Expression Of Endoplasmic Reticulum Stress Markers Research Articles

4-PBA improves lithium-induced nephrogenic diabetes insipidus by attenuating ER stress.

Endoplasmic reticulum (ER) stress has been implicated in some types of glomerular and tubular disorders. The objectives of this study were to elucidate the role of ER stress in lithium-induced nephrogenic diabetes insipidus (NDI) and to investigate whether attenuation of ER stress by 4-phenylbutyric acid (4-PBA) improves urinary concentrating defect in lithium-treated rats. Wistar rats received lithium (40 mmol/kg food), 4-PBA (320 mg/kg body wt by gavage every day), or no treatment (control) for 2 wk, and they were dehydrated for 24 h before euthanasia. Lithium treatment resulted in increased urine output and decreased urinary osmolality, which was significantly improved by 4-PBA. 4-PBA also prevented reduced protein expression of aquaporin-2 (AQP2), pS256-AQP2, and pS261-AQP2 in the inner medulla of kidneys from lithium-treated rats after 24-h dehydration. Lithium treatment resulted in increased expression of ER stress markers in the inner medulla, which was associated with dilated cisternae and expansion of ER in the inner medullary collecting duct (IMCD) principal cells. Confocal immunofluorescence studies showed colocalization of a molecular chaperone, binding IgG protein (BiP), with AQP2 in principal cells. Immunohistochemistry demonstrated increased intracellular expression of BiP and decreased AQP2 expression in IMCD principal cells of kidneys from lithium-treated rats. 4-PBA attenuated expression of ER stress markers and recovered ER morphology. In IMCD suspensions isolated from lithium-treated rats, 4-PBA incubation was also associated with increased AQP2 expression and ameliorated ER stress. In conclusion, in experimental lithium-induced NDI, 4-PBA improved the urinary concentrating defect and increased AQP2 expression, likely via attenuating ER stress in IMCD principal cells.

Salubrinal improves cardiac function in rats with heart failure post myocardial infarction through reducing endoplasmic reticulum stress-associated apoptosis

Endoplasmic reticulum (ER) stress plays an important role in ischemia-mediated cell death. The aim of the current study is to investigate the effects of salubrinal (Sal), a selective eIF2a dephosphorylation inhibitor, on heart failure rats and related mechanisms. Heart failure was induced by coronary artery ligation (MI) in adult male Sprague-Dawley rats. To ensure comparable MI sizes post coronary artery ligation on various groups, echocardiography examination was performed before and 30 minutes after ligation in MI groups. Then rats were randomly assigned to 4 groups: Sham group (n=12), MI group (n=10), MI plus vehicle injections group (DMSO group, n=12) and MI plus Sal injection group (Sal group, n=12). Sal (1 mg/kg) or DMSO was injected via the tail vein daily for the first 3 days (starting at 30 minutes after ligation of the left coronary artery), followed by subcutaneous injections twice per week for 8 weeks. Cardiac function was assessed by echocardiography and cell apoptosis assessed by flow cytometric analysis after 8 weeks. Protein and mRNA levels of ER stress markers were evaluated by immunohistochemistry and real time RT-PCR respectively. Eight weeks later, LVEF was significantly higher, while LVESD and LVEDD values were significantly lower in Sal group compared to MI and DMSO groups (all P<0.05); LV/BW ratio was significantly higher in MI group than in Sham group ((2.30±0.40) mg/g vs.(1.78±0.31) mg/g, P<0.05), which was significantly reduced in Sal group ((1.88±0.25) mg/g), but not in DMSO group((2.25±0.36) mg/g, P<0.05 vs. MI). In addition, flow cytometric analysis showed that Sal treatment significantly reduced apoptosis but not necrosis in post MI. Immunohistochemistry and real time PCR analysis showed that the myocardial protein and mRNA expression of ER stress markers were significantly lower in Sal group than in MI group, myocardial caspase-12 expression was significantly upregulated in MI group and significantly reduced by Sal treatment. Our results suggest that reduction of ER stress and myocardial apoptosis through inhibition of eIF2α dephosphorylation may serve as the potential mechanisms for the improved cardiac function and attenuated cardiac remodeling post Sal treatment in this heart failure rat model.

Lipopolysaccharide-Induced Behavioral Alterations Are Alleviated by Sodium Phenylbutyrate via Attenuation of Oxidative Stress and Neuroinflammatory Cascade

Oxido-nitrosative stress, neuroinflammation, and reduced level of neurotrophins are implicated in the pathophysiology of anxiety and depressive illness. A few recent studies have revealed the role of endoplasmic reticulum (ER) stress in the pathophysiology of stress and depression. The aim of the present study is to investigate the neuroprotective potential of sodium phenylbutyrate (SPB), an ER stress inhibitor against lipopolysaccharide (LPS)-induced anxiety and depressive-like behavior in Swiss albino mice. Anxiety and depressive-like behavior was induced by LPS (0.83mg/kg; i.p.) administration. Various behavioral tests were conducted to evaluate the anxiety and depressive-like behavior in mice. Real-time PCR was employed for the detection and expression of ER stress markers (78-kDa glucose-regulated protein (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)). Pretreatment with SPB significantly ameliorated the LPS-induced anxiety and depressive-like behavior as revealed by behavioral paradigm results. LPS-induced oxidative stress was ameliorated by SPB pretreatment in hippocampus (HC) and prefrontal cortex (PFC) region. Neuroinflammation was significantly reduced by SPB pretreatment in LPS-treated mice as evident from reduction in proinflammatory cytokines (IL-1β and TNF-α). Importantly, LPS administration significantly up-regulated the GRP78 mRNA expression level in the HC which suggests the involvement of unfolded protein response (UPR) in LPS-evoked behavioral anomalies. These results highlight the neuroprotective potential of SPB in LPS-induced anxiety and depressive illness model which may be partially due to inhibition of oxidative stress-neuroinflammatory cascade.

Inhibition of endoplasmic reticulum stress improves coronary artery function in type 2 diabetic mice

What is the central question of this study? Endoplasmic reticulum (ER) stress has been reported to be involved in type 2 diabetes; however, the role of exacerbated ER stress in vascular dysfunction in type 2 diabetes remains unknown. What is the main finding and its importance? The main findings of this study are that ER stress is increased in the coronary arteries in type 2 diabetes, and inhibition of ER stress using taurine-conjugated ursodeoxycholic acid improves vascular function, which is associated with normalization of the myogenic response and endothelium-dependent relaxation. Vascular dysfunction is a major complication in type 2 diabetes. Although endoplasmic reticulum (ER) stress has been suggested to be a contributory factor in cardiovascular diseases, the relationship between ER stress and vascular dysfunction in type 2 diabetes remains unclear. Thus, in the present study, we examined whether ER stress contributes to coronary artery dysfunction and whether inhibition of ER stress ameliorates vascular function in type 2 diabetes. Type 2 diabetic mice and their control counterparts were treated with an ER stress inhibitor (taurine-conjugated ursodeoxycholic acid, 150mgkg(-1) day(-1) , by i.p. injection) for 2weeks or not treated. The myogenic response and endothelium-dependent relaxation were measured in pressurized coronary arteries. In type 2 diabetic mice, blood glucose and body weight were elevated compared with control mice. The myogenic response was potentiated and endothelium-dependent relaxation impaired in coronary arteries from the type 2 diabetic mice. Interestingly, treatment with the ER stress inhibitor normalized the myogenic responses and endothelium-dependent relaxation. These data were associated with an increase in ER stress marker expression or phosphorylation (IRE1-XBP-1 and PERK-eIF2α) in type 2 diabetic mice, which were reduced by treatment with the ER stress inhibitor. Inhibition of ER stress normalizes the myogenic response and improves vascular function in type 2 diabetes. Therefore, ER stress could be a potential target for cardiovascular diseases in diabetes mellitus.

Ca2+-Dependent Endoplasmic Reticulum Stress Regulates Mechanical Stress–Mediated Cartilage Thinning

Our previous study identified that endoplasmic reticulum stress (ERS) plays a critical role in chondrocyte apoptosis and mandibular cartilage thinning in response to compressive mechanical force, although the underlying mechanisms remain elusive. Because the endoplasmic reticulum (ER) is a primary site of intracellular Ca2+ storage, we hypothesized that Ca2+-dependent ERS might be involved in mechanical stress–mediated mandibular cartilage thinning. In this study, we used in vitro and in vivo models to determine Ca2+ concentrations, histological changes, subcellular changes, apoptosis, and the expression of ERS markers in mandibular cartilage and chondrocytes. The results showed that in chondrocytes, cytosolic Ca2+ ([Ca2+]i) was dramatically increased by compressive mechanical force. Interestingly, the inhibition of Ca2+ channels by ryanodine and 2-aminoethoxydiphenyl borate, inhibitors of ryanodine receptors and inositol trisphosphate receptors, respectively, partially rescued mechanical force–mediated mandibular cartilage thinning. Furthermore, chondrocyte apoptosis was also compromised by inhibiting the increase in [Ca2+]i that occurred in response to compressive mechanical force. Mechanistically, the ERS induced by compressive mechanical force was also repressed by [Ca2+]i inhibition, as demonstrated by a decrease in the expression of the ER stress markers 78 kDa glucose-regulated protein (GRP78) and 94 kDa glucose-regulated protein (GRP94) at both the mRNA and protein levels. Collectively, these data identified [Ca2+]i as a critical mediator of the pathological changes that occur in mandibular cartilage under compressive mechanical force and shed light on the treatment of mechanical stress–mediated cartilage degradation.

Renoprotective Effects of 4‐Phenylbutyrate Against Palmitate‐Induced Renal Cell Injury

BackgroundIncreased dietary intake of fats rich in saturated fatty acids and dyslipidemia have been shown to significantly aggravate the risk of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Although multiple mechanisms including endoplasmic reticulum (ER) stress and oxidative stress are implicated in the pathophysiology of CKD, the effects of elevated free fatty acid (FFA) levels per se on renal cells is still unclear. Hence, we investigated the effects of palmitic acid (a saturated fatty acid) on rat renal proximal tubular cells in vitro, and tested the renoprotective effects of 4‐phenylbutyrate (4‐PBA), a chemical chaperone shown to inhibit ER stress in vitro and in vivo, in our model.MethodsSubconfluent rat renal proximal tubular (NRK‐52E) cells were exposed to 100 mM palmitic acid in presence and absence of 5 mM 4‐PBA for 24 hours. Cells were then assessed for alterations in cell viability (using MTT assay), oxidative stress (via Dichlorofluorescein (DCF) staining) and the expression of ER stress markers (i.e., glucose‐regulated protein, GRP78 and C/EBP homologous protein, CHOP) and apoptosis (caspase‐3 and ‐12) using western blotting. Flow cytometry for cell cycle analysis and immunoblotting for p27 (an inhibitor of cell cycle) were performed to determine the effects of palmitate and 4‐PBA on the cell cycle in renal cells.ResultsRenal cells exposed to palmitate for 24 hours showed decreased viability (76.9% to that of control), increased DCF staining (1.3‐fold increase over control), and marked upregulation of GRP78 and CHOP. Similarly, the expression of active forms of caspase‐3 (1.8‐fold increase over control) and caspase‐12 (3‐fold increase over control) were also increased in renal cells treated with palmitate. These changes were significantly attenuated in cells co‐treated with 4‐PBA. Intriguingly, exposure to palmitate did not alter the cell cycle in renal cells, whereas treatment with 4‐PBA caused downregulation of p27 (54% to that of control) and increased the transition of cells from G1 to S‐phase and G2/M phase.ConclusionsOur findings indicate that exposure to palmitate induces ER stress as well as oxidative stress in renal cells, and results in renal cell apoptosis. Co‐treatment with 4‐PBA protects renal cells against the perturbations induced by palmitate and improves renal cell viability, which underscores the renoprotective effects of 4‐PBA against fatty acid‐induced renal cell injury. Further studies are required to understand the mechanisms by which 4‐PBA prevents oxidative stress and alters cell cycle in renal cells.Support or Funding InformationSupported by student research grants # QUST‐CPH‐FALL‐12/13‐6 and QUST‐CPH‐SPR‐12/13‐3 from the Office of Academic Research, Qatar University.

Schisandra chinensis extract ameliorates nonalcoholic fatty liver via inhibition of endoplasmic reticulum stress

Ethnopharmacological relevanceSchisandra chinensis (Turcz.) Baill. (SC) is a traditional Chinese herbal medicine with diverse pharmacological activities for treatment of various human diseases. Endoplasmic reticulum (ER) stress is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the protective effects of methanol extract of Schisandra chinensis (SC extract) against ER stress-induced NAFLD in vitro and in vivo. Material and methodsThe protective effects of SC extract were examined in tunicamycin- or palmitate-treated HepG2 cells in vitro, and in tunicamycin-injected mice or high fed diet (HFD) obese mice in vivo. Expression of ER stress markers including glucose regulated protein 78 (GRP78), C/EBP homolog protein (CHOP), and X-box-binding protein-1 (XBP-1), and triglyceride accumulation were measured in HepG2 cells and in the liver of mice. ResultsSC extract significantly inhibited expression of tunicamycin-induced ER stress markers in tunicamycin-treated HepG2 cells and in the liver of tunicamycin-injected mice, and it also inhibited tunicamycin-induced triglyceride accumulation. Similar observations were made under physiological ER stress conditions such as in palmitate-treated HepG2 cells and in the liver of HFD obese mice. In addition, SC extract repressed the expression of inflammatory genes and lipogenic genes in palmitate-treated HepG2 cells. Schisandrin, an abundant bioactive lignan in SC extract, inhibited the expression of ER stress markers in tunicamycin-or palmitate-treated HepG2 cells, whereas Gomisin J did not affect ER stress markers. ConclusionsSC attenuates ER stress and prevents development of NAFLD. SC may be useful as a pharmacological agent for protection against ER stress-induced human diseases.

Nickel chloride (NiCl2) induces endoplasmic reticulum (ER) stress by activating UPR pathways in the kidney of broiler chickens.

It has been known that overexposure to Ni can induce nephrotoxicity. However, the mechanisms of underlying Ni nephrotoxicity are still elusive, and also Ni- and Ni compound-induced ER stress has been not reported in vivo at present. Our aim was to use broiler chickens as animal model to test whether the ER stress was induced and UPR was activated by NiCl2 in the kidney using histopathology, immunohistochemistry and qRT-PCR. Two hundred and eighty one-day-old broiler chickens were divided into 4 groups and fed on a control diet and the same basal diet supplemented with 300 mg/kg, 600mg/kg and 900mg/kg of NiCl2 for 42 days. We found that dietary NiCl2 in excess of 300 mg/kg induced ER stress, which was characterized by increasing protein and mRNA expression of ER stress markers, e.g., GRP78 and GRP94. Concurrently, all the three UPR pathways were activated by dietary NiCl2. Firstly, the PERK pathway was activated by increasing eIF2a and ATF4 mRNA expression. Secondly, the IRE1 pathway was activated duo to increase in IRE1 and XBP1 mRNA expression. And thirdly, the increase of ATF6 mRNA expression suggested that ATF6 pathway was activated. The findings clearly demonstrate that NiCl2 induces the ER stress through activating PERK, IRE1 and ATF6 UPR pathways, which is proved to be a kind of molecular mechanism of Ni- or/and Ni compound-induced nephrotoxicity.

Endoplasmic reticulum stress is involved in the colonic epithelium damage induced by maternal separation

BackgroundMaternal separation (MS) leads to intestinal barrier dysfunction in neonatal mice. Endoplasmic reticulum (ER) stress is associated with apoptosis and pro-inflammatory response induction. We hypothesized that MS induced gut damage is associated with ER stress and that administration of an ER stress inhibitor protects gut damage. MethodsC57BL/6 mice received intraperitoneal PBS (n=10) or Salubrinal (1mg/kg/day, n=10). MS was performed soon after treatment for 3h daily between P5 and P9. Ten untreated neonatal mice served as control. The colon was harvested on P9 and analyzed for ER stress markers (BiP, CHOP), apoptosis (CC3), goblet cell number per crypt and crypt length (Alcian blue, hematoxylin/eosin), and transcellular permeability (Ussing chamber). Groups were compared using one-way ANOVA with Bonferroni post-test. ResultsCompared to controls, MS mice had higher relative protein expression of ER stress and apoptosis markers (p<0.05) and reduced goblet cell number per crypt and crypt length (p<0.001). In comparison to PBS mice, Salubrinal treated mice had higher goblet cell number (p<0.05), crypt length (p<0.001), and lower transcellular permeability (p<0.05). ConclusionsMaternal separation induces ER stress and causes colon damage, but ER stress inhibitor protects morphology and permeability. This provides insights on bowel pathogenesis and potential novel treatments for diseases such as necrotizing enterocolitis.

Celecoxib induces apoptosis but up-regulates VEGF via endoplasmic reticulum stress in human colorectal cancer in vitro and in vivo.

In our previous study, we found that celecoxib, a kind of COX-2 inhibitor, led to cell apoptosis while up-regulating the expression of vascular endothelial growth factor (VEGF) in colorectal cancer HCT116 cells (COX-2 deficient), and endoplasmic reticulum (ER) stress was involved in the mechanism. Thus, we would like to explore whether these results are universal for other colorectal cancer cells, especially for COX-2-expressing ones, and whether the results in vitro and in vivo are matched. HT29 cells (COX-2 expressing) were treated with celecoxib under different conditions to evaluate cell apoptosis, VEGF expression and the activation of ER stress. HT29 and HCT116 xenograft tumor models were established to evaluate anti-tumor effects and verify the experiment results we obtained in vitro. Celecoxib (≥60µM) up-regulated the expression of ER stress markers (GRP78 and CHOP) and induced cell apoptosis accompanying with a correlated increased expression of VEGF in HT29 cells. Celecoxib-induced gene expression and cell apoptosis were inhibited by an ER stress inhibitor, PBA. In xenograft models, celecoxib treatment inhibited tumor growth with increased GRP78 and VEGF, which was consistent with the results in vitro. Celecoxib, both in vitro and in vivo, induced apoptosis of colorectal cancer cells but increased the VEGF levels at the same time in a COX-2-independent manner, namely by activating ER stress. The increased VEGF would impair the effect of celecoxib and bring drug resistant; hence, the optimal schedule of the combination of celecoxib with anti-VEGF drugs needs to be explored.

Knockdown of RTN1A attenuates ER stress and kidney injury in albumin overload-induced nephropathy.

Our previous studies have suggested a critical role of reticulon (RTN)1A in mediating endoplasmic reticulum (ER) stress in kidney cells of animal models and humans with kidney diseases. A large body of evidence suggests that proteinuria itself can cause tubular cell injury leading to the progression of kidney disease. In the present study, we determined whether RTN1A mediates proteinuria-induced tubular cell injury through increased ER stress. We found that incubation of HK2 cells with human serum albumin induced the expression of RTN1A and ER stress markers, whereas knockdown of RTN1A expression attenuated human serum albumin-induced ER stress and tubular cell apoptosis in vitro. In vivo, we found that tubular cell-specific RTN1 knockdown resulted in a significant attenuation of tubular cell ER stress, apoptosis, and renal fibrosis in a model of albumin overload nephropathy. Based on these findings, we conclude that RTN1A is a key mediator for proteinuria-induced tubular cell toxicity and renal fibrosis.

Expression of ER stress markers (GRP78/BiP and PERK) in patients with tongue cancer.

The glucose-regulated protein (GRP78/BiP) and PKR-like endoplasmic reticulum kinase (PERK) plays a crucial role in the endoplasmic reticulum (ER) stress response. GRP78/BiP is highly elevated in various human cancers. Our study is to examine the clinicopathological significance of GRP78/BiP and PERK expression in patients with tongue cancer. A total of 85 tongue cancer patients were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP, PERK, GLUT1, Ki-67 and microvessel density (MVD) determined by CD34.GRP78/BiP and PERK were highly expressed in 47% and 35% of all patients, respectively. GRP78/BiP disclosed a significant relationship with PERK expression, lymphatic permeation, vascular invasion, glucose metabolism and cell proliferation. The expression of GRP78/BiP was significantly higher in metastatic sites than in primary sites (79% vs. 47%, p=0.003). We found that the high expression of GRP78/BiP was proven to be an independent prognostic factor for predicting poor outcome in patients with tongue cancer. In the analysis of PFS, PERK was identified as an independent predictor. The increased GRP78/BiP expression was clarified as an independent prognostic marker for predicting worse outcome. Our study suggests that the expression of GRP78/BiP as ER stress marker is important in the pathogenesis and development of tongue cancer.

Glutathione Peroxidase-1 Suppresses the Unfolded Protein Response upon Cigarette Smoke Exposure.

Oxidative stress provokes endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the lungs of chronic obstructive pulmonary (COPD) subjects. The antioxidant, glutathione peroxidase-1 (GPx-1), counters oxidative stress induced by cigarette smoke exposure. Here, we investigate whether GPx-1 expression deters the UPR following exposure to cigarette smoke. Expression of ER stress markers was investigated in fully differentiated normal human bronchial epithelial (NHBE) cells isolated from nonsmoking, smoking, and COPD donors and redifferentiated at the air liquid interface. NHBE cells from COPD donors expressed heightened ATF4, XBP1, GRP78, GRP94, EDEM1, and CHOP compared to cells from nonsmoking donors. These changes coincided with reduced GPx-1 expression. Reintroduction of GPx-1 into NHBE cells isolated from COPD donors reduced the UPR. To determine whether the loss of GPx-1 expression has a direct impact on these ER stress markers during smoke exposure, Gpx-1 −/− mice were exposed to cigarette smoke for 1 year. Loss of Gpx-1 expression enhanced cigarette smoke-induced ER stress and apoptosis. Equally, induction of ER stress with tunicamycin enhanced antioxidant expression in mouse precision-cut lung slices. Smoke inhalation also exacerbated the UPR response during respiratory syncytial virus infection. Therefore, ER stress may be an antioxidant-related pathophysiological event in COPD.

Knockdown of histidine-rich calcium-binding protein (HRC) suppresses liver fibrosis by inhibiting the activation of hepatic stellate cells.

ABSTRACTThe histidine-rich calcium-binding protein (HRC) is a regulator of Ca2+ homeostasis and it plays a significant role in hepatocellular carcinoma (HCC) progression. However, the relationship between HRC and liver fibrogenesis is still unknown. Our data demonstrates that HRC was upregulated in fibrotic liver and activated hepatic stellate cells (HSCs). TGF-β treatment increased α-SMA and HRC expression dose-dependently in HSCs. Repression of HRC reduced α-SMA, CTGF and collagen expression, and inhibited HSC proliferation and migration. In addition, we found that the anti-fibrosis effect of HRC knockdown was associated with endoplasmic reticulum (ER) stress. Silencing of HRC decreased the expression of ER stress and autophagy markers. Moreover, ER stress agonist thapsigargin (TG) enhanced, whereas ER stress antagonist 4-phenylbutyric acid (4-PBA) alleviated HSCs activation and autophagy. In conclusion, these data indicate that depletion of HRC inhibited HSC activation through the ER stress pathway, and HRC may be a potential regulator of liver fibrosis.

4‑Phenylbutyrate protects rat skin flaps against ischemia‑reperfusion injury and apoptosis by inhibiting endoplasmic reticulum stress.

4-phenylbutyrate (4-PBA) is a low molecular weight fatty acid, which has been demonstrated to regulate endoplasmic reticulum (ER) stress. ER stress-induced cell apoptosis has an important role in skin flap ischemia; however, a pharmacological approach for treating ischemia-induced ER dysfunction has yet to be reported. In the present study, the effects of 4-PBA-induced ER stress inhibition on ischemia-reperfusion injury were investigated in the skin flap of rats, and transcriptional regulation was examined. 4-PBA attenuated ischemia-reperfusion injury and inhibited cell apoptosis in the skin flap. Furthermore, 4-PBA reversed the increased expression levels of two ER stress markers: CCAAT/enhancer-binding protein-homologous protein and glucose-regulated protein 78. These results suggested that 4-PBA was able to protect rat skin flaps against ischemia-reperfusion injury and apoptosis by inhibiting ER stress marker expression and ER stress-mediated apoptosis. The beneficial effects of 4-PBA may prove useful in the treatment of skin flap ischemia-reperfusion injury.

114 ENDOPLASMIC RETICULUM (ER) STRESS IN HYPOXIA-INDUCED DIABETES MELLITUS MODEL

Endoplasmic reticulum (ER) regulates calcium ion concentration as a reservoir in the cell. ER stress is a cellular stress response related to the endoplasmic reticulum. At the initial stage of ER stress, ER tries to restore normal function by halting protein translation, degrading misfolded proteins, and increasing production of chaperones involved in protein folding. If ER fails to restore ER stress, ER stress can lead cells to apoptosis. To study the signaling between ER stress and calcium channels under ER-stressed circumstances, we designed a hypoxia-induced diabetic model. Nine-week-old male mice were chosen, maintained under hypoxic condition under 10% O2, 5% CO2 for 10 days, and the expression of ER stress markers and calcium channel gene expression were examined by real-time PCR. By maintaining hypoxic condition, the mice showed high glucose levels. Under this diabetic condition, in pancreatic beta cells, ER stress markers were elevated. This tendency showed an increase in calbindin-D9k KO mice. Chaperones such as calreticulin and calnexin were decreased, but in calbindin-D9k KO mice chaperone calnexin was not decreased. Interestingly, the calbindin-D9k KO normoxia mice showed increased glucose level compared with wild-type normoxia mice. Also, calnexin expression of pancreas was decreased in calbindin-D9k KO normoxia mice. This result indicates that pancreas cells were under endoplasmic reticulum stress. Taken together, calbindin may play an important role in endoplasmic reticulum stress in pancreas. This work was supported by the National Research Foundation of Korea (NRF) grant of Korean government (MEST) (No. 2013-010514).

Protective Effects of Alisma orientale Extract against Hepatic Steatosis via Inhibition of Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion.

4-PBA prevents pressure overload-induced myocardial hypertrophy and interstitial fibrosis by attenuating endoplasmic reticulum stress

Our previous study indicated that attenuation of endoplasmic reticulum (ER) stress by administration of 4-phenylbutyric acid (4-PBA) could prevent cardiac rupture and remodeling in a mouse model of myocardial infarction (MI). However, whether 4-PBA is protective in hypertrophic heart disease is unclear. Thus, we tested the therapeutic effect of 4-PBA on pressure-overload induced myocardial hypertrophy. Transverse aortic constriction (TAC) was used to create myocardial hypertrophy in C57BL/6 male mice for 4 weeks. Immediately after surgery, the mice were administrated either 4-PBA (20 mg/kg/day) or 0.9% NaCl by intraperitoneal injection. At the end of 4 weeks, the mice underwent high-resolution echocardiographic imaging. Our results showed that both the left ventricular posterior wall thickness at end systole (LVPWs) and diastole (LVPWd) were increased in the TAC group, compared to control. 4-PBA administration attenuated hypertrophy and decreased the heart weight over body weight ratio. Masson's trichrome staining showed that myocardial interstitial fibrosis and collagen deposition were also decreased by 4-PBA. We next detected the ER stress response in the heart tissues of TAC mice in different time points. Western blotting showed that the expression of ER stress marker, GRP78, CHOP and phosphor-PERK, were persistently increased 4 weeks after TAC. The treatment of 4-PBA inhibited the expression of ER stress markers. We also demonstrated that the 4-PBA at 20 mg/kg/day had no effect on histone 3 deacetylation inhibition, while attenuating ER stress and TAC-induced hypertrophy. These findings suggest that 4-PBA may be a therapeutic strategy to consider in preventing pressure-overload induced myocardial hypertrophy and interstitial fibrosis by selectively attenuating ER stress.

Valsartan protects HK-2 cells from contrast media-induced apoptosis by inhibiting endoplasmic reticulum stress.

Contrast-induced acute kidney injury (CI-AKI) is associated with increasing in-hospital and long-term adverse clinical outcomes in high-risk patients undergoing percutaneous coronary intervention (PCI). Contrast media (CM)-induced renal tubular cell apoptosis is reported to participate in this process by activating endoplasmic reticulum (ER) stress. An angiotensin II type 1 receptor (AT1R) antagonist can alleviate ER stress-induced renal apoptosis in streptozotocin (STZ)-induced diabetic mice and can reduce CM-induced renal apoptosis by reducing oxidative stress and reversing the enhancement of bax mRNA and the reduction of bcl-2 mRNA, but the effect of the AT1R blocker on ER stress in the pathogenesis of CI-AKI is still unknown. In this study, we explored the effect of valsartan on meglumine diatrizoate-induced human renal tubular cell apoptosis by measuring changes in ER stress-related biomarkers. The results showed that meglumine diatrizoate caused significant cell apoptosis by up-regulating the expression of ER stress markers, including glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP) and caspase 12, in a time- and dose-dependent manner, which could be alleviated by preincubation with valsartan. In conclusion, valsartan had a potential nephroprotective effect on meglumine diatrizoate-induced renal cell apoptosis by inhibiting ER stress.

ER Stress Mediates TiAl6V4 Particle-Induced Peri-Implant Osteolysis by Promoting RANKL Expression in Fibroblasts.

Wear particle-induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the synovial fibroblasts present in the periprosthetic membrane are important targets of wear debris during osteolysis. However, the interaction mechanisms between the wear debris and fibroblasts remain largely unknown. In the present study, we investigated the effect of ER (endoplasmic reticulum) stress induced by TiAl6V4 particles (TiPs) in human synovial fibroblasts and calvarial resorption animal models. The expression of ER stress markers, including IRE1-α, GRP78/Bip and CHOP, were determined by western blot in fibroblasts that had been treated with TiPs for various times and concentration. To address whether ER stress was involved in the expression of RANKL, the effects of ER stress blockers (including 4-PBA and TUDCA) on the expression of RANKL in TiPs-treated fibroblasts were examined by real-time PCR, western blot and ELISA. Osteoclastogenesis was assessed by tartrate resistant acid phosphatase (TRAP) staining. Our study demonstrated that ER stress markers were markedly upregulated in TiPs-treated fibroblasts. Blocking ER stress significantly reduced the TiPs-induced expression of RANKL both in vitro and in vivo. Moreover, the inhibition of ER stress ameliorated wear particle-induced osteolysis in animal models. Taken together, these results suggested that the expression of RANKL induced by TiPs was mediated by ER stress in fibroblasts. Therefore, down regulating the ER stress of fibroblasts represents a potential therapeutic approach for wear particle-induced periprosthetic osteolysis.