PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.

Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perkf/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.

Curcumin Inhibits the PERK-eIF2α-CHOP Pathway through Promoting SIRT1 Expression in Oxidative Stress-induced Rat Chondrocytes and Ameliorates Osteoarthritis Progression in a Rat Model.

Oxidative stress plays a crucial role in the occurrence and development of osteoarthritis (OA) through the activation of endoplasmic reticulum (ER) stress. Curcumin is a polyphenolic compound with significant antioxidant and anti-inflammatory activity among various diseases. To elucidate the role of curcumin in oxidative stress-induced chondrocyte apoptosis, this study investigated the effect of curcumin on ER stress-related apoptosis and its potential mechanism in oxidative stress-induced rat chondrocytes. The results of flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining showed that curcumin can significantly attenuate ER stress-associated apoptosis. Curcumin inhibited the expression of cleaved caspase3, cleaved poly (ADP-ribose) polymerase (PARP), C/EBP homologous protein (CHOP), and glucose-regulated protein78 (GRP78) and upregulated the chondroprotective protein Bcl2 in TBHP-treated chondrocytes. In addition, curcumin promoted the expression of silent information regulator factor 2-related enzyme 1 (SIRT1) and suppressed the expression of activating transcription factor 4 (ATF4), the ratio of p-PERK/PERK, p-eIF2α/eIF2α. Our anterior cruciate ligament transection (ACLT) rat OA model research demonstrated that curcumin (50 mg/kg and 150 mg/kg) ameliorated the degeneration of articular cartilage and inhibited chondrocyte apoptosis in ACLT rats in a dose-dependent manner. By applying immunohistochemical analysis, we found that curcumin enhanced the expression of SIRT1 and inhibited the expression of CHOP and cleaved caspase3 in ACLT rats. Taken together, our present findings firstly indicate that curcumin could inhibit the PERK-eIF2α-CHOP axis of the ER stress response through the activation of SIRT1 in tert-Butyl hydroperoxide- (TBHP-) treated rat chondrocytes and ameliorated osteoarthritis development in vivo.

Inhibition of the dephosphorylation of eukaryotic initiation factor 2α ameliorates murine experimental pancreatitis.

BACKGROUND: Endoplasmic reticulum (ER) stress in the pancreas is closely associated with the development of acute pancreatitis. However, the role of the protein kinase RNA-like ER kinase (PERK) in this disease is not fully understood. We investigated whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, could improve murine experimental pancreatitis through the amelioration of ER stress. METHODS: Acute pancreatitis was induced by the intraperitoneal administration of cerulein (50 µg/kg) six times at 1-h intervals followed by lipopolysaccharide (10 mg/kg). Salubrinal was administered intraperitoneally immediately after lipopolysaccharide injection and 3 h later. Mice were sacrificed 24 h after the first injection of cerulein, and serum amylase and proinflammatory cytokines were measured. The severity of pancreatitis was evaluated histologically using a scoring system. The expression levels of ER stress-related proteins were evaluated by Western blotting. RESULTS: The administration of salubrinal significantly attenuated the increase in serum amylase levels and improved histologically assessed pancreatitis. The serum levels of proinflammatory cytokines were significantly suppressed in salubrinal-treated mice, as was the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein, and cleaved caspase-3. CONCLUSIONS: The amelioration of ER stress through augmentation of the PERK-signaling pathway may be a therapeutic target for the treatment of acute pancreatitis.

GCN2 reduces inflammation by p-eIF2α/ATF4 pathway after intracerebral hemorrhage in mice.

Intracerebral hemorrhage (ICH) is a common and severe neurological disorder, which is associated with high rates of mortality and morbidity. This study aimed to evaluate whether general control non-derepressible-2 (GCN2) stimulation ameliorated neuroinflammation after ICH. Male CD-1 mice were subjected to experimental ICH by infusion of bacterial collagenase. Post-ictus assessment included neurobehavioral tests, brain edema measurement, quantification of neutrophil infiltration and microglia activation, and measurement of TNF-α and IL-1ß expression at 24h after ICH. Furthermore, we tested the long-term neurological improvement by GCN2 at 21 days after ICH. Our results showed that GCN2 improved neurological function and reduced brain edema at 24 and 72 h following experimental ICH in CD-1 mice in contrast to the vehicle administration alone. GCN2 was also found to decrease levels of IL-1ß and TNF-α, and inhibit neutrophil infiltration activation. In addititon, GCN2 also alleviated long-term neurological impairment after ICH. However, inhibition of eIF2α or ATF4 abolished the protective effects of GCN2, indicating eIF2α/ATF4 signaling pathway as the downstream mediator of GCN2.

Boric Acid Activation of eIF2α and Nrf2 Is PERK Dependent: a Mechanism that Explains How Boron Prevents DNA Damage and Enhances Antioxidant Status.

Boron is abundant in vegetables, nuts, legumes, and fruit and intake is associated with reduced risk of cancer and DNA damage and increased antioxidant status. Blood boric acid (BA) levels are approximately 10 µM BA in men at the mean US boron intake. Treatment of DU-145 human prostate cancer cells with 10 µM BA stimulates phosphorylation of elongation initiation factor 2α (eIF2α) at Ser51 leading to activation of the eIF2α/ATF4 pathway which activates the DNA damage-inducible protein GADD34. In the present study, we used MEF WT and MEF PERK (±) cells to test the hypothesis that BA-activated eIF2α phosphorylation requires protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activates Nrf2 and the antioxidant response element (ARE). BA (10 µM) increased phosphorylation of eIF2α Ser51 in MEF WT cells at 1 h, but not in MEF Perk -/- cells exposed for as long as 6 h. GCN2 kinase-dependent phosphorylation of eIF2α Ser51 was activated in MEF PERK -/- cells by amino acid starvation. Nrf2 phosphorylation is PERK dependent and when activated is translocated from the cytoplasm to the nucleus where it acts as a transcription factor for ARE. DU-145 cells were treated with 10 µM BA and Nrf2 measured by immunofluorescence. Cytoplasmic Nrf2 was translocated to the nucleus at 1.5-2 h in DU-145 and MEF WT cells, but not MEF PERK -/- cells. Real-time PCR was used to measure mRNA levels of three ARE genes (HMOX-1, NQO1, and GCLC). Treatment with 10 µM BA increased the mRNA levels of all three genes at 1-4 h in DU-145 cells and HMOX1 and GCLC in MEF WT cells. These results extend the known boric acid signaling pathway to ARE-regulated genes. The BA signaling pathway can be expressed using the schematic [BA + cADPR → cADPR-BA → [[ER]i Ca2+↓] → 3 pathways: PERK/eIF2αP → pathways ATF4 and Nrf2; and [[ER]i Ca2+↓] → ER stress → ATF6 pathway. This signaling pathway provides a framework that links many of the molecular changes that underpin the biological effects of boron intake.

The effect of metformin treatment on endoplasmic reticulum (ER) stress induced by status epilepticus (SE) via the PERK-eIF2α-CHOP pathway.

Status epilepticus (SE) is defined as continuous seizure activity lasting more than 5 minutes. It results in neuronal cell death, mediated by endoplasmic reticulum (ER) stress response. Previously, metformin demonstrated neuroprotective effects in primary cortical neurons. In this study, we analyzed the effect of metformin on ER stress via the pro-apoptotic protein kinase RNA-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (eIF2α)-C/EBP homologous protein (CHOP) pathway. SE was induced in rats by pentylenetetrazole. Following SE, the rats were treated with salubrinal, GSK2656157, or metformin. In a control group (normal saline) SE was not induced. CHOP, eIF2α, and PERK expression was determined by Western blot; apoptosis was analyzed by TUNEL assay. CHOP expression was significantly increased at 6 and 24 hours following SE. At both time points, eIF2α and PERK levels were also increased. At 6 hours, CHOP expression was significantly reduced in salubrinal, GSK2656157 and metformin groups versus SE group. eIF2α and PERK levels were decreased in metformin compared to SE group. eIF2α expression was markedly decreased in salubrinal versus SE group, while PERK expression was markedly reduced in GSK2656157 versus SE group. At 6 and 24 hours, the apoptosis rate was significantly increased in SE versus control group, while it was significantly reduced in salubrinal, GSK2656157, and metformin groups compared to SE group. The apoptosis rate also decreased in salubrinal group at 24 hours, although not to the extent observed in metformin group. Overall, CHOP expression and apoptosis induced by SE in rats were reduced with metformin. Further studies are required to evaluate the clinical relevance of metformin for patients with SE.

Neuroprotective Effects of Protein Tyrosine Phosphatase 1B Inhibition against ER Stress-Induced Toxicity.

Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Protein tyrosine phosphatase 1B (PTP1B) is known to regulate the ER stress signaling pathway, but its role in neuronal systems in terms of ER stress remains largely unknown. Here, we showed that rotenone-induced toxicity in human neuroblastoma cell lines and mouse primary cortical neurons was ameliorated by PTP1B inhibition. Moreover, the increase in the level of ER stress markers (eIF2α phosphorylation and PERK phosphorylation) induced by rotenone treatment was obviously suppressed by concomitant PTP1B inhibition. However, the rotenone-induced production of reactive oxygen species (ROS) was not affected by PTP1B inhibition, suggesting that the neuroprotective effect of the PTP1B inhibitor is not associated with ROS production. Moreover, we found that MG132-induced toxicity involving proteasome inhibition was also ameliorated by PTP1B inhibition in a human neuroblastoma cell line and mouse primary cortical neurons. Consistently, downregulation of the PTP1B homologue gene in Drosophila mitigated rotenone- and MG132-induced toxicity. Taken together, these findings indicate that PTP1B inhibition may represent a novel therapeutic approach for ER stress-mediated neurodegenerative diseases.

Involvement of the agmatinergic system in the depressive-like phenotype of the Crtc1 knockout mouse model of depression.

Recent studies implicate the arginine-decarboxylation product agmatine in mood regulation. Agmatine has antidepressant properties in rodent models of depression, and agmatinase (Agmat), the agmatine-degrading enzyme, is upregulated in the brains of mood disorder patients. We have previously shown that mice lacking CREB-regulated transcription coactivator 1 (CRTC1) associate behavioral and molecular depressive-like endophenotypes, as well as blunted responses to classical antidepressants. Here, the molecular basis of the behavioral phenotype of Crtc1(-/-) mice was further examined using microarray gene expression profiling that revealed an upregulation of Agmat in the cortex of Crtc1(-/-) mice. Quantitative polymerase chain reaction and western blot analyses confirmed Agmat upregulation in the Crtc1(-/-) prefrontal cortex (PFC) and hippocampus, which were further demonstrated by confocal immunofluorescence microscopy to comprise an increased number of Agmat-expressing cells, notably parvalbumin- and somatostatin-positive interneurons. Acute agmatine and ketamine treatments comparably improved the depressive-like behavior of male and female Crtc1(-/-) mice in the forced swim test, suggesting that exogenous agmatine has a rapid antidepressant effect through the compensation of agmatine deficit because of upregulated Agmat. Agmatine rapidly increased brain-derived neurotrophic factor (BDNF) levels only in the PFC of wild-type (WT) females, and decreased eukaryotic elongation factor 2 (eEF2) phosphorylation in the PFC of male and female WT mice, indicating that agmatine might be a fast-acting antidepressant with N-methyl-D-aspartate (NMDA) receptor antagonist properties. Collectively, these findings implicate Agmat in the depressive-like phenotype of Crtc1(-/-) mice, refine current understanding of the agmatinergic system in the brain and highlight its putative role in major depression.

Regulation of eIF2α expression and renal interstitial fibrosis by resveratrol in rat renal tissue after unilateral ureteral obstruction.

PURPOSE: This study was performed to assess the effect of resveratrol on the expression of eukaryotic initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4) in renal tissues of rats with unilateral ureteral obstruction (UUO). METHODS: Using UUO animal model, after 14 days of surgery, pathological changes were detected by HE staining, renal tubular damage index, renal interstitial collagen deposition area were evaluated by Masson staining, in situ cell apoptosis in renal tissue was analyzed by TUNEL assay, and protein expression of eIF2α and ATF4 in renal tissue was analyzed using western blot detection. RESULTS: After comparison of the treatment groups with model group, we observed that the degree of renal tubular damage, relative area of renal interstitial collagen and eIF2α, ATF4 protein expression were also significantly reduced (p<0.05, p <0.01) in the high-dose resveratrol group. CONCLUSION: Resveratrol can reduce the level of eIF2α protein expression, which further reduces the ATF4 levels.

The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly.

Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.

Inhibition of the dephosphorylation of eukaryotic initiation factor 2α ameliorates murine experimental colitis.

BACKGROUND/AIMS: Endoplasmic reticulum (ER) stress in the intestine is closely associated with the development of inflammatory bowel disease (IBD). However, the role of the protein kinase RNA-like ER kinase in this disease is not fully known. We studied whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, improves murine experimental colitis through the amelioration of ER stress. METHODS: Colitis was induced by the administration of 3% dextran sulfate sodium (DSS) for 5 days. Mice were injected salubrinal intraperitoneally from the commencement of DSS treatment and were sacrificed on day 10. The severity of colitis was evaluated histologically using a scoring system.Myeloperoxidase activity and the expression of proinflammatory cytokine genes in the colon were analyzed. The expression levels of ER stress-related proteins were evaluated by Western blotting. RESULTS: The administration of salubrinal significantly attenuated body weight loss and improved colitis, as assessed histologically. The elevation of myeloperoxidase activity and the expression of proinflammatory cytokine genes were suppressed in salubrinal-treated mice. The expression of glucose-regulated protein 78, activating translation factor 4, and heat-shock protein 70 was elevated in mice treated with salubrinal. CONCLUSION: The amelioration of ER stress may be a therapeutic target for the treatment of IBD.

Arsenic trioxide induced endoplasmic reticulum stress in laryngeal squamous cell line Hep-2 cells.

Arsenic trioxide (As2O3) has been used in the treatment of acute promyelocytic leukemia (APL) and many malignant solid tumors. Recently, endoplasmic reticulum (ER) stress plays an important role in As2O3-treated laryngeal squamous cell line Hep-2 cells. In the present work, the expression of ER stress-related proteins was investigated in As2O3-treated Hep-2 cells. The results showed that As2O3 increased the expression of GRP78, CHOP, phosphorylated eIF2α and ATF4, all of which are the molecule of ER stress. Therefore, As2O3 induced ER stress in Hep-2 cells.

Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine-induced cytotoxicity and ECM degradation. MATERIAL AND METHODS: Cytotoxicity and cell death were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT-PCR and western blot analysis. RESULTS: Treatment with nicotine reduced cell viability and increased the proportion of annexin V-negative, propidium iodide-positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α and glucose-regulated protein-78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP-1, MMP-2, MMP-8 and MMP-9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine-induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor-κB. CONCLUSION: These results indicate that nicotine-induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.

Effects of salubrinal on cadmium-induced apoptosis in HK-2 human renal proximal tubular cells.

Cadmium exposure is known to cause endoplasmic reticulum (ER) stress. In our current study, we examined the effects of salubrinal, a selective inhibitor of eukaryotic translation initiation factor 2 subunit α (eIF2α) dephosphorylation, on apoptotic cell death and ER stress-signaling events in HK-2 human renal proximal tubular cells exposed to cadmium chloride (CdCl(2)). Using phase-contrast microscopy and a cell viability assay, we observed that salubrinal suppressed CdCl(2)-induced cellular damage and cell death. Treatment with salubrinal reduced the number of TUNEL-positive cells and the cleavages of caspase-3 and poly(ADP-ribose) polymerase, but not the cleavage of light chain 3B, indicating protection from CdCl(2)-induced apoptosis but not autophagy. Although eIF2α remained phosphorylated after CdCl(2) exposure to salubrinal-treated HK-2 cells, the expression of activating transcription factor 4 (ATF4) and the 78 kDa glucose-regulated protein (GRP78) was not increased. On the other hand, CdCl(2)-induced expression of C/EBP homologous protein (CHOP) was reduced by salubrinal treatment. Expression of ATF4, an upstream regulator of GRP78 and CHOP, appeared to be a prerequisite for full protection by salubrinal against cadmium cytotoxicity, because CdCl(2)-induced cellular damage was not fully suppressed in ATF4-deficient cells. Phosphorylated forms of mitogen-activated protein kinases (MAPKs), including c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal-regulated protein kinase (ERK), increased after CdCl(2) exposure, whereas salubrinal suppressed the phosphorylation of JNK and p38 but not ERK. These results suggest that salubrinal protects CdCl(2)-exposed HK-2 cells from apoptosis by suppressing cell death signal transduction pathways.

Sorafenib with doxorubicin augments cytotoxicity to renal cell cancer through PERK inhibition.

Although cytokine therapy involving interleukin-2 or interferon-alpha has been employed for metastatic renal cell cancer (RCC) treatment, these therapies yielded limited response and benefit. Recently, several molecular-targeted agents have become available, and one newly developed anti-RCC agent, sorafenib (BAY 43-9006), is known to target multiple kinases. In this study, sorafenib was found to inhibit phosphorylation of the eukaryotic initiation factor-2alpha (eIF2alpha) and induce cell cycle arrest at G2/M phase and increase cell death. One of eIF2alpha kinases, PERK was responsible for eIF2alpha phosphorylation in RCC cells and PERK knockdown induced cell death similar to sorafenib treatment. The efficiency of sorafenib treatment correlated with phosphorylation level of eIF2alpha and nuclear Nrf2 expression level in eight RCC cell lines. Furthermore, sorafenib made Caki-1 and 786-O cells, but not ACHN cells sensitive to oxidative stress exerted by both hydrogen peroxide and doxorubicin. In addition, PERK knockdown sensitized Caki-1 and 786-O cells, but not ACHN cells to oxidative stress. In conclusion, levels of phospho-eIF2alpha and nuclear Nrf2 expression level in RCC might be a predictor of outcome in sorafenib treatment. In addition, PERK inhibition as well as sorafenib plus doxorubicin might be a promising therapeutic approach for RCC characterized by high levels of phosphorylated-eIF2alpha and nuclear Nrf2.

The induction of G2/M cell-cycle arrest and apoptosis by cucurbitacin E is associated with increased phosphorylation of eIF2alpha in leukemia cells.

The antiproliferative and apoptotic effects of cucurbitacin E, a natural product isolated from Cucurbitaceae, were determined in human leukemia HL-60 cells. Cucurbitacin E at low concentrations (3-50 nmol/l) inhibited the growth of HL-60 cells, which was associated with G2/M cell-cycle arrest, decrease in the levels of cyclin-dependent kinase1, and increase in the levels of p21. Cucurbitacin E at high concentrations (1-10 mol/l) induced apoptosis of HL-60 cells and activation of caspase-3, caspase-8, and caspase-9. Jurkat leukemia cells with or without caspase-8 expression were nearly equally sensitive to cucurbitacin E-induced apoptosis. Cucurbitacin E did not increase the levels of reactive oxygen species and antioxidants, N-acetylcysteine and catalase, did not block cucurbitacin E-induced apoptosis. Cucurbitacin E decreased the levels of the antiapoptotic proteins XIAP, survivin, and Mcl-1, but increased the level of the proapoptotic protein, Bax. The levels of phosphorylated eukaryotic translation initiation factor 2 subunit (eIF2) were induced in cells undergoing both apoptosis and cell-cycle arrest. As phosphorylated eIF2 is an inhibitor of protein translation initiation, our data suggest that cucurbitacin E induces cell growth arrest and apoptosis through the induction of eIF2 phosphorylation, which leads to the inhibition of cyclin-dependent kinase 1, Mcl-1, survivin, and/or XIAP protein synthesis and that cucurbitacin E induces apoptosis mainly through a mitochondrial-mediated pathway.

Attenuation of skeletal muscle atrophy in cancer cachexia by D-myo-inositol 1,2,6-triphosphate.

PURPOSE: To determine the effectiveness of the polyanionic, metal binding agent D-myo-inositol-1,2,6-triphosphate (alpha trinositol, AT), and its hexanoyl ester (HAT), in tissue wasting in cancer cachexia. METHODS: The anti-cachexic effect was evaluated in the MAC16 tumour model. RESULTS: Both AT and HAT attenuated the loss of body weight through an increase in the nonfat carcass mass due to an increase in protein synthesis and a decrease in protein degradation in skeletal muscle. The decrease in protein degradation was associated with a decrease in activity of the ubiquitin-proteasome proteolytic pathway and caspase-3 and -8. Protein synthesis was increased due to attenuation of the elevated autophosphorylation of double-stranded RNA-dependent protein kinase, and of eukaryotic initiation factor 2alpha together with hyperphosphorylation of eIF4E-binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2. In vitro, AT completely attenuated the protein degradation in murine myotubes induced by both proteolysis-inducing factor and angiotensin II. CONCLUSION: These results show that AT is a novel therapeutic agent with the potential to alleviate muscle wasting in cancer patients.

Role of endoplasmic reticulum stress in acrolein-induced endothelial activation.

Acrolein is a ubiquitous environmental pollutant and an endogenous product of lipid peroxidation. It is also generated during the metabolism of several drugs and amino acids. In this study, we examined the effects of acrolein on endothelial cells. Treatment of human umbilical vein endothelial cells (HUVECs) with 2 to 10 microM acrolein led to an increase in the phosphorylation of eIF-2alpha within 10 to 30 min of exposure. This was followed by alternate splicing of XBP-1 mRNA and an increase in the expression of the endoplasmic reticulum (ER) chaperone genes Grp78 and Herp. Within 2-4 h of treatment, acrolein also increased the abundance and the nuclear transport of the transcription factors ATF3, AFT4, and CHOP. Acrolein-induced increase in ATF3 was prevented by treating the cells with the chemical chaperone - phenylbutyric acid (PBA). Treatment with acrolein increased phosphorylation of ERK1/2, p38, and JNK. The increase in JNK phosphorylation was prevented by PBA. Acrolein treatment led to activation and nuclear translocation of the transcription factor NF-kappaB and an increase in TNF-alpha, IL-6 and IL-8, but not MCP-1, mRNA. Increased expression of cytokine genes and NF-kappaB activation were not observed in cells treated with PBA. These findings suggest that exposure to acrolein induces ER stress and triggers the unfolded protein response and that NF-kappaB activation and stimulation of cytokine production by acrolein could be attributed, in part, to ER stress. Chemical chaperones of protein-folding may be useful in treating toxicological and pathological states associated with excessive acrolein exposure or production.

A chemical compound commonly used to inhibit PKR, {8-(imidazol-4-ylmethylene)-6H-azolidino[5,4-g] benzothiazol-7-one}, protects neurons by inhibiting cyclin-dependent kinase.

Activation of the double-stranded RNA-dependent protein kinase (PKR) has been implicated in the pathogenesis of several neurodegenerative diseases. We find that a compound widely used as a pharmacological inhibitor of this enzyme, referred to as PKR inhibitor (PKRi), {8-(imidazol-4-ylmethylene)-6H-azolidino[5,4-g]benzothiazol-7-one}, protects against the death of cultured cerebellar granule and cortical neurons. PKRi also prevents striatal neurodegeneration and improves behavioral outcomes in a chemically induced mouse model of Huntington's disease. Surprisingly, PKRi fails to block the phosphorylation of eIF2alpha, a downstream target of PKR, and does not reduce the autophosphorylation of PKR enzyme immunoprecipitated from neurons. Furthermore, neurons lacking PKR are fully protected from apoptosis by PKRi, demonstrating that neuroprotection by this compound is not mediated by PKR inhibition. Using in vitro kinase assays we investigated whether PKRi affects any other protein kinase. These analyses demonstrated that PKRi has no major inhibitory effect on pro-apoptotic kinases such as the c-Jun N-terminal kinases, the p38 MAP kinases and the death-associated protein kinases, or on other kinases including c-Raf, MEK1, MKK6 and MKK7. PKRi does, however, inhibit the activity of certain cyclin-dependent kinases (CDKs), including CDK1, CDK2 and CDK5 both in vitro and in low potassium-treated neurons. Consistent with its inhibitory action on mitotic CDKs, the treatment of HT-22 and HEK293T cell lines with PKRi sharply reduces the rate of cell cycle progression. Taken together with the established role of CDK activation in the promotion of neurodegeneration, our results suggest that PKRi exerts its neuroprotective action by inhibiting CDK.

The G1-S checkpoint in fission yeast is not a general DNA damage checkpoint.

Inhibitory mechanisms called checkpoints regulate progression of the cell cycle in the presence of DNA damage or when a previous cell-cycle event is not finished. In fission yeast exposed to ultraviolet light the G1-S transition is regulated by a novel checkpoint that depends on the Gcn2 kinase. The molecular mechanisms involved in checkpoint induction and maintenance are not known. Here we characterise the checkpoint further by exposing the cells to a variety of DNA-damaging agents. Exposure to methyl methane sulphonate and hydrogen peroxide induce phosphorylation of eIF2alpha, a known Gcn2 target, and an arrest in G1 phase. By contrast, exposure to psoralen plus long-wavelength ultraviolet light, inducing DNA adducts and crosslinks, or to ionizing radiation induce neither eIF2alpha phosphorylation nor a cell-cycle delay. We conclude that the G1-S checkpoint is not a general DNA-damage checkpoint, in contrast to the one operating at the G2-M transition. The tight correlation between eIF2alpha phosphorylation and the presence of a G1-phase delay suggests that eIF2alpha phosphorylation is required for checkpoint induction. The implications for checkpoint signalling are discussed.