Hypoxic preconditioning protects microvascular endothelial cells against hypoxia/reoxygenation injury by attenuating endoplasmic reticulum stress

Endothelial cells (ECs) are directly exposed to hypoxia and contribute to injury during myocardial ischemia/reperfusion. Hypoxic preconditioning (HPC) protects ECs against hypoxia injury. This study aimed to explore whether HPC attenuates hypoxia/reoxygenation (H/R) injury by suppressing excessive endoplasmic reticulum stress (ERS) in cultured microvascular ECs (MVECs) from rat heart. MVECs injury was measured by lactate dehydrogenase (LDH) leakage, cytoskeleton destruction, and apoptosis. Expression of glucose regulating protein 78 (GRP78) and C/EBP homologous protein (CHOP), activation of caspase-12 (pro-apoptosis factors) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) were detected by western blot analysis. HPC attenuated H/R-induced LDH leakage, cytoskeleton destruction, and cell apoptosis, as shown by flow cytometry, Bax/Bcl-2 ratio, caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. HPC suppressed H/R-induced ERS, as shown by a decrease in expression of GRP78 and CHOP, and caspase-12 activation. HPC enhanced p38 MAPK phosphorylation but decreased that of protein kinase R-like ER kinase (PERK, upstream regulator of CHOP). SB202190 (an inhibitor of p38 MAPK) abolished HPC-induced cytoprotection, downregulation of GRP78 and CHOP, and activation of caspase-12, as well as PERK phosphorylation. HPC may protect MVECs against H/R injury by suppressing CHOP-dependent apoptosis through p38 MAPK mediated downregulation of PERK activation.     

Delayed cytoprotection induced by hypoxic preconditioning in cultured neonatal rat cardiomyocytes: role of GRP78

Hypoxic preconditioning (HPC) has been well demonstrated to have potent protective effects in many cell types; however, the mechanisms responsible for this phenomenon are not fully understood. Recently, glucose-regulated protein 78 (GRP78), an inducible molecular chaperon, was indicated to be associated with ischemic preconditioning. We hypothesized that HPC protects cardiomyocytes against hypoxia by inducing GRP78 in cultured neonatal rat cardiomyocytes. HPC was induced by exposing cardiomyocytes to brief hypoxia (1% O(2), 30 min) followed by reoxygenation. GRP78 was expressed constitutively in cultured cardiomyocytes and its expression was enhanced at 12 h, peaked at 24 h (207.3+/-23.6% of the baseline), and was sustained for up to 72 h after HPC. Twenty-four hours after HPC, the myocytes were subjected to prolonged hypoxia (1% O(2), 12 h). The lactic dehydrogenase (LDH) release and malondialdehyde (MDA) content were reduced, while cell viability and superoxide dismutase (SOD) activity were increased in the preconditioned cells compared with the non-HPC cells. The GRP78 protein level was higher in cells exposed to both HPC and hypoxia than in the cells exposed to HPC alone or hypoxia alone. Heat shock protein 70 (HSP70) was induced in parallel by late HPC. Transfection of GRP78 antisense oligonucleotides blocked GRP78 expression but not HSP70, resulting in attenuated cardioprotection afforded by late HPC. Furthermore, inducing GRP78 by gene transfer protected cardiomyocytes from hypoxic injury. These findings demonstrate that the induction of GRP78 partially mediates the late HPC, suggesting that GRP78 is a novel mechanism responsible for the late cytoprotection of HPC.     

Peptide-binding GRP78 protects neurons from hypoxia-induced apoptosis

Brain ischemia has major consequences leading to the apoptosis of astrocytes and neurons. Glucose-regulated protein 78 (GRP78) known for its role in endoplasmic reticulum stress alleviation was discovered on several cell surfaces acting as a receptor for signaling pathways. We have previously described peptides that bind cell surface GRP78 on endothelial cells to induce angiogenesis. We have also reported that ADoPep1 binds cardiomyocytes to prevent apoptosis of ischemic heart cells. In this study we describe the effect of hypoxia on astrocytes and neurons cell surface GRP78. Under hypoxic conditions, there was an increase of more than fivefold in GRP78 on cell surface of neurons while astrocytes were not affected. The addition of the GRP78 binding peptide, ADoPep1, to neurons decreased the percentage of GRP78 positive cells and did not change the percent of astrocytes. However, a significant increase in early and late apoptosis of both astrocytes and neurons under hypoxia was attenuated in the presence of ADoPep1. Intravitreal administration of ADoPep1 to mice in a model of optic nerve crush significantly reduced retinal cell loss after 21 days compared to the crush-damaged eyes without treatment or by control saline vehicle injection. Histological staining demonstrated reduced GRP78 after ADoPep1 treatment. The mechanism of peptide neuroprotection was demonstrated by the inhibition of hypoxia induced caspase 3/7 activity, cytochrome c release and p38 phosphorylation. This study is the first report on hypoxic neuronal and astrocyte cell surface GRP78 and suggests a potential therapeutic target for neuroprotection.     

Cellular and Subcellular Localization of Endoplasmic Reticulum Chaperone GRP78 Following Transient Focal Cerebral Ischemia in Rats

The 78-kDa glucose-regulated protein (GRP78), a chaperone protein located in the endoplasmic reticulum (ER), has been reported to have neuroprotective effects in the injured central nervous system. Our aim was to examine the expression profiles and subcellular distributions of GRP78 and its association with the neuroglial reaction in the rat striatum after transient, focal cerebral ischemia. In sham-operated rats, constitutive, specific immunoreactivity for GRP78 was almost exclusively localized to the rough ER of striatal neurons, with none in the resting, ramified microglia or astrocytes. At 1 day post reperfusion, increased expression was observed in ischemia-resistant cholinergic interneurons, when most striatal neurons had lost GRP78 expression (this occurred earlier than the loss of other neuronal markers). By 3 days post reperfusion, GRP78 expression had re-emerged in association with the activation of glial cells in both infarct and peri-infarct areas but showed different patterns in the two regions. Most of the expression induced in the infarct area could be attributed to brain macrophages, while expression in the peri-infarct area predominantly occurred in neurons and reactive astrocytes. A gradual, sustained induction of GRP78 immunoreactivity occurred in reactive astrocytes localized to the astroglial scar, lasting for at least 28 days post reperfusion. Using correlative light- and electron-microscopy, we found conspicuous GRP78 protein localized to abnormally prominent, dilated rough ER in both glial cell types. Thus, our data indicate a link between GRP78 expression and the activated functional status of neuroglial cells, predominantly microglia/macrophages and astrocytes, occurring in response to ischemia-induced ER stress.     

A 78-kDa glucose-regulated protein is involved in the decrease of interleukin-6 secretion by lead treatment from astrocytes

Interleukin (IL)-6 is a cytokine produced mainly by microglia and astrocytes and plays a pleiotropic role in the central nervous system. In this study, we cloned rat IL-6 cDNA into an enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRed2) vector and rat 78-kDa glucose-regulated protein (GRP78) cDNA into an EGFP vector to construct IL-6-EGFP, IL-6-DsRed2, and GRP78-EGFP chimeras for the investigation of the mechanism of IL-6 secretion from astrocytes. The data showed that constructed IL-6-EGFP and IL-6-DsRed2 chimeras retained the secretory property, and the secretion of IL-6-EGFP from astrocytes could be attenuated by GRP78 depletion with double-stranded RNA interference. Coexpression of IL-6-DsRed2 and dysfunctional GRP78-EGFP abolished IL-6-DsRed2 secretion, and two chimeric proteins colocalized inside living astrocytes. Coimmunoprecipitation analysis indicated that IL-6 and GRP78 resided in the same complex. The data further revealed that IL-6-EGFP secretion from astrocytes was blocked by the heavy metal lead (Pb) in a concentration-dependent manner. Analysis of the Pb interaction with protein on a Pb-affinity column demonstrated that Pb bound to GRP78 but failed to bind to IL-6. Therefore, these data suggest that IL-6-EGFP or IL-6-DsRed2 chimeras can be used as imaging probes to study IL-6 secretion from living cells, that GRP78 is involved in IL-6 secretion from astrocytes, and that Pb can block IL-6 secretion from astrocytes via targeting GRP78. chaperone; cytokine; protein-protein interactions     

Lead-induced endoplasmic reticulum (ER) stress responses in the nervous system

Lead (Pb) poisoning continues to be a significant health risk because of its pervasiveness in the environment, its known neurotoxic effects in children, and potential endogenous exposure from Pb deposited in bone. New information about mechanisms by which Pb enters cells and its organelle targets within cells are briefly reviewed. Toxic effects of Pb on the endoplasmic reticulum (ER) are considered in detail, based on recent evidence that Pb induces the expression of the gene for 78-kD glucose-regulated protein (GRP78) and other ER stress genes. GRP78 is a molecular chaperone that binds transiently to proteins traversing through the ER and facilitates their folding, assembly, and transport. Models are presented for the induction of ER stress by Pb in astrocytes, the major cell type of the central nervous system, in which Pb accumulates. A key feature of the models is disruption of GRP78 function by direct Pb binding. Possible pathways by which Pb-bound GRP78 stimulates the unfolded protein response (UPR) in the ER are discussed, specifically transduction by IRE1/ATF6 and/or IRE1/JNK. The effect of Pb binding to GRP78 in the ER is expected to be a key component for understanding mechanisms of Pb-induced ER stress gene expression.     

Myocardial ER chaperone activation and protein degradation occurs due to synergistic,not individual,cold and hypoxic stress

Environmental stress at high altitude affects the myocardium at the physiological and molecular level. Characterized by hypobaric hypoxia and low temperatures, the cumulative impact of these stressors on the protein folding homeostasis in the heart is yet unexplored. The present study evaluates the collective effect of cold and hypoxia on the myocardial protein oxidation and activation of the endoplasmic reticulum (ER) stress response. Adult rats were exposed to either a singular acute stress of cold (10 °C; C), hypobaric hypoxia (7620 m; H) or simultaneously to both cold and hypobaric hypoxia (CH) for 6 h. Hypoxic stress amplified the free radical generation in H and CH groups, leading to enhanced HIF-1α expression. Coupled to cold stress, reduced oxygen availability caused substantial protein oxidative modifications, as well as cardiac tissue injury and matrix remodeling, evident in the histological staining. Presence of oxidized proteins caused a significant upregulation in expression of ER chaperones GRP78 and PDI in the cold hypoxia exposed animals. Enhanced proteolytic activity signaled the removal of misfolded proteins. Linked intricately to cellular stress response, cell survival kinases were expressed higher in CH group; however apoptotic CHOP (C/EBP homologous protein) expression remained unaltered. Administration of ER stress inducer, tunicamycin along with cold hypoxic stress, caused a discernible increase in protein oxidation and GRP78 expression, along with a significant elevation in proteasome and apoptotic activity. Highlighting the significance of a synergistic, rather than individual, effect of low oxygen and temperature on the protein folding machinery, our study provides evidence for the activation of ER stress response in the myocardium under acute high altitude stress.     

GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis

The chaperone glucose-regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non-small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and suppressed phosphorylation of Raf-1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf-1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf-1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf-1 to maintain mitochondrial permeability and thus protect cells from ER stress-induced apoptosis.     

Pre-conditioning induces the precocious differentiation of neonatal astrocytes to enhance their neuroprotective properties

Hypoxic preconditioning reprogrammes the brain''s response to subsequent H/I (hypoxia–ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O₂). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.     

内质网应激预处理对人正常肝细胞缺氧再复氧损伤的保护作用

目的:研究内质网应激预处理对人肝细胞缺氧复氧损伤的保护作用。方法:将培养的人肝细胞分为4组:正常对照(C)组、细胞缺氧复氧损伤(H/R)组、内质网应激(ER)组、内质网应激预处理(ERP+H/R)组。收集各组细胞,以流式细胞仪检测细胞凋亡,Western-bloting及RT-PCR检测内质网应激特异蛋白GRP78表达水平,并通过透射电镜观察各组细胞超微结构改变。结果:ERP+H/R组细胞凋亡率明显低于H/R组(P<0.05),ER及ERP+H/R组GRP78蛋白表达明显高于H/R组(P<0.05)。结论:内质网应激预处理对肝细胞缺氧复氧损伤具有明显的保护作用,内质网应激特异性蛋白GRP78可能在肝细胞缺氧复氧损伤中作为一种关键性的保护蛋白出现。     

Metabolic and Biosynthetic Alterations in Cultured Astrocytes Exposed to Hypoxia/Reoxygenation

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to ± 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.     

低氧联合LPS刺激对星形胶质细胞中炎性因子和BNIP3表达的影响*

目的:探讨在低氧联合脂多糖(LPS)作用下,星形胶质细胞中B淋巴细胞瘤-2/腺病毒E1B 19-kD相互作用蛋白3(BNIP3)的表达和炎症反应变化。方法:将体外培养的原代星形胶质细胞和神经元进行下列分组:常氧组、LPS组、低氧组和LPS+低氧组(每组设置3个复孔)。LPS处理后,低氧组和LPS+低氧组放入低氧细胞孵箱,LPS组和常氧组放入正常的细胞孵箱。LPS浓度:100 ng/ml,氧气浓度为0.3%。处理时间为24 h。原代的星形胶质细胞进行上述的分组,时间点设为6 h、12 h和24 h。Western blot检测BNIP3的表达变化,RT-PCR和ELISA分别检测星形胶质细胞的肿瘤坏死因子-ɑ(TNF-ɑ)、白细胞介素-1β(IL-1β)和白细胞介素6(IL-6)mRNA水平变化和分泌情况。结果:与常氧组比较,低氧组炎症因子的表达没有变化,LPS组和LPS＋低氧组的炎症因子TNF-ɑ、IL-1β和IL-6 mRNA水平升高(P＜0.01);与LPS组比较,LPS＋低氧组炎症因子IL-1β和IL-6 mRNA水平进一步升高(P＜0.05,P＜0.01)。与常氧组比较,低氧组炎症因子的分泌水平没有变化,LPS组和LPS＋低氧组的炎症因子TNF-ɑ和IL-6 分泌水平升高(P＜0.01),IL-1β的水平没有变化;与LPS组比较,LPS＋低氧组炎症因子TNF-ɑ和IL-6分泌水平没有进一步升高。BNIP3在体外培养的神经元和星型胶质细胞中都有表达;在星形胶质细胞中,与常氧组比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达明显增加(P＜0.01);在神经元中,与常氧组比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达增加(P＜0.05,P＜0.01);与神经元的低氧组比较,星形胶质细胞的低氧组BNIP3的表达增加更明显(P＜0.01)。在星形胶质细胞中LPS联合低氧刺激6、12、24 h后BNIP3蛋白的表达,与常氧组相同时间点比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达增加(P＜0.05,P＜0.01);与低氧组相同时间点比较,6 h和12 h的LPS＋低氧组BNIP3的表达增加的更高(P＜0.01)。结论:低氧联合LPS刺激可以增强星形胶质细胞的炎症反应,LPS能增加低氧下星形胶质细胞中BNIP3的表达,提示BNIP3在星形胶质细胞的炎性反应中可能具有一定的调节作用。     

GRP78 effectively protect hypoxia/reperfusion‐induced myocardial apoptosis via promotion of the Nrf2/HO‐1 signaling pathway

Myocardial infarction is a major cause of death worldwide. Despite our understanding of the pathophysiology of myocardial infarction and the therapeutic options for treatment have improved substantially, acute myocardial infarction remains a leading cause of morbidity and mortality. Recent findings revealed that GRP78 could protect myocardial cells against ischemia reperfusion injury‐induced apoptosis, but the exact function and molecular mechanism remains unclear. In this study, we aimed to explore the effects of GRP78 on hypoxia/reperfusion (H/R)‐induced cardiomyocyte injury. Intriguingly, we first observed that GRP78 overexpression significantly protected myocytes from H/R‐induced apoptosis. On mechanism, our work revealed that GRP78 protected myocardial cells from hypoxia/reperfusion‐induced apoptosis via the activation of the Nrf2/HO‐1 signaling pathway. We observed the enhanced expression of Nrf2/HO‐1 in GRP78 overexpressed H9c2 cell, while GRP78 deficiency dramatically antagonized the expression of Nrf2/HO‐1. Furthermore, we found that blocked the Nrf2/HO‐1 signaling by the HO‐1 inhibitor zinc protoporphyrin IX (Znpp) significantly retrieved H9c2 cells apoptosis that inhibited by GRP78 overexpression. Taken together, our findings revealed a new mechanism by which GRP78 alleviated H/R‐induced cardiomyocyte apoptosis in H9c2 cells via the promotion of the Nrf2/HO‐1 signaling pathway.     

Neuritin Attenuates Neuronal Apoptosis Mediated by Endoplasmic Reticulum Stress In Vitro

Neuritin is an extracellular glycophosphatidylinositol-linked protein that promotes neuronal survival, differentiation, function, and repair, but the exact mechanism of this neuroprotective effect remains unclear. Meanwhile, endoplasmic reticulum stress (ERS) induced apoptosis is attracting increased attention. In this work, we hypothesized that neuritin inhibited ERS to protect cortical neurons. To check this hypothesis, we exposed primary cultured cortical neurons to oxygen and glucose deprivation (OGD) for 45 min followed by reperfusion (R) to activate ERS. We then performed resuscitation for 6, 12, 24, and 48 h. ERS-related factors such as glucose-regulated protein 78 (GRP78), caspase-12 and CHOP were detected by Western blotting and quantitative real-time polymerase chain reaction assay. Apoptosis was assessed by Annexin V binding and propidium iodide staining. Ultrastructural changes of endoplasmic reticulum were observed under a transmission electron microscope. Results showed that GRP78 expression significantly increased at 12, 24, and 48 h and peaked at 24 h. Caspase-12 and CHOP expression significantly increased in a time-dependent manner at 12, 24, and 48 h. GRP78, caspase-12 and CHOP expression as well as apoptosis rate of primary cultured neurons and the ultrastructural changes of endoplasmic reticulum in the OGD/R?+?neuritin group significantly improved compared with the OGD/R group. In conclusion, the neuroprotection function of neuritin may be involved in ERS pathways.     

Astrocytes aged in vitro show a decreased neuroprotective capacity

Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100beta increased at 90 days when cells were positive for the senescence beta-galactosidase marker. In long-term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn-superoxide dismutase (SOD-1) and haeme oxygenase-1 (HO-1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90-DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H2O2 exposure. Enhanced glutamate uptake was probably because of up-regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age-related neurodegenerative processes.     

Expression of glucose-regulated proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.     

Activated protein C and thrombin participate in the regulation of astrocyte functions

Protein C anticoagulant system is a multifunctional cofactor-dependent system. In addition to anticoagulant function, activated protein C (APC) also exhibits neuroprotective activity in hypoxia and stroke, but there are no data on potential effects of APC on astrocytes. In the present work we have studied the influence of APC and thrombin on rat astrocytes in primary culture. It was found that thrombin at concentrations above 10 nM (1 U/mL) induced significant activation in the cultured astrocytes resulting in reactive astrogliosis. The cultures exposed to thrombin for 24 h demonstrated a significant increase in proliferation and the S100b protein expression. Thrombin at high concentrations produced visible changes in the cytoskeleton of astrocytes, in particular, an increase in the number of stress fibers in the cultured cells. Moreover, thrombin apparently affected astrocyte migration. Thus, the treatment of serum-starved astrocytes with thrombin resulted in changes in cell monolayer uniformity and formation of “free fields”. APC prevented thrombin-induced proliferation of astrocytes and the S100b protein expression, reducing the parameters under study to the control values. In addition, APC reduced thrombin-induced disorganization of fibrils and formation of “free fields”. The results have demonstrated a new aspect of the protective effect of APC, which suppresses astrocyte activation induced by the proinflammatory effect of thrombin. It suggests a potential application of APC as a regulator of astrogliosis in pathological brain conditions.     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

78-kilodalton glucose-regulated protein is induced in Rous sarcoma virus-transformed cells independently of glucose deprivation.

To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast (CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and rat amino acid sequences). In contrast to previous observations, we found that GRP78 was induced in RSV-transformed cells in the absence of glucose deprivation. When cells were grown in glucose-supplemented medium, the level of GRP78 mRNA was approximately fivefold higher in RSV-transformed CEF than in transformation-defective virus-infected or uninfected CEF. Similar changes in GRP78 protein content were also found. Using a temperature-sensitive mutant of RSV and supplemental glucose, we found a gradual increase in the level of GRP78 mRNA beginning at 4 h after shiftdown to permissive temperature. Uridine supplementation did not block the induction seen in CEF infected with a temperature-sensitive mutant. These results indicate that GRP78 is induced by p60v-src in the absence of glucose deprivation.     

Epidermal growth factor regulates astrocyte expression of the interleukin-4 receptor via a MAPK-independent pathway

Human astrocytes express the interleukin (IL)-4 receptor alpha chain (IL-4R alpha) in vitro and in vivo but mechanisms governing astrocyte IL-4R alpha expression have not been established. We hypothesized that epidermal growth factor (EGF) and IL-4, agents that profoundly affect astrocyte proliferation, might also alter IL-4R alpha expression. Exposure to EGF for 24 h enhanced IL-4R alpha mRNA levels; in contrast, IL-4 yielded no increase. Immunoblotting demonstrated that EGF but not IL-4 increased astrocyte IL-4R alpha protein after 2--4 days of exposure. Similarly, EGF but not IL-4 strongly activated phosphorylation of p42/p44 extracellular regulated kinase isoforms, a reaction blocked by the mitogen-activated protein kinase (MAPK) inhibitor, PD98059. PD98059 also blocked EGF-stimulated DNA synthesis but not IL-4R alpha mRNA levels, while antibody to the EGF receptor (erbB1) blocked both EGF effects. Data suggest that astrocyte IL-4R alpha expression is upregulated by EGF but not by IL-4 in an EGF-receptor-dependent manner and that mechanisms are independent of MAPK activation.