Expression Levels Of BiP Research Articles

Deficiency of Jamc Leads to Congenital Nuclear Cataract and Activates the Unfolded Protein Response in Mouse Lenses.

PurposeThe malfunction of junctional adhesion molecule C (JAM-C) has been reported to induce congenital cataract in humans and mice; however, specific characters and the mechanism of this cataract are still unclear. This study aimed to characterize abnormal lens development in Jamc knockout mice and clarify the underlying mechanism.Methods Jamc knockout mice backcrossed onto the C57BL/6 genetic background were used for this research. Slit-lamp and darkfield images showed the cataract phenotype of Jamc−/− mice. Hematoxylin and eosin staining was performed to visualize the morphological and histological features. RNA sequencing was applied to detect differentially expressed genes. Quantitative RT-PCR, western blot, and immunofluorescence were used to determine the level of unfolded protein response (UPR)-related genes. TUNEL staining was utilized to label cell death.Results Jamc knockout mice exhibited nuclear cataract with abnormal lens morphology and defective degradation of nuclei and organelles in lens fiber cells. Compared with wild-type control mice, the expression level of BiP, CHOP, TRIB3, and CHAC1, genes involved in endoplasmic reticulum stress and the UPR, were highly upregulated in Jamc−/− lenses, suggesting that abnormal lens development was accompanied by UPR activation. Moreover, increased cell death was also found in Jamc−/− lenses.ConclusionsCongenital nuclear cataract caused by Jamc deficiency is accompanied by defective degradation of nuclei and organelles in lens fiber cells, lens structure disorder, and UPR activation, suggesting that JAM-C is required for maintaining normal lens development and that UPR activation is involved in cataract formation in Jamc-deficient lenses.

Inhibition of extracellular signal-regulated kinase downregulates endoplasmic reticulum stress-induced apoptosis and decreases brain injury in a cardiac arrest rat model.

Cerebral ischemia-reperfusion injury (CIRI) is the predominant cause of neurological disability after cardiac arrest/cardiopulmonary resuscitation (CA/CPR). The endoplasmic reticulum stress (ERs)-induced apoptosis plays an important role in neuronal survival/death in CIRI. Our previous studies reported that the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, alleviates CIRI after CA/CPR. Whether ERs-induced apoptosis is involved in the neuroprotection of PD98059 remains unknown. This study aims to investigate the effects of ERK inhibition by PD98059 on ERs-induced apoptosis after CIRI in the CA/CPR rat model. The baseline characteristics of male adult Sprague-Dawley (SD) rats in all groups were evaluated before CA/CPR. The SD rats that survived from CA/CPR were randomly divided into 3 groups (n=12/group): normal saline group (1 ml/kg), dimethylsulfoxide (DMSO, the solvent of PD98059, 1 ml/kg) group, PD98059 group (0.3 mg/kg). Another 12 SD rats were randomly selected as the Sham group. Twenty-four hours after resuscitation, neural injury was assessed by survival rate, neurological deficit scores (NDS) and Nissl staining; apoptosis of brain cells was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining; mRNA expression and protein levels of ERs-related protein BIP, PERK, ATF4 and CHOP were checked with RT-PCR and Western Blot. The results showed that there were no significant differences in baseline characteristics before CA/CPR among all groups. PD98059 significantly improved survival rate and NDS, increased the Nissl bodies in neurons, reduced apoptosis, downregulated the mRNA transcription and expression levels of BIP, PERK, ATF4 and CHOP at 24 h after CA/CPR. Our results demonstrate that inhibition of ERK by PD98059 alleviates ERs-induced apoptosis via BIP-PERK-ATF4-CHOP signaling pathway and mitigates CIRI in the CA/CPR rat model.

4-Phenylbutyric acid protects islet \u03b2 cell against cellular damage induced by glucocorticoids

This study, using the MIN6 cell line, examines the effect of glucocorticoids (GCs) on the expression and protein levels of endoplasmic reticulum stress (ERS) related genes. Furthermore, we evaluated the protective role of 4-phenylbutyric acid (4-PBA) on the aforesaid GCs induced changes. Pancreatic islet MIN6 cells were treated with dexamethasone (DEX) at distinct concentrations (0.1 μmol/L and 0.5 μmol/L) for different periods (1 h, 4 h, 12 h, and 24 h). The mRNA and protein levels of ERS related genes were measured using real-time qPCR (qRT-PCR) and western blotting. Similar evaluations were also carried out for the cells treated with 4-PBA combined with DEX. Upon DEX intervention which induces the unfolded protein response (UPR), the expression levels of BIP, ATF6, IRE1, and PERK increased in the MIN6 cells, both in concentration and time-dependent manner. Similarly, ERS associated gene CHOP, which is involved in the apoptotic pathway, also showed increased levels both in concentration and time-dependent manner. However, treatment with 4-PBA decreased the expression levels of ERS related proteins. Quantitative analysis found that all these results were statistically significant (P < 0.05). GCs markedly activates the ERS in the MIN6 cell line in vitro, however, this effect can be significantly alleviated upon treatment with 4-PBA.

Starvation Induces Endoplasmic Reticulum Stress and Autophagy in Malpighian Tubules of Two-Spotted Field Crickets Gryllus bimaculatus

We investigated gene expression related to endoplasmic reticulum (ER) stress, apoptosis, and autophagy to understand how Gryllus bimaculatus (two-spotted field cricket) overcomes starvation stress. Male adults were used in starvation experiments. Expression levels of genes related to ER stress, apoptosis, and autophagy were investigated for each tissue after starvation stress (consisting of 1, 3, and 6 days of starvation, and 1 and 2 days of refeeding after 6 days of starvation) by reverse-transcription PCR. Furthermore, Malpighian tubules were stained with LysoTracker Red to observe autophagy. Starvation significantly upregulated gene expression of ER chaperones in the Malpighian tubules of G. bimaculatus. Gene expression levels of BiP, ERap2, and SERCA were upregulated up to 2.5-fold, ATF6 was upregulated up to 2-fold, and PDI and Derl2 were upregulated up to 1.5-fold in the starvation group compared to controls. Although autophagy was induced by starvation, apoptosis was not. Notably, gene expression of ATG9 was upregulated up to 15-fold in the Malpighian tubules of G. bimaculatus, which is the highest among the autophagy-related genes. These results suggest that ER stress-induced autophagy appears to be acutely involved in the mechanism by which insects overcome starvation stress.

Attenuation of Endoplasmic Reticulum Stress, Impaired Calcium Homeostasis, and Altered Bioenergetic Functions in MPP+-Exposed SH-SY5Y Cells Pretreated with Rutin.

Parkinson's disease (PD) is a common neurodegenerative disorder that affects approximately 1% of the population over the age of 65years. While treatment options for PD are limited, reports show that plant-derived bioactive compounds such as rutin possess numerous pharmacological benefits, including antioxidant and antiapoptotic activities. This study aimed to investigate the potential role of rutin in MPP+-treated SH-SY5Y neuroblastoma cells, an established cell model of PD. Our findings reveal increased concentrations of Ca2+ and endoplasmic reticulum (ER) stress as well as impaired mitochondrial membrane potential and bioenergetic status in SH-SY5Y cells treated with MPP+ only. This is demonstrated by a significant reduction in the expression levels of BiP, significantly reduced basal respiration, maximal respiration, and spare respiratory capacity as well as a significant increase in the expression levels of CHOP; however, these effects were significantly attenuated following pretreatment with rutin. Also, rutin significantly improved basal and compensatory glycolysis as a response to an impaired oxidative phosphorylation system triggered by MPP+, characterized by deficient ATP production. In conclusion, our findings provide the first evidence on the ability of rutin to maintain Ca2+ homeostasis, inhibit ER stress, and protect the mitochondria in MPP+-treated SH-SY5Y cells.

Vitamin D3 Alleviates High-Fat Diet–Induced Endoplasmic Reticulum Stress and Insulin Resistance in Mice

Endoplasmic reticulum stress (ERs) may be a key mechanism for the connection of obesity, chronic inflammation, and insulin resistance (IR). As we know, vitamin D3 deficiency is prevalent in obese patients, and it may be an independent risk factor for obesity-induced IR and type 2 diabetes mellitus (T2DM). However, the effects of vitamin D3 on ERs, inflammatory response, and IR were still unknown. This study investigate the effects of vitamin D3 on ERs, inflammatory response, and IR in high-fat-diet (HFD) induced obese mice. Eighty male C57BL/6 mice were randomly divided into four group, including control group (n = 20), HFD group, HFD + low dose (50 IU/kg) of vitamin D3 group (VitD-L), HFD + high dose (250 IU/kg) of vitamin D3 group (VitD-H). The control group was fed with common feed, and the other groups were fed with HFD. After the establishment of the obesity model, the mice in experimental group was fed with different dose of vitamin D3 for 1 week. Then western blot was used to detect the protein expression level of Bip, IRE1, PERK and NF-κB in adipose tissue; serum levels of TNF-α, IL-6, serum 25-hydroxyvitamin D3, fasting blood glucose, glycosylated hemoglobin, triacylglyceride and fasting insulin were measured and the insulin resistance index was calculated. The results indicated that vitamin D3 in both low and high dose could significantly down-regulate the expression level of Bip, IRE1 and PERK, and decrease the expression levels of NF-κB, TNF-α, and IL-6 (p &amp;lt; 0.05). It could also significantly decrease the levels of fasting blood glucose, glycosylated hemoglobin, triacylglyceride and fasting insulin in obese mice (p &amp;lt; 0.05), and significantly alleviate the insulin resistance phenomenon in obese mice. The results indicated that vitamin D3 supplementation could reduce the synthesis and release of inflammatory factors, alleviate endoplasmic reticulum stress and insulin resistance. Disclosure L. Liu: None. X. Li: None.

Abstract 1241: Podophyllotoxin acetate (PA) as an anti-cancer drug candidate regulates various malignancy traits of non-small cell lung cancer

Abstract Here, we reported that podophyllotoxin acetate (PA) showed various aniti-cancer effects: induction of several cell death pathways, inhibition of γ-ionizing radiation (IR)-induced cancer invasion, chemo-sensitizer effect with topoisomerase (TOP) inhibitors against non-small cell lung cancer (NSCLC) cell lines as follows. (1) Stimulation of cell death via various mechanisms. First, PA dose-dependently induced cell cycle arrest at G2/M phase, as shown by accumulation of the mitosis-related proteins, p21, survivin and aurora B. This arrest was due to the PA-induced inhibition of microtubule polymerization that related to DNA damage (reflected by accumulation of γ-H2AX) resulting induction of intrinsic/extrinsic damage apoptotic pathways. PA also activated the pro-apoptotic ER stress pathway, as evidenced by increased expression levels of BiP, CHOP, IRE1-a, phospho-PERK, and phospho-JNK. Next, PA induced autophagy that reflected the expression of beclin-1, Atg3, Atg5 and Atg7, and the cleavage of LC3. (2) Inhibition of IR-induced cancer invasion. IR increased the invasion/migration of A549 cells, and this effect was decreased by PA treatment that the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). In this study, we also identified IR-induced new signaling pathway, EGFR - p38/ERK - CREB-1/STAT3 - EMT pathway, that was inhibited by PA. (3) Chemo-sensitizer effect conjugated with TOP inhibitors - etoposide (Eto) and camptothecin (Cpt). Combination Index (CI) values of the combinations showed synergistic effects between PA and topoisomerase inhibitors. Combination with PA and Eto/Cpt promotes disruption of dynamics of actin filaments, which subsequently enhanced apoptotic cell death that accompanied to increased phosphorylation of p38. We also observed these combinations also inhibited activation of CREB-1, synergistically. Therefore, this study indicated that PA functions as a chemosensitizer by enhancing apoptosis through activation of p38/caspases apoptotic axis and suppression of CREB-1.Taken together; these results suggest PA is a new anti-cancer drug candidate that could control several malignancy traits of cancer including uncontrolled cell cycle and IR-induced invasion. Citation Format: Jeong Hyun Cho, Wan Gi Hong, Sang-Gu Hwang, Hong-Duck Um, Jong Kuk Park. Podophyllotoxin acetate (PA) as an anti-cancer drug candidate regulates various malignancy traits of non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1241.

Functional proteomic analysis revels that the ethanol extract of Annona muricata L. induces liver cancer cell apoptosis through endoplasmic reticulum stress pathway

Ethnopharmacological relevanceAnnona muricata L. is used to treat cancer in some countries. Extracts of Annona muricata have been shown to cause apoptosis of various cancer cells in vitro, and inhibit tumor growth in vivo in animal models. However, the molecular mechanisms underlying its anti-cancer and apoptotic effects of the herb remain to be explored. Aim of studyThe study investigated the molecular mechanisms underlying liver cancer cell apoptosis triggered by the ethanol extract of leaves of Annona muricata L. Materials and methodsLiver cancer HepG2 cells were used as experimental model. MTT assay was employed to evaluate cell viability. Flow cytometry and TUNEL assays were performed to confirm apoptosis. We employed functional proteomic analysis to delineate molecular pathways underlying apoptosis triggered by the herbal extract. ResultsWe showed that the extract was able to reduce viability and trigger apoptosis of the cancer cells. Proteomic analysis identified 14 proteins associated with the extract-elicited apoptosis, which included the increased expression levels of HSP70, GRP94 and DPI-related protein 5. Western blot analysis confirmed that the extract did up-regulated the protein levels of HSP70 and GRP94. Results from bioinformatic annotation pulled out two molecular pathways for the extract, which, notably, included endoplasmic reticulum (ER) stress which was evidenced by the up-regulation of HSP70, GRP94 and PDI-related protein 5. Further examinations of typical protein signaling events in ER stress using western blot analysis have shown that the extract up-regulated the phorsphorelation of PERK and eIF2α as well as the expression level of Bip and CHOP. ConclusionOur results indicate that the ethanol extract of leaves of Annona muricata L. causes apoptosis of liver cancer cells through ER stress pathway, which supports the ethnomedicinal use of this herb as an alternative or complementary therapy for cancer.

Induction of HaCaT Cell Apoptosis by Sodium Nitroprusside

Nitric Oxide (NO) has been known to play important physiological and pathological roles. In this study, Sodium nitroprusside (SNP), NO donor, induced the apoptosis of HaCaT cell, human spontaneous immortal keratinocyte, which was investigated through DAPI staining and cleavage of PARP and caspase-3 protein. However, the expression level of Bip and CHOP, involved in ER stress, was not significantly changed as compared to the control cell group. Recent studies have showed that SIRT1, NAD+-dependent deacetylase, is the key protein that controls cell survival and death. SNP treatment suppressed the SIRT1 gene expression, which indicated that apoptosis induced by SNP could be implicated in SIRT1 down-regulation.

Duhuo Jisheng decoction inhibits endoplasmic reticulum stress in chondrocytes induced by tunicamycin through the downregulation of miR-34a.

Our previous study showed that Duhuo Jisheng decoction (DHJSD) inhibited chondrocyte apoptosis by the mitochondria-dependent signaling pathway. Endoplasmic reticulum (ER) stress is upstream of the mitochondria-dependent signaling pathway and has been shown to promote chondrocyte apoptosis that occurs in osteoarthritis (OA). The present study aimed to evaluate whether DHJSD inhibits the chondrocyte apoptosis by regulating ER stress. DHJSD enhanced the viability of tunicamycin (TM)‑exposed chondrocytes, a model of ER stress-induced apoptosis, in a dose‑ and time‑dependent manner, as shown by MTT assay. The present results showed that DHJSD and sodium 4-phenylbutyrate (PBA), an ER stress inhibitor, reduced TM‑induced chondrocyte apoptosis by 4',6-diamidino‑2-phenylindole staining. To gain insight into the mechanisms of DHJSD that are responsible for enhancing the viability and inhibiting TM‑induced chondrocyte apoptosis, the associated mRNA expressions and protein levels were detected by reverse transcription‑polymerase chain reaction (RT‑PCR) and western blot analysis, respectively. The results showed that the expression levels of Xbp1, Xbp1s and Bcl‑2 were increased, and the expression levels of Bip, Atf4, Chop, Bax, caspase‑9 and ‑3 were decreased in the TM‑exposed chondrocytes treated with DHJSD or PBA compared with that in the TM‑exposed chondrocytes. To identify the possible mechanisms, the expression of miR‑34a was examined by the TaqMan microRNA assay, and was downregulated in the TM‑exposed chondrocytes treated with DHJSD or PBA compared with that in the TM-exposed chondrocytes. DHJSD inhibits ER stress in chondrocytes induced by exposure to TM by downregulating miR‑34a, suggesting that DHJSD may be a potential therapeutic agent for OA.

Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress

In the endoplasmic reticulum (ER), secretory and membrane proteins are properly folded and modified, and the failure of these processes leads to ER stress. At the same time, unfolded protein response (UPR) genes are activated to maintain homeostasis. Despite the thorough characterization of the individual gene regulation of UPR genes to date, further investigation of the mutual regulation among UPR genes is required to understand the complex mechanism underlying the ER stress response. In this study, we aimed to reveal a gene regulatory network formed by UPR genes, including immunoglobulin heavy chain-binding protein (BiP), X-box binding protein 1 (XBP1), C/EBP [CCAAT/enhancer-binding protein]-homologous protein (CHOP), PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring 1 (IRE1), activating transcription factor 6 (ATF6), and ATF4. For this purpose, we focused on promoter-luciferase reporters for BiP, XBP1, and CHOP genes, which bear an ER stress response element (ERSE), and p5 × ATF6-GL3, which bears an unfolded protein response element (UPRE). We demonstrated that the luciferase activities of the BiP and CHOP promoters were upregulated by all the UPR genes, whereas those of the XBP1 promoter and p5 × ATF6-GL3 were upregulated by all the UPR genes except for BiP, CHOP, and ATF4 in HeLa cells. Therefore, an ERSE- and UPRE-centered gene regulatory network of UPR genes could be responsible for the robustness of the ER stress response. Finally, we revealed that BiP protein was degraded when cells were treated with DNA-damaging reagents, such as etoposide and doxorubicin; this finding suggests that the expression level of BiP is tightly regulated at the post-translational level, rather than at the transcriptional level, in the presence of DNA damage.

Effect of Broussonetia papyrifera ( L. ) Vent aqueous extract on the ability of space learning and memory in the rats complex model of Alzheimer's disease

Objective To observe the effect of aqueous extract of Broussonetia papyrifera ( L. ) Vent on the ability of space learning and memory in the rats with Alzheimer' s disease (AD) induced by Aβ 25-35 and Dgalactose and to explore the mechanisms underlying those improvements. Methods The animal model of AD was established by Aβ 25-35 stereotactic injection into the hippocampus of rats in 5 minutes,while long-term intraperitoneal injection with D-gal. After the injection of Aβ25-35,rats were treated with aqueous extract of Broussonetia papyrifera ( L. ) Vent for the next 30 days. Morris water maze with computer system and the spatial exploration experiments were used to assess the behavior performances of the rats. Immunohistochemical technique was used to detect the expression of BiP, PERK and CHOP. Results The ability of space learning and memory of rats complex model of Alzheimer's disease induced by Aβ25-35 and D-gal was damaged,while escape latency was (20.90± 9.16 ) s,and the proportion of original platform quadrant was ( 11.05 ± 4.43 ) %. The expression level of Bip was reduced ,while the mean gray was ( 139.71 ± 3.47 ). The expression level of PERK and CHOP was increased,while the mean gray were (97.96 ± 5.97 ), ( 110.93±4.91 )separately. The escape latency of rats in the aqueous extract of Broussonetia papyrifera ( L. ) Vent treated groups was ( 5.41 ± 3.47 ) s and shorter than the model group,while the proportion of original platform quadrant was (48.28 ± 7.03 )% and higher than the model group.The expression level of Bip in the treated group was higher than the model group, while the mean gray were ( 121.17 ±4.76). The expression level of PERK and CHOP in the treated group was lower than the model group,while the mean gray were ( 122.11 ± 4.73 ), ( 123.34 ± 7.73 ) separately. Significant differences were observed between model group and aqueous extract of Broussonetia papyrifera ( L. ) Vent treated groups (P＜ 0. 05 ～ 0. 01 ).Conclusion Aqueous extract of Broussonetia papyrifera ( L. ) Vent can improve learning and memory disorders of the model rats induced by Aβ25-35 and D-galactose. ER (endoplasmic reticulum) stress and correlated apoptosis pathway might be involved in the underlying mechanisms. Key words: Alzheimer's disease; β-Amyloidal peptide25-35; D-galactose; ER stress; Broussonetia papyrifera (L.) Vent

Down‐regulation of BiP/GRP78 sensitizes resistant prostate cancer cells to gene‐therapeutic overexpression of REIC/Dkk‐3

We have recently shown that an adenovirus carrying REIC/Dkk-3 (Ad-REIC) exhibits a potent tumor-specific cell-killing function for various human cancers. It has also become evident that some human cancers are resistant to Ad-REIC-induced apoptosis. The aim of the present study was to determine the molecular mechanisms of resistance to Ad-REIC. First, we isolated resistant clones from a human prostate cancer cell line, PC3, after repeated exposure to Ad-REIC. Infection efficiency of the adenovirus vector and expression level of REIC/Dkk-3 in the resistant clones were similar to those in the parental PC3 cells. By screening for alteration in levels and functional status of proteins involved in Ad-REIC-induced apoptosis, we found that BiP/GRP78, an ER-residing chaperone protein, was expressed at higher levels consistently among resistant cells. Expression levels of BiP and rates of apoptosis induced by Ad-REIC were inversely correlated. Down-regulation of BiP with siRNA sensitized the resistant cells to Ad-REIC in vivo as well as in culture. These results indicate that BiP is a major determinant of resistance to Ad-REIC-induced apoptosis. Thus BiP is useful for diagnosis of inherent and acquired resistance of cancers and also as a target molecule to overcome resistance to the gene therapeutic Ad-REIC.

Protection of pancreatic β-cells by exendin-4 may involve the reduction of endoplasmic reticulum stress; in vivo and in vitro studies

The aim of this study was to investigate the in vivo and in vitro effects of exendin-4, a potent glucagon-like peptide 1 agonist, on the protection of the pancreatic beta-cells against their cell death. In in vivo experiments, we used beta-cell-specific calmodulin-overexpressing mice where massive apoptosis takes place in their beta-cells, and we examined the effects of chronic treatment with exendin-4. Chronic and s.c. administration of exendin-4 reduced hyperglycemia. The treatment caused significant increases of the insulin contents of the pancreas and islets, and retained the insulin-positive area. Dispersed transgenic islet cells lived only shortly, and several endoplasmic reticulum (ER) stress-related molecules such as immunoglobulin-binding protein (Bip), inositol-requiring enzyme-1alpha, X-box-binding protein-1 (XBP-1), RNA-activated protein kinase-like endoplasmic reticulum kinase, activating transcription factor-4, and C/EBP-homologous protein (CHOP) were more expressed in the transgenic islets. We also found that the spliced form of XBP-1, a marker of ER stress, was also increased in beta-cell-specific calmodulin-overexpressing transgenic islets. In the quantitative real-time PCR analyses, the expression levels of Bip and CHOP were reduced in the islets from the transgenic mice treated with exendin-4. These findings suggest that excess of ER stress occurs in the transgenic beta-cells, and the suppression of ER stress and resultant protection against cell death may be involved in the anti-diabetic effects of exendin-4.