78-kDa Glucose-regulated Protein Research Articles

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.

GRP78 Inhibitor YUM70 Suppresses SARS-CoV-2 Viral Entry, Spike Protein Production and Ameliorates Lung Damage.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has given rise to many new variants with increased transmissibility and the ability to evade vaccine protection. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) chaperone that has been recently implicated as an essential host factor for SARS-CoV-2 entry and infection. In this study, we investigated the efficacy of YUM70, a small molecule inhibitor of GRP78, to block SARS-CoV-2 viral entry and infection in vitro and in vivo. Using human lung epithelial cells and pseudoviral particles carrying spike proteins from different SARS-CoV-2 variants, we found that YUM70 was equally effective at blocking viral entry mediated by original and variant spike proteins. Furthermore, YUM70 reduced SARS-CoV-2 infection without impacting cell viability in vitro and suppressed viral protein production following SARS-CoV-2 infection. Additionally, YUM70 rescued the cell viability of multi-cellular human lung and liver 3D organoids transfected with a SARS-CoV-2 replicon. Importantly, YUM70 treatment ameliorated lung damage in transgenic mice infected with SARS-CoV-2, which correlated with reduced weight loss and longer survival. Thus, GRP78 inhibition may be a promising approach to augment existing therapies to block SARS-CoV-2, its variants, and other viruses that utilize GRP78 for entry and infection.

Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication.

Calcium (Ca2+), a ubiquitous second messenger, plays a crucial role in many cellular functions. Viruses often hijack Ca2+ signaling to facilitate viral processes such as entry, replication, assembly, and egress. Here, we report that infection by the swine arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV), induces dysregulated Ca2+ homeostasis, subsequently activating calmodulin-dependent protein kinase-II (CaMKII) mediated autophagy, and thus fueling viral replication. Mechanically, PRRSV infection induces endoplasmic reticulum (ER) stress and forms a closed ER-plasma membrane (PM) contacts, resulting the opening of store operated calcium entry (SOCE) channel and causing the ER to take up extracellular Ca2+, which is then released into the cytoplasm by inositol trisphosphate receptor (IP3R) channel. Importantly, pharmacological inhibition of ER stress or CaMKII mediated autophagy blocks PRRSV replication. Notably, we show that PRRSV protein Nsp2 plays a dominant role in the PRRSV induced ER stress and autophagy, interacting with stromal interaction molecule 1 (STIM1) and the 78 kDa glucose-regulated protein 78 (GRP78). The interplay between PRRSV and cellular calcium signaling provides a novel potential approach to develop antivirals and therapeutics for the disease outbreaks.

COLEC10 Induces Endoplasmic Reticulum Stress by Occupying GRP78 and Inhibits Hepatocellular Carcinoma

Collectin subfamily member 10 (COLEC10), a C-type lectin mainly expressed in the liver, is involved in the development of hepatocellular carcinoma (HCC). However, its underlying molecular mechanism in HCC progression remains unknown. In this study, reduced COLEC10 expression in tumor tissues was validated using various HCC cohorts and was associated with poor patient prognosis. COLEC10 overexpression attenuated HCC cell growth and migration abilities in vitro and in vivo. We identified that COLEC10 was a novel interactor of 78-kDa glucose-regulated protein (GRP78), a master modulator of the unfolded protein response in the endoplasmic reticulum (ER). COLEC10 overexpression potentiated ER stress in HCC cells, as demonstrated by elevated expression levels of phosphorylated protein kinase RNA-like ER kinase, phosphorylated inositol-requiring protein 1α, activating transcription factor 4, DNA damage-inducible transcript 3, and X-box-binding protein 1s. The ER in COLEC10-overexpressing cells also showed a dilated and fragmented pattern. Mechanistically, COLEC10 overexpression increases GRP78 occupancy through direct binding by the C-terminal carbohydrate recognition domain in the ER, which released and activated the ER stress transducers protein kinase RNA-like ER kinase and phosphorylated inositol-requiring protein 1α, triggering the unfolded protein response activity. COLEC10-overexpressing HCC cells generated a relatively high reactive oxygen species level and switched to apoptotic cell death under sorafenib-treated conditions. Our study provides the first novel view that COLEC10 inhibits HCC progression by regulating GRP78-mediated ER stress signaling and may serve as a promising therapeutic and prognostic biomarker.

Arbutin abrogates testicular ischemia/reperfusion injury in rats through repression of inflammation and ER stress

The aim of this study was to investigate the effects of arbutin (ARB) administration on oxidative stress, inflammation, endoplasmic reticulum (ER) stress and apoptosis in an experimental testicular torsion/detorsion (T/D)-induced testicular injury model for the first time. A total of 24 male Sprague-Dawley rats were divided into four groups with six rats in each group: sham control, T/D, T/D+ARB (50 mg/kg) and T/D+ARB (100 mg/kg). Torsion and detorsion times were applied as 4 h and 2 h, respectively. The levels of lipid peroxidation [malondialdehyde (MDA)] and oxidative stress [total oxidant status (TOS) and total antioxidant status (TAS)] in testicular tissues were determined using colorimetric methods. The levels of DNA damage [8-hydroxy-2′-deoxyguanosine (8-OHdG)], antioxidant system [superoxide dismutase (SOD) and catalase (CAT)], pro-inflammatory cytokines [high mobility group box 1 (HMGB1), nuclear factor kappa B protein 65 (NF-κB p65), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and myeloperoxidase (MPO)], ER stress [78-kDa glucose-regulated protein (GRP78), activating transcription factor 6 (ATF6) and CCAAT-enhancer-binding protein homologous protein (CHOP)] and apoptosis (caspase-3) markers in testicular tissues were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits. Johnsen's testicle scoring system was used for histological evaluation. In the T/D group, it was determined that statistically significant increasing in the levels of oxidative stress, inflammation, ER stress and apoptosis compared with sham control group (p < 0.05). ARB administrations statistically significantly restored testicular I/R damage in a dose dependent manner (p < 0.05). In addition, it was determined that the data of histological examinations supported the biochemical results. Our findings support the hypothesis that ARB may be used as a protective agent against T/D-induced testicular damage.

Porcine Epidemic Diarrhea Virus and Its nsp14 Suppress ER Stress Induced GRP78.

Porcine epidemic diarrhea virus (PEDV), a member of the α-coronavirus genus, can cause vomiting, diarrhea, and dehydration in piglets. Neonatal piglets infected with PEDV have a mortality rate as high as 100%. PEDV has caused substantial economic losses to the pork industry. Endoplasmic reticulum (ER) stress, which can alleviate the accumulation of unfolded or misfolded proteins in ER, involves in coronavirus infection. Previous studies have indicated that ER stress could inhibit the replication of human coronaviruses, and some human coronaviruses in turn could suppress ER stress-related factors. In this study, we demonstrated that PEDV could interact with ER stress. We determined that ER stress could potently inhibit the replication of GⅠ, GⅡ-a, and GⅡ-b PEDV strains. Moreover, we found that these PEDV strains can dampen the expression of the 78 kDa glucose-regulated protein (GRP78), an ER stress marker, while GRP78 overexpression showed antiviral activity against PEDV. Among different PEDV proteins, PEDV non-structural protein 14 (nsp14) was revealed to play an essential role in the inhibition of GRP78 by PEDV, and its guanine-N7-methyltransferase domain is necessary for this role. Further studies show that both PEDV and its nsp14 negatively regulated host translation, which could account for their inhibitory effects against GRP78. In addition, we found that PEDV nsp14 could inhibit the activity of GRP78 promotor, helping suppress GRP78 transcription. Our results reveal that PEDV possesses the potential to antagonize ER stress, and suggest that ER stress and PEDV nsp14 could be the targets for developing anti-PEDV drugs.

Astaxanthin treatment ameliorates ER stress in polycystic ovary syndrome patients: a randomized clinical trial

Astaxanthin (ASX), as a natural carotenoid compound, exists in various types of seafood and microorganisms. It has several possible beneficial therapeutic effects for patients with polycystic ovary syndrome (PCOS). Patients with PCOS also suffer from endoplasmic reticulum (ER) stress. In the present work, it was hypothesized that ER stress could be improved by ASX in PCOS patients. Granulosa cells (GCs) were obtained from 58 PCOS patients. The patients were classified into ASX treatment (receiving 12 mg/day for 60 days) and placebo groups. The expression levels of ER stress pathway genes and proteins were explored using Western blotting and quantitative polymerase chain reaction. To assess oxidative stress markers, follicular fluid (FF) was gained from all patients. The Student’s t test was used to perform statistical analysis. After the intervention, ASX led to a considerable reduction in the expression levels of 78-kDa glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and X-box-binding protein 1 compared to the placebo group, though the reduction in the messenger RNA (mRNA) expression level of activating transcription factor 6 was not statistically significant. However, ASX significantly increased the ATF4 expression level. GRP78 and CHOP protein levels represented a considerable decrease in the treatment group after the intervention. In addition, a statistically significant increase was found in the FF level of total antioxidant capacity in the treatment group. Based on clinical outcomes, no significant differences were found between the groups in terms of the oocyte number, fertilization rate, and fertility rate, but the ASX group had higher rates of high-quality oocytes, high-quality embryo, and oocyte maturity compared to the placebo group. Our findings demonstrated that ER stress in the GCs of PCOS patients could be modulated by ASX by changing the expression of genes and proteins included in the unfolding protein response.Trial registration This study was retrospectively registered on the Iranian Registry of Clinical Trials website (www.irct.ir; IRCT-ID: IRCT20201029049183N, 2020-11-27).

Endoplasmic Reticulum Stress-Activated Neuronal and Microglial Autophagy Contributes to Postoperative Cognitive Dysfunction in Neonatal rats.

Surgery and anesthesia in neonates may lead to cognitive impairment or abnormal behaviors. It has been shown that autophagy plays a critical role in neuropsychiatric disorders, while the role of autophagy in postoperative cognitive impairment in neonates is not known. Here, we determined this role and the involvement of endoplasmic reticulum (ER) stress in regulating brain cell autophagy after surgery. Seven-day old neonatal rats (P7) had right common carotid artery exposure under anesthesia with 3% sevoflurane for 2h. Learning and memory were tested using Barnes maze (BM) and fear conditioning (FC) on P31-42 and P42-44, respectively. In another experiment, rat brains were harvested for biochemical studies. The ratio of microtubule-associated protein 1 light chain 3 (LC3) BII/I was increased and sequestosome 1 (P62/SQSTM1) levels were decreased in the brain 24h after surgery and anesthesia in neonatal rats. Immunofluorescent staining of LC3B was co-localized with a neuronal or a microglial marker but was not co-localized with a marker for astrocytes in rats with surgery. These rats had a poorer performance in the BM and FC tests than control rats when they were adolescent. Pretreatment with an autophagy inhibitor, 3-methyladenine, attenuated the poor performance. Surgery and anesthesia increased the expression of 78kDa glucose-regulated protein (BIP/GRP78), an indicator of ER stress, 6h after surgery and anesthesia. The ER stress activator tunicamycin and inhibitor tauroursodeoxycholic acid increased the markers for autophagy in control rats and decreased the autophagy markers in rats with surgery, respectively. Our results suggest that surgery in neonatal rats induces ER stress that then activates neuronal and microglial autophagy, which contributes to learning and memory impairment later in life.

Pharmacological inhibition of lysosomal two-pore channel 2 (TPC2) confers neuroprotection in stroke via autophagy regulation

Lysosomal function and organellar Ca2+ homeostasis become dysfunctional in Stroke causing disturbances in autophagy, the major process for the degradation of abnormal protein aggregates and dysfunctional organelles. However, the role of autophagy in Stroke is controversial since excessive or prolonged autophagy activation exacerbates ischemic brain injury. Of note, glutamate evokes NAADP-dependent Ca2+ release via lysosomal TPC2 channels thus controlling basal autophagy. Considering the massive release of excitotoxins in Stroke, autophagic flux becomes uncontrolled with abnormal formation of autophagosomes causing, in turn, disruption of excitotoxins clearance and neurodegeneration. Here, a fine regulation of autophagy via a proper pharmacological modulation of lysosomal TPC2 channel has been tested in preclinical Stroke models. Primary cortical neurons were subjected to oxygen and glucose deprivation+reoxygenation to reproduce in vitro brain ischemia. Focal brain ischemia was induced in rats by transient middle cerebral artery occlusion (tMCAO). Under these conditions, TPC2 protein expression as well as autophagy and endoplasmic reticulum (ER) stress markers were studied by Western blotting, while TPC2 localization and activity were measured by immunocytochemistry and single-cell video-imaging, respectively. TPC2 protein expression and immunosignal were highly modulated in primary cortical neurons exposed to extreme hypoxic conditions causing dysfunction in organellar Ca2+ homeostasis, ER stress and autophagy-induced cell death. TPC2 knocking down and pharmacological inhibition by Ned-19 during hypoxia induced neuroprotection. The effect of Ned-19 was reversed by the permeable form of TPC2 endogenous agonist, NAADP-AM. Of note, Ned-19 prevented ER stress, as measured by GRP78 (78 kDa glucose-regulated protein) protein reduction and caspase 9 downregulation. In this way Ned-19 restored organellar Ca2+ level. Interestingly, Ned-19 reduced the infarct volume and neurological deficits in rats subjected to tMCAO and prevented hypoxia-induced cell death by blocking autophagic flux. Collectively, the pharmacological inhibition of TPC2 lysosomal channel by Ned-19 protects from focal ischemia by hampering a hyperfunctional autophagy.

Secreted glucose regulated protein78 ameliorates DSS-induced mouse colitis.

The secreted form of 78-kDa glucose-regulated protein (sGRP78) has been widely reported for its property in aiding resolution of inflammatory. However, little is known on its potential in the treatment of colitis. To investigate the expression pattern and functional outcome of GRP78 in ulcerative colitis, its expression was measured in human and murine colitis samples. It was found that GRP78 was spontaneously secreted to a high level in gut, which is a physiological site of immune tolerance. During the active phase of DSS-induced colitis, the sGRP78 level was significantly reduced but rebounded quickly during resolving phase, making it a potential candidate for the treatment of colitis. In the following experiments, the administration of sGRP78 was proved to decrease susceptibility to experimental colitis, as indicated by an overall improvement of intestinal symptoms, restoration of TJ integrity, decreased infiltration of immune cells and impaired production of inflammatory cytokines. And specific cleavage of endogenous sGRP78 could aggravate DSS colitis. Adoptive transfer of sGRP78-conditioned BMDMs reduced inflammation in the gut. We linked sGRP78 treatment with altered macrophage biology and skewed macrophage polarization by inhibiting the TLR4-dependent MAP-kinases and NF-κB pathways. Based on these studies, as a naturally occurring immunomodulatory molecule, sGRP78 might be an attractive novel therapeutic agent for acute intestinal inflammation.

Induction of autophagy improves skin and hair conditions in dogs with underlying diseases.

Autophagy was reported to play a crucial role in maintaining general and skin health. The study used a synthesized autophagy inducer (AI) (Aquatide™ cospharm Inc.; Daejeon, Korea), for evaluating the effects of autophagy on skin and hair in dogs. Twenty-two dogs with poor skin and hair which were diagnosed with canine atopic dermatitis (CAD) or pituitary-dependent hyperadrenocorticism (PDH) were included. Clinical scores using Canine Atopic Dermatitis Extent and Severity Index-04 (CADESI-04), Pruritus Visual Analog Scale (PVAS) and skin barrier function using measurement of transepidermal water loss (TEWL) were evaluated and canine keratinocytes were also used in vitro investigation of pro-inflammatory cytokines after AI treatment. In the AI group, clinical scores and skin barrier function were improved at week 8 significantly compared to in the other groups. In particular, the AI significantly improved the hair surface damage at 8 weeks compared to the baseline. In vitro, the AI reduced pro-inflammatory cytokines by activating the 78-kDa glucose-regulated protein (GRP78). AI improve skin barrier function and hair damage and reduce pro-inflammatory cytokines by inhibiting reactive oxygen species (ROS) production in dogs.

Syringic acid ameliorates ischemia/reperfusion-induced testicular injury in rats via suppressing of HMGB1/NF-κB axis and endoplasmic reticulum stress.

To investigate the possible protective role of syringic acid on torsion/detorsion-induced testicular injury using biochemical and histopathological approaches for the first time. A total of 24 rats were divided into 4 groups: sham control, torsion/detorsion, torsion/detorsion + syringic acid (50mg/kg and 100mg/kg). Tissue malondialdehyde, total oxidant status and total antioxidant status levels were determined using colorimetric methods. Tissue 8-hydroxy-2'-deoxyguanosine, superoxide dismutase, catalase, high mobility group box 1, nuclear factor kappa B protein 65, tumor necrosis factor-alpha, interleukin-6, myeloperoxidase, 78-kDa glucose-regulated protein, activating transcription factor-6, C/EBP homologous protein and caspase-3 levels were determined using commercial enzyme-linked immunosorbent assay kits. Johnsen's testicle scoring system was used for histological evaluation. Compared with the control group, the levels of oxidative stress, inflammation, endoplasmic reticulum stress and apoptosis were significantly increased in the torsion/detorsion group (p < 0.05). Syringic acid administrations statistically significantly restored these damage in a dose dependent manner (p < 0.05). Moreover, it was found that the results of histological examinations supported the biochemical results to a statistically significant extent. The overall results suggest that syringic acid emerges as a potential compound for the treatment of testicular torsion and may be subject to clinical trials.

Endoplasmic reticulum stress and unfolded protein accumulation correlate to seizure recurrence in focal cortical dysplasia patients.

Epileptic seizures occur due to an imbalance between excitatory and inhibitory neurosignals. The excitotoxic insults promote the accumulation of reactive oxygen species (ROS), unfolded proteins (UFP) aggregation, and sometimes even cell death. The epileptic brain samples in our study showed significant changes in the quantity of UFP accumulation. This part explored the efficiency of ER stress and autophagy responses at neutralizing the UFP using resected epileptic brain tissue samples. Meanwhile, we regularly observed these patients' post-surgical clinical data to find the recurrence of seizures. According to International League against Epilepsy (ILAE) suggestions, we classified the patients (n = 26) as class 1 (completely seizure-free), class 2 (less frequent seizures or auras), and class 3 (auras with < 3 seizures per year). The classification helped us understand the reason for variations in the UFP accumulation in patient samples. We have observed the protein levels of ER chaperone, glucose-regulated protein 78kDa (GRP78/BiP), inositol-requiring enzyme 1α (IRE1α), X box-binding protein 1s (XBP1s), eukaryotic translation initiation factor 2α (peIF2α), C/EBP homologous protein (CHOP), NADPH oxidase (NOX2), and autophagy proteins like BECLIN1, ATG 7, 12, 5, 16, p62, and LC3. Our results suggested that ER stress response limitation may contribute to seizure recurrence in epilepsy patients, particularly in classes 2 and 3. In addition, we have observed significant upregulation of ER stress-dependent apoptosis initiation factor CHOP in these patients. These results indicate that understanding the ER stress response pattern infers the possibility of post-surgical outcomes in focal cortical dysplasia (FCD) patients.

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

Abstract Background Endoplasmic-reticulum-stress (ER Stress) and associated chaperones like the main ER-Stress moderator GRP78 (glucose-regulated-protein, 78kDa) are involved in the pathogenesis of coronary artery disease (CAD). In addition to their intracellular localization and effects, secretion and extracellular properties of chaperones, including GRP78, were recently described. However, a diagnostic or prognostic relevance of serum ER-chaperones in patients with CAD has not been established yet. Purpose To investigate the significance of serum GRP78 in patients undergoing coronary angiography for suspected CAD. Methods Serum concentration of GRP78 was measured by ELISA in 789 patients with indication for coronary angiography (both ACS and CCS). CAD was defined as &amp;gt;50% stenosis in any major coronary artery. Clinical endpoint was one-year all-cause mortality. Results Mean age was 70.8±11.9 years and 65% of patients were male. 192 (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±7.1 kg/m2 vs. 27.0±6.0 kg/m2, 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. GRP78 levels were increased in patients with CAD when compared to patients without CAD (2640 ng/ml [95% CI: 2415–2864] vs. 2178 ng/ml [95% CI: 1893–2463], p=0.013, Fig A). Interestingly, GRP78 levels were lower in patients with ACS than in patients with CCS (2284 ng/ml [95% CI: 1944–2642] vs. 2822 [95% CI: 2531–3113], p=0.018, Fig B). To further elucidate these findings, we analyzed available proteomics of patients with hypertrophic cardiomyopathy undergoing septal ablation as a model for a planned myocardial infarction (PMI)1. Coronary sinus blood samples drawn 10 and 60 min after PMI showed a downregulation of GRP78 levels compared to baseline. Finally, we assessed prognostic relevance of GRP78 in patients with CAD. Increased GRP78 levels were associated with lower one-year mortality (5.3% vs. 10.6% vs. p=0.016). Increased GRP78 levels were associated with reduced one-year mortality in both continuous (HR: 0.48 [95% CI: 0.25–0.92]) and categorial analyses stratified by median GRP78. After adjusting for age, sex, BMI, diabetes, CKD and ACS, GRP78 remained an independent predictor of one-year mortality (HR: 0.51 [0.26–0.96], p=0.039). Conclusion GRP78 serum levels are generally elevated in patients with CAD but 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. Further mechanistic studies are warranted to foster our understanding of the effects of extracellular GRP78 and to aid in the identification of modifying therapeutic targets. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University hospital Bonn

Activation of PPARγ Protects Obese Mice from Acute Lung Injury by Inhibiting Endoplasmic Reticulum Stress and Promoting Mitochondrial Biogenesis.

Objective Obesity-induced endoplasmic reticulum (ER) stress plays a role in increased susceptibility to acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The activation of peroxisome proliferator-activated receptor-γ (PPARγ) is associated with lung protection and is effective in ameliorating ER stress and mitochondrial dysfunction. The aim of this study was to investigate the expression of PPARγ in the lung tissues of obese mice and explore whether the PPARγ-dependent pathway could mediate decreased ALI/ARDS by regulating ER stress and mitochondrial biogenesis. Methods We determined PPARγ expression in the lung tissues of normal and obese mice. ALI models of alveolar epithelial cells and of obese mice were used and treated with either PPARγ activator rosiglitazone (RSG) or PPARγ inhibitor GW9662. Lung tissue and cell samples were collected to assess lung inflammation and apoptosis, and ER stress and mitochondrial biogenesis were detected. Results PPARγ expression was significantly decreased in the lung tissue of obese mice compared with that in normal mice. Both in vivo and in vitro studies have shown that activation of PPARγ leads to reduced expression of the ER stress marker proteins 78-kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), and Caspase12. Conversely, expression of the mitochondrial biogenesis-related proteins peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1α), nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM) increased. Furthermore, activation of PPARγ is associated with decreased levels of lung inflammation and epithelial apoptosis and increased expression of adiponectin (APN) and mitofusin2 (MFN2). GW9662 bound to PPARγ and blocked its transcriptional activity and then diminished the protective effect of PPARγ on lung tissues. Conclusions PPARγ activation exerts anti-inflammation effects in alveolar epithelia by alleviating ER stress and promoting mitochondrial biogenesis. Therefore, lower levels of PPARγ in the lung tissues of obese mice may lead to an increased susceptibility to ALI.

Targeting GRP78 suppresses oncogenic KRAS protein expression and reduces viability of cancer cells bearing various KRAS mutations

KRAS is the most commonly mutated oncogene in human cancers with limited therapeutic options, thus there is a critical need to identify novel targets and inhibiting agents. The 78-kDa glucose-regulated protein GRP78, which is upregulated in KRAS cancers, is an essential chaperone and the master regulator of the unfolded protein response (UPR). Following up on our recent discoveries that GRP78 haploinsufficiency suppresses both KRASG12D-driven pancreatic and lung tumorigenesis, we seek to determine the underlying mechanisms. Here, we report that knockdown of GRP78 via siRNA reduced oncogenic KRAS protein level in human lung, colon, and pancreatic cancer cells bearing various KRAS mutations. This effect was at the post-transcriptional level and is independent of proteasomal degradation or autophagy. Moreover, targeting GRP78 via small molecule inhibitors such as HA15 and YUM70 with anti-cancer activities while sparing normal cells significantly suppressed oncogenic KRAS expression in vitro and in vivo, associating with onset of apoptosis and loss of viability in cancer cells bearing various KRAS mutations. Collectively, our studies reveal that GRP78 is a previously unidentified regulator of oncogenic KRAS expression, and, as such, augments the other anti-cancer activities of GRP78 small molecule inhibitors to potentially achieve general, long-term suppression of mutant KRAS-driven tumorigenesis.

Enhanced tumor targeting and timely viral release of mesenchymal stem cells/oncolytic virus complex due to GRP78 and inducible E1B55K expressions greatly increase the antitumor effect of systemic treatment

Systemic delivery of oncolytic viruses has been widely regarded as an impractical option for antitumor treatment. Here, we selected two target genes as leading components, and significant therapeutic effects were obtained by simultaneously reducing the expression of transforming growth factor β 1 (TGF-β1) and heat shock protein 27 (HSP27) in various cancer cell types. Downregulation of HSP27 reduced the cellular levels of tumor progression-related proteins, and the simultaneous downregulation of HSP27 and TGF-β1 increased tumor cell death beyond that observed with TGF-β1 downregulation alone. To increase the potential for systemic administration, we generated modified mesenchymal stem cells (MSCs) to act as oncolytic adenovirus factories and carriers and assessed bioavailability in tumors after MSC injection. The MSCs were modified to express 78-kDa glucose-regulated protein (GRP78) and adenovirus early-region 1B 55 kDa (E1B55K). The tightly controlled inducible system permitted selective timing of viral release from carrier MSCs within the tumor. This approach significantly improved viral production, tumor targeting, timely viral release at the tumor site, and antitumor efficacy of the oncolytic adenovirus. These combined results demonstrate that engineered MSCs can significantly enhance the antitumor effects of oncolytic viruses without adverse safety issues, which may greatly extend the clinical applicability of oncolytic adenoviruses.

Roles of C/EBP-homologous protein and histone H3 lysine 4 methylation in arsenic-induced mitochondrial apoptosis in hepatocytes.

C/EBP-homologous protein (CHOP) and histone H3 lysine 4 (H3K4) methylation have been verified to be correlated with apoptosis, whereas their biological function in arsenic-induced hepatocyte apoptosis through the mitochondrial pathway is still unclear. This study aimed to explore the specific regulatory mechanism of CHOP and H3K4me1/2 in arsenic-induced mitochondrial apoptosis in hepatocytes. Apoptosis and proliferation results showed arsenic promoted apoptosis and inhibited cell growth in BRL-3A cells. Meanwhile, arsenic treatment significantly upregulated the 78-kDa glucose-regulated protein (GRP78), CHOP, su(var)-3-9,enhancer-of-zeste,trithorax (SET) domain containing 7/9 (SET7/9), H3K4me1/2, BIM and BAX expression, while markedly downregulated lysine-specific histone demethylase 1 (LSD1) and BCL2 expression. After down-regulating CHOP, LSD1, and (su(var)-3-9,enhancer-of-zeste,trithorax) domain-containing protein 7/9 (SET7/9) in BRL-3A cells by siRNA, silencing CHOP and SET7/9 notably attenuated the pro-apoptotic and anti-proliferative effects of arsenic treatment on BRL-3A cells, which was reversed after inhibiting LSD1. In addition, our results suggested that knockdown of CHOP altered the expression of mitochondrial-associated proteins BCL2 and BIM, whereas knockdown of LSD1 and SET7/8 regulated the level of H3K4me1/2 modification and BAX protein. Coupled with chromatin immunoprecipitation results, we found that the level of CHOP in the promoter regions of BCL2 and BIM was significantly increased in BRL-3A cells exposed to 30µmol/L NaAsO2 for 24h, whereas the levels of H3K4me1/2 in the promoter regions of BAX were unchanged. Collectively, these data indicated that arsenic triggered the mitochondrial pathway to induce hepatocyte apoptosis by up-regulating the levels of CHOP and H3K4me1/2.

Zanthoxylum Alatum Attenuates Chronic Restraint Stress Adverse Behavioral Effects Via the Mitigation of Oxidative Stress and Modulating the Expression of Genes Involved in Endoplasmic Reticulum Stress in Mice.

The functions of the endoplasmic reticulum (ER) are important, particularly in the proteins' synthesis, folding, modification, and transport. Based on traditional medicine and our previous studies on Zanthoxylum alatum in lipopolysaccharide-induced depressive behavior and scopolamine-induced impaired memory, the present study explored the role of hydroalcoholic extract of Z. alatum (ZAHA) seeds in reducing the ER stress in mice. The mice were restrained for 28 days in polystyrene tubes. ZAHA (100 and 200 mg/kg, PO) and imipramine (10 mg/kg, IP) were administered daily, 45 min before restraint from day 22 to 28. The mice were assessed by the forced swim test. Also, the antioxidant enzyme levels of Superoxide Dismutase (SOD), reduced glutathione (GSH), and lipid peroxidation (LPO) were measured in the hippocampus of mice. The expression of 78 kDa glucose-regulated protein (GRP78), 94 kDa Glucose-Regulated Protein (GRP94), and C/EBPhomologous protein (CHOP) genes was assessed by real-time PCR to explore the molecular mechanism. ZAHA (100 and 200 mg/kg, PO, and imipramine, IP) counteracted the stress by significantly reducing the immobility time in the force swimming test, receding oxidative stress and lipid peroxidation. The antioxidant enzyme (SOD and GSH) levels were elevated in the restraint stress group. Down-regulation of genes (GRP78, GRP94, and CHOP) compared to the chronic restraint stress group indicated stress modulating properties of the seeds in ER stress. Hesperidin, magnoflorine, melicopine, and sesamin, isolated from the active extract, were hypothesized to exert the activity. It can be concluded that Z. alatum reverted chronic restraint stress through its antioxidant properties and down-regulation of genes involved in ER stress.