Expression Of Endoplasmic Reticulum Stress Proteins Research Articles

Effect of sulfur dioxide on endoplasmic reticulum stress protein expression in myocardial tissue of rats with left to right shunt

Objective To explore the possible role of sulfur dioxide(SO2) in endoplasmic reticulum stress(ERS) protein expression in myocardial tissue of rats with left to right shunt. Methods A total of 24 male Sprague Dawley rats were randomly divided into sham group(n=8), shunt group(n= 8)and shunt plus SO2 donor group(n= 8). The rats of shunt group and shunt plus SO2 donor group, an abdominal aorta and inferior vena cava shunting was created.Na2SO3/NaHSO3 was administrated intra-peritoneally in rats of shunt plus SO2 donor group.Eight weeks later, the ratio of quantity of pulmonary to quantity of system(Qp/Qs) was measured; Heart weight/body weight(HW/BW) and right ventricle/left ventricle+ ventricular septum [RV/(LV+ SP)] were detected; Expressions of collagen type Ⅰ in right ventricle myocardial tissue was measured by enzyme linked immunosorbent assay(ELISA); High performance liquid chromatography was used to measure SO2 content in right ventricle myocardial tissue.Glucose regulated protein 78(GRP78), c-jun N-terminal kinase(JNK) and phosphorylation-c-jun N-termina kinase(p-JNK) mRNA and protein expressions in RV myocardial tissue were detected by Western blot and real time-polymerase chain reaction(RT-PCR). Results After 8-weeks shunting, Qp/Qs in the shunt and shunt plus SO2 groups were higher significantly than those of the sham group(all P<0.05). Compared with sham group, HW/BW, RV/LV+ SP and expressions of collagen type Ⅰ in the shunt group were significantly higher(all P<0.05). Treatment of SO2, HW/BW and RV/LV+ SP and collagen type Ⅰ in the shunt group were lower significantly(all P<0.05). The level of SO2 of RV in shunt group increased significantly compared with sham group(all P<0.05), but significantly lower than that of shunt plus SO2 donor group(P<0.05). Compared with sham group, the expressions of GRP78, JNK and p-JNK mRNA and protein in RV increased significantly in shunt group(P<0.05). After treatment with SO2, the expressions of GRP78, JNK and p-JNK mRNA and protein decreased significantly(P<0.05). Conclusion SO2 might protect RV myocardial tissue of the rats with left-right shunt by regulating the expressions of ERS. Key words: Sulfur dioxide; Endoplasmic reticulum stress; Glucose regulated protein 78; c-jun N-terminal kinase; Phosphorylation-c-jun N-terminal kinase

Expression of Endoplasmic Reticulum Stress Markers GRP78 and CHOP Induced by Oxidative Stress in Blue Light-Mediated Damage of A2E-Containing Retinal Pigment Epithelium Cells

Aims: Age-related lipofuscin N-retinylidene-N-retinylethanolamine (A2E) accumulated in human retinal pigment epithelium (RPE) cells confers susceptibility to blue light-mediated damage, which represents one pathogenesis of age-related macular degeneration. This study investigated the expression of 2 best-characterized endoplasmic reticulum (ER) stress markers, glucose-related protein 78 (GRP78) and C/EBP homologous protein (CHOP), as well as their regulation by oxidative stress after blue light-mediated damage of A2E-containing RPE cells. Methods: ARPE-19 cells were incubated with A2E (10, 25, 50 μ<smlcap>M</smlcap>) for 2 h and exposed to blue light for 20 min. A2E distributions in RPE cells were assessed via laser scanning confocal microscope and liquid chromatography-mass spectrometry. Cell viability was measured by a Cell Titer 96 Aqueous One Solution cell proliferation assay. The quantity of intracellular reactive oxygen species (ROS) was detected by dihydroethidium fluorescence using flow cytometry. Expressions of GRP78 and CHOP were measured at both mRNA and protein levels. To examine the role of oxidative stress in regulating GRP78 and CHOP expression, RPE cells were pretreated with the antioxidant N-acetylcysteine (NAC) for 2 h. RNA interference of GRP78 performed by short hairpin RNA was used to evaluate the effect of GRP78 in blue light-mediated damage of RPE cells. Results: After blue light exposure, A2E-treated RPE cells showed a gradual decrease in cell viability and a particular increase in ROS levels. Meanwhile, the expressions of GRP78 and CHOP in A2E-treated RPE cells were significantly increased at different time points after illumination. Pretreatment with NAC attenuated the expression of 2 ER stress markers, especially CHOP in A2E and blue light-treated RPE cells. Silencing of GRP78 by RNA interference upregulated CHOP and caspase-12 expression as well as aggravated the blue light-mediated damage of A2E-laden RPE cells. Conclusion: RPE cells exhibited ROS accumulation and subsequent elevation of GRP78 and CHOP expression after A2E and blue light-induced damage. The ROS scavenger NAC diminished ER stress protein expression, suggesting a connection between ER and oxidative stress in blue light-mediated damage of A2E-containing RPE cells. Besides, GRP78 may play a protective role in it.

Selective Modulation of Endoplasmic Reticulum Stress Markers in Prostate Cancer Cells by a Standardized Mangosteen Fruit Extract

The increased proliferation of cancer cells is directly dependent on the increased activity of the endoplasmic reticulum (ER) machinery which is responsible for protein folding, assembly, and transport. In fact, it is so critical that perturbations in the endoplasmic reticulum can lead to apoptosis. This carefully regulated organelle represents a unique target of cancer cells while sparing healthy cells. In this study, a standardized mangosteen fruit extract (MFE) was evaluated for modulating ER stress proteins in prostate cancer. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells (PrECs) procured from two patients undergoing radical prostatectomy were treated with MFE. Flow cytometry, MTT, BrdU and Western blot were used to evaluate cell apoptosis, viability, proliferation and ER stress. Next, we evaluated MFE for microsomal stability and anti-cancer activity in nude mice. MFE induced apoptosis, decreased viability and proliferation in prostate cancer cells. MFE increased the expression of ER stress proteins. Interestingly, MFE selectively promotes ER stress in prostate cancer cells while sparing PrECs. MFE suppressed tumor growth in a xenograft tumor model without obvious toxicity. Mangosteen fruit extract selectively promotes endoplasmic reticulum stress in cancer cells while sparing non-tumorigenic prostate epithelial cells. Furthermore, in an in vivo setting mangosteen fruit extract significantly reduces xenograft tumor formation.

Asbestos-Induced Alveolar Epithelial Cell Apoptosis. The Role of Endoplasmic Reticulum Stress Response

Asbestos exposure results in pulmonary fibrosis (asbestosis) and malignancies (bronchogenic lung cancer and mesothelioma) by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) apoptosis is important in the development of pulmonary fibrosis after exposure to an array of toxins, including asbestos. An endoplasmic reticulum (ER) stress response and mitochondria-regulated (intrinsic) apoptosis occur in AECs of patients with idiopathic pulmonary fibrosis, a disease with similarities to asbestosis. Asbestos induces AEC intrinsic apoptosis, but the role of the ER is unclear. The objective of this study was to determine whether asbestos causes an AEC ER stress response that promotes apoptosis. Using human A549 and rat primary isolated alveolar type II cells, amosite asbestos fibers increased AEC mRNA and protein expression of ER stress proteins involved in the unfolded protein response, such as inositol-requiring kinase (IRE) 1 and X-box-binding protein-1, as well as ER Ca²(2+) release ,as assessed by a FURA-2 assay. Eukarion-134, a superoxide dismutase/catalase mimetic, as well as overexpression of Bcl-XL in A549 cells each attenuate asbestos-induced AEC ER stress (IRE-1 and X-box-binding protein-1 protein expression; ER Ca²(2+) release) and apoptosis. Thapsigargin, a known ER stress inducer, augments AEC apoptosis, and eukarion-134 or Bcl-XL overexpression are protective. Finally, 4-phenylbutyric acid, a chemical chaperone that attenuates ER stress, blocks asbestos- and thapsigargin-induced AEC IRE-1 protein expression, but does not reduce ER Ca²(2+) release or apoptosis. These results show that asbestos triggers an AEC ER stress response and subsequent intrinsic apoptosis that is mediated in part by ER Ca²(2+) release.

Advanced stent coating for drug delivery and in vivo biocompatibility

As an effort to alleviate stent-induced cardiovascular injury including restenosis and thrombosis, advanced drug-eluting stent (ADES) with a bilayer construct composed of a top-coat made of collagen and a base-coat incorporated with N-nitrosomelatonin (NOMela)-loaded PLGA nanoparticles has been developed. NOMela is a hydrophobic prodrug of nitric oxide (NO) that is an endogenous anti-platelet compound. ADES was coated with PLGA nanoparticles via either electrophoretic deposition (EPD) technique or dip-coating technique, and their coating characteristics and efficacies were compared. The drug-loading efficacy and in vitro drug-release profiles from ADES were expressed with various variables including the additives to the collagen layer, the number of layers of the collagen top-coat, the hydrophobicity/hydrophilicity of the loaded drug, the coating technique of nanoparticles, and the concentration of coating emulsions in the EPD method. The morphological status of cross-section and surface of ADES was evaluated by laser scanning confocal microscope and scanning electronic microscope. The real-time release profiles of NO were assessed using the NO-microbiosensor. The anti-platelet activity of ADES was evaluated on the rabbit whole blood using an aggregometer. The intima formation and protein expression in aorta were examined using an in vivo rat model. Both collagen and PLGA used in ADES are biodegradable polymers that fully degrade and consequently produce less inflammation responses. NO released from ADES significantly reduced platelet aggregation in the rabbit blood as compared with those exposed to the control stents. ADES coated with a double layer consisted of collagen and PLGA and containing NOMela was less antigenic at the implanted sites and alleviating intima formation and thrombosis. An external exposure of aorta to NO elicits distinct and specific effects on mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) activities which evoke the endoplasmic reticulum (ER) stress response. These findings elucidated that coordinate and reciprocal alterations in the protein kinases followed by the ER stress protein expression are an integral feature of the in-stent-mediated cardiovascular injury.

Sigma 1 Receptor plays a prominent role in IL-24-induced cancer-specific apoptosis

Interleukin-24 (IL-24), a member of the IL-10 cytokine family, is an immunomodulatory cytokine that also displays broad cancer-specific suppressor effects. The tumor suppressor activities of IL-24 include inhibition of angiogenesis, sensitization to chemotherapy, and cancer-specific apoptosis. We show that Sigma 1 Receptor (S1R), a ligand-regulated protein chaperone contributes to IL-24 induction of apoptosis. IL-24 generated from an adenovirus expressing IL-24 (Ad.IL-24) induces cancer-specific apoptosis by inducing an endoplasmic reticulum (ER) stress, reactive oxygen species production, and calcium mobilization. The present studies reveals that S1R is required for Ad.IL-24-induced cell death. We provide several lines of evidence to confirm a physical and functional interaction between IL-24 and S1R including: (a) S1R and IL-24 co-localize, as judged by immunocytochemical analysis studies; (b) S1R and IL-24 co-immunoprecipitate using either S1R or IL-24 antibody; (c) S1R agonist (+)-SKF10047 inhibits apoptosis by Ad.IL-24; (d) (+)-SKF10047-mediated inhibition of Ad.IL-24 results in: diminished ER stress protein expression; (e) Calcium mobilization; and (f) ROS production. Collectively, these data demonstrate that S1R interacts with IL-24 and suggest that IL-24:S1R interaction determines apoptosis induction by Ad.IL-24. These studies define Sigma 1 Receptor as a key initial mediator of IL-24 induction of cancer-specific killing. These findings have important implications for our understanding of IL-24 as a tumor suppressor protein as well as an immune modulating cytokine.

Abstract 3676: Selective modulation of CHOP/GADD153 in prostate cancer cells by alpha-mangostin promotes cell death.

Abstract Background: It is well-established that during endoplasmic reticulum (ER) stress cells utilize several cell signal pathways collectively named “unfolded protein reaction (UPR)” to re-establish homeostasis and promote cell survival. The upregulation of UPR proteins to an extent is thought to be a growth advantage for cancer cells. However, chronic ER stress or prolonged UPR will lead to apoptosis. Alpha-mangostin, a xanthone from mangosteen fruit, has been reported to effectively induce prostate cancer cell apoptosis in vitro and suppress xenograft tumor growth. Thus, we investigated the alteration of ER stress-related proteins in mangosteen fruit extract (MFE)- or α-Mangostin-treated prostate cancer cells as well as its correlation with apoptosis. Methods: Two human prostate cancer cell lines, 22Rv1 and LNCaP, and a prostate epithelial cell line were treated with MFE or α-mangostin. Flow cytometry, MTT and BrdU were used to evaluate cell apoptosis, viability and proliferation. Western blot and fluorescent microscopy were used to detect proteins involved in apoptosis or ER stress. We also evaluated the anti-cancer activity of MFE and α-mangostin in nude mice inoculated with 22Rv1 cells. In addition, pharmacokinetics data of α-mangostin was collected on wild type C57BL6 mice for toxicity concern. Results: MFE increased apoptosis, decreased viability and inhibited proliferation in prostate cancer cell lines. Both MFE and α-mangostin significantly upregulated the expression of ER stress proteins, such as PERK, CHOP and IRE1α. Both MFE and α-mangostin also effectively suppressed tumor growth in a xenograft tumor model without affecting body weight. Interestingly, CHOP knockdown enhanced apoptosis in α-mangostin-treated prostate cancer cells; both MFE and α-mangostin decreased PERK expression and did not induce ER stress in prostate epithelial cells (PrECs) from prostate cancer patients. Conclusions: Both MFE and α-mangostin selectively promote ER stress in prostate cancer cells and inhibit prostate cancer cell growth in a xenograft mouse model without observable toxicity, suggesting further research is needed to evaluate its potential as an anti-cancer agent. Citation Format: Gongbo Li, Sakina Petiwala, Dana Pierce, Jeremy Johnson. Selective modulation of CHOP/GADD153 in prostate cancer cells by alpha-mangostin promotes cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3676. doi:10.1158/1538-7445.AM2013-3676

Activation of AMP-Activated Protein Kinase Inhibits Albumin-Induced Endoplasmic Reticulum Stress and Apoptosis through Inhibition of Reactive Oxygen Species

Background: Endoplasmic reticulum (ER) stress induced by urinary albumin plays an important role in tubulointerstitial injury. We have shown that albumin-induced ER stress is regulated through reactive oxygen species (ROS)-c-Src kinase-mTOR signaling pathways. We postulated that peroxisome proliferator-activated receptor-γ (PPAR-γ) might also act as an upstream signaling molecule between c-Src kinase and mTOR. It has been suggested that AMP-activated protein kinase (AMPK) is involved in attenuation of ER stress. We examined whether and how activation of AMPK suppressed the albumin-induced ER stress and apoptosis in tubular epithelial cells. Method: HK-2 cells, a proximal tubular cell line, were used. Protein expressions were measured by Western blot analysis. Intracellular ROS and apoptosis were analyzed by flow cytometry. Results: Albumin-induced PPAR-γ expression and PPAR-γ inhibitor (GW9662) suppressed the albumin-induced ER stress. c-Src kinase inhibitor and GW9662 reduced the albumin-induced PPAR-γ and mTOR, respectively. Metformin (the best known clinical activator of AMPK) and another AMPK activator (AICAR) suppressed the albumin-induced ER stress via inhibition of ROS through induction of endogenous antioxidant thioredoxin. AMPK inhibitor blocked the effect of metformin and AICAR. Our in vivo animal study showed that metformin reduced the renal cortical expression of ER stress protein (GRP78) in protein-overload proteinuria rats. Metformin also reduced the caspase 3-dependent apoptosis induced by albumin. Conclusion: PPAR-γ was involved in albumin-induced ER stress as an upstream signaling molecule between c-Src kinase and mTOR. AMPK activation might be beneficial in attenuating the tubulointerstitial injury induced by albumin.

β-Adrenergic receptor stimulation induces endoplasmic reticulum stress in adult cardiac myocytes: role in apoptosis

Accumulation of misfolded proteins and alterations in calcium homeostasis induces endoplasmic reticulum (ER) stress, leading to apoptosis. In this study, we tested the hypothesis that β-AR stimulation induces ER stress, and induction of ER stress plays a pro-apoptotic role in cardiac myocytes. Using thapsigargin and brefeldin A, we demonstrate that ER stress induces apoptosis in adult rat ventricular myocytes (ARVMs). β-AR-stimulation (isoproterenol; 3h) significantly increased expression of ER stress proteins, such as GRP-78, Gadd-153, and Gadd-34, while activating caspase-12 in ARVMs. In most parts, these effects were mimicked by thapsigargin. β-AR stimulation for 15 min increased PERK and eIF-2α phosphorylation. PERK phosphorylation remained higher, while eIF-2α phosphorylation declined thereafter, reaching to ~50% below basal levels at 3 h after β-AR stimulation. This decline in eIF-2α phosphorylation was prevented by β1-AR, not by β2-AR antagonist. Forskolin, adenylyl cyclase activator, simulated the effects of ISO on eIF-2α phosphorylation. Salubrinal (SAL), an ER stress inhibitor, maintained eIF-2α phosphorylation and inhibited β-AR-stimulated apoptosis. Furthermore, inhibition of caspase-12 using z-ATAD inhibited β-AR-stimulated and thapsigargin-induced apoptosis. In vivo, β-AR stimulation induced ER stress in the mouse heart as evidenced by increased expression of GRP-78 and Gadd-153, activation of caspase-12, and dephosphorylation of eIF-2α. SAL maintained phosphorylation of eIF-2α, inhibited activation of caspase-12, and decreased β-AR-stimulated apoptosis in the heart. Thus, β-AR stimulation induces ER stress in cardiac myocytes and in the heart, and induction of ER stress plays a pro-apoptotic role.

Activation of c-Jun N-terminal kinase is required for the regulation of endoplasmic reticulum stress response in the rat dorsal striatum following repeated cocaine administration

Repeated exposure to cocaine upregulates endoplasmic reticulum (ER) stress response and c-Jun N-terminal kinase (JNK) phosphorylation is associated with the ER stress response in neurons. In this study, we investigated the involvement of JNK in the regulation of the ER stress response following repeated cocaine administration in the dorsal striatum in vivo. The results showed that systemic injections of cocaine (20 mg/kg) for seven consecutive days increased the induction of p46 JNK (JNK) phosphorylation, immunoglobulin heavy chain binding protein (BiP), the ER stress-associated protein caspase-12, and behavioral locomotor activity. This enhancement of BiP and caspase-12 expression and locomotor response was reduced by inhibiting JNK. Similar reduction of elevated JNK phosphorylation was induced by blocking dopamine D1 receptors, N-methyl-D-aspartate (NMDA) receptors, and group I metabotropic glutamate receptors (mGluRs). These data suggest that JNK activation following repeated cocaine administration is required for the regulation of the ER stress protein expression and behavioral alteration in the dorsal striatum. Stimulation of dopamine D1 receptors, NMDA receptors or group I mGluRs participates in the regulation of JNK activation.

P38 Mitogen-Activated Protein Kinase is Involved in Endoplasmic Reticulum Stress-Induced Cell Death and Autophagy in Human Gingival Fibroblasts

Inflammation or hypoxia in gingival tissue can induce endoplasmic reticulum (ER) stress, which is related with autophagy. The autophagy is a catabolic process involving the degradation of a cell's own components. Although autophagy resulting in the total destruction of the cell is one of cell death types, no conclusive evidence exists for such a process. In order to examine the association of ER stress and autophagy in gingival system, ER stress agents brefeldin A, thapsigargin, and tunicamycin were exposed to human gingival cells. The ER stress agents induced cell death and the expression of ER stress proteins, glucose-regulated protein of 78 kDa (GRP78) and CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP). ER stress also increased the formation of autophagic vesicles with the expression of beclin and LC-3 (microtubule-associated protein1 light chain 3) II, two autophagic markers. ER stress induced the phosphorylation of p38MAPK (mitogen-activated protein kinase), and the p38MPAK inhibitor, SB203580, inhibited the resulting cell death and autophagy. In summary, ER stress induces cell death and autophagy through p38MAPK in human gingival cells.

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

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

Short-Term Exercise Does Not Increase ER Stress Protein Expression in Cardiac Muscle

Both short-term (three to five consecutive days) and long-term (weeks to months) endurance exercise training provides cardioprotection against ischemia-reperfusion (IR)-induced injury. However, the mechanisms responsible for exercise-induced cardioprotection are not well understood. Emerging evidence indicates that endoplasmic reticulum (ER) damage contributes to IR-induced myocardial injury. It follows that exercise-induced expression of ER stress proteins could serve as the mediators of exercise-induced cardioprotection against IR injury. Hence, these experiments tested the hypothesis that exercise training is associated with an increase in ER stress proteins in the heart. Adult male Sprague-Dawley rats (N=13) were habituated to treadmill running for 5 d, followed by five 60-min exercise bouts (approximately 70% of VO2max) on consecutive days. Infarct area resulting from IR was determined by a standard histological (triphenyltetrazolium chloride (TTC)) method. Cardiac levels of ER stress proteins Grp78, Grp94, and calreticulin were analyzed via Western blot. Moreover, we determined myocardial levels of heat shock protein 72 (HSP72) along with ER proteins associated with cellular injury, including CHOP, caspase 12, Puma, Noxa, and ATF3. Our exercise protocol resulted in cardioprotection as evidenced by reduced infarct size (P<0.05) and increased myocardial HSP72 levels (+227%; P<0.01) in the exercise-trained animals. Nonetheless, exercise training did not increase (P>0.05) cardiac levels of the ER stress proteins, Grp78, Grp94, and calreticulin. Moreover, exercise did not alter myocardial levels of CHOP, caspase 12, Puma, Noxa, or ATF3. These data reveal that short-term exercise training does not elevate ER stress proteins in the heart. Hence, the cardioprotective effect of short-term exercise training does not seem to be linked to ER stress adaptation.

Cocaine increases endoplasmic reticulum stress protein expression in striatal neurons

Cocaine administration upregulates the levels of extracellular glutamate and dopamine in the striatum. Activation of the receptors alters calcium homeostasis in striatal neurons leading to the expression of the endoplasmic reticulum (ER) stress proteins. It was therefore hypothesized that cocaine upregulates the expression of the ER stress proteins, immunoglobulin heavy chain binding protein (BiP), Ire1α and perk via glutamate and dopamine receptor activation. A novel glutamate microbiosensor and Western immunoblot analyses were mainly performed to test the hypothesis in the rat dorsal striatum. The results showed that i.p. injection of repeated cocaine (20 mg/kg) for nine consecutive days significantly increased extracellular glutamate levels while acute cocaine injection did not. However, the immunoreactivities (IR) of the ER stress proteins in the dorsal striatum were significantly increased by either acute or repeated cocaine injections as compared with saline controls. Intrastriatal injection (i.s.) of the selective group I metabotropic glutamate receptor (mGluR) antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 25 nmol) or the mGluR5 subtype antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 2 and 25 nmol) significantly decreased repeated cocaine-induced increases in the IR of the ER stress proteins in the injected dorsal striatum. Similarly, the selective D1 antagonist ( R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrochloride (SCH23390; 0.1 mg/kg, i.p.) or the N-methyl- d-aspartate antagonist dizocilpine/(5 S,10 R)-(+)-5-methyl-10,11-dihydro-5 H-ibenzo[a,d]cyclohepten-5,10-imine maleate (MK801; 2 nmol, i.s.) decreased acute or repeated cocaine-induced the IR of the ER stress proteins in the dorsal striatum. These data suggest that cocaine upregulates expression of the ER stress proteins in striatal neurons via a mechanism involving activation of glutamate and dopamine receptors.

A rat model of human FENIB (familial encephalopathy with neuroserpin inclusion bodies)

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is caused by intracellular accumulation/polymerization of mutant neuroserpins in the endoplasmic reticulum (ER). Transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic–acid Schiff (PAS)-positive inclusions distributed throughout deeper layers of cerebral cortex, CA1 of the hippocampus, and substantia nigra. Hippocampal extracts from Tg meg rats showed increased expression of ER stress proteins, and activation of caspases-12 and -3, associated with decreased neuronal density. Enhanced ER stress was also observed in dopaminergic neurons in the substantia nigra, in parallel with decreased neuronal viability and motor coordination. In each case, PAS-positive inclusions were also positive for megsin. These data suggest that overexpression of megsin results in ER stress, eventuating in the formation of PAS-positive inclusions. Tg meg rats provide a novel model of FENIB, where accumulation of serpins in the ER induces selective dysfunction/loss of specific neuronal populations.

Short‐term exercise does not affect ER stress protein expression in cardiac muscle

Cardiac muscle damage resulting from ischemia-reperfusion (IR) injury remains a major health problem around the world. We and others have shown that short-term exercise training provides protection against IR-induced cardiac injury. However, the mechanisms responsible for this protection are not well understood. Emerging evidence indicates that the endoplasmic reticulum (ER) stress is involved in IR-induced myocardial injury. Indeed IR-induced ER dysfunction can initiate cell death in the event of prolonged ER stress. Therefore, this study investigated the effect of short-term exercise training on ER stress protein levels in the heart. Adult male Sprague-Dawley rats (n=10) were habituated to treadmill running for 5 days, followed by three 60-minute exercise bouts (@~70% of VO2 max) on consecutive days. The trained animals, along with sedentary controls, were sacrificed 24 hours after the last exercise bout and the hearts were excised following a sham surgery. Cardiac levels of ER stress proteins Grp78, Grp94 along with the 72kDa Heat Shock Protein (HSP72) and PKR were analyzed via Western blot. Although our exercise protocol elevated myocardial HSP72 levels by 227% compared to controls (p<0.01), cardiac levels of Grp78, Grp94 and PKR were unaltered (p>0.05) by exercise. These data reveal that short-term exercise training does not elevate ER stress proteins in the heart. Hence the cardioprotective effect of exercise training does not appear to be linked to ER stress adaptation. Supported by NIH HL072789 (SKP)

Cytoprotective gene bi-1 is required for intrinsic protection from endoplasmic reticulum stress and ischemia-reperfusion injury

Ischemia-reperfusion (IR) injury induces endoplasmic reticulum (ER) stress and cell death. Bax Inhibitor-1 (BI-1) is an evolutionarily conserved ER protein that suppresses cell death and that is abundantly expressed in both liver and kidney. We explored the role of BI-1 in protection from ER stress and IR injury by using bi-1 knockout mice, employing models of transient hepatic or renal artery occlusion. Compared to wild-type bi-1 mice, bi-1 knockout mice subjected to hepatic IR injury exhibited these characteristics: (i) increased histological injury; (ii) increased serum transaminases, indicative of more hepatocyte death; (iii) increased percentages of TUNEL-positive hepatocytes; (iv) greater elevations in caspase activity; and (v) more activation of ER stress proteins inositol-requiring enzyme 1 and activating transcription factor 6 and greater increases in expression of ER stress proteins C/EBP homologous protein and spliced XBP-1 protein. Moreover, hepatic IR injury induced elevations in bi-1 mRNA in wild-type liver, suggesting a need for bi-1 gene induction to limit tissue injury. Similar sensitization of kidney to ER stress and IR injury was observed in bi-1(-/-) mice. We conclude that bi-1 provides endogenous protection of liver and kidney from ER stress and IR injury. Analysis of components of the bi-1-dependent pathway for protection from IR injury may therefore reveal new strategies for organ preservation.

Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation

An imbalance between protein load and folding capacity is referred to as endoplasmic reticulum (ER) stress. As a defense mechanism, cells express ER stress inducible chaperons, such as oxygen-regulated proteins 150 (ORP150) and glucose-regulated proteins (GRPs). While ER stress is important in various diseases, a pathophysiologic role for ER stress in kidney disease remains elusive. Here we investigate expression of ER stress proteins in cultured rat podocytes as well as in our recently developed animal model of abnormal protein retention within the ER of podocytes (i.e., megsin transgenic rat). The expression of ER stress inducible proteins (ORP150, GRP78, or GRP94) in cultured podocytes treated with tunicamycin, A23187, SNAP, hypoxia, or hyperglycemia, and the renal tissues or isolated glomeruli from megsin transgenic rats was analyzed by Western blotting analysis, immunohistochemistry, or confocal microscopy. Cultured podocytes demonstrated that treatment with tunicamycin, A23187, and SNAP, but not hypoxia or hyperglycemia, up-regulate expression of ER stress proteins. Extracts of isolated glomeruli from megsin transgenic rats reveal marked up-regulation of ER stress chaperones in podocytes, which was supported by immunohistochemical analysis. Confocal microscopy revealed that ER stress in podocytes was associated with cellular injury. Podocytes of transgenic rats overexpressing a mutant megsin, without the capacity for polymerization within the ER, do not exhibit ER stress or podocyte damage, suggesting a pathogenic role of ER retention of polymerized megsin. This paper implicates a crucial role for the accumulation of excessive proteins in the podocyte ER in the induction of ER stress and associated podocyte injury.

Role of the Endoplasmic Reticulum Unfolded Protein Response in Glomerular Epithelial Cell Injury

C5b-9-induced glomerular epithelial cell (GEC) injury in vivo (in passive Heymann nephritis) and in culture is associated with damage to the endoplasmic reticulum (ER) and increased expression of ER stress proteins. Induction of ER stress proteins is enhanced via cytosolic phospholipase A(2) (cPLA(2)) and limits complement-dependent cytotoxicity. The present study addresses another aspect of the ER unfolded protein response, i.e. activation of protein kinase R-like ER kinase (PERK or pancreatic ER kinase), which phosphorylates eukaryotic translation initiation factor 2-alpha (eIF2alpha), thereby generally suppressing translation and decreasing the protein load on a damaged ER. Phosphorylation of eIF2alpha was enhanced significantly in glomeruli of proteinuric rats with passive Heymann nephritis, compared with control. In cultured GECs, complement induced phosphorylation of eIF2alpha and reduced protein synthesis, and complement-stimulated phosphorylation of eIF2alpha was enhanced by overexpression of cPLA(2). Ischemia-reperfusion in vitro (deoxyglucose plus antimycin A followed by glucose re-exposure) also stimulated eIF2alpha phosphorylation and reduced protein synthesis. Complement and ischemia-reperfusion induced phosphorylation of PERK (which correlates with activation), and fibroblasts from PERK knock-out mice were more susceptible to complement- and ischemia-reperfusion-mediated cytotoxicity, as compared with wild type fibroblasts. The GEC protein, nephrin, plays a key role in maintaining glomerular permselectivity. In contrast to a general reduction in protein synthesis, translation regulated by the 5'-end of mouse nephrin mRNA during ER stress was paradoxically maintained, probably due to the presence of short open reading frames in this mRNA segment. Thus, phosphorylation of eIF2alpha and consequent general reduction in protein synthesis may be a novel mechanism for limiting complement- or ischemia-reperfusion-dependent GEC injury.

Complement C5b-9 Membrane Attack Complex Increases Expression of Endoplasmic Reticulum Stress Proteins in Glomerular Epithelial Cells

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of cytosolic phospholipase A(2) (cPLA(2)). This study addresses the role of endoplasmic reticulum (ER) stress proteins (bip, grp94) in GEC injury. GEC that overexpress cPLA(2) (produced by transfection) and "neo" GEC (which expresses cPLA(2) at a lower level) were incubated with complement (40 min), and leakage of constitutively expressed bip and grp94 from ER into cytosol was measured to monitor ER injury. Greater leakage of bip and grp94 occurred in complement-treated GEC that overexpress cPLA(2), as compared with neo, implying that cPLA(2) activation perturbed ER membrane integrity. After chronic incubation (4-24 h), C5b-9 increased bip and grp94 mRNAs and proteins, and the increases were dependent on cPLA(2). Expression of bip-antisense mRNA reduced stimulated bip protein expression and enhanced complement-dependent GEC injury. Glomerular bip and grp94 proteins were up-regulated in proteinuric rats with PHN, as compared with normal control. Pretreatment of rats with tunicamycin or adriamycin, which increase ER stress protein expression, reduced proteinuria in PHN. Thus, C5b-9 injures the ER and enhances ER stress protein expression, in part, via activation of cPLA(2). ER stress protein induction is a novel mechanism of protection from complement attack.