Glucose Response Protein Research Articles

Regulation of glucose responsive protein (GRP) gene expression by insulin

While screening for insulin-induced genes, we identified two members of a family of stress-induced genes referred to as glucose-regulated proteins (GRPs). GRPs are members of the stress-responsive superfamily of genes which also includes heat shock proteins (HSPs). The GRP proteins are not normally heat-inducible, but are overproduced when cells are starved of glucose. The two major GRP proteins, GRP78 and GRP94, are highly conserved among vertebrates. We have found that physiological concentrations of insulin stimulate the transcription of GRP78 and GRP94 in rat H4IIE hepatoma cells. The regulation of GRP78 transcription was rapid, with the first induction within minutes, and a further induction after several hours, and both occurred in the presence of glucose. GRP78 transcription was more greatly induced by insulin in the presence of SB202190, a specific p38-MAPK inhibitor. Transcription of GRP94 was also induced, but only after several hours. Calcimycin (A23187) and anisomycin were used to induce endoplasmic reticulum (ER)/cellular stress, and both induced GRP78 and GRP94 transcription.

Calcium mitigates fluoride-induced kallikrein 4 inhibition via PERK/eIF2α/ATF4/CHOP endoplasmic reticulum stress pathway in ameloblast-lineage cells

ObjectivesThe present study aimed to investigated the effect and mechanism of Ca2+ treatment on fluoride in ameloblast-lineage cells (ALCs). Materials and methodsThe effects of fluoride and different Ca2+ levels treatment on the proliferative activity, cell apoptosis, cell cycle, intracellular free Ca2+, were firstly determined. Kallikrein 4 (KLK4), glucose-responsive protein 78 (GRP78), Protein kinase R -like endoplasmic reticulum kinase (PERK), the α subunit of eukaryotic initiation factor 2 (eIF2α), activating transcription factor 4 (ATF4), CCAAT enhancer-binding protein homologous protein (CHOP), were investigated in ALCs. ResultsThe proliferative activity was obviously inhibited under concentrations of single fluoride high than 1 mM, and indicated highest proliferation at single 2.5 mM Ca2+ concentration in ALC cells. In addition, we found that single fluoride markedly induced intracellular free Ca2+ increasing, G2/M phase arrest, apoptosis. GRP78 and endoplasmic reticulum stress pathway of PERK/eIF2α/ATF4/CHOP were significantly increased, while the proliferation and KLK4 were markedly reduced in ALCs. Ca2+ additional treatment can obviously reverse the effect of fluoride-induced apoptosis and inhibition of KLK4. The effect of GRP78 and endoplasmic reticulum stress pathway of PERK/eIF2α/ATF4/CHOP were also alleviated under Ca2+ additional treatment in ALCs. More important, the results of 2.5 mmol/L Ca2+ treatment on the proliferation, cell cycle and apoptosis suggest this concentration is relatively better to mediate the intracellular Ca2+ homeostasis in ALCs. ConclusionsIn sum, Ca2+-supplementation exerts antagonistic the toxic effects on fluoride and this inhibitory effect suggests the potential implications for Ca2+-supplementation on fluorosis.

51-OR: Loss of Critical ER Chaperone GRP78 Leads to Beta-Cell Death by a JNK Dependent Mechanism

Endoplasmic reticulum (ER) health is an important determinant of pancreatic beta cell function or failure. Adaptive ER stress leads to expansion of beta cell number through proliferation, but decompensated ER stress results in beta cell loss and contributes to both T1D and T2D. Therefore, understanding the mechanisms of ER-stress-induced beta cell death could help identify novel strategies for the prevention or treatment of diabetes. Glucose responsive protein GRP78/HSPA5 is a key ER chaperone. Loss of GRP78 causes cell death in many cell types; including, in our own prior work, the beta cell. To study the mechanism by which GRP78 loss leads to beta cell death we devised an ex vivo model of GRP78 knockdown by transducing Grp78-flox mouse islets with adenovirus expressing Cre recombinase. To study human islets, we used adenovirus expressing shRNA against GRP78. Using these tools, we successfully reduced GRP78 RNA and protein in primary mouse and human islet cells. Loss of GRP78 strongly activated the ATF6, IRE1 and PERK pathways of the unfolded protein response (UPR). GRP78 knockdown decreased beta cell number due to increased cell death as measured by Annexin V and TUNEL staining. Probing the mechanisms of cell loss, we found that loss of GRP78 activated multiple death-related pathways, notably autophagy and apoptosis; cell death effectors Chop, caspase 9 and cleaved caspase 3 were increased. Additionally, GRP78 deletion increased stress kinase JNK (c-Jun-N-terminal kinase) which has been implicated in cellular proliferation as well as cell death. Inhibition of JNK kinase using JNK-IN-8 inhibitor during GRP78 loss conditions decreased phosphorylation of JNK and rescued beta cells from cell death as measured with Annexin V and TUNEL. Therefore, we conclude that ER stress related cell death induced by loss of GRP78 (ER stress) involves JNK activation, and that modulation of JNK under certain circumstances may help maintain beta cell mass to protect against diabetes. Disclosure R.B. Sharma: None. C.O. Darko: None. B. Gablaski: None. A.S. Lee: None. L.C. Alonso: Consultant; Self; Fairbanks Pharmaceuticals Inc. Research Support; Self; American Diabetes Association. Funding National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK113300-02)

Metformin Suppresses Expression of Pre-Fibrotic Enzyme Lysyl-Oxidase Levels through Inhibition of UPR in MPN Cells

The endoplasmic reticulum (ER) is a cytoplasmic organelle required for proper protein folding and sorting. When protein load exceeds the capacity of the ER, the accumulation of the misfolded proteins triggers a quality control response known as the unfolded protein response (UPR). Recent studies have revealed that increased activation of the UPR is one of the hallmarks of hematological malignancies. In addition, excess UPR is associated with pro-fibrotic conditions, i.e., pulmonary fibrosis, renal fibrosis and kidney fibrosis.Lysyl-oxidase (LOX) is an enzyme that regulates the crosslinking of extracellular matrix proteins, such as collagen, and enhances a fibrotic phenotype. Recent studies revealed that the genes of LOX family members are upregulated in myeloproliferative neoplasms (MPN). Furthermore, megakaryocytes and platelets derived from MPN patients had increased LOX protein concentrations in comparison with healthy subjects. It is also reported that the UPR is one of the important regulators of LOX. Based on this knowledge, we speculated that the enhanced UPR might be involved in the elevation of LOX in JAK2V617F harboring MPN and may contribute to the development of myelofibrosis. To study this hypothesis, we initially confirmed that JAK2V617F-positive cell lines(HEL and SET-2) show enhanced activation of UPR, including phosphorylation of eIF2-alpha, nuclear localization of ATF6 and XBP1s and upregulation of glucose response protein 78 (GRP78). We also confirmed the presence of elevated levels of LOX in these cells. Treatment of the cells with a chemical chaperone, namely, tauroursodeoxycholic acid (TUDCA), suppressed UPR and decreased expression of LOX, suggesting that UPR is responsible for elevation of LOX in MPN cells. To analyze whether JAK2V617F is responsible for the enhanced UPR and expression of LOX in MPN cells, we treated the cells with JAK1/JAK2 inhibitor ruxolitinib. Unexpectedly, ruxolitinib did not block UPR nor expression of LOX. Previously, we reported that metformin, which is a member of the biguanide family, inhibited growth of MPN cells through activation of AMPK and PP2A (Kawashima et al. Exp.Hematol, 2016). Furthermore, a number of studies demonstrated that metformin suppressed UPR in several cancer cells through activation of AMPK. Therefore, we investigated whether metformin could block UPR and LOX levels in these cells. As expected, metformin reduced the phosphorylation levels of eIF2alpha and nuclear localization of XBP1 and ATF6. Expression of GRP78 was also blocked by metformin. These results clearly indicated that metformin suppressed UPR in MPN cells. Metformin also blocked the activation of the molecules required for proteins synthesis including p70S6kinase, mTOR and 4E-PB1. These results indicated that suggested that metformin blocked aberrant UPR through inhibition of enhanced protein synthesis. Importantly, we also confirmed that LOX expression was also suppressed by metformin in MPN cells.Our observations indicated that the UPR is enhanced in MPN cells irrespective of JAK2V617F, and metformin could block this process leading to suppression of LOX levels. Combination of ruxolitinib with metformin might be a new and attractive method to suppress myelofibrosis in MPN. DisclosuresKirito:Novartis Pharma KK: Honoraria.

2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells

The glycolytic inhibitor 2-deoxy-d-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells.

Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Chitosan Microgels Embedded with Catalase Nanozyme-Loaded Mesocellular Silica Foam for Glucose-Responsive Drug Delivery.

A glucose-responsive closed-loop insulin delivery system represents an ideal form of treatment for type 1 diabetes mellitus. Here, we develop a glucose-responsive protein delivery system based on chitosan microgels loaded with enzyme-mimicking inorganic nanoparticles. The pH-sensitive chitosan microgels, integrated with glucose-mediated pH-lowering enzymatic large-pore mesoporous silica (MCF), were fabricated via an electrospray process. Ceria nanoparticles (CeNPs), which is a catalase-mimicking inorganic artificial enzyme with a substantial stability compared to that of catalase, were incorporated into the MCF along with glucose oxidase. In hyperglycemic conditions, CeNPs successfully decomposed the toxic hydrogen peroxide that was generated from the glucose oxidation reaction mediated by glucose oxidase and regenerate oxygen; this protected glucose oxidase from denaturation. The pH-lowering induced by the enzymatic MCF in high glucose concentration resulted in swelling of the chitosan microgels and the subsequent release of the encapsulated model protein drug, such as bovine serum albumin and insulin. Finally, we successfully demonstrated self-regulated repetitive protein release from the chitosan microgels, which showed a basal release rate under normoglycemic conditions and an enhanced release rate under hyperglycemic conditions.

Glucose-Mediated N-glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth

Tumorigenesis is associated with increased glucose consumption and lipogenesis, but how these pathways are interlinked is unclear. Here, we delineate a pathway in which EGFR signaling, by increasing glucose uptake, promotes N-glycosylation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and consequent activation of SREBP-1, an ER-bound transcription factor with central roles in lipid metabolism. Glycosylation stabilizes SCAP and reduces its association with Insig-1, allowing movement of SCAP/SREBP to the Golgi and consequent proteolytic activation of SREBP. Xenograft studies reveal that blocking SCAP N-glycosylation ameliorates EGFRvIII-driven glioblastoma growth. Thus, SCAP acts as key glucose-responsive protein linking oncogenic signaling and fuel availability to SREBP-dependent lipogenesis. Targeting SCAP N-glycosylation may provide a promising means of treating malignancies and metabolic diseases.

Label-free profiling of white adipose tissue of rats exhibiting high or low levels of intrinsic exercise capacity.

Divergent selection has created rat phenotypes of high- and low-capacity runners (HCR and LCR, respectively) that have differences in aerobic capacity and correlated traits such as adiposity. We analyzed visceral adipose tissue of HCR and LCR using label-free high-definition MS (elevated energy) profiling. The running capacity of HCR was ninefold greater than LCR. Proteome profiling encompassed 448 proteins and detected 30 significant (p <0.05; false discovery rate <10%, calculated using q-values) differences. Approximately half of the proteins analyzed were of mitochondrial origin, but there were no significant differences in the abundance of proteins involved in aerobic metabolism. Instead, adipose tissue of LCR rats exhibited greater abundances of proteins associated with adipogenesis (e.g. cathepsin D), ER stress (e.g. 78 kDa glucose response protein), and inflammation (e.g. Ig gamma-2B chain C region). Whereas the abundance antioxidant enzymes such as superoxide dismutase [Cu-Zn] was greater in HCR tissue. Putative adipokines were also detected, in particular protein S100-B, was 431% more abundant in LCR adipose tissue. These findings reveal low running capacity is associated with a pathological profile in visceral adipose tissue proteome despite no detectable differences in mitochondrial protein abundance.

CRH knockout inhibits the murine innate immune responses in association with endoplasmic reticulum stress after thermal injury

Previous studies in our laboratory have demonstrated the hypothalamus destruction and adrenalectomy could blunt the innate immunity while boosting the excessive inflammation after injury. We aimed to investigate the effects of corticotrophin-releasing hormone knockout (CRH KO) on the innate immune responses in macrophages as well as to elucidate the underlying mechanism. The chemotaxis and phagocytosis activities of macrophages, bacteria translocation, plasma tumor necrosis factor (TNF)-α secretion, and intestinal injury were observed in the presence of the endoplasmic reticulum stress after thermal injury in CRH KO mice. Meanwhile, the messenger RNA (mRNA) and protein expression of glucose response protein 78 (GRP78), X-box binding protein 1 (XBP1), and activating transcription factor 6 (ATF6) in macrophages was also determined. After thermal injury, the chemotaxis and phagocytosis of peritoneal macrophages were increased, which were both reversed by CRH gene deficiency. The gut-derived bacteria translocation to liver tissues, lung tissues and mesenteric lymph nodes was significantly strengthened in CRH KO mice compared with CRH wild-type littermates. Circulating TNF-α level was increased markedly in response to thermal injury and CRH KO further increased its secretion. Furthermore, the mRNA and protein levels of GRP78, XBP1, and ATF6 in peritoneal macrophages increased, while their expressions in CRH KO mice all decreased significantly. CRH KO mice showed enhancement of inflammatory responses and severe tissue injuries after thermal injury. CRH exerted immune defensive actions on immune cells and organs in the early phase of injury, suggesting that the underlying mechanisms are related to endoplasmic reticulum stress.

Abstract 3035: Cripto/GRP78 signaling promotes the stem cell phenotype in normal and neoplastic mammary epithelial cells

Abstract Microenvironmental factors are critical regulators of stem cell and tumor cell behavior. However, few stem cell factors have been identified and targeted in breast and other cancers. Using a highly enriched stem cell pool isolated from fetal mammary rudiments and single cell analyses, we have identified the onco-fetal protein Cripto as a potent soluble factor that regulates the mammary stem cell state. Cripto is a GPI-anchored/secreted signaling protein and a known regulator of PI3K/AKT and TGF-beta pathways. Here, we develop a novel Cripto antagonist, ALK4L75A-Fc, which selectively blocks the growth factor-like effects of soluble Cripto. We show that this antagonist promotes the differentiation of mammary stem cells cultured ex vivo while Cripto treatment maintains the stem cell phenotype and yields colonies with enhanced mammary gland reconstitution capacity. We previously discovered that Cripto signaling requires binding to cell surface Glucose Response Protein 78kDa (GRP78). GRP78 is an HSP70 family member that is induced by stresses including hypoxia and glucose deprivation and it is highly expressed in tumors where these conditions prevail. We show here that cell surface GRP78 marks fetal mouse mammary stem cells and a population of bipotent adult mammary epithelial cells that are selectively responsive to soluble Cripto in vitro. GRP78high mammary epithelial cells also harbor substantially higher stem cell activity upon transplantation than GRP78low cells. Consistently, we further show that deletion of GRP78 from adult mammary epithelial cells ex vivo blocks mammary gland reconstitution. Finally, we find that Cripto antagonism with ALK4L75A-Fc inhibits the proliferation of triple negative breast cancer cell lines in vitro and that this antagonist can also inhibit tumor growth in vivo. Since Cripto and GRP78 are both induced by hypoxia and other stressful conditions found in tumors, our data raise the possibility that Cripto/GRP78 signaling may exacerbate breast and other cancers by increasing the number of stem cell-like tumor cells in tumor microenvironments where these conditions predominate. Citation Format: Benjamin T. Spike, Jonathan A. Kelber, Evan Booker, Madhuri Kalathur, Rose Rodewald, Julia Lipianskaya, Justin La, Marielle He, Tracy Wright, Richard Klemke, Geoffrey Wahl, Peter C. Gray. Cripto/GRP78 signaling promotes the stem cell phenotype in normal and neoplastic mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3035. doi:10.1158/1538-7445.AM2014-3035

Prevention of Doxorubicin Cardiopathic Changes by a Benzyl Styryl Sulfone in Mice

Cardiac toxicity is a major limitation in the use of doxorubicin (and related anthracyclins). ON 1910.Na (Estybon, Rogersitib, or 1910), a substituted benzyl styryl sulfone, is equally active as doxorubicin against MCF-7 human mammary carcinoma xenografted into nude mice. 1910 augments the antitumor activity of doxorubicin when given simultaneously. Furthermore, when given in combination, 1910 protects against cardiac weight loss and against morphological damage to cardiac tissues. Doxorubicin induces inactivation of glucose response protein 78 (GRP78), a principal chaperone that serves as the master regulator of the unfolded protein response (UPR). Inactivated GRP78 leads to an increase in misfolded proteins, endoplasmic reticulum (ER) stress, activation of UPR sensors, and increased CHOP expression. 1910 prevents the inactivation of GRP78 by doxorubicin, and the combination, while more active against the tumor, protects against cardiac weight loss.

Corticosterone exerts immunostimulatory effects on macrophages via endoplasmic reticulum stress

: Glucocorticoids are the central effector hormones for the hypothalamic-pituitary-adrenal axis. However, the effects of endogenous glucocorticoids on the immune system are not understood completely. : Macrophage function (adherence, chemotaxis and cytokine production) was assessed in the presence of increasing concentrations of corticosterone. The role of endoplasmic reticulum (ER) stress in corticosterone immunoregulation was determined with thapsigargin and plasmid pGCL-GFP-siXBP1. Mifepristone was used to determine the role of glucocorticoid receptor in the corticosterone-induced ER stress response. : Corticosterone exerted immunostimulatory effects on macrophage function at low concentrations. No effects were observed at high concentrations in the absence of immunological stimulation. Low-dose corticosterone induced ER stress, which was correlated to the corticosterone immunostimulatory activities. Expression of X box-binding protein (XBP) 1, but not activating transcription factor 6, was significantly increased at both mRNA and protein levels only in the presence of low-dose corticosterone. Inhibition of XBP1 expression with small interfering RNA significantly inhibited the corticosterone immunostimulatory effects. In addition, pretreatment of macrophages with mifepristone significantly inhibited the expression of glucose response protein 78 and XBP1 in macrophages by low-dose corticosterone. : At low concentrations, endogenous glucocorticoids exert immunostimulatory actions on macrophages. The underlying mechanisms may be correlated to ER stress via the glucocorticoid receptor, in which XBP1 plays an important role.

Exogenous Norepinephrine Correlates with Macrophage Endoplasmic Reticulum Stress Response in Association with XBP-1

The regulation of neuroendocrine hormones on the innate immune responses remains controversial. This report investigated the effects of exogenous norepinephrine with respect to macrophage function as well as to elucidate the underlying mechanism. The adherence, chemotaxis, phagocytosis, and cytokine production of macrophages were observed in the presence of increasing concentrations of norepinephrine. The expression of macrophage glucose response protein 78 (GRP78), X-box binding protein 1 (XBP1), activating transcription factor 6(ATF6), and C-EBP homologous protein (CHOP) in macrophages was determined. The lentiviral vector pGCL-GFP-siXBP1 was cloned by inserting the annealed oligonucleotides encoding shRNAs specific for XBP1. Norepinephrine exerted immunostimulatory effects on macrophage at low concentrations, while partial effects were observed at high concentrations. Low-dose norepinephrine induced an endoplasmic reticulum stress response, which was correlated with the immunostimulatory activities of norepinephrine. Levels of mRNA expression of XPB1, but not ATF6 or CHOP, was significantly increased only by low concentrations of norepinephrine. Inhibition of XBP1 expression with siRNA treatment significantly inhibited the immunostimulatory effects of low concentrations of norepinephrine. Our data convincingly indicated that norepinephrine exerted immunostimulatory actions on macrophages at low concentrations, suggesting that the underlying mechanisms are related to endoplasmic reticulum stress via XBP1.

ENDOPLASMIC RETICULUM STRESS INVOLVED IN HEART AND LIVER INJURY IN IRON‐LOADED RATS

1. Iron overload contributes to the pathogenesis of various diseases and directly induces tissue injury. In the present study, we investigated the relationship between heart and liver injury induced by iron overload and cellular endoplasmic reticulum (ER) stress to explore the molecular mechanism of iron overload-induced cellular injury. 2. Iron overload in rats was generated by intraperitoneal injection of iron-dextran chronically (30 mg/kg per day for 9 weeks) or acutely (300 mg/kg once). Tissue injury was assessed by determining serum lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, as well as malondialdehyde (MDA) content in the heart and liver. The ER stress response was analysed by expression of glucose-response protein 78 (GRP78) and activation of caspase 12. 3. In chronic iron-loaded rats, iron levels in the heart and liver were higher, by approximately 2- and 7.8-fold, respectively (P < 0.01), compared with control. Serum LDH, ALT and AST activity, as well as MDA content, GRP78 expression and caspase 12 activity in the heart and liver, were upregulated in chronically iron-loaded rats. In acute iron-loaded rats, iron content in the heart and liver was 51% and 63% higher than in controls (both P < 0.01). Serum LDH, ALT and AST activity, MDA content in the heart and liver and levels of ER stress markers were all increased in acute iron-loaded rats. N-Acetylcysteine (150 mg/kg, s.c.) lowered the levels of these parameters in acute iron-loaded rats. 4. The results of the present study indicate that ER stress may play an important role in iron-induced tissue injury and that reactive oxygen species may mediate the ER stress response in the pathogenesis of iron-overload cellular injury.

Apigenin protects HT22 murine hippocampal neuronal cells against ER stress‐induced apoptosis

Endoplasmic reticulum (ER)‐stress is known to induce neuronal cell death and is involved in neurodegenerative diseases including Alzheimer's diseases, Parkinson diseases, and cerebral ischemia. Apigenin, a bioactive flavonoid, is shown to have anti‐tumorigenic and anti‐inflammatory properties in various cell types. In the present study, we investigated the effect of apigenin on ER stress‐induced apoptosis and action mechanism involved in murine HT22 hippocampal neuronal cells. Apigenin blocked apoptotic cell death of HT22 cells induced by ER stress inducers, thapsigargin (TG) and brefeldin A (BFA). We showed that apigenin reduced the expression levels of unfolded protein response (UPR) proteins including C/EBP homologous protein (CHOP) and glucose response protein 78 (GRP78) and phosphorylations of MAP kinases in TG or BFA‐treated HT22 cells. We also showed that TG or BFA increased intracellular ROS accumulation and apigenin blocked ROS accumulation induced by TG or BFA. Moreover, we found that TG or BFA decreased the mitochondrial membrane potential (MMP) and apigenin inhibited this decrease in MMP by TG or BFA. These results suggest that apigenin inhibits ER stress‐induced apoptosis by reducing ROS accumulation, mitochondrial dysfunction, and CHOP induction in HT22 hippocampal neuronal cells.This work was supported by grants from KOSEF of Korean Government.

Subcellular stress response and induction of molecular chaperones and folding proteins after transient global ischemia in rats

Brain ischemia induces the toxic accumulation of unfolded proteins in vulnerable neurons. This cellular event can trigger the unfolded protein response (UPR) and activate the expression of a number of genes involved in pro-survival pathways. One of the pro-survival pathways involves the sequestration and elimination of misfolded and aggregated proteins. Recent evidence suggests that the endoplasmic reticulum (ER), mitochondria, and cytoplasm respond individually to the accumulation of unfolded proteins by induction of organelle specific molecular chaperones and folding enzymes. This study utilized a rat model of transient (15 min) global ischemia (2-vessel occlusion) to investigate the regional and temporal induction of some of these key stress proteins after ischemia. Electron microscopy demonstrated that visible protein aggregates accumulated predominately in the cytoplasm. We used in situ hybridization (forebrain structures) and western blot (hippocampus) analysis to measure changes in expression of heat shock protein 70 (HSP70 cytoplasmic), HSP60 (mitochondrial), ER luminal proteins glucose response proteins GRP78 and GRP94, protein disulphide isomerase (PDI), homocysteine-inducible, endoplasmic reticulum stress-inducible protein (HERP), and calnexin. Induction of mRNA for HSP70 occurred earlier (beginning at 30 min) and at a higher level relative to the delayed (4–24 h) and more moderate induction of mRNAs for mitochondrial matrix HSP60 and the ER lumen HERP, GRP78, GRP94, calnexin and PDI. Increases in hippocampal proteins were observed at 4 h (HSP70) and 24 h (HSP60, GRP78, GRP94) after reperfusion. These results demonstrate that after a transient ischemic insult, the subcellular responses to the accumulation of unfolded proteins varies between cellular compartments and are most prevalent in the cytoplasm and, to a lesser degree, in the mitochondrial matrix and ER lumen.

Bax Inhibitor-1 Regulates Endoplasmic Reticulum Stress-associated Reactive Oxygen Species and Heme Oxygenase-1 Expression

The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced apoptosis. The ER is associated with generation of reactive oxygen species (ROS) through oxidative protein folding. This study examined the role of BI-1 in the regulation of ER stress-induced accumulation of ROS and expression of unfolded protein response-associated proteins. BI-1 reduced the expression levels of glucose response protein 78, C/EBP homologous protein, phospho-eukaryotic initiation factor 2alpha, IRE1alpha, XBP-1, and phospho-JNK and inhibited the cleavage of ATF-6alpha p-90, leading to the inhibition of ROS. Although ROS scavengers offer some protection against ER stress-induced apoptosis, the expression of pro-apoptotic ER stress proteins was not affected. This study shows that the response of unfolded proteins is followed by ROS accumulation under ER stress, which is regulated in BI-1 cells. The mechanism for these BI-1-associated functions involves the expression of heme oxygenase-1 (HO-1) through nuclear factor erythroid 2-related factor 2. In BI-1 cells, the transfection of HO-1 small interfering RNA completely abolished the BI-1-induced protection. The endogenous expression of HO-1 through ER stress-initiated ROS is believed to be as a protection signal. In conclusion, these observations suggest that BI-1 can inhibit the ER stress proteins as well as the accumulation of ROS, thereby protecting the cells. Moreover, HO-1 plays an important role in the BI-1-associated protection against ER stress.

Up-regulation of GRP78 and antiapoptotic signaling in murine peritoneal macrophages exposed to insulin

The unfolded protein response pathway (UPR) compensates for excessive protein accumulation in the endoplasmic reticulum (ER). As insulin induces global protein synthesis, it may cause accumulation of unfolded proteins in the ER, thus triggering UPR. We assessed UPR activation in insulin-treated murine peritoneal macrophages using a number of markers including 78 kDa glucose response protein (GRP78), X-box-binding protein (XBP)-1, pancreatic ER kinase (PERK), eukaryotic initiation factor 2 (eIF2)alpha, and growth arrest and DNA damage (GADD)34. Exposure of cells to insulin activated UPR, as evidenced by an increased expression of GRP78, XBP-1, phosphorylated PERK (p-PERK), and p-eIF2alpha. The insulin-induced, elevated expression of GRP78 was comparable with that observed with tunicamycin, a classical inducer of ER stress. Concomitantly, insulin also up-regulated prosurvival mechanisms by elevating GADD34 and elements of the antiapoptotic pathway including Bcl-2, X-linked inhibitor of apoptosis, and phosphorylated forkhead transcription factor. In conclusion, we show here that insulin treatment does cause ER stress in macrophages, but insulin-dependent mechanisms overcome this ER stress by up-regulating UPR and the antiapoptotic pathway to promote cell survival.

Ge-Jee-Bok-Ryung-Hwan induces apoptosis in human cervical carcinoma HeLa cells: -An endoplasmic reticulum stress pathway-

Ge-Jee-Bok-Ryung-Hwan (GJBRH), a commonly used herb formulation in Korea, Japan and China, caused a decrease of viability in HeLa human cervical carcinoma cells. The treatment of GJBRH resulted in genomic DNA fragmentation as well as the increase of Sub-G 1 portion in cell cycle analysis. In this study, GFP-Bax over-expression system showed that Bax, pro-apoptotic Bcl-2 family protein, was translocated to mitochondria by the presence of GJBRH. The treatment of BAPTA-AM, permeable endogenous calcium chelator, inhibited GJBRH-induced caspase-3 and -9 activations, the release of cytochrome c and Smac/DIABLO into cytoplasm and the resultant cell death in HeLa human cervical carcinoma cells. The treatment of BAPTA-AM increased the expression of XIAP, which mediates binding to and inhibiting caspases and showed protective effect, in GJBRH-treated cells. GJBRH induced the expression of Glucose Response Protein 78 (GRP 78), a positive ER stress marker protein. However, BAPTA-AM did not interfere with the ER-stress response pathway that triggers the expression of GRP 78. This study showed that GJBRH induces cell death, which occurs downstream of or parallel to this point in the ER-stress pathway linked to apoptosis. In conclusion, GJBRH induces apoptosis in HeLa cells via ER stress-pathway associated mitochondria-dependent apoptosis mechansim.