78-kDa Glucose-regulated Protein Research Articles

GRP78 recognizes EV-F 3D protein and activates NF-κB to repress virus replication by interacting with CHUK/IKBKB.

Enteroviruses are the causative agents associated with several human and animal diseases, posing a significant threat to human and animal health. As one of the host immune defense strategies, innate immunity plays a crucial role in defending against invading pathogens, where the host utilizes a variety of mechanisms to inhibit or eliminate the pathogen. Here, we report a new strategy for the host to repress enterovirus replication by the 78 kDa glucose-regulated protein (GRP78), also known as heat shock protein family A member 5 (HSPA5). The GRP78 recognizes the EV-encoded RNA-dependent RNA polymerases (RdRPs) 3D protein and interacts with the nuclear factor kappa B kinase complex (CHUK) and subunit beta gene (IKBKB) to facilitate the phosphorylation and nuclear translocation of NF-κB, which induces the production of inflammatory factors and leads to a broad inhibition of enterovirus replication. These findings demonstrate a new role of GRP78 in regulating host innate immunity in response to viral infection and provide new insights into the mechanism underlying enterovirus replication and NF-κB activation.IMPORTANCEGRP78 is known as a molecular chaperone for protein folding and plays a critical role in maintaining protein folding and participating in cell proliferation, cell survival, apoptosis, and metabolism. However, the functions of GRP78 to participate in enterovirus genome replication and innate immune responses are rarely documented. In this study, we explored the functions of the EV-3D-interacting protein GRP78 and found that GRP78 inhibits enterovirus replication by activating NF-κB through binding to EV-F 3D and interacting with the NF-κB signaling molecules CHUK/IKBKB. This is the first report that GRP78 interacts with CHUK/IKBKB to activate the NF-κB signaling pathway, which leads to the expression of the proinflammatory cytokines and inhibition of enterovirus replication. These results demonstrate a unique mechanism of virus replication regulation by GRP78 and provide insights into the prevention and treatment of viral infections.

TEMPORARY REMOVAL: Ip3r-Grp75-Vdac and Relevant Ca2+ Signaling Regulate Dietary Palmitic Acid-Induced De Novo Lipogenesis by Mitochondria-Associated ER Membrane (MAM) Recruiting Seipin in Yellow Catfish

BackgroundThe mitochondria-associated endoplasmic reticulum membrane (MAM) is the central hub for endoplasmic reticulum and mitochondria functional communication. It plays a crucial role in hepatic lipid homeostasis. However, even though MAM has been acknowledged to be rich in enzymes that contribute to lipid biosynthesis, no study has yet investigated the exact role of MAM on hepatic neutral lipid synthesis. ObjectivesTo address these gaps, this study investigated the systemic control mechanisms of MAM on neutral lipids synthesis by recruiting seipin, focusing on the role of the inositol trisphosphate receptor-1,4,5(Ip3r)-75 kDa glucose-regulated protein (Grp75)-voltage-dependent anion channel (Vdac) complex and their relevant Ca2+ signaling in this process. MethodsTo this end, a model animal for lipid metabolism, yellow catfish (Pelteobagrus fulvidraco), were fed 6 different diets containing a range of palmitic acid (PA) concentrations from 0–150 g/kg in vivo for 10 wk. In vitro, experiments were also conducted to intercept the MAM-mediated Ca2+ signaling in isolated hepatocytes by transfecting them with si-mitochondrial calcium uniporter (mcu). Because mcu was placed in the inner mitochondrial membrane (IMM), si-mcu cannot disrupt MAM’s structural integrity. Results1. Hepatocellular MAM subproteome analysis indicated excessive dietary PA intake enhanced hepatic MAM structure joined by activating Ip3r-Grp75-Vdac complexes. 2. Dietary PA intake induced hepatic neutral lipid accumulation through MAM recruiting Seipin, which activated lipid droplet biogenesis. Our findings also revealed a previously unidentified mechanism whereby MAM-recruited seipin and controlled hepatic lipid homeostasis, depending on Ip3r-Grp75-Vdac-controlled Ca2+ signaling and not only MAM’s structural integrity. ConclusionsThese results offer a novel insight into the MAM-recruited seipin in controlling hepatic lipid synthesis in a MAM structural integrity-dependent and Ca2+ signaling-dependent manner, highlighting the critical contribution of MAM in maintaining hepatic neutral lipid homeostasis.

GRP78 Contributes to the Beneficial Effects of SGLT2 Inhibitor on Proximal Tubular Cells in DKD.

Beneficial effects of SGLT2 inhibitors on kidney function are well-known; however, their molecular mechanisms are not fully understood. We focused on 78 kDa glucose-regulated protein (GRP78) and its interaction with SGLT2 and Integrin ß1 beyond the chaperone property of GRP78. In STZinduced diabetic mouse kidneys, GRP78, SGLT2, and Integrin ß1 increased in the plasma membrane fraction, while they were suppressed by canagliflozin. The altered subcellular localization of GRP78/Integrin ß1 in STZ mice promoted epithelial mesenchymal transition (EMT) and fibrosis, which were mitigated by canagliflozin. High glucose conditions reduced intracellular GRP78, increased its secretion, and caused EMT-like changes in cultured HK2 cells, which were again inhibited by canagliflozin. Urinary GRP78 increased in STZ mice, and in vitro experiments with recombinant GRP78 suggested that inflammation spread to surrounding tubular cells and canagliflozin reversed this effect. Under normal glucose culture, canagliflozin maintained SERCA activity, promoted endoplasmic reticulum (ER) robustness, reduced ER stress response impairment, and protected proximal tubular cells. In conclusion, canagliflozin restored subcellular localization of GRP78, SGLT2 and Integrin ß1 and inhibited EMT and fibrosis in DKD. In non-diabetic CKD, canagliflozin promoted ER robustness by maintaining SERCA activity and preventing ER stress response failure, and contribute to tubular protection.

Gallic acid attenuates torsion/detorsion-induced testicular injury in rats through suppressing of HMGB1/NF-κB axis and endoplasmic reticulum stress.

It was aimed to evaluate whether gallic acid (GA) have a beneficial effect in the testicular ischemia/reperfusion injury (IRI) model in rats for the first time. Testicular malondialdehyde, 8-hydroxy-2'-deoxyguanosine, superoxide dismutase, catalase, high mobility group box 1 protein, nuclear factor kappa B, tumor necrosis factoralpha, interleukin-6, myeloperoxidase, 78-kDa glucose-regulated protein, activating transcription factor 6, CCAAT-enhancer-binding protein homologous protein and caspase-3 levels were determined using colorimetric methods. The oxidative stress, inflammation, endoplasmic reticulum stress and apoptosis levels increased statistically significantly in the IRI group compared with the sham operated group (p < 0.05). GA application improved these damage significantly (p < 0.05). Moreover, it was found that the results of histological examinations supported the biochemical results to a statistically significant extent. Our findings suggested that GA may be evaluated as a protective agent against testicular IRI.

GRP78 improves the therapeutic effect of mesenchymal stem cells on hemorrhagic shock-induced liver injury: Involvement of the NF-кB and HO-1/Nrf-2 pathways.

Mesenchymal stem cells (MSCs) are a popular cell source for repairing the liver. Improving the survival rate and colonization time of MSCs may significantly improve the therapeutic outcomes of MSCs. Studies showed that 78-kDa glucose-regulated protein (GRP78) expression improves cell viability and migration. This study aims to examine whether GRP78 overexpression improves the efficacy of rat bone marrow-derived MSCs (rBMSCs) in HS-induced liver damage. Bone marrow was isolated from the femurs and tibias of rats. rBMSCs were transfected with a GFP-labeled GRP78 expression vector. Flow cytometry, transwell invasion assay, scratch assay immunoblotting, TUNEL assay, MTT assay, and ELISA were carried out. The results showed that GRP78 overexpression enhanced the migration and invasion of rBMSCs. Moreover, GRP78-overexpressing rBMSCs relieved liver damage, repressed liver oxidative stress, and inhibited apoptosis. We found that overexpression of GRP78 in rBMSCs inhibited activation of the NLRP3 inflammasome, significantly decreased the levels of inflammatory factors, and decreased the expression of CD68. Notably, GRP78 overexpression activated the Nrf-2/HO-1 pathway and inhibited the NF-κB pathway. High expression of GRP78 efficiently enhanced the effect of rBMSC therapy. GRP78 may be a potential target to improve the therapeutic efficacy of BMSCs.

Salivary proteomic profile of young healthy subjects

Background: The incidence of noncommunicable diseases (NCDs) has been rapidly ramped up worldwide. Hence, there is an urgent need to non-invasively detect NCDs possibly by exploiting saliva as a ‘liquid biopsy’ to identify biomarkers of the health status. Since, the absence of standardized procedures of collection/analysis and the lack of normal ranges makes the use of saliva still tricky, our purpose was to outline a salivary proteomic profile which features healthy individuals.Methods: We collected saliva samples from 19 young blood donors as reference population and the proteomic profile was investigated through mass-spectrometry.Results: We identified 1,004 proteins of whose 243 proteins were shared by all subjects. By applying a data clustering approach, we found a set of six most representative proteins across all subjects including Coronin-1A, F-actin-capping protein subunit alpha, Immunoglobulin J chain, Prosaposin, 78 kDa glucose-regulated protein and Heat shock 70 kDa protein 1A and 1B.Conclusion: All of these proteins are involved in immune system activation, cellular stress responses, proliferation, and invasion thus suggesting their use as biomarkers in patients with NCDs.

Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.

The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.

Serum levels of the endoplasmic-reticulum-stress chaperone GRP78 are associated with inflammation, identify patients with coronary artery disease and predict mortality

Abstract Introduction Endoplasmic-reticulum-stress (ER Stress) and associated chaperones like the ER-Stress moderator GRP78 (glucose-regulated-protein, 78kDa) are involved in the pathogenesis of coronary artery disease (CAD). In addition to their intracellular effects, secretion and extracellular properties of chaperones, including GRP78, were recently described. In a recent study using proteomic data, we found coronary endothelial cells to secrete GRP78 into the extracellular space. Here, we investigated the significance of serum GRP78 in patients undergoing coronary angiography for suspected CAD. Methods Serum concentration of GRP78 was measured by ELISA in an all-comers cohort of 789 patients with clinical indication for coronary angiography. Samples were drawn from the arterial sheath. CAD was defined as &amp;gt;50% stenosis in any major epicardial coronary artery. Patients were stratified according to their clinical presentation (CCS: chronic coronary syndrome or ACS). Clinical endpoint was all-cause mortality after one year. Patients with malignant or active auto-immune diseases were excluded. Results Mean age was 70.8 ± 11.9 years and 65% of patients were male. 24% patients presented with acute coronary syndrome (ACS). CAD was found in 72.4% of patients. Mean GRP78 serum concentration was 2492 ng/ml. Increased levels of GRP78 (&amp;gt; median) were associated with diabetes (29.9% vs. 23.1%, p = 0.03), a higher BMI (28.9 kg/m2 ± 7.1 vs. 27.0 kg/m2 ± 6.0, p &amp;lt; 0.0001) and chronic kidney disease (CKD: 23.3 % vs. 17.1 %, p = 0.03). There was no difference regarding age or sex. We further compared GRP78 levels to other biomarkers including WBC, Hemoglobin, Troponin, Creatinine, and Interleukin-6 (IL-6). There was no association between GRP78 levels and the mentioned biomarkers, except for a positive correlation between GRP78 and levels of Interleukin-6 (Pearson r =0.14, p = 0.004). GRP78 levels were increased in patients with CAD when compared to patients without CAD (2640 ng/ml vs. 2178 ng/ml, p = 0.013). Interestingly, GRP78 levels were lower in patients with ACS than in patients with CCS (2284 ng/ml vs. 2822 ng/ml, p = 0.018). There was no difference between NSTEMI and STEMI patients. Finally, we assessed prognostic relevance of GRP78 in patients with CAD. Increased GRP78 levels were associated with lower occurrence of the clinical endpoint of one-year mortality (5.3% vs. 10.6 % vs. p = 0.016). Increased GRP78 levels were associated with reduced one-year mortality (HR: 0.48 [95% CI: 0.25 - 0.92]). After adjusting for IL-6, age, sex, BMI, diabetes, CKD, and ACS, GRP78 remained an independent predictor of one-year mortality in patients with CAD. Conclusion GRP78 serum levels are generally elevated in patients with CAD but are downregulated during ACS. Reduced levels are associated with increased one-year mortality. These results support previous findings that GRP78 secretion is a protective mechanism in cardiovascular diseases.

Protein misfolding: an additional player in the stroke-heart syndrome

Abstract Background Patients suffering from acute ischemic stroke experience an increased risk of major adverse cardiovascular events, introducing the term of stroke-heart syndrome (SHS). Beside common risk factors, the so-called brain-heart axis represents a relevant yet not fully understood biological matrix driving this multi-organ condition. Together with inflammatory and neurohormonal signals, protein misfolding appears as an additional component of this matrix. In fact, previous studies employing animal models of cerebral ischemia identified wrongly arranged toxic proteins (oligomers). In light of the spreading capabilities of such proteins, we questioned whether protein misfolding plays a role in the SHS. Purpose The purpose of this study is to investigate the occurrence of protein misfolding in the post-stroke cardiac dysfunction by assessing presence, origin, localization and routes of oligomers in the heart following an acute ischemic stroke. Its biological and physiological relevance are evaluated by in vivo and in vitro assessments of the myocardium. Methods Acute cerebral ischemic injury was induced in the mouse by left transient middle cerebral artery occlusion for 45 minutes followed by 48 hours of reperfusion in 3-4 month-old C57BL/6J wild type males. Sham operated littermate mice were used as controls. Forty-eight hours after acute cerebral ischemia, cardiac function was evaluated by electrocardiography and compared to pre-operative assessment. Organs were harvested after the abovementioned assessment for the localization and semi-quantification of oligomers in heart and lymph nodes. Furthermore, biological responses related to protein homeostasis in the myocardium were assessed on mRNA and protein levels. Results Our preliminary results show that acute ischemic stroke is associated with an increased presence of oligomers in the heart. In addition, higher levels of oligomers were detected in axillary lymph nodes isolated from stroke mice, suggesting a possible route of oligomers spreading (Fig 1A). Correlating with oligomers occurrence, molecular analyses showed significant upregulation of the inositol-requiring enzyme 1 (IRE1), and catalase at mRNA level (Fig 1B). On the other hand, the expression of the 78-kDa glucose-regulated protein (GRP78), and catalase significantly decreased at protein level, indicating impaired myocardial protein quality control and oxidative stress after acute ischemic stroke (Fig 1C). Stroke-induced cardiac dysfunction was presented by decreased heart weight to tibia length ratio as well as slower and irregular heart rhythm suggesting signs of arrhythmia (Fig 2). Conclusions Acute ischemic stroke is associated with an increased oligomers occurrence in the myocardium and ultimately correlates with altered cardiac resistance against proteotoxicity. Further experiments will be conducted in order to elucidate the role of oligomers as active players or bystanders in the stroke-heart syndrome.

Dengue virus NS1 secretion is regulated via importin-subunit β1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin.

Dengue virus (DENV) is a major human pathogen that can cause hemorrhagic fever and shock syndrome. One important factor of DENV pathogenicity is non-structural protein 1 (NS1), a glycoprotein that is secreted from infected cells. Here we study the mode of action of the widely used drug ivermectin, used to treat parasitic infections and recently shown to lower NS1 blood levels in DENV-infected patients. We found that ivermectin blocks the nuclear transport of transcription factors required for the expression of chaperones that support the folding and secretion of glycoproteins, including NS1. Impairing nuclear transport of these transcription factors by ivermectin or depleting them from infected cells dampens NS1 folding and thus its secretion. These results reveal a novel mode of action of ivermectin that might apply to other flaviviruses as well.

MiR-199a-5p inhibits aortic valve calcification by targeting ATF6 and GRP78 in valve interstitial cells.

Calcific aortic valve disease (CAVD) is an important cause of disease burden among aging populations. Excessive active endoplasmic reticulum stress (ERS) was demonstrated to promote CAVD. The expression level of miR-199a-5p in patients with CAVD was reported to be downregulated. In this article, we aimed to investigate the function and mechanism of miR-199a-5p in CAVD. The expression level of miR-199a-5p and ERS markers was identified in calcific aortic valve samples and osteogenic induction by real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry, and western blotting (WB). Alizarin red staining, RT-qPCR, and WB were used for the verification of the function of miR-199a-5p. The dual luciferase reporter assay and rescue experiment were conducted to illuminate the mechanism of miR-199a-5p. In our study, the expression level of miR-199a-5p was significantly decreased in calcified aortic valves and valve interstitial cells' (VICs) osteogenic induction model, accompanying with the upregulation of ERS markers. Overexpression of miR-199a-5p suppressed the osteogenic differentiation of VICs, while downregulation of miR-199a-5p promoted this function. 78 kDa glucose-regulated protein (GRP78) and activating transcription factor 6 (ATF6), both of which were pivotal modulators in ERS, were potential targets of miR-199a-5p. miR-199a-5p directly targeted GRP78 and ATF6 to modulate osteoblastic differentiation of VICs. miR-199a-5p inhibits osteogenic differentiation of VICs by regulating ERS via targeting GRP78 and ATF6.

Effects of Social Defeat Stress on Microtubule Regulating Proteins and Tubulin Polymerization.

: Microtubule (MT) stability in neurons is vital for brain development; instability is associated with neuropsychiatric disorders. The present study examined the effects of social defeat stress (SDS) on MT-regulating proteins and tubulin polymerization. : After 10 days of SDS, defeated mice were separated into susceptible (Sus) and unsusceptible (Uns) groups based on their performance in a social avoidance test. Using extracted brain tissues, we measured the expression levels of α-tubulin, acetylated α-tubulin, tyrosinated α-tubulin, MT-associated protein-2 (MAP2), stathmin (STMN1), phospho stathmin serine 16 (p-STMN1 [Ser16]), phospho stathmin serine 25 (p-STMN1 [Ser25]), phospho stathmin serine 38 (p-STMN1 [Ser38]), stathmin2 (STMN2), phospho stathmin 2 serine 73 (p-STMN2 [Ser73]), 78-kDa glucose-regulated protein (GRP-78), and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) using Western blot assay. The tubulin polymerization rate was also measured. : We observed increased and decreased expression of acetylated and tyrosinated α-tubulin, respectively, decreased expression of p-STMN1 (Ser16) and increased expression of p-STMN1 (Ser25), p-STMN2 (Ser73) and GRP-78 and CHOP in the prefrontal cortex and/or hippocampus of defeated mice. A reduced tubulin polymerization rate was observed in the Sus group compared to the Uns and Con groups. : Our findings suggest that SDS has detrimental effects on MT stability, and a lower tubulin polymerization rate could be a molecular marker for susceptibility to SDS.

Leishmania (L.) amazonensis LaLRR17 increases parasite entry in macrophage by a mechanism dependent on GRP78.

Leishmaniases affect 12 million people worldwide. They are caused by Leishmania spp., protozoan parasites transmitted to mammals by female phlebotomine flies. During the life cycle, promastigote forms of the parasite live in the gut of infected sandflies and convert into amastigotes inside the vertebrate macrophages. The parasite evades macrophage's microbicidal responses due to virulence factors that affect parasite phagocytosis, survival and/or proliferation. The interaction between Leishmania and macrophage molecules is essential to phagocytosis and parasite survival. Proteins containing leucine-rich repeats (LRRs) are common in several organisms, and these motifs are usually involved in protein–protein interactions. We have identified the LRR17 gene, which encodes a protein with 6 LRR domains, in the genomes of several Leishmania species. We show here that promastigotes of Leishmania (L.) amazonensis overexpressing LaLRR17 are more infective in vitro. We produced recombinant LaLRR17 protein and identified macrophage 78 kDa glucose-regulated protein (GRP78) as a ligand for LaLRR17 employing affinity chromatography followed by mass spectrometry. We showed that GRP78 binds to LaLRR17 and that its blocking precludes the increase of infection conferred by LaLRR17. Our results are the first to report LRR17 gene and protein, and we hope they stimulate further studies on how this protein increases phagocytosis of Leishmania.

A computational peptide model induces cancer cells’ apoptosis by docking Kringle 5 to GRP78

BackgroundCells can die through a process called apoptosis in both pathological and healthy conditions. Cancer development and progression may result from abnormal apoptosis. The 78-kDa glucose-regulated protein (GRP78) is increased on the surface of cancer cells. Kringle 5, a cell apoptosis agent, is bound to GRP78 to induce cancer cell apoptosis. Kringle 5 was docked to GRP78 using ClusPro 2.0. The interaction between Kringle 5 and GRP78 was investigated.ResultsThe interacting amino acids were found to be localized in three areas of Kringle 5. The proposed peptide is made up of secondary structure amino acids that contain Kringle 5 interaction residues. The 3D structure of the peptide model amino acids was created using the PEP-FOLD3 web tool.ConclusionsThe proposed peptide completely binds to the GRP78 binding site on the Kringle 5, signaling that it might be effective in the apoptosis of cancer cells.

Effects of endoplasmic reticulum stress on erectile function in rats with cavernous nerve injury.

Erectile dysfunction (ED) occurs in an increasing number of patients after radical prostatectomy and cystectomy, and the phenotypic modulation of corpus cavernosum smooth muscle cells is closely related to ED. To determine whether endoplasmic reticulum stress (ERS) is implicated in the phenotypic modulation of ED induced by bilateral cavernous nerve injury (BCNI). In total, 36 Sprague-Dawley rats were randomly divided into 3 groups: sham, in which rats received sham surgery with bilateral cavernous nerve exposure plus phosphate-buffered saline; control, in which rats received BCNI plus phosphate-buffered saline; and experimental, in which rats received BCNI plus 4-phenylbutyric acid. Analysis of variance and a Bonferroni multiple-comparison test were utilized to evaluate differences among groups. Erectile function, smooth muscle/collagen ratios, and the expression levels of phenotypic modulation and ERS were measured. Two ratios-maximum intracavernosal pressure/mean arterial pressure and smooth muscle/collagen-were decreased in the control group as compared with the sham group. In penile tissue, there was increased expression of GRP78 (78-kDa glucose-regulated protein), p-PERK/PERK (phosphorylated protein kinase R-like endoplasmic reticulum kinase/protein kinase R-like endoplasmic reticulum kinase), caspase 3, CHOP (C/EBP homologous protein), and OPN (osteopontin) but decreased expression of nNOS (neuronal nitric oxide synthase) and α-SMA (α-smooth muscle actin). As compared with the control group, erectile function was improved and pathologic changes were partially recovered in the experimental group. The present study demonstrated that ERS is involved in ED caused by cavernous nerve injury, thereby providing a new target and theoretical basis for clinical treatment. The present study demonstrated for the first time that ERS is related to ED caused by cavernous nerve injury. Inhibition of ERS reverses phenotypic modulation and improves erectile function in rats with BCNI. Additional in vitro studies should be performed to verify these conclusions and explore the specific mechanism of phenotypic modulation. The present study demonstrated that inhibiting ERS reverses phenotypic modulation and enhances erectile function in rats with BCNI.

Effects of Brimonidine, Omidenepag Isopropyl, and Ripasudil Ophthalmic Solutions to Protect against H2O2-Induced Oxidative Stress in Human Trabecular Meshwork Cells

Purpose We investigated whether hydrogen peroxide (H2O2)-induced oxidative stress causes human trabecular meshwork (HTM) cell dysfunction observed in open angle glaucoma (OAG) in vitro, and the effects of topical glaucoma medications on oxidative stress in HTM cells. Methods We used commercially available ophthalmic solutions of brimonidine, omidenepag isopropyl, and ripasudil in the study. HTM cells were exposed to H2O2 for 1 h, with or without glaucoma medications. We assessed cell viability and senescence via WST-1 and senescence-associated-β-galactosidase (SA-β-Gal) activity assays. After exposure to H2O2 and glaucoma medications, we evaluated changes in markers of fibrosis and stress by using real-time quantitative polymerase chain reaction (qPCR) to measure the mRNA levels of collagen type I alpha 1 chain (COL1A1), fibronectin, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), endoplasmic reticulum stress markers of C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and splicing X-box binding protein-1 (sXBP-1). Results HTM cell viability decreased and SA-β-Gal activity increased significantly after exposure to H2O2. Treatment with three ophthalmic solutions attenuated these changes. Real-time qPCR revealed that H2O2 upregulated the mRNA levels of COL1A1, fibronectin, α-SMA, CHOP, GRP78, and sXBP-1, whereas it downregulated MMP-2 mRNA expression significantly. Brimonidine suppressed the upregulation of stress markers CHOP and GRP78. Additionally, omidenepag isopropyl and ripasudil decreased the upregulation of COL1A1 and sXBP-1. Furthermore, ripasudil significantly suppressed fibrotic markers fibronectin and α-SMA, compared with the other two medications. Conclusion In vitro, H2O2 treatment of HTM cells induced characteristic changes of OAG, such as fibrosis changes and the upregulation of stress markers. These glaucomatous changes were attenuated by additional treatments with brimonidine, omidenepag isopropyl, and ripasudil ophthalmic solutions.

CARD6: Warm Ischemic Time, Cold Static Storage and Impairment of Cardiac Endoplasmic Reticular Stress in Human Hearts Donated After Circulatory Death and Brain Death

Background: Endoplasmic reticulum (ER) plays an important role in maintaining normal myocardial function at the cellular level. Overexpression of ER stress-associated proteins during ischemia impact cardiac function. Initial result of heart transplantation using donation after circulatory death (DCD) heart with short warm ischemic time (WIT, less than 30 minutes) was encouraging. However, little is known about the impact of various WIT on donor heart and with subsequent cold storage as used in normothermic regional perfusion(NRP) on the DCD hearts. In this study we investigated expression of myocardial ER stress response proteins in the DCD hearts to evaluate the impact of warm ischemic time (WIT) and subsequent cold storage. We also studied expression of myocardial ER stress response proteins in the brain death donor (DBD) hearts as a control group. Methods: A total of 23 human hearts, DCD group, n=17, with 3 WIT sub-groups [WIT 20 (n=6), 40 (n=6), ≥60 (n=5) minutes] and DBD group, n=6, no warm ischemia (control: WIT:0) were studied. All the DCD hearts received del Nido cardioplegia and all DBD hearts received Celsior solution before they were placed at cold storage for 6 hours. Left ventricular biopsies were performed at hour 0 (T0), 2 (T2), 4 (T4) and 6 (T6). Myocardial ER stress response proteins ATF6 (Activating Transcription Factor 6) and GRP78 (78-kDa glucose-regulated protein) were measured through immunoblotting after normalizing to GAPDH loading control in all cardiac biopsies. Results: Representative western blots showing the protein expressions patterns among the DBD and three different groups of DCD hearts under increasing time points (T0:0hrs, T2:2hrs, T4:4hrs, T6:6hrs) of cold static storage conditions (Fig.1A). At baseline, the proteins expression found to be comparable between DBD, DCD:20 and 40 groups, while DCD:≥60 group remained significantly higher (Fig.1B,C). We noticed differential temporal increase of ER stress proteins after 2 hours of cold storage and became prominent at hour 6 in DCD groups when compared to DBD group; while the DCD:60 group always remained highly significant throughout the cold storage compared to all other groups (Fig.1D,E). Conclusions: Our study concluded that duration of warm ischemia and subsequent cold storage condition in DCD hearts were associated with elevated ER stress response in myocardium. The length of cold storage was also found to be linked with elevated ER stress in DCD hearts. The DCD hearts with more than 60 min warm ischemic has the highest ES stress response suggesting profound alteration in ER homeostasis which may promote cardiac damage. Such findings are important in guiding clinical decision making on selecting DCD donor hearts. On the other hand the DBD hearts with up to 6 hour cold storage exerted minimal ER stress, supporting current DBD heart transplant donor preservation practice.

Tumor necrosis factor-α reduces adiponectin production by decreasing transcriptional activity of peroxisome proliferator-activated receptor-γ in calf adipocytes

Adiponectin (encoded by ADIPOQ) is an adipokine that orchestrates energy homeostasis by modulating glucose and fatty acid metabolism in peripheral tissues. During the periparturient period, dairy cows often develop adipose tissue inflammation and decreased plasma adiponectin levels. Proinflammatory cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role in regulating the endocrine functions of adipocytes, but whether it affects adiponectin production in calf adipocytes remains obscure. Thus, the present study aimed to determine whether TNF-α could affect adiponectin production in calf adipocytes and to identify the underlying mechanism. Adipocytes isolated from Holstein calves were differentiated and used for (1) BODIPY493/503 staining; (2) treatment with 0.1 ng/mL TNF-α for different times (0, 8, 16, 24, or 48 h); (3) transfection with peroxisome proliferator-activated receptor-γ (PPARG) small interfering RNA for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h; and (4) overexpression of PPARG for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h. After differentiation, obvious lipid droplets and secretion of adiponectin were observed in adipocytes. Treatment with TNF-α did not alter mRNA abundance of ADIPOQ but reduced the total and high molecular weight (HMW) adiponectin content in the supernatant of adipocytes. Quantification of mRNA abundance of endoplasmic reticulum (ER)/Golgi resident chaperones involved in adiponectin assembly revealed that ER protein 44 (ERP44), ER oxidoreductase 1α (ERO1A), and disulfide bond-forming oxidoreductase A-like protein (GSTK1) were downregulated in TNF-α-treated adipocytes, while 78-kDa glucose-regulated protein and Golgi-localizing γ-adaptin ear homology domain ARF binding protein-1 were unaltered. Moreover, TNF-α diminished nuclear translocation of PPARγ and downregulated mRNA abundance of PPARG and its downstream target gene fatty acid synthase, suggesting that TNF-α suppressed the transcriptional activity of PPARγ. In the absence of TNF-α, overexpression of PPARG enhanced the total and HMW adiponectin content in supernatant and upregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. However, knockdown of PPARG reduced the total and HMW adiponectin content in supernatant and downregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. In the presence of TNF-α, overexpression of PPARG decreased, while knockdown of PPARG further exacerbated TNF-α-induced reductions in total and HMW adiponectin secretion and gene expression of ERP44, ERO1A, and GSTK1. Overall, TNF-α reduces adiponectin assembly in the calf adipocyte, which may be partly mediated by attenuation of PPARγ transcriptional activity. Thus, locally elevated levels of TNF-α in adipose tissue may be one reason for the decrease in circulating adiponectin in periparturient dairy cows.

THU-264 - Alpha-1 antitrypsin inclusions sequester 78 kDa glucose-regulated protein in a bile-acid inducible manner

THU-264 - Alpha-1 antitrypsin inclusions sequester 78 kDa glucose-regulated protein in a bile-acid inducible manner

Cholesterol induces inflammation and reduces glucose utilization.

Cholesterol stimulates inflammation and affects the normal function of islet tissues. However, the precise mechanism underlying the effects of cholesterol on islet cells requires clarification. In this study, we explored the role of cholesterol in glucose utilization in pancreatic cells. Beta-TC-6 cells and mice were treated with cholesterol. We used glucose detection kits to identify the glucose content in the cell culture supernatant and mouse serum and an enzyme-linked immunosorbent assay was used to detect insulin levels in the serum. Glucose-6-phosphatase catalytic subunit 2 (G6PC2), 78 kDa glucose-regulated protein (GRP78), 94 kDa glucose-regulated protein (GRP94), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1 (casp1), and interleukin-1β (IL-1β) expression levels were detected using immunofluorescence, immunohistochemistry, western blotting, and reverse transcription-quantitative polymerase chain reaction. Hematoxylin-eosin staining was used to detect the histological alterations in pancreatic tissues. Cholesterol decreased beta-TC-6 cell glucose utilization; enhanced pancreatic tissue pathological alterations; increased glucose and insulin levels in mouse serum; increased G6PC2, GRP78, GRP94, and NLRP3 expression levels; and elevated casp1 and pro-IL-1β cleavage. Cholesterol can attenuate glucose utilization efficiency in beta-TC-6 cells and mice, which may be related to endoplasmic reticulum stress and inflammation.