Thapsigargin Research Articles

Lithium ameliorates spinal cord injury through endoplasmic reticulum stress-regulated autophagy and alleviated apoptosis through IRE1 and PERK/eIF2α signaling pathways

ObjectiveThis study aims to investigate the role of apoptosis and autophagy under endoplasmic reticulum (ER) stress in a lithium-treated SCI model. MethodsWe established a rat thoracic 10 (T10) spinal cord contusion model and observed its therapeutic effect by intraperitoneal (IP) injection of lithium. Histological and behavioral recovery with or without lithium injection were evaluated after rat spinal cord injury. In addition, we employed an oxygen-glucose deprivation (OGD)-PC12 cell model to study the effects of lithium on OGD-PC12 cell apoptosis, autophagy and ER stress. ResultsWe found that lithium administration to SCI rats reduced neuronal apoptosis and autophagy, restored rat locomotor function by reducing ER stress via IRE1 and PERK/eIF2α pathways. In vitro experiments confirmed that upon lithium treatment, OGD-PC12 cells resisted ER stress caused by thapsigargin (TG) via the IRE1 and PERK/eIF2α signaling pathways. ConclusionLithium attenuated neuronal apoptosis and autophagy, and facilitates the recovery after spinal cord injury through ameliorating ER stress, providing a new therapeutic mechanism for lithium to treat SCI.

Translational control of murine adiponectin expression by an upstream open reading frame element

ABSTRACT Adiponectin, an adipocyte-specific secretory protein encoded by the ADIPOQ gene has a causal role in insulin resistance. Anti-diabetic drugs increase plasma adiponectin by a poorly understood, post-transcriptional mechanism enhancing insulin sensitivity. Deletion analysis of a reporter bearing the mouse Adipoq mRNA 5’-leader identified an inhibitory cis-regulatory sequence. The 5’-leader harbours two potential upstream open reading frames (uORFs) overlapping the principal downstream ORF. Mutation of the uORF ATGs increased reporter translation ~3-fold, indicative of a functional uORF. uORFs are common in mammalian mRNAs; however, only a select group resist translational repression by the integrated stress response (ISR). Thapsigargin (TG), which induces endoplasmic reticulum (ER) stress and the ISR, enhanced expression of a reporter bearing the Adipoq 5’-leader; polysome profiling verified translation-stimulation. TG-stimulated translation was absent in cells defective in Ser51 phosphorylation of eukaryotic initiation factor 2α (eIF2α), required for the ISR. To determine its role in expression and function of endogenous adiponectin, the upstream uORF was disrupted by CRISPR-Cas9-mediated mutagenesis of differentiated mouse 3T3-L1 adipocytes. uORF disruption in adipocytes increased adiponectin expression, triacylglycerol accumulation, and glucose uptake, and inhibited paracrine muscle and liver cell expression of gluconeogenic enzymes, establishing an important role of the uORF in adiponectin-mediated responses to stress.

Selenium preserves cytosolic-Ca2+ homeostasis in olive callus cells during oxidative stress

Selenium (Se) is not essential for plant nutrition, however, growing evidence suggests a role of this element against oxidative stress. Here, we investigated the antioxidant effect of Se in an experimental system consisting of suspensions of olive callus cells exposed to hydrogen peroxide. Due to the disruptive capacity of oxidative stress on cytosolic Ca2+ homeostasis, Se treatments were simply and effectively assessed with cytosolic Ca2+ measurements. Se did not alter cell morphology, nor interfered with fluorometric determinations, suggesting a suitable model for monitoring alterations of cytosolic Ca2+. Furthermore, we used thapsigargin (TG), a known inhibitor of the Ca2+-ATPases of the endoplasmic reticulum, to establish whether Se had a specific impact on oxidative stress. We found that Se antagonized H2O2-mediated perturbations of cytosolic Ca2+, but was unable to offset TG-mediated disruptions. We conclude that Se merits consideration in strategies designed to tackle abiotic stress leading to oxidative insults.

1491-P: ER Stress Drives the VEGF Expression in Monocytic Cells by Mechanisms Involving CHOP/ROS/HIF-1alpha/NRF2 and NF-kappaB/p38 MAPK Pathways

Obesity is marked by metabolic inflammation and by metabolic impairment caused by increased endotoxin, free fatty acids, and vascular endothelial growth factor (VEGF) levels; and involves the endoplasmic reticulum (ER) stress as well. However, it remains unclear whether the ER stress can induce/amplify VEGF expression in metabolically-stressed monocytic cells; and if so, by which mechanism(s). To test this, metabolic stress was induced in THP-1 monocytic cells by treating cells separately with lipopolysaccharide (LPS), palmitic acid (PA), and oleic acid (OA), in presence/absence of ER stressor thapsigargin (TG). Gene expression of VEGF/ER stress markers was assessed by qRT-PCR, protein expression by ELISA, ROS by DCFH-DA assay, phosphorylation of HIF-1α, NF-κB, ERK1/2, and p38 MAPK by immunoblotting, and the insulin response in stressed cells by glucose-uptake assay. Regarding clinical analyses, adipose VEGF gene and protein expression was detected using qRT-PCR and IHC, respectively, while plasma hs-CRP, TNF-α, MDA, and OX-LDL levels were measured by ELISA. The experimental data show that a cooperative interaction between the metabolic and ER stresses led to the expression of VEGF, ROS, CHOP, ATF6, SOD2, and NRF2 (P˂0.05), as well as stimulated the CHOP and NRF2 promoter activities in reporter cells (P˂0.05). However, the glucose uptake was not impaired. The VEGF expression was dependent on phosphorylation of HIF-1α, NF-κB, and p38 MAPK; and the inhibitors of NF-κB/MAPK pathways as well as antioxidants or ROS scavengers suppressed the VEGF production. Further, individuals with obesity showed increased VEGF expression which associated positively with plasma levels of hs-CRP, TNF-α, MDA, and OX-LDL (P≤0.05). Overall, our findings support a cooperativity model in which the ER and metabolic stresses interact to augment VEGF expression in monocytic cells via the mechanisms involving CHOP/ROS/HIF-1α/NRF2 and NF-κB/p38 MAPK pathways. Disclosure S.A.K.Sindhu: None. F.Alzaid: None. F.Almulla: None. R.Ahmad: None. N.Akhter: None. A.Wilson: None. H.Arefanian: None. A.Al madhoun: None. R.S.Thomas: None. S.P.Kochumon: None. F.Alrashed: None. F.Bahman: None. Funding Kuwait Foundation for the Advancement of Sciences (RA2015-027, RA2010-003, RAHM-2019-022)

Functional differences in agonist-induced plasma membrane expression of Orai1α and Orai1β.

Orai1 is the pore-forming subunit of the store-operated Ca2+ release-activated Ca2+ (CRAC) channels involved in a variety of cellular functions. Two Orai1 variants have been identified, the long form, Orai1α, containing 301 amino acids, and the short form, Orai1β, which arises from alternative translation initiation from methionines 64 or 71, in Orai1α. Orai1 is mostly expressed in the plasma membrane, but a subset of Orai1 is located in intracellular compartments. Here we show that Ca2+ store depletion leads to trafficking and insertion of compartmentalized Orai1α in the plasma membrane via a mechanism that is independent on changes in cytosolic free-Ca2+ concentration, as demonstrated by cell loading with the fast intracellular Ca2+ chelator dimethyl BAPTA in the absence of extracellular Ca2+ . Interestingly, thapsigargin (TG) was found to be unable to induce translocation of Orai1β to the plasma membrane when expressed individually; by contrast, when Orai1β is co-expressed with Orai1α, cell treatment with TG induced rapid trafficking and insertion of compartmentalized Orai1β in the plasma membrane. Translocation of Orai1 forms to the plasma membrane was found to require the integrity of the actin cytoskeleton. Finally, expression of a dominant negative mutant of the small GTPase ARF6, and ARF6-T27N, abolished the translocation of compartmentalized Orai1 variants to the plasma membrane upon store depletion. These findings provide new insights into the mechanism that regulate the plasma membrane abundance of Orai1 variants after Ca2+ store depletion.

Mechanisms of autophagy and endoplasmic reticulum stress in the reversal of platinum resistance of epithelial ovarian cancer cells by naringin.

Our previous studies showed that naringin (Nar) can effectively reverse the cisplatin resistance of ovarian cancer cells. This study aims to explore the potential mechanism by which Nar reverses cisplatin resistance in ovarian cancer. The proliferative activity of cells was evaluated using CCK8 and cell clone formation assays. Autophagic flux in cells was evaluated via LC3B immunofluorescence and monodansylcadaverine (MDC) staining. The expression levels of autophagy, endoplasmic reticulum (ER) stress, and apoptosis-related proteins were detected via Western blotting. Autophagy and ER stress were regulated using siATG5, siLC3B, rapamycin (Rap), chloroquine (CQ), 4-phenylbutyric acid (4-PBA), and thapsigargin (TG). siATG5 and siLC3B are short interfering RNAs (siRNAs) used to knock down the expression of ATG5 and LC3B genes, respectively. Nar inhibited autophagy in SKOV3/DDP cells by activating the PI3K/AKT/mTOR pathway. And Nar increased the levels of ER stress-related proteins, namely, P-PERK, GRP78, and CHOP, and promoted apoptosis in SKOV3/DDP cells. Moreover, treatment with the inhibitor of ER stress alleviated apoptosis induced by Nar in SKOV3/DDP cells. In addition, compared to cisplatin or naringin alone, the combination of Nar and cisplatin significantly reduced the proliferative activity of SKOV3/DDP cells. And siATG5, siLC3B, CQ or TG pretreatment further inhibited the proliferative activity of SKOV3/DDP cells. Conversely, Rap or 4-PBA pretreatment alleviated the cell proliferation inhibition caused by Nar combined with cisplatin. Nar not only inhibited the autophagy in SKOV3/DDP cells by regulating the PI3K/AKT/mTOR signalling pathway, but also promoted apoptosis in SKOV3/DDP cells by targeting ER stress. Nar can reverse the cisplatin resistance in SKOV3/DDP cells through these two mechanisms.

Effects of GRP78 on endoplasmic reticulum stress and inflammation induced by Thapsigargin in macrophages of large yellow croaker (Larimichthys crocea)

The endoplasmic reticulum (ER) homeostasis plays a pivotal role in cell physiological functions. Various pathological, physiological, or pharmacological stimuli can destroy ER homeostasis and cause an adaptive response noted as ER stress. Meanwhile, ER stress is frequently associated with inflammatory response. GRP78 (Glucose-regulated protein 78) is crucial to maintaining the homeostasis of cells. Nonetheless, the potential mechanisms underlying the impact of GRP78 on ER stress and inflammation remain incompletely elucidated in fish. In the current research, the ER stress and inflammation were induced by Thapsigargin (TG) treatment in large yellow croaker macrophages. Then, the macrophages were incubated with agonist/inhibitor of GRP78 before or after the TG incubation to figure out the effects of GRP78. The results demonstrated that exposure to TG could significantly induce ER stress and inflammation in large yellow croaker macrophages, while the overexpression of GRP78 attenuated the ER stress and inflammation induced by TG. Furthermore, the inhibition of GRP78 further induced the ER stress and inflammation induced by TG. In conclusion, the above results indicated that GRP78 could regulate the ER stress and inflammation induced by TG in large yellow croaker.

Complement dependent TNFα production in neutrophil-like HL60 cells

Neutrophils develop in the bone marrow (BM) from hematopoietic stem cells (HSCs) through a series of progenitor cells and mature neutrophils play a critical role in the human immune system. Previous studies revealed that tumor necrosis factor α (TNFα) produced by immature neutrophils contributes to HSCs development and vascular regeneration in the BM niche. However, the precise mechanism of TNFα production in immature neutrophils remains unclear. This study aims to assess the relationship between complement C3 activation and TNFα production from immature neutrophils. We investigated the regulatory mechanism of TNFα production by complement components in neutrophil-like HL60 cells. Flow cytometric analysis showed that C3a receptor (C3aR) and C3bi receptor (CR3, Mac-1, CD11b/CD18, integrin αMβ2) are expressed on the surface of neutrophil-like HL60 cells. We found that zymosan-treated human serum leads to TNFα production in neutrophil-like HL60 cells, but not in human polymorphonuclear cells (PMNs). A C3-convertase inhibitor, compstatin suppresses TNFα production. These data suggest that the TNFα production is mediated by complement C3 activation. Furthermore, the TNFα production is enhanced by Ca2+ elevating agents, thapsigargin (TG), but is suppressed by treatment with Ca2+ chelators, EGTA, or BAPTA-AM. In addition, the soluble TNFα production is suppressed by treatment with immobilized-fibrinogen or -fibronectin. Thus, the TNFα production is enhanced by intracellular Ca2+ elevation and is negatively regulated by the interaction between the neutrophil-like HL60 cells and fibrinogen or fibronectin.

Kinetics of the thapsigargin-induced Ca2+ mobilisation: A quantitative analysis in the HEK-293 cell line.

Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump and, when applied acutely, it initiates a Ca2+ mobilisation that begins with the loss of Ca2+ from the endoplasmic reticulum (ER) and culminates with store-operated Ca2+ entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca2+ levels using FURA-2 and the FRET-based ER Ca2+ sensor D1ER, respectively. Our analysis predicts an ER Ca2+ leak of 5-6µM⋅s-1 for the typical basal ER Ca2+ level of 335-407µM in HEK-293 cells. The resulting cytosolic Ca2+ transients reached peak amplitudes of 0.6-1.0µM in the absence of external Ca2+ and were amplified by SOCE that amounted to 28-30nM⋅s-1 in 1mM external Ca2+. Additionally, cytosolic Ca2+ transients were shaped by a Ca2+ clearance of 10-13nM⋅s-1. Using puromycin (PURO), which enhances the ER Ca2+ leak, we show that TG-induced cytosolic Ca2+ transients are directly related to ER Ca2+ levels and to the ER Ca2+ leak. A one-compartment model incorporating ER Ca2+ leak and cytosolic Ca2+ clearance accounted satisfactorily for the basic features of TG-induced Ca2+ transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca2+ transients most likely reflects an increase in ER Ca2+ leak.

Sitagliptin attenuates neuronal apoptosis via inhibiting the endoplasmic reticulum stress after acute spinal cord injury.

Regulation of endoplasmic reticulum stress (ER) stress-induced apoptosis and nerve regeneration is a hopeful way for acute spinal cord injury (SCI). Sitagliptin (Sita) is one of dipeptidyl peptidase-4 (DPP-4) inhibitor, which is beneficial neurons damaged diseases. However, its protective mechanisms of avoiding nerve injury remain unclear. In this study, we further investigated the mechanism of the anti-apoptotic and neuroprotective effects of Sita in promoting locomotor recovery from SCI. In vivo results showed that Sita treatment reduced neural apoptosis caused by SCI. Moreover, Sita effectively attenuated the ER tress and associated apoptosis in rats with SCI. A striking feature was the occurrence of nerve fiber regeneration at the lesion site, which eventually led to significant locomotion recovery. In vitro results showed that the PC12 cell injury model induced by Thapsigargin (TG) also showed similar neuroprotective effects. Overall, sitagliptin showed potent neuroprotective effects by targeting the ER stress-induced apoptosis both in vivo and vitro, thus facilitating the regeneration of the injured spinal cord.

Identification of long non-coding RNA and circular RNA associated networks in cellular stress responses.

Mammalian cells employ various adaptive responses to cope with multiple stresses to maintain homeostasis. Functional roles of non-coding RNAs (ncRNAs) in response to cellular stresses have been proposed, and systematical investigations about the crosstalk among distinct types of RNAs are required. Here, we challenged HeLa cells with thapsigargin (TG) and glucose deprivation (GD) treatments to induce endoplasmic reticulum (ER) and metabolic stresses, respectively. Ribosomal RNA (rRNA)-depleted RNA sequencing (RNA-seq) was then performed. Characterization of the RNA-seq data revealed a series of differentially expressed long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) with parallel changes responsive to both stimuli. We further constructed the lncRNA/circRNA-mRNA co-expressing network, competing endogenous RNA (ceRNA) network in the lncRNA/circRNA-miRNA-mRNA axis, and lncRNA/circRNA-RNA binding protein (RBP) interactome map. These networks indicated the potential cis and/or trans regulatory roles of lncRNAs and circRNAs. Moreover, Gene Ontology analysis demonstrated that these identified ncRNAs were associated with several essential biological processes known to be related to cellular stress responses. In conclusion, we systematically established functional regulatory networks of lncRNA/circRNA-mRNA, lncRNA/circRNA-miRNA-mRNA and lncRNA/circRNA-RBP to perceive the potential interactions and biological processes during cellular stresses. These results provided insights in ncRNA regulatory networks of stress responses and the basis for further identification of pivotal factors involved in cellular stress responses.

Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging.

Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (15N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the 15N/14N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.

The metallic compound promotes primordial follicle activation and ameliorates fertility deficits in aged mice.

Background: Aged women and premature ovarian insufficiency (POI) patients have residual dormant primordial follicles that are hard to be activated through a physiological process. However, there are no effective and safe drugs to help them. Methods: We used the in vitro culture model of newborn mouse ovaries to identify the drugs that promote primordial follicle activation and study its mechanisms. It was verified by in vivo injection model of newborn mice and in vitro culture model of human ovarian tissue. In addition, we used the aged mice as a low infertility model to verify the effects of primordial follicle activation, and fertility by drugs. Results: Eleven metallic compounds activated mouse primordial follicles, and the five most effective compounds were selected for further study. Thapsigargin (TG), CrCl3, MnCl2, FeCl3 and ZnSO4 increased the levels of the glycolysis-related proteins (glucose transporter type 4, GLUT4; hexokinase 1, HK1; pyruvate kinase M2, PKM2; phosphofructokinase, liver type, PFKL), phosphorylated mammalian target of rapamycin (p-mTOR) in cultured mouse ovaries. The compound-promoted p-mTOR levels could be completely blocked by 2-DG (the inhibitor of glycolysis). The compounds also increased the levels of phosphorylated protein kinase B (p-Akt). TG-, CrCl3- and FeCl3-promoted p-Akt levels, but not MnCl2- and ZnSO4- promoted p-Akt levels, could be completely blocked by ISCK03 (the inhibitor of proto-oncogenic receptor tyrosine kinase, KIT). The injection of newborn mice with the compounds also activated primordial follicles and increased the levels of the glycolysis-related proteins, p-mTOR, and p-Akt. The oral administration of the compounds in adolescent and aged mice promoted primordial follicle activation, and had no obvious side effect. Importantly, ZnSO4 also increased ovulated oocytes, oocyte quality and offspring in aged mice. Furthermore, the compounds promoted human primordial follicle activation and increased the levels of the glycolysis-related proteins, p-mTOR, and p-Akt. Conclusion: The metallic compounds activate primordial follicles through the glycolysis-dependent mTOR pathway and/or the PI3K/Akt pathway, and the oral administration of ZnSO4 enhances fertility in aged mice. We suggest that these metallic compounds may be oral drugs to ameliorate fertility deficits in aged women and POI patients.

Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Cannabidiol Protects Striatal Neurons by Attenuating Endoplasmic Reticulum Stress.

Introduction: The aggregation of misfolded proteins in the endoplasmic reticulum (ER) is a pathological trait shared by many neurodegenerative disorders. This aggregation leads to the persistent activation of the unfolded protein response (UPR) and ultimately apoptosis as a result of ER stress. Cannabidiol (CBD) has been demonstrated to be neuroprotective in various cellular and animal models of neurodegeneration, which has been attributed to its antioxidant and anti-inflammatory properties. However, little is known about the role of CBD in the context of protein folding and ER stress. The purpose of this study was to investigate whether CBD is neuroprotective against an in vitro model of ER stress. Materials and Methods: Using different exposure models, mouse striatal STHdhQ7/Q7 cells were exposed to either the ER stress inducer thapsigargin (TG) and/or CBD. Cell viabilities assays were used to investigate the effect of CBD pre-treatment, co-treatment, and post-treatment on TG-induced cell death. Real-time quantitative polymerase chain reaction was used to measure changes in ER stress regulators and UPR genes such as glucose-regulated protein-78 (GRP78), mesencephalic astrocyte-derived neurotrophic factor (MANF), B cell lymphoma 2 (BCL-2), BCL-2 interacting mediator of cell death (BIM), and caspase-12. Results: Cell viability increased significantly when cells were pre-treated with CBD before TG exposure. An increase in the gene expression of pro-survival ER chaperone GRP78 and ER-resident neurotrophic factor MANF coincided with this effect and decreased ER-mediated pro-apoptotic markers such as BIM, and caspase-12 was observed. Conclusions: These data suggest that CBD pre-treatment is neuroprotective against TG-induced cell death. Understanding the role of ER stress in CBD-driven neuroprotection provides insight into the therapeutic potential of CBD and the role of ER dysfunction in neurodegenerative disorders.

Icaritin, a metabolite of Icarrin, Alleviates non-alcoholic fatty liver disease via inhibition of lipogenesis and ER stress

Non-alcoholic fatty liver disease (NAFLD) is one of the most serious global public health concerns. However, there are currently no effective drugs for treatment of this disease. Icariin (ICA), a small-molecule natural product extracted from Epimedium brevicornu Maxim, offers various pharmacological activities. In the present work, we wondered whether ICA can attenuate NAFLD in db/db mice treated with ICA for 8 weeks and how ICA exerts an influence on NAFLD. In db/db mice, ICA treatment had a robust effect on inhibition of lipogenesis associated with NAFLD amelioration by decreasing liver lipid deposition, together with ameliorating insulin sensitivity, glucose tolerance, and fasting serum glucose. Of note, ICA-treated rats showed a much higher concentration of icaritin (ICT) in plasma, a major metabolite of ICA, about 2000 times higher than that of ICA by liquid chromatography mass spectrometry (LC-MS). Interestingly, ICT, rather than ICA, can dramatically decrease hepatic lipogenesis-related markers in oleate acid/palmitate acid (OA/PA)-induced steatosis in primary hepatocytes (PH) and HepG2 cells, and hepatic lipid accumulation in db/db mice, demonstrating the inhibitory effect of ICT on lipogenesis. Mechanistically, we found that anti-lipogenic activities of ICT were related to reducing endoplasmic reticulum (ER) stress as evidenced by Western blot, qPCR, and other assays in thapsigargin (THP) induced-ER stress models. To our knowledge, this is the first report showing the unexpected and key role for ICT on the prevention of NAFLD in db/db mice through an ER stress mechanism.

The calcium-antagonist activity of the material released by olive pollen (PMR), tested on Ca2+-cytosolic of PE/CA-PJ15 cells

Inhalation of olive pollen (Olea europaea L.) is one of the main causes of allergy in Mediterranean countries and some areas of North America. The response to allergens consists in the production of inflammatory cytokines which is mediated by the deregulation of Ca2+ signals. In this study, the biological activity of the material released in olive pollen hydration (PMR) was tested on Ca2+ cytosolic of PE/CA-PJ15 cells (PJ-15). Ca2+ cytosolic was determined by fluorometric assay with the cell line PE/CA-PJ15 (PJ-15) labeled with the fluorescent probe FURA 2 AM. The material released in olive pollen hydration (PMR) was analyzed by HPLC for the determination of phenolic acids. PMR was subjected to fractionation by gel filtration, and the fractions with Ca2+-chelating activity were tested with SDS-PAGE and the single bands characterized by proteomic analysis. PMR showed high Ca2+-chelating activity and is able of blocking the increase Ca2+-cytosolic produced by thapsigargin (TG). PMR then restored Ca2+ homeostasis in PJ-15 cells deregulated by the endoplasmic reticulum Ca2+-ATPases inhibitor. It is therefore possible that PMR can antagonize the effects of allergens on Ca2+ cytosolic. The analytical characterization of the material released by the pollen highlighted in the pollen allergen Ole e 3 and in the p-coumaric acid the possible culprits of the Ca2+-antagonist activity of PMR. Furthermore, the sequence of Ole e 3 could provide information for the possible construction of a synthetic peptide to be used in an allergy-targeted Ca2+-antagonist therapy.

IRE1α-XBP1 regulates PDK1-dependent induction of epithelial-mesenchymal transition in non-small cell lung cancer cells

Mounting evidence indicates that activation of unfolded protein response (UPR) and metabolic reprogramming contribute to cancer cell migration and invasion, but the molecular mechanism of pro-EMT program through a coordinated action of UPR with metabolism has not been defined. In this study, we utilized ER stress-inducing reagent, thapsigargin (TG), to induced pharmacologic ER stress in lung cancer cells. Here. We report that the branch of UPR, IRE1α-XBP1 pathway plays a pivotal role in reprogramming lung cancer cell metabolism. At the molecular level, the expression of pyruvate dehydrogenase kinase-1 (PDK-1) is directly induced by XBP1 as a consequence of UPR activation, thus facilitating aerobic glycolysis and lactate production. We also demonstrated that PDK1 serves as a downstream element of UPR activation in induction of Snail and EMT program. In addition, PDK1-induced Snail was dependent on the lactate production derived from metabolic reprogramming. Our findings reveal a critical role of lactate in pro-invasion events and establishes a direct connection between ER-stress and metabolic reprogramming in facilitating cancer cell progression.

Orientin downregulating oxidative stress-mediated endoplasmic reticulum stress and mitochondrial dysfunction through AMPK/SIRT1 pathway in rat nucleus pulposus cells in vitro and attenuated intervertebral disc degeneration in vivo.

Although considered as a major contributor to low back pain (LBP), intervertebral disc degeneration (IVDD) has poor medical and surgical treatments. Various studies have revealed that endoplasmic reticulum (ER) stress and extracellular matrix (ECM) degeneration play a vital role in initiating and developing the progression of IVDD. Moreover, restoration of SIRT1/AMPK was confirmed to prevent IVDD and damage via maintaining ER and extracellular homeostasis. In addition, orientin (Ori) has been shown to upregulate SIRT1. However, the effect of Ori in nucleus pulposus cells (NPCs) is not determined. Hence, in this study we aim to explore the function of Ori in IVDD pathological model. The results demonstrate that Ori treatment in vitro increased SIRT1/AMPK in NPCs, maintained ECM and ER balance and decreased oxidative stress (OS) response. Ori rescued the disordered homeostasis stimulated by tert-butyl hydroperoxide (TBHP), and its function can be inhibited by thapsigargin (TG). Compound C and EX-527, inhibitors of AMPK and SIRT1 counteracted the Ori-mediated ER stress elimination. These results confirm that Ori exerts its effects by upregulating AMPK and SIRT1. Puncture-stimulated IVDD rats were used to show that Ori attenuates the pathological development in vivo. In all, we partly unveil the underlying mechanisms of Ori in IVDD.

Transcription factor EB protects against endoplasmic reticulum stress in human coronary artery endothelial cells

Oxidative stress and endoplasmic reticulum (ER) stress promote atherogenesis while transcription factor EB (TFEB) inhibits atherosclerosis. Since reducing oxidative stress with antioxidants have failed to reduce atherosclerosis possibly because of aggravation of ER stress, we studied the effect of TFEB on ER stress in human coronary artery endothelial cells. ER stress was measured using the secreted alkaline phosphatase assay. Expression and phosphorylation of key mediators of unfolded protein response (UPR). TFEB, inositol-requiring enzyme 1α (IRE1α), phospho-IRE1α, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phospho-PERK, and activating transcription factor 6 (ATF6) expression were measured by Western blot. The effect of TFEB gain- and loss-of-function on ER stress were assessed with a plasmid expressing a constitutively active form of TFEB and via siRNA-mediated silencing, respectively. Treatment with tunicamycin (TM) and thapsigargin (TG) increased TFEB expression by 42.8% and 42.3%, respectively. In HCAEC transfected with the TFEB siRNA, treatment with either TM, TG or high-dextrose increased IRE1α and PERK phosphorylation and ATF6 levels significantly more compared to cells transfected with the control siRNA and treated similarly. Furthermore, transient transfection with a plasmid expressing a constitutively active form of TFEB reduced ER stress. Increased expression of TFEB inhibited ER stress in HCAEC treated with pharmacologic (TM and TG) and physiologic (high-dextrose) ER stress inducers, while TFEB knockout aggravated ER stress caused by these ER stress inducers. TFEB-mediated ER stress reduction may contribute to its anti-atherogenic effects in HCAEC and may be a novel target for drug development.