ATF6 supports lysosomal function in tumor cells to enable ER stress-activated macroautophagy and CMA: impact on mutant TP53 expression.

The inhibition of the unfolded protein response (UPR), which usually protects cancer cells from stress, may be exploited to potentiate the cytotoxic effect of drugs inducing ER stress. However, in this study, we found that ER stress and UPR activation by thapsigargin or tunicamycin promoted the lysosomal degradation of mutant (MUT) TP53 and that the inhibition of the UPR sensor ATF6, but not of ERN1/IRE1 or EIF2AK3/PERK, counteracted such an effect. ATF6 activation was indeed required to sustain the function of lysosomes, enabling the execution of chaperone-mediated autophagy (CMA) as well as of macroautophagy, processes involved in the degradation of MUT TP53 in stressed cancer cells. At the molecular level, by pharmacological and genetic approaches, we demonstrated that the inhibition of ATF6 correlated with the activation of MTOR and with TFEB and LAMP1 downregulation in thapsigargin-treated MUT TP53 carrying cells. We hypothesize that the rescue of MUT TP53 expression by ATF6 inhibition, could further activate MTOR and maintain lysosomal dysfunction, further inhibiting MUT TP53 degradation, in a vicious circle. The findings of this study suggest that the presence of MUT TP53, which often exerts oncogenic properties, should be considered before approaching treatments combining ER stressors with ATF6 inhibitors against cancer cells, while it could represent a promising strategy against cancer cells that harbor WT TP53.

1,25-dihydroxyvitamin D3 affects thapsigargin-induced endoplasmic reticulum stress in 3T3-L1 adipocytes.

BACKGROUND/OBJECTIVES: Endoplasmic reticulum (ER) stress in adipose tissue causes an inflammatory response and leads to metabolic diseases. However, the association between vitamin D and adipose ER stress remains poorly understood. In this study, we investigated whether 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) alleviates ER stress in adipocytes. MATERIALS/METHODS: 3T3-L1 cells were treated with different concentrations (i.e., 10-100 nM) of 1,25(OH)2D3 after or during differentiation (i.e., on day 0-7, 3-7, or 7). They were then incubated with thapsigargin (TG, 500 nM) for an additional 24 h to induce ER stress. Next, we measured the mRNA and protein levels of genes involved in unfold protein response (UPR) and adipogenesis using real-time polymerase chain reaction and western blotting and quantified the secreted protein levels of pro-inflammatory cytokines. Finally, the mRNA levels of UPR pathway genes were measured in adipocytes transfected with siRNA-targeting Vdr. RESULTS: Treatment with 1,25(OH)2D3 during various stages of adipocyte differentiation significantly inhibited ER stress induced by TG. In fully differentiated 3T3-L1 adipocytes, 1,25(OH)2D3 treatment suppressed mRNA levels of Ddit3, sXbp1, and Atf4 and decreased the secretion of monocyte chemoattractant protein-1, interleukin-6, and tumor necrosis factor-α. However, downregulation of the mRNA levels of Ddit3, sXbp1, and Atf4 following 1,25(OH)2D3 administration was not observed in Vdr-knockdown adipocytes. In addition, exposure of 3T3-L1 preadipocytes to 1,25(OH)2D3 inhibited transcription of Ddit3, sXbp1, Atf4, Bip, and Atf6 and reduced the p-alpha subunit of translation initiation factor 2 (eIF2α)/eIF2α and p-protein kinase RNA-like ER kinase (PERK)/PERK protein ratios. Furthermore, 1,25(OH)2D3 treatment before adipocyte differentiation reduced adipogenesis and the mRNA levels of adipogenic genes. CONCLUSIONS: Our data suggest that 1,25(OH)2D3 prevents TG-induced ER stress and inflammatory responses in mature adipocytes by downregulating UPR signaling via binding with Vdr. In addition, the inhibition of adipogenesis by vitamin D may contribute to the reduction of ER stress in adipocytes.

N-recognins UBR1 and UBR2 as central ER stress sensors in mammals.

In eukaryotes, a primary protein quality control (PQC) process involves the destruction of conformationally misfolded proteins through the ubiquitin-proteasome system. Because approximately one-third of eukaryotic proteomes fold and assemble within the endoplasmic reticulum (ER) before being sent to their destinations, the ER plays a crucial role in PQC. The specific functions and biochemical roles of several E3 ubiquitin ligases involved in ER-associated degradation in mammals, on the other hand, are mainly unknown. We identified 2 E3 ligases, ubiquitin protein ligase E3 component N-recognin 1 (UBR1) and ubiquitin protein ligase E3 component N-recognin 2 (UBR2), which are the key N-recognins in the N-degron pathway and participate in the ER stress response in mammalian cells by modulating their stability. Cells lacking UBR1 and UBR2 are hypersensitive to ER stress-induced apoptosis. Under normal circumstances, these proteins are polyubiquitinated through Lys48-specific linkages and are then degraded by the 26S proteasome. In contrast, when cells are subjected to ER stress, UBR1 and UBR2 exhibit greater stability, potentially as a cellular adaptive response to stressful conditions. Although the precise mechanisms underlying these findings require further investigation, our findings show that cytoplasmic UBR1 and UBR2 have anti-ER stress activities and contribute to global PQC in mammals. These data also reveal an additional level of complexity within the mammalian ER-associated degradation system, implicating potential involvement of the N-degron pathway.

Cyclin-dependent kinase inhibitor 1 plays a more prominent role than activating transcription factor 4 or the p53 tumour suppressor in thapsigargin-induced G1 arrest.

Background: Thapsigargin (Tg) is a compound that inhibits the SERCA calcium transporter leading to decreased endoplasmic reticulum (ER) Ca2+ levels. Many ER chaperones are required for proper folding of membrane-associated and secreted proteins, and they are Ca2+ dependent. Therefore, Tg leads to the accumulation of misfolded proteins in the ER, activating the unfolded protein response (UPR) to help restore homeostasis. Tg reportedly induces cell cycle arrest and apoptosis in many cell types but how these changes are linked to the UPR remains unclear. The activating transcription factor 4 (ATF4) plays a key role in regulating ER stress-induced gene expression so we sought to determine if ATF4 is required for Tg-induced cell cycle arrest and apoptosis using ATF4-deficient cells. Methods: Two-parameter flow cytometric analysis of DNA replication and DNA content was used to assess the effects of Tg on cell cycle distribution in isogenic HCT116-derived cell lines either expressing or lacking ATF4. For comparison, we similarly assessed the Tg response in isogenic cell lines deleted of the p53 tumour suppressor and the p53-regulated p21WAF1 cyclin-dependent kinase inhibitor important in G1 and G2 arrests induced by DNA damage. Results: Tg led to a large depletion of the S phase population with a prominent increase in the proportion of HCT116 cells in the G1 phase of the cell cycle. Importantly, this effect was largely independent of ATF4. We found that loss of p21WAF1 but not p53 permitted Tg treated cells to enter S phase and synthesize DNA. Therefore, p21WAF1plays an important role in these Tg-induced cell cycle alterations while ATF4 and p53 do not. Remarkably, the ATF4-, p53-and p21WAF1-deficient cell lines were all more sensitive to Tg-induced apoptosis. Taken together, p21WAF1 plays a larger role in regulating Tg-induced G1 and G2 arrests than ATF4 or p53 but these proteins similarly contribute to protection from Tg-induced apoptosis. This work highlights the complex network of stress responses that are activated in response to ER stress.

Synergistic lethality in chronic myeloid leukemia - targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment.

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

Intracellular remodeling associated with endoplasmic reticulum stress modifies biomechanical compliance of bladder cells.

Bladder cells face a challenging biophysical environment: mechanical cues originating from urine flow and regular contraction to enable the filling voiding of the organ. To ensure functional adaption, bladder cells rely on high biomechanical compliance, nevertheless aging or chronic pathological conditions can modify this plasticity. Obviously the cytoskeletal network plays an essential role, however the contribution of other, closely entangled, intracellular organelles is currently underappreciated. The endoplasmic reticulum (ER) lies at a crucial crossroads, connected to both nucleus and cytoskeleton. Yet, its role in the maintenance of cell mechanical stability is less investigated. To start exploring these aspects, T24 bladder cancer cells were treated with the ER stress inducers brefeldin A (10-40nM BFA, 24 h) and thapsigargin (0.1-100nM TG, 24 h). Without impairment of cell motility and viability, BFA and TG triggered a significant subcellular redistribution of the ER; this was associated with a rearrangement of actin cytoskeleton. Additional inhibition of actin polymerization with cytochalasin D (100nM CytD) contributed to the spread of the ER toward cell periphery, and was accompanied by an increase of cellular stiffness (Young´s modulus) in the cytoplasmic compartment. Shrinking of the ER toward the nucleus (100nM TG, 2 h) was related to an increased stiffness in the nuclear and perinuclear areas. A similar short-term response profile was observed also in normal human primary bladder fibroblasts. In sum, the ER and its subcellular rearrangement seem to contribute to the mechanical properties of bladder cells opening new perspectives in the study of the related stress signaling cascades. Video Abstract.

Translocation of IGF-1R in endoplasmic reticulum enhances SERCA2 activity to trigger Ca2+ER perturbation in hepatocellular carcinoma.

The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rß is translocated into the ER by ß-arrestin2 (ß-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rß. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rß levels. ER IGF-1Rß phosphorylates SERCA2 on Tyr990 to enhance its activity. Mutation of SERCA2-Tyr990 disrupted the interaction of ER IGF-1Rß with SERCA2, and therefore ER IGF-1Rß failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca2+ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rß and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca2+ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr990 in HCC.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity.

AIMS: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. MATERIALS AND METHODS: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. KEY FINDINGS: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SIGNIFICANCE: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

Obtaining Functional Proteomics Insights From Thermal Proteome Profiling Through Optimized Melt Shift Calculation and Statistical Analysis With InflectSSP.

Thermal proteome profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein-ligand, protein-protein, and protein-RNA interaction dynamics along with changes in protein stability resulting from cellular signaling. The increasing number of reports employing this assay has not been met concomitantly with new approaches leading to advancements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is important to fill as TPP findings have reported subtle melt shift changes related to signaling events such as protein posttranslational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP, available at https://CRAN.R-project.org/package=InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. InflectSSP incorporates calculation of a "melt coefficient" into the pipeline with production of average melt curves for biological replicate studies to aid in identification of proteins with significant melts. To benchmark InflectSSP, we have reanalyzed two previously reported datasets to demonstrate the performance of our publicly available R-based program for TPP data analysis. We report new findings following temporal treatment of human cells with the small molecule thapsigargin that induces the unfolded protein response as a consequence of inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase 2A. InflectSSP analysis of our unfolded protein response study revealed highly reproducible and statistically significant target engagement over a time course of treatment while simultaneously providing new insights into the possible mechanisms of action of the small molecule thapsigargin.

Role of Nitric Oxide in the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis.

INTRODUCTION: Previously, we found that intracellular calcium (Ca2+) homeostasis is altered in platelets from an experimental model of liver cirrhosis, namely the bile-duct-ligated (BDL) rat. These alterations are compatible with the existence of a hypercoagulable state. OBJECTIVE: In the present study, we analyzed the role of nitric oxide in the abnormal calcium signaling responses of an experimental cirrhosis model, the bile duct-ligated rat. METHODS: Chronic treatment with L-NAME was used to inhibit NO production in a group of control and BDL animals, and the responses compared to those obtained in a control and BDL untreated group (n = 6 each). The experiments were conducted on isolated platelets loaded with fura-2, using fluorescence spectrometry. RESULTS: Chronic treatment with L-NAME increased thrombin-induced Ca2+ release from internal stores in both control and BDL rats. However, the effect was significantly greater in the BDL rats (p < 0.05). Thrombin-induced calcium entry from the extracellular space was also elevated but at lower doses and, similarly in both control and BDL platelets, treated with the NO synthesis inhibitor. Capacitative calcium entry was also enhanced in the control platelets but not in platelets from BDL rats treated with L-NAME. Total calcium in intracellular stores was elevated in untreated platelets from BDL rats, and L-NAME pretreatment significantly (p < 0.05) elevated these values both in controls and in BDL but significantly more in the BDL rats (p < 0.05). CONCLUSIONS: Our results suggest that nitric oxide plays a role in the abnormal calcium signaling responses observed in platelets from BDL rats by interfering with the mechanism that releases calcium from the internal stores.

Effects of multiple stressors on pancreatic human islets proteome reveal new insights into the pathways involved.

Pancreatic ß-cell dysfunction is an early hallmark of type 1 diabetes mellitus. Among the potentially critical factors that cause ß-cell dysfunction are cytokine attack, glucotoxicity, induction of endoplasmic reticulum (ER) or mitochondria stress. However, the exact molecular mechanism underlying ß-cell's inability to maintain glucose homeostasis under severe stresses is unknown. This study used proinflammatory cytokines, thapsigargin, and rotenone in the presence of high concentration glucose to mimicking the conditions experienced by dysfunctional ß-cells in human pancreatic islets, and profiled the alterations to the islet proteome with TMT-based proteomics. The results were further verified with label-free quantitative proteomics. The differentially expressed proteins under stress conditions reveal that immune related pathways are mostly perturbed by cytokines, while the respiratory electron transport chains and protein processing in ER pathways by rotenone. Thapsigargin together with high glucose induces dramatic increases of proteins in lipid synthesis and peroxisomal protein import pathways, with energy metabolism and vesicle secretion related pathways downregulated. High concentration glucose, on the other hand, alleviated complex I inhibition induced by rotenone. Our results contribute to a more comprehensive understanding of the molecular events involved in ß-cell dysfunction.

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 5-6 µM⋅s-1 for the typical basal ER Ca2+ level of 335-407 µM in HEK-293 cells. The resulting cytosolic Ca2+ transients reached peak amplitudes of 0.6-1.0 µM in the absence of external Ca2+ and were amplified by SOCE that amounted to 28-30 nM⋅s-1 in 1 mM external Ca2+. Additionally, cytosolic Ca2+ transients were shaped by a Ca2+ clearance of 10-13 nM⋅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.

CAPN1 (Calpain1)-Dependent Cleavage of STIM1 (Stromal Interaction Molecule 1) Results in an Enhanced SOCE (Store-Operated Calcium Entry) in Human Neonatal Platelets.

BACKGROUND: Altered intracellular Ca2+ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca2+ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. METHODS: Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca2+ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. RESULTS: Altered TG (thapsigargin)-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor) were found in neonatal platelets as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca2+ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca2+-dependent inactivation, so reproducing the Ca2+-homeostasis pattern observed in neonatal platelets. CONCLUSIONS: CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. s-STIM1 may avoid slow Ca2+-dependent inactivation and, subsequently, results in an enhanced TG-evoked SOCE as observed in neonatal platelets.

Elevation of Cytoplasmic Calcium Suppresses Microtentacle Formation and Function in Breast Tumor Cells.

Cytoskeletal remodeling in circulating tumor cells (CTCs) facilitates metastatic spread. Previous oncology studies examine sustained aberrant calcium (Ca2+) signaling and cytoskeletal remodeling scrutinizing long-term phenotypes such as tumorigenesis and metastasis. The significance of acute Ca2+ signaling in tumor cells that occur within seconds to minutes is overlooked. This study investigates rapid cytoplasmic Ca2+ elevation in suspended cells on actin and tubulin cytoskeletal rearrangements and the metastatic microtentacle (McTN) phenotype. The compounds Ionomycin and Thapsigargin acutely increase cytoplasmic Ca2+, suppressing McTNs in the metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-436. Functional decreases in McTN-mediated reattachment and cell clustering during the first 24 h of treatment are not attributed to cytotoxicity. Rapid cytoplasmic Ca2+ elevation was correlated to Ca2+-induced actin cortex contraction and rearrangement via myosin light chain 2 and cofilin activity, while the inhibition of actin polymerization with Latrunculin A reversed Ca2+-mediated McTN suppression. Preclinical and phase 1 and 2 clinical trial data have established Thapsigargin derivatives as cytotoxic anticancer agents. The results from this study suggest an alternative molecular mechanism by which these compounds act, and proof-of-principle Ca2+-modulating compounds can rapidly induce morphological changes in free-floating tumor cells to reduce metastatic phenotypes.

Peculiar Ca2+ Homeostasis, ER Stress, Autophagy, and TG2 Modulation in Celiac Disease Patient-Derived Cells.

Celiac disease (CD) is an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals by genetically predisposed individuals. Constitutive differences between cells from CD patients and control subjects, including levels of protein phosphorylation, alterations of vesicular trafficking, and regulation of type 2 transglutaminase (TG2), have been reported. In the present work, we investigated how skin-derived fibroblasts from CD and control subjects responded to thapsigargin, an endoplasmic reticulum ER stress inducer, in an attempt to contribute to the comprehension of molecular features of the CD cellular phenotype. We analyzed Ca2+ levels by single-cell video-imaging and TG2 activity by a microplate assay. Western blots and PCR analyses were employed to monitor TG2 levels and markers of ER stress and autophagy. We found that the cytosolic and ER Ca2+ level of CD cells was lower than in control cells. Treatments with thapsigargin differently activated TG2 in control and CD cells, as well as caused slightly different responses regarding the activation of ER stress and the expression of autophagic markers. On the whole, our findings identified further molecular features of the celiac cellular phenotype and highlighted that CD cells appeared less capable of adapting to a stress condition and responding in a physiological way.

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.

TRPC6 involves in endoplasmic reticulum stress induced apoptosis of glomerular mesangial cells / 西安交通大学学报(医学版)

【Objective】 To explore the role and mechanism of TRPC6 in apoptosis of glomerular mesangial cells (HBZY-1) induced by endoplasmic reticulum stress (ERS). 【Methods】 The experiment groups were classified as follows: normal control (NC), thapsigargin (TG), TG+SKF96365, and TG+TRPC6 siRNA groups. Transcription and protein expressions of TRPC6 and ERS related proteins (GRP78 and Caspase12) were detected by qRT-PCR and Western blotting. Additionally, cell apoptosis was measured by flow cytometry and Hoechst33258. Finally, Fluo-4 AM Ca2+ imaging technique was used to determine changes of intracellular calcium ( [Ca2+] i) by laser scanning confocal microscope. 【Results】 Morphological changes of apoptotic cells were characterized by nuclear enrichment or nuclear fragmentation, and the apoptosis rate was increased after TG stimulation. The expressions of TRPC6 and ERS related proteins (GRP78 and Caspase12) were elevated in TG group compared with NC group (P<0.05). Pre-incubation of HBZY-1 cells with SKF96365 and TRPC6 siRNA decreased cell apoptosis (P<0.05). The entry of [Ca2+] i also increased after TG stimulation (P<0.05). The expressions of TRPC6, GRP78 and Caspase12 were downregulated compared with TG group after treatment with SKF96365 and TRPC6 siRNA accompanied by decreased [Ca2+] i (P<0.05). 【Conclusion】 Taken together, this study suggests that inhibition of TRPC6 can alleviate TG-induced HBZY-1 cell apoptosis.

Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro: A potential antagonist of corticotropin-releasing factor (CRF).

Teneurin C-terminal associated peptides (TCAP) are natural bioactive peptides that possess anxiety-reducing roles in animals, in vivo, and increase cell viability, in vitro. Although these peptides have some primary structural similarity to corticotropin-releasing factor (CRF), they are derived from the distal extracellular region of the teneurin transmembrane protein where they may act as separate soluble peptides after auto-catalytic cleavage from the teneurin protein following interaction with the cognate teneurin receptor, latrophilin (ADGRL), or expressed as a separate mRNA. However, although the signal transduction mechanism of TCAP in neurons has not been established, previous studies indicate an association with the intracellular calcium flux. Therefore, in this study, we have characterized the TCAP-mediated calcium response in hypothalamic cell lines using single-cell calcium methods with pharmacological antagonists to identify potential calcium channels, in vitro. Under normal circumstances, TCAP-1 reduces cytosolic calcium concentrations by uptake into the mitochondria and efflux through the plasma membrane independently of the teneurins. In doing so, TCAP-1 could inhibit the potential 'stress' -inducing actions of CRF.

Blocking the cytohesin-2/ARF1 axis by SecinH3 ameliorates osteoclast-induced bone loss via attenuating JNK-mediated IRE1 endoribonuclease activity.

cytohesin-2 is a guanine nucleotide exchange factor to activate ARF1 and ARF6, which are involved in various biological processes, including signal transduction, cell differentiation, cell structure organization, and survival. Nevertheless, there is a lack of evidence revealing the role of cytohesin-2 in osteoclast differentiation and in the development of osteoporosis. In this study, we find cytohesin-2 and ARF1 positively regulate osteoclast differentiation and function. Blocking the cytohesin-2 /ARF1 axis with SecinH3 or by genetic silencing of cytohesin-2 inhibits osteoclast formation and function in vitro. In vivo treatment with SecinH3 ameliorates ovariectomy-induced osteoporosis. Mechanistically, RNA-sequencing combined with molecular biological methodologies reveal that the regulatory function of cythohesin-2/ARF1 axis in osteoclast differentiation is mainly dependent on activating the JNK pathway. Further, in addition to the common viewpoint that JNK is activated by IRE1 via its kinase activity, we found that JNK can act upstream and regulate the endoribonuclease activity of IRE1 to promote XBP1 splicing. Both SecinH3 and silencing of cytohesin-2 inhibit JNK activation and IRE1 endoribonuclease activity, leading to the suppression of osteoclast differentiation. Taken together, our findings add new insights into the regulation between JNK and IRE1, and reveal that inhibiting the cytohesin-2/ARF1/JNK/IRE1 axis might represent a potential new strategy for the treatment of post-menopause osteoporosis.

Antagonizing Sec62 function in intracellular Ca2+ homeostasis represents a novel therapeutic strategy for head and neck cancer.

Various cancer types including head and neck squamous cell carcinomas (HNSCC) show a frequent amplification of chromosomal region 3q26 that encodes, among others, for the SEC62 gene. Located in the ER membrane, this translocation protein is known to play a critical role as a potential driver oncogene in cancer development. High SEC62 expression levels were observed in various cancer entities and were associated with a poor outcome and increased metastatic burden. Because of its intracellular localization the SEC62 protein is poorly accessible for therapeutic antibodies, therefore a functional SEC62 knockdown represents the most promising mechanism of a potential antineoplastic targeted therapy. By stimulating the Ca2+ efflux from the ER lumen and thereby increasing cellular stress levels, a functional inhibition of SEC62 bears the potential to limit tumor growth and metastasis formation. In this study, two potential anti-metastatic and -proliferative agents that counteract SEC62 function were investigated in functional in vitro assays by utilizing an immortalized human hypopharyngeal cancer cell line as well as a newly established orthotopic murine in vivo model. Additionally, a CRISPR/Cas9 based SEC62 knockout HNSCC cell line was generated and functionally characterized for its relevance in HNSCC cell proliferation and migration as well as sensitivity to SEC62 targeted therapy in vitro.