Glucose-related Protein 78 Expression Research Articles

Targeting endoplasmic reticulum stress, Nrf-2/HO-1, and NF-κB by myristicin and its role in attenuation of ulcerative colitis in rats

AimsUlcerative colitis (UC) is characterized by the up-regulation of pro-inflammatory mediators, apoptotic signals, and oxidative stress that can lead to an increased risk of colorectal cancer. The present study aims to investigate the possible role of myristicin in modulating endoplasmic reticulum stress (ERS) and risk-associated conditions in acetic acid (AA)-induced UC. Materials and methodsAdult male rats were treated with 150 mg/kg body weight of myristicin or mesalazine orally either as pre/post treatment or post-treatment only. The gene expression of glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and nuclear factor kappa B (NF-κB), percentage of DNA fragmentation, and serum levels of some oxidative and inflammatory markers were measured. Key findingsThe results indicated the potential upregulation of ERS, pro-apoptotic, lipid peroxidation, and pro-inflammatory cascades by induction of UC in rats. However, myristicin could effectively reverse the deteriorated effects of ulceration in colonic mucosa. It was mediated through downregulation of the ERS markers GRP78 and CHOP genes expression, reduction of NF-κB mRNA expression, DNA fragmentation, reduced lipid peroxidation, myeloperoxidase (MPO) activity and pro-inflammatory markers (Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β) and cyclo‑oxygenase (COX-2) activity). Accompanied by elevated levels of IL-10, colonic Nuclear erythroid factor (Nrf-2) and Heme oxygenase (HO-1) activity as well as blood antioxidant enzymes activity. Results of docking might confirm the biological results of our study. SignificanceMyristicin could effectively modulate important stress, and inflammatory effectors and protect mucosal DNA from oxidative damage which can serve as a promising candidate for the treatment of ulcerative colitis.

Dexmedetomidine Preconditioning Reduces Myocardial Ischemia-Reperfusion Injury in Rats by Inhibiting the PERK Pathway.

ResumoFundamento A cardiopatia isquêmica atraiu muito atenção devido às altas taxas de mortalidade, custos do tratamento e a crescente morbidade na população jovem. Estratégias de reperfusão reduziram a mortalidade. Porém, a reperfusão pode levar à morte do cardiomiócito e subsequente dano irreversível ao miocárdio. No momento, não há um tratamento eficiente e direcionado para a lesão de isquemia-reperfusão (I/R).Objetivos Avaliar se a dexmedetomidina (DEX) tem efeito protetivo na I/R do miocárdio e explorar os possíveis mecanismos por trás dela.Métodos Corações de ratos foram perfundidos com o sistema de perfusão de Langendorff e aleatoriamente distribuídos em cinco grupos: grupo controle, perfundido com solução de Krebs-Henseleit (K-H) por 205 minutos sem isquemia; e quatro grupos de teste que foram submetidos a 40 minutos de isquemia global e 120 minutos de reperfusão. O Grupo DEX, o grupo ioimbina (IO) e o grupo DEX + IO foram perfundidos com DEX (10 nM), IO (1 μM) ou a combinação de DEX e IO antes da reperfusão, respectivamente. A hemodinâmica cardíaca, o tamanho do infarto do miocárdio e a histologia do miocárdio foram avaliados. A expressão da proteína-78 regulada pela glicose (GRP78), a proteína quinase do retículo endoplasmático (PERK), a PERK fosforilada, o fator de iniciação eucariótico 2α (eIF2α), eIF2α fosforilado, o fator de transcrição 4 (TCF-4) e a proteína homóloga à proteína ligadora do acentuador CCAAT (CHOP) foram avaliados. P< 0,05 foi considerado para indicar a diferença estatisticamente significativa.Resultados O pré-condicionamento com DEX melhorou a função cardíaca nos corações com I/R, reduziu o infarto do miocárdio, a apoptose do miocárdio e a expressão de GRP78, p-PERK, eIF2α, p-eIF2α, TCF-4 e CHOP.Conclusões O pré-tratamento com DEX reduziu a lesão de I/R no miocárdio ao suprimir a apoptose, o que foi induzido pela via PERK.

The effect of nicotine and dextrose on endoplasmic reticulum stress in human coronary artery endothelial cells

Cigarette smoking is one of the major causes of coronary artery disease (CAD) as is diabetes. However, nicotine has been generally regarded as safe and is used in smoking cessation programs. This presumption of nicotine safety was examined in human coronary artery endothelial cells (HCAEC). Endoplasmic reticulum (ER) stress was measured using the secreted alkaline phosphatase (SAP) assay. The ER stress markers inositol-requiring enzyme 1α (IRE1α), phospho-IRE1α, double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), phospho-PERK, activating transcription factor 6 (ATF6), and glucose-related protein 78 (GRP78) were measured by western blot. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet staining. Intact and cleaved caspase 3, BH3 interacting-domain death agonist (BID), and B-cell lymphoma 2 (Bcl2) were measured by western blot. In cells transfected with the SAP expression plasmid, treatment with nicotine resulted in a dose-dependent decrease in SAP expression with no noticeable toxicity. Nicotine (10nM) also increased IRE1α and PERK phosphorylation, and ATF6 and GRP78 expression. Although nicotine at concentrations up to 10μM did not cause cell death, treatment of HCAEC with 10nM nicotine in the presence of 13.8mM dextrose aggravated ER stress, increased cell death, increased cleaved caspase 3 and BID, and decreased BCL2. Nicotine at concentrations commonly achieved in nicotine-replacement therapy (NRT) significantly increased ER stress in HCAEC and aggravated dextrose-induced ER stress and cell apoptosis. People using electronic cigarettes and on NRT may be at increased risk for CAD.

Effects of thyroid hormone on ovarian cell apoptosis in the rat.

Follicle development is a complicated process regulated by thyroid hormone (TH). TH dysregulation is associated with reproductive disorders; however, the mechanism underlying these relationships remains unclear. Glucose-related protein 78 (GRP78) is a well-characterised endoplasmic reticulum stress protein related to ovarian cell apoptosis. To clarify whether GRP78 is regulated by TH and the involvement of GRP78 in follicle development, we established rat models of hypothyroidism and hyperthyroidism and investigated the effects of TH dysregulation on levels of GRP78, C/EBP homologous protein (CHOP) and cleaved caspase-3. TH dysregulation decreased levels of GRP78 and increased those of CHOP and cleaved caspase-3 in both rat models. However, treatment with equine chorionic gonadotrophin reversed these effects, as well as granulosa cell apoptosis induced by TH dysregulation. Together, these results provide evidence that TH dysregulation alters the GRP78 expression profile, triggering the apoptotic signalling pathway, and suggest that GRP78 is a novel mediator of TH in follicle development.

Febuxostat, a novel inhibitor of xanthine oxidase, reduces ER stress through upregulation of SIRT1-AMPK-HO-1/thioredoxin expression.

Endoplasmic reticulum (ER) stress has been implicated in the development of various renal diseases. Thus, inhibition of ER stress using pharmacological agents may serve as a promising therapeutic approach. We postulated that febuxostat, a novel xanthine oxidase inhibitor, could suppress the ER stress through upregulation of SIRT1 (silent mating type information regulation 2 homolog 1)-AMPK (AMP activated protein kinase)-HO-1 (heme oxygenase-1)/thioredoxin expression. We examined the effect of febuxostat on the ER stress induced by a chemical inducer, tunicamycin and non-chemical agents such as angiotensin II, aldosterone, high glucose, and albumin in renal tubular cells. We further examined the in vivo effects of febuxostat using mouse model of kidney disease induced by unilateral ureteral obstruction (UUO). Expression of ER stress was measured by western blot analysis and immunohistochemical stain. Febuxostat suppressed the ER stress induced by tunicamycin and non-chemical agents, as shown by inhibition of increased GRP78 (glucose-related protein78) and p-eIF2α (phosphospecific-eukaryotic translation initiation factor 2α) expression. Inhibitory effect of febuxostat was mediated through upregulation of SIRT1-AMPK followed by induction of HO-1 and thioredoxin. In animal model of UUO, febuxostat reduced the UUO-induced ER stress, which was abolished by pretreatment with SIRT1 inhibitor (sirtinol) and AMPK inhibitor (compound C). Febuxostat could suppress the ER stress caused by various ER stress inducers through upregulation of SIRT1-AMPK-HO-1/thioredoxin expression. Targeting these pathways might serve as one of the possible therapeutic approaches in kidney diseases under excessive ER stress.

Plasminogen kringle 5 suppresses gastric cancer via regulating HIF-1\u03b1 and GRP78

Inhibition of tumour angiogenesis has an important role in antitumour therapy. However, a recent study indicates that antiangiogenesis therapy may lead to glucose-related protein 78 (GRP78) associated antiapoptotic resistance. The present study aims to elucidate the dual effects of plasminogen kringle 5 (K5) on tumour angiogenesis and apoptosis induction by targeting hypoxia-inducible factor 1α (HIF-1α) and GRP78. Co-immunoprecipitation and western blotting were used for examining the ubiquitination of HIF-1α and analysing angiogenesis and apoptosis-associated proteins. K5 promoted the sumo/ubiquitin-mediated proteasomal degradation of HIF-1α by upregulating von Hippel-Lindau protein under hypoxia, resulting in the reduction of vascular endothelial growth factor and thus suppressing tumour angiogenesis. Furthermore, K5 decreased GRP78 expression via downregulation of phosphorylated extracellular-regulated protein kinase, leading to caspase-7 cleavage and tumour cell apoptosis. Blocking voltage-dependent anion channel abrogated the effects of K5 on both HIF-1α and GRP78. K5 significantly inhibited the growth of gastric carcinoma xenografts by inhibiting both angiogenesis and apoptosis. The dual effects suggest that K5 might be a promising bio-therapeutic agent in the treatment of gastric cancer, particularly in patients who exhibit the induction of GRP78.

Anti-Fibrotic Effect of Losartan, an Angiotensin II Receptor Blocker, Is Mediated through Inhibition of ER Stress via Up-Regulation of SIRT1, Followed by Induction of HO-1 and Thioredoxin.

Endoplasmic reticulum (ER) stress is increasingly identified as modulator of fibrosis. Losartan, an angiotensin II receptor blocker, has been widely used as the first choice of treatment in chronic renal diseases. We postulated that anti-fibrotic effect of losartan is mediated through inhibition of ER stress via SIRT1 (silent mating type information regulation 2 homolog 1) hemeoxygenase-1 (HO-1)/thioredoxin pathway. Renal tubular cells, tunicamycin (TM)-induced ER stress, and unilateral ureteral obstruction (UUO) mouse model were used. Expression of ER stress was assessed by Western blot analysis and immunohistochemical stain. ER stress was induced by chemical ER stress inducer, tunicamycin, and non-chemical inducers such as TGF-β, angiotensin II, high glucose, and albumin. Losartan suppressed the TM-induced ER stress, as shown by inhibition of TM-induced expression of GRP78 (glucose related protein 78) and p-eIF2α (phosphospecific-eukaryotic translation initiation factor-2α), through up-regulation of SIRT1 via HO-1 and thioredoxin. Losartan also suppressed the ER stress by non-chemical inducers. In both animal models, losartan reduced the tubular expression of GRP78, which were abolished by pretreatment with sirtinol (SIRT1 inhibitor). Sirtinol also blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis. These findings provide new insights into renoprotective effects of losartan and suggest that SIRT1, HO-1, and thioredoxin may be potential pharmacological targets in kidney diseases under excessive ER stress condition.

Liver metabolic disruption induced after a single exposure to PCB126 in rats.

Polychlorinated biphenyls (PCBs) have been recognized as metabolic disruptors. The liver plays a pivotal role in detoxification of an organism. Fatty liver results from altered intra-, and extra-hepatic mediators and is associated with increased glucose-related protein 78 (GRP78), commonly used as a marker for endoplasmic reticulum (ER) stress signaling. This pilot study aimed to study the effects of a single exposure on fatty liver metabolic parameters. The objective of the study is to characterize the effects of 3,3',4,4',5-pentachlorobiphenyl (PCB126) on ER stress protein chaperon GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP) and intra-hepatic mediators such as microsomal triglyceride transfer protein (MTP), sterol regulatory element-binding protein 1c (SREBP1c), and peroxisome proliferator-activated receptor alpha (PPARα), as well as extra-hepatic factors such as non-esterified fatty acid (NEFA) and tumor necrosis factor alpha (TNFα). Hepatic GRP78 mRNA and protein levels, indicating the presence of ER stress, were significantly increased following a single PCB126 exposure in rats. Intra-hepatic mechanisms such as lipoprotein secretion pathway (i.e., MTP), lipogenesis de novo (i.e., SREBP1c), and oxidation (i.e., PPARα) were altered in PCB126-treated rats. In addition, a state of inflammation measured by higher TNFα plasma levels was present in contaminated rats. These data indicate that a single injection of PCB126-modulated expression of GRP78 associated with hepatic ER stress and systemic inflammation in rats.

Up-Regulation of SIRT1 Reduces Endoplasmic Reticulum Stress and Renal Fibrosis

Background: Endoplasmic reticulum (ER) stress is emerging as an important factor in the development of organ fibrosis. Therefore, modulation of ER stress may serve as one of the possible therapeutic approaches to renal fibrosis. SIRT1, a class III histone deacetylase, has been found to exert beneficial effects in kidney diseases. However, it is largely unknown whether and how SIRT1 suppresses the ER stress. We postulated that upregulation of SIRT1 would suppress the ER stress through induction of heme oxygenase-1 (HO-1) and thioredoxin. Methods: HK-2 tubular cells, experimental mouse models of tunicamycin (TM)-induced ER stress and unilateral ureteral obstruction (UUO) were used. Expression of ER stress-induced protein was measured by Western blot analysis and immunohistochemical staining. ER stress was induced by chemical ER stress inducers [TM [,]thapsigargin (TG)] and non-chemical inducers such as angiotensin II, aldosterone, high glucose and albumin. Results: SIRT1 activator (SRT1720) induced SIRT1 expression in a time- and dose-dependent manner in HK-2 cells. SRT1720 suppressed the TM- or TG-induced ER stress, as shown by inhibition of TM- or TG-induced upregulation of glucose-related protein 78 (GRP78), phosphor-specific eukaryotic translation initiation factor-2α and C/EBP homologous protein through HO-1 and thioredoxin, which were abolished by pretreatment with SIRT1 inhibitor (sirtinol). SRT1720 also suppressed the ER stress induced by angiotensin II, aldosterone, high glucose and albumin. In animal studies, treatment with SRT1720 reduced the tubular expression of GRP78 and increased the expression of HO-1 and thioredoxin. SRT1720 also reduced the UUO-induced renal fibrosis. Conclusion: SIRT1 may serve as a promising therapeutic target by reducing ER stress and fibrosis.

Autophagy activation prevents sevoflurane-induced neurotoxicity in H4 human neuroglioma cells.

The inhaled anesthetic sevoflurane may induce cognitive impairment in both animals and humans. Previous study has shown that sevoflurane triggers ER stress and may lead to apoptosis in rat hippocampal neurons. In this study, we examined whether sevoflurane caused autophagy and its contributions to sevoflurane induced neuronal cell injury. H4 human neuroglioma cells were exposed to 4.1% sevoflurane for 6 h. Cell viability and apoptosis ratio were assessed using a CCK8 kit and flow cytometry, respectively. Autophagosomes in the cells were detected using GFP-LC3 plasmid transfection or transmission electronic microscopy. The expression of LC3B, p62/SQSTM, C/EBP homologous protein (CHOP) and glucose-related protein 78 (GRP78) was assessed with Western blotting. Sevoflurane treatment induced apoptosis and markedly increased the LC3-II level and GFP-LC3 puncta number, decreased p62 expression in H4 cells. Activation of autophagy by rapamycin (1 μmol/L) significantly reduced sevoflurane-induced apoptosis and increased cell viability, whereas inhibition of autophagy with 3-MA (5 mmol/L) caused the opposite effects. Furthermore, sevoflurane treatment markedly increased the expression of CHOP and GRP78, two hallmark proteins of ER stress. Inhibition of ER stress by 4-phenylbutyrate (500 μmol/L) abrogated sevoflurane-induced autophagy and apoptosis, and improved the viability. Moreover, sevoflurane-stimulated expression of CHOP and GRP78 was inhibited by rapamycin, but further enhanced by 3-MA. Sevoflurane treatment induces ER stress and activates autophagy, which antagonizes sevoflurane-induced apoptosis in H4 human neuroglioma cells. The results suggest that autophagy may be a potential therapeutic target in preventing sevoflurane-induced neurotoxicity.

Scutellaria baicalensis Georgi extract protects against alcohol‑induced acute liver injury in mice and affects the mechanism of ER stress.

The aims of the present study were to examine the hepatoprotective effect of Scutellaria baicalensis Georgi extract (Scutellariae Radix extract; SRE) against acute alcohol-induced liver injury in mice, and investigate the mechanism of endoplasmic reticulum (ER) stress. High performance liquid chromatography was used for the phytochemical analysis of SRE. Animals were administered orally with 50% alcohol (12 ml/kg) 4 h following administration of doses of SRE every day for 14 days, with the exception of normal control group. The protective effect was investigated by measuring the levels of aspartate transaminase (AST), alanine transferase (ALT) and triglyceride (TG) in the serum, and the levels of glutathione (GSH) and malondialdehyde (MDA) in liver tissues. The levels of glucose-related protein 78 (GRP78) were detected using immunohistochemical localization and an enzyme-linked immunosorbent assay. Hepatocyte apoptosis was assessed using terminal-deoxynucleoitidyl transferase mediated nick end labeling. The SRE contained 31.2% baicalin. Pretreatment with SRE had a marked protective effect by reversing the levels of biochemical markers and levels of GRP78 in a dose-dependent manner. The results of the present study demonstrated that pretreatment with SRE exerted a marked hepatoprotective effect by downregulating the expression of GRP78, which is a marker of ER stress.

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.

A role for MT1-MMP as a cell death sensor/effector through the regulation of endoplasmic reticulum stress in U87 glioblastoma cells

Recent findings in cell death signalling show that membrane type 1 matrix metalloproteinase (MT1-MMP), an MMP known for its involvement in cancer cell invasion and metastasis, can act as a "bioswitch" in the invasion versus cell death decision in brain tumour cells. Given that the endoplasmic reticulum (ER) is a subcellular compartment involved in metabolic control and cell death signalling and that cytoskeleton disruption, as encountered during cancer cell invasion, can lead to ER stress, we questioned whether MT1-MMP contributes to ER stress. We found that MT1-MMP gene silencing or pharmacological inhibition of vesicular trafficking with Brefeldin-A abrogated MT1-MMP cell surface-mediated proMMP-2 activation by the lectin Concanavalin-A (ConA) in U87 glioblastoma cells. ConA, also known to trigger the expression of pro-inflammatory cyclooxygenase (COX)-2 through MT1-MMP signalling from the plasma membrane, failed to do so when MT1-MMP was prevented from reaching the cell surface by Brefeldin-A. Gene silencing of MT1-MMP antagonized the expression of ConA-induced COX-2 and of the ER stress marker glucose-related protein 78 (GRP78), further suggesting that plasma membrane localization of MT1-MMP contributes to signalling ER stress. MT1-MMP maturation, which partially occurs during its trafficking from the ER to the plasma membrane, showed correlation of the 60kDa MT1-MMP with GRP78 expression. Finally, Brefeldin-A treatment of glioblastoma cells led to Akt dephosphorylation; this effect was reversed when MT1-MMP was silenced. Collectively, our results provide a molecular rationale for a new role for MT1-MMP in the regulation of cancer cell death processes through ER stress signalling.