of eukaryotic initiation factor-2α and mitogen-activated protein kinases, such as p38, JNK, and ERK. Knock-down of CHOP expression by siRNA transfection and specific inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) as well as anti-oxidant (N-acetylcysteine) reduced TG- or BFA-induced cell death. Baicalein also reduced TG- and BFA-induced ROS accumulation and MMP reduction. Taken together, these results suggest that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.

Metabolic Remodeling Induced by Mitochondrial Aldehyde Stress Stimulates Tolerance to Oxidative Stress in the Heart

Aldehyde accumulation is regarded as a pathognomonic feature of oxidative stress-associated cardiovascular disease. We investigated how the heart compensates for the accelerated accumulation of aldehydes. Aldehyde dehydrogenase 2 (ALDH2) has a major role in aldehyde detoxification in the mitochondria, a major source of aldehydes. Transgenic (Tg) mice carrying an Aldh2 gene with a single nucleotide polymorphism (Aldh2*2) were developed. This polymorphism has a dominant-negative effect and the Tg mice exhibited impaired ALDH activity against a broad range of aldehydes. Despite a shift toward the oxidative state in mitochondrial matrices, Aldh2*2 Tg hearts displayed normal left ventricular function by echocardiography and, because of metabolic remodeling, an unexpected tolerance to oxidative stress induced by ischemia/reperfusion injury. Mitochondrial aldehyde stress stimulated eukaryotic translation initiation factor 2alpha phosphorylation. Subsequent translational and transcriptional activation of activating transcription factor-4 promoted the expression of enzymes involved in amino acid biosynthesis and transport, ultimately providing precursor amino acids for glutathione biosynthesis. Intracellular glutathione levels were increased 1.37-fold in Aldh2*2 Tg hearts compared with wild-type controls. Heterozygous knockout of Atf4 blunted the increase in intracellular glutathione levels in Aldh2*2 Tg hearts, thereby attenuating the oxidative stress-resistant phenotype. Furthermore, glycolysis and NADPH generation via the pentose phosphate pathway were activated in Aldh2*2 Tg hearts. (NADPH is required for the recycling of oxidized glutathione.) The findings of the present study indicate that mitochondrial aldehyde stress in the heart induces metabolic remodeling, leading to activation of the glutathione-redox cycle, which confers resistance against acute oxidative stress induced by ischemia/reperfusion.

Blockade of GRP78 sensitizes breast cancer cells to microtubules‐interfering agents that induce the unfolded protein response

Taxane and vinblastine represent two classes of microtubules-targeted agents for cancer chemotherapy. Although taxol and vinblastine are widely used for cancer treatment, resistance to these agents is frequently encountered in the clinic. An ongoing question has been what mechanisms are involved in the resistance of tumour cells to microtubules-targeted agents or how the clinical effectiveness can be improved. There is increasing evidence that microtubules interact with the endoplasmic reticulum (ER). Here, we have shown that taxol and vinblastine induce multiple arms of the ER stress response, including up-regulation of glucose-regulated protein 78 (GRP78) expression, X-box binding protein 1 splicing and eukaryotic initiation factor 2α phosphorylation. Abrogation of GRP78 induction sensitizes breast cancer cells to taxol and vinblastine. Treatment with (-)-epigallocatechin gallate (EGCG), a known GRP78 inhibitor, synergistically promotes taxol- and vinblastine-induced cell death. GRP78 knockdown or EGCG potentiates taxol- and vinblastine-induced activation of pro-apoptosis arms of the ER stress response, such as JNK phosphorylation, caspase-7 and PARP cleavage. Inhibition of JNK and caspase-7 abrogates EGCG sensitization of breast cancer cells to taxol and vinblastine. We conclude that induction of the unfolded protein response represents a novel mechanism underlying the efficacy and resistance to microtubules-targeted agents. Combination of compounds capable of suppressing GRP78 might be a novel approach for improving the effectiveness of microtubules-targeted chemotherapy.

Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that delta(9)-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.

Mutation of eukaryotic initiation factor‐2 α at serine 51 inhibits foam cell formation

We previously reported that ApoE‐deficient, ApoB48‐containing (E?/B48) lipoprotein‐induced foam cell formation was associated with an increased phosphorylation of eukaryotic initiation factor‐2α (eIF‐2α), a hallmark feature of endoplasmic reticulum stress. The objective of this study is to examine the impact of a nonphosphorylatable eIF‐2α mutant on E?/B48 lipoprotein‐induced changes in gene expression and foam cell formation. We observed that incubation of mouse peritoneal macrophages (MPMs) with E?/B48 lipoproteins reduced the protein levels of lysosomal hydrolases and cholesterol transporters, and enhanced eIF‐2α phosphorylation. Transfection of MPMs with an eIF‐2α mutant, in which serine 51 was replaced by alanine, alleviated the suppressive effect of E?/B48 lipoproteins on lysosomal hydrolase and cholesterol transporter expression, increased macrophage degradation of E?/B48 lipoproteins, enhanced cholesterol efflux, and suppressed foam cell formation. These observations suggest that induction of eIF‐2α phosphorylation is a mechanism by which ApoE‐deficient lipoproteins transform macrophages into foam cells. NIH grant support: K01HL076623 and RR003032 (H.Y.), R01ES014471 (Z.G.).

Effect of BSA-induced ER stress on SGLT protein expression levels and α-MG uptake in renal proximal tubule cells

Recent studies demonstrated that endoplasmic reticulum (ER) stress regulates glucose homeostasis and that ER stress preconditioning which induces an adaptive, protective unfolded protein response (UPR) offers cytoprotection against nephrotoxins. Thus the aim of the present study was to use renal proximal tubule cells (PTCs) to further elucidate the link between the BSA-induced ER stress and alpha-methyl-d-glucopyranoside (alpha-MG) uptake and to identify related signaling pathways. Among ER stress inducers such as high glucose, BSA, H2O2, or tumicamycin, BSA pretreatment ameliorated the reduction of Na(+)-glucose cotransporter (SGLT) expression and alpha-MG uptake by gentamicin or cyclosporine A. Immunofluorescence studies revealed that BSA (10 mg/ml) stimulated the expression of glucose-regulated protein 78 (GRP78), an ER stress biomarker. In addition, BSA increased levels of GRP78 protein expression and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation in a time-dependent manner. Furthermore, transfection with a GRP78-specific small interfering RNA (siRNA) inhibited BSA-stimulated SGLT expression and alpha-MG uptake. In experiments designed to unravel the mechanisms underlying BSA-induced ER stress, BSA stimulated the production of cellular reactive oxygen species (ROS), and antioxidants such as ascorbic acid or N-acetylcysteine (NAC) blocked BSA-induced increases in GRP78 activation, eIF2alpha phosphorylation, SGLT expression, and alpha-MG uptake. Moreover, the cells upregulated peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA levels in response to BSA or troglitazone (a PPARgamma agonist), but BSA was ineffective in the presence of GW9662 (a PPARgamma antagonist). In addition, both BSA and troglitazone stimulated GRP78 and eIF2alpha activation, SGLT expression, and alpha-MG uptake, whereas GW9662 inhibited the effects of BSA. BSA also stimulated phosphorylation of JNK and NF-kappaB, and GW9662 or GRP78 siRNA attenuated this response. Moreover, SP600125 or SN50 effectively blocked SGLT expression and alpha-MG uptake in BSA- or PPARgamma agonists (troglitazone or PGJ2)-treated PTCs. We conclude that BSA induces ER stress through ROS production and PPARgamma activation, which subsequently activates JNK/NF-kappaB signaling to enhance glucose uptake in renal PTCs.

Oxidized Low-Density Lipoproteins Trigger Endoplasmic Reticulum Stress in Vascular Cells

Oxidized low-density lipoproteins (oxLDLs) trigger various biological responses potentially involved in atherogenesis. Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response, which tends to restore ER homeostasis but switches to apoptosis when ER stress is prolonged. We aimed to investigate whether ER stress is induced by oxLDLs and can be prevented by the ER-associated chaperone ORP150 (150-kDa oxygen-regulated protein). oxLDLs and the lipid oxidation products 7-ketocholesterol and 4-hydroxynonenal induce ER stress in human endothelial cells (HMEC-1), characterized by the activation of ER stress sensors (phosphorylation of Ire1alpha and PERK, nuclear translocation of ATF6) and of their subsequent pathways (eukaryotic initiation factor 2alpha phosphorylation, expression of XBP1/spliced XBP1, CHOP, and KDEL chaperones GRP78, GRP94, ORP150). ER stress was inhibited by the antioxidant N-acetylcysteine. In advanced atherosclerotic lesions, phospho-Ire1alpha, KDEL, and ORP150 staining were localized in lipid-rich areas with 4-hydroxynonenal adducts and CD68-positive macrophagic cells. By comparison, staining for 4-hydroxynonenal, phospho-Ire1alpha, KDEL, and ORP were faint and more diffuse in intimal hyperplasia. ER stress takes part in the apoptotic effect of oxLDLs, through the Ire1alpha/c-Jun N-terminal kinase pathway, as assessed by the protective effect of specific small interfering RNAs and c-Jun N-terminal kinase inhibitor. Forced expression of the chaperone ORP150 reduced both oxLDL-induced ER stress and apoptosis. ER stress markers and ORP150 chaperone are expressed in areas containing oxLDLs in atherosclerotic lesions and are induced by oxLDLs and oxidized lipids in cultured cells. The forced expression of ORP150 highlights its new protective role against oxLDL-induced ER stress and subsequent apoptosis.

Roles of IP3R and RyR Ca2+ Channels in Endoplasmic Reticulum Stress and β-Cell Death

OBJECTIVE—Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca2+ release channels in the ER stress–associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis.RESEARCH DESIGN AND METHODS—Kinetics of β-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca2+ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)–based probes were used to measure Ca2+ in ER and mitochondria.RESULTS—Neither RyR nor IP3R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca2+ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)–associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP3Rs and RyRs. Conversely, stimulation of ER Ca2+ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization.CONCLUSIONS—This study demonstrates that the activity of ER Ca2+ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca2+ homeostasis and ER stress.

Stimulation of Ebola virus production from persistent infection through activation of the Ras/MAPK pathway

Human infections with Ebola virus (EBOV) result in a deadly viral disease known as Ebola hemorrhagic fever. Up to 90% of infected patients die, and there is no available treatment or vaccine. The sporadic human outbreaks are believed to result when EBOV "jumps" from an infected animal to a person and is subsequently transmitted between persons by direct contact with infected blood or body fluids. This study was undertaken to investigate the mechanism by which EBOV can persistently infect and then escape from model cell and animal reservoir systems. We report a model system in which infection of mouse and bat cell lines with EBOV leads to persistence, which can be broken with low levels of lipopolysaccharide or phorbol-12-myristate-13-acetate (PMA). This reactivation depends on the Ras/MAPK pathway through inhibition of RNA-dependent protein kinase and eukaryotic initiation factor 2alpha phosphorylation and occurs at the level of protein synthesis. EBOV also can be evoked from mice 7 days after infection by PMA treatment, indicating that a similar mechanism occurs in vivo. Our findings suggest that EBOV may persist in nature through subclinical infection of a reservoir species, such as bats, and that appropriate physiological stimulation may result in increased replication and transmission to new hosts. Identification of a presumptive mechanism responsible for EBOV emergence from its reservoir underscores the "hit-and-run" nature of the initiation of human and/or nonhuman primate EBOV outbreaks and may provide insight into possible countermeasures to interfere with transmission.

Novel Function of PERK as a Mediator of Force-induced Apoptosis

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.

GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2α in Arabidopsis

The yeast regulatory protein kinase, general control non-derepressible-2 (GCN2) plays a key role in general amino acid control. GCN2 phosphorylates the α subunit of the trimeric eukaryotic translation initiation factor-2 (eIF2), bringing about a decrease in the general rate of protein synthesis but an increase in the synthesis of GCN4, a transcription factor that promotes the expression of genes encoding enzymes for amino acid biosynthesis. The present study concerned the phosphorylation of Arabidopsis eIF2α (AteIF2α) by the Arabidopsis homologue of GCN2, AtGCN2, and the role of AtGCN2 in regulating genes encoding enzymes of amino acid biosynthesis and responding to virus infection. A null mutant for AtGCN2 called GT8359 was obtained and western analysis confirmed that it lacked AtGCN2 protein. GT8359 was more sensitive than wild-type Arabidopsis to herbicides that affect amino acid biosynthesis. Phosphorylation of AteIF2α occurred in response to herbicide treatment but only in wild-type Arabidopsis, not GT8359, showing it to be AtGCN2-dependent. Expression analysis of genes encoding key enzymes for amino acid biosynthesis and nitrate assimilation revealed little effect of loss of AtGCN2 function in GT8359 except that expression of a nitrate reductase gene, NIA1, was decreased. Analysis of wild-type and GT8359 plants infected with Turnip yellow mosaic virus or Turnip crinkle virus showed that AteIF2α was not phosphorylated.

2-Aminopurine Inhibits Lipid Accumulation Induced by Apolipoprotein E-Deficient Lipoprotein in Macrophages: Potential Role of Eukaryotic Initiation Factor-2α Phosphorylation in Foam Cell Formation

We previously reported that apolipoprotein (Apo) E-deficient, ApoB48-containing (E^–/B48) lipoproteins inhibited expression of lysosomal hydrolase and transformed mouse peritoneal macrophages (MPMs) into foam cells. The present study examined the effect of 2-aminopurine (2-AP), an inhibitor of eukaryotic initiation factor (eIF)-2α phosphorylation, on E^–/B48 lipoprotein-induced changes in gene expression and foam cell formation. Our data demonstrated that E^–/B48 lipoproteins enhanced phosphorylation of eIF-2α in macrophages. Incubation of MPMs with E^–/B48 lipoproteins inhibited the translation efficiency of mRNAs encoding lysosomal acid lipase, cathepsin B, and cation-dependent mannose 6 phosphate receptor, with a parallel reduction in the level of these proteins. Addition of 2-AP to the culture media alleviated the suppressive effect of E^–/B48 lipoproteins on lysosomal hydrolase mRNA translation, increased macrophage degradation of E^–/B48 lipoproteins, and inhibited foam cell formation. Transfection of MPMs with a nonphosphorylatable eIF-2α mutant also attenuated the suppressive effect of E^–/B48 lipoproteins on expression of lysosomal acid lipase, associated with a reduced accumulation of cellular cholesterol esters. This is the first demonstration that ApoE-deficient lipoproteins inhibit lysosomal hydrolase synthesis and transform macrophages into foam cells through induction of eIF-2α phosphorylation.

A decrease in cellular energy status stimulates PERK-dependent eIF2α phosphorylation and regulates protein synthesis in pancreatic β-cells

In the present study, we demonstrate that, in pancreatic beta-cells, eIF2alpha (eukaryotic initiation factor 2alpha) phosphorylation in response to a decrease in glucose concentration is primarily mediated by the activation of PERK [PKR (protein kinase RNA activated)-like endoplasmic reticulum kinase]. We provide evidence that this increase in PERK activity is evoked by a decrease in the energy status of the cell via a potentially novel mechanism that is independent of IRE1 (inositol requiring enzyme 1) activation and the accumulation of unfolded nascent proteins within the endoplasmic reticulum. The inhibition of eIF2alpha phosphorylation in glucose-deprived cells by the overexpression of dominant-negative PERK or an N-terminal truncation mutant of GADD34 (growth-arrest and DNA-damage-inducible protein 34) leads to a 53% increase in the rate of total protein synthesis. Polysome analysis revealed that this coincides with an increase in the amplitude but not the number of ribosomes per mRNA, indicating that eIF2alpha dephosphorylation mobilizes hitherto untranslated mRNAs on to polysomes. In summary, we show that PERK is activated at low glucose concentrations in response to a decrease in energy status and that this plays an important role in glucose-regulated protein synthesis in pancreatic beta-cells.

Double-Stranded RNA Induces Pancreatic β-Cell Apoptosis by Activation of the Toll-Like Receptor 3 and Interferon Regulatory Factor 3 Pathways

Viral infections contribute to the pathogenesis of type 1 diabetes. Viruses, or viral products such as double-stranded RNA (dsRNA), affect pancreatic beta-cell survival and trigger autoimmunity by unknown mechanisms. We presently investigated the mediators and downstream effectors of dsRNA-induced beta-cell death. Primary rat beta-cells and islet cells from wild-type, toll-like receptor (TLR) 3, type I interferon receptor (IFNAR1), or interferon regulatory factor (IRF)-3 knockout mice were exposed to external dsRNA (external polyinosinic-polycytidylic acid [PICex]) or were transfected with dsRNA ([PICin]). TLR3 signaling mediated PICex-induced nuclear factor-kappaB (NF-kappaB) and IRF-3 activation and beta-cell apoptosis. PICin activated NF-kappaB and IRF-3 in a TLR3-independent manner, induced eukaryotic initiation factor 2 alpha phosphorylation, and triggered a massive production of interferon (IFN)-beta. This contributed to beta-cell death, as islet cells from IFNAR1(-/-) or IRF-3(-/-) mice were protected against PICin-induced apoptosis. PICex and PICin trigger beta-cell apoptosis via the TLR3 pathway or IRF-3 signaling, respectively. Execution of PICin-mediated apoptosis depends on autocrine effects of type I IFNs.

Activation and Dysregulation of the Unfolded Protein Response in Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) are associated with known triggers of the unfolded protein response (UPR). The aims were to (1) evaluate the activity of UPR in NAFL and NASH and (2) correlate expression of UPR pathways with liver histology. Messenger RNA (mRNA) and protein expression were measured by quantitative real-time PCR and Western blot, respectively. Apoptosis was assessed by TUNEL assay. Liver histology was scored using the NASH clinical research network criteria. Compared with subjects with the metabolic syndrome and normal liver histology (n = 17), both NAFL (n = 21) and NASH (n = 21) were associated with increased eukaryotic initiation factor-2alpha (eIF-2alpha) phosphorylation. Activating transcription factor 4 (ATF4) mRNA and protein, C/EBP homologous protein (CHOP), and growth arrest, DNA damage-34 (GADD34) mRNA were not increased in NAFL or NASH. Whereas immunoglobulin heavy chain binding protein mRNA was significantly increased in NASH, unspliced X-box protein-1 (XBP-1) protein did not increase. Also, endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein mRNA levels were inversely related to spliced XBP-1 mRNA in NASH. NASH was specifically associated with low sXBP-1 protein and increased JNK phosphorylation. This correlated with increased TUNEL activity in NASH. The histologic severity correlated with sXBP-1 mRNA and JNK phosphorylation. There is a variable degree of UPR activation in NAFL and NASH. Although both NAFL and NASH are associated with eIF-2alpha phosphorylation, there is a failure to activate downstream recovery pathways, ie, ATF4-CHOP-GADD34. NASH is specifically associated with (1) failure to generate sXBP-1 protein and (2) activation of JNK.

Translational Inhibition of Colonic Epithelial Heat Shock Proteins by IFN-γ and TNF-α in Intestinal Inflammation

Inducible heat shock proteins (iHsp), Hsp25/27 and Hsp70, play essential roles in protecting cells against stress and, in intestinal mucosal inflammation, potentially lessening the extent and severity of injury. We examined the expression and regulation of iHsp in human and experimental inflammatory bowel diseases (IBD) and in vitro. iHsp expression and regulation were assessed in normal and IBD colonic biopsy specimens, IL-10(-/-) mice, and young adult mouse colonic epithelial cells by immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR). Phosphorylation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2alpha (eIF-2alpha) was determined by Western blot. Hsp25/27 and Hsp70 levels were selectively reduced in areas of active mucosal inflammation associated with human IBD and IL-10(-/-) mice with colitis. Wild-type mice treated in vivo with interferon (IFN)-gamma + tumor necrosis factor (TNF)-alpha also demonstrated reduced colonic Hsp25/27 and Hsp70. In young adult mouse colonic epithelial cells, IFN-gamma+TNF-alpha inhibited heat induction of Hsp25/27 and Hsp70, an effect not associated with changes in iHsp messenger RNA or protein half-lives but caused by suppressed de novo iHsp synthesis. IFN-gamma+TNF-alpha cotreatment activated PKR, resulting in phosphorylation and inactivation of eIF-2alpha, an essential factor in protein translation. These effects were not due to induced apoptosis and could be negated by PKR-inhibitor and short interfering RNA to PKR. Increased phosphorylation of PKR and eIF-2alpha were also observed in active IBD tissues. Mucosal inflammation is associated with iHsp down-regulation, an effect that appears mediated by translational down-regulation by proinflammatory cytokines. In the context of IBD, we propose that this mechanism contributes to the severity, extent, and persistence of inflammation-induced mucosal injury.