thapsigargin and salubrinal

To investigate the impact of alpha subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation on liver regeneration.. Male BALB/c mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce liver injury. Human hepatocyte LO2 cells were incubated with thapsigargin to induce endoplasmic reticulum (ER) stress. Salubrinal, integrated stress response inhibitor (ISRIB), and DnaJC3 overexpression were used to alter eIF2α phosphorylation levels.. CCl4 administration induced significant ER stress and eIF2α phosphorylation, and increased hepatocyte proliferation proportionally to the extent of injury. Inhibiting eIF2α dephosphorylation with salubrinal pretreatment significantly mitigated liver injury and hepatocyte proliferation. In LO2 cells, thapsigargin induced significant eIF2α phosphorylation and inhibited proliferation. Inhibiting eIF2α dephosphorylation partly restored cell proliferation during ER stress.. In acute liver injury, inhibiting eIF2α dephosphorylation protects injured hepatocytes and reduces hepatocyte proliferation.

Topics: Animals; Apoptosis; Carbon Tetrachloride; Cell Proliferation; Chemical and Drug Induced Liver Injury; Cinnamates; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Hepatocytes; HSP40 Heat-Shock Proteins; Humans; Liver Regeneration; Male; Mice; Mice, Inbred BALB C; Phosphorylation; Thapsigargin; Thiourea

This study aimed to investigate the differential responses of trabecular meshwork stem cells (TMSCs) and trabecular meshwork (TM) cells to endoplasmic reticulum (ER) stress inducers.. Human TM cells and TMSCs were exposed to tunicamycin, brefeldin A, or thapsigargin. Cell apoptosis was evaluated by flow cytometry. ER stress markers were detected by quantitative PCR, Western blotting, and immunostaining. Morphologic changes were evaluated by transmission electron microscopy. Cells were treated with the PERK inhibitor GSK2606414 or the elF2α dephosphorylation inhibitor Salubrinal together with tunicamycin to evaluate their effects on ER stress.. Both TMSCs and TM cells underwent apoptosis after 48- and 72-hour treatment with ER stress inducers. ER stress triggered the unfolded protein response (UPR) with increased expression of GRP78, sXBP1, and CHOP, which was significantly lower in TMSCs than TM cells. Swollen ER and mitochondria were detected in both TMSCs and TM cells. Neither GSK2606414 nor salubrinal alone activated UPR. GSK2606414 significantly reduced cell survival rates after tunicamycin treatment, and salubrinal increased cell survival rates. The increased expression of GRP78, sXBP1, CHOP, and GADD34 peaked at 6 or 12 hours and lasted longer in TM cells than TMSCs. Salubrinal treatment dramatically increased OCT4 and CHI3L1 expression in TMSCs.. In response to ER stress inducers, TMSCs activated a lower level of UPR and lasted shorter than TM cells. Inhibition of elF2α dephosphorylation had a protective mechanism against cell death. Stem cells combined with salubrinal may be a more effective way for TM regeneration in glaucoma.

Topics: Adenine; Anti-Bacterial Agents; Apoptosis; Biomarkers; Blotting, Western; Brefeldin A; Cell Survival; Cinnamates; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Indoles; Microscopy, Electron, Transmission; Real-Time Polymerase Chain Reaction; Stem Cells; Thapsigargin; Thiourea; Trabecular Meshwork; Tunicamycin; Unfolded Protein Response

Accumulation of misfolded proteins and alterations in calcium homeostasis induces endoplasmic reticulum (ER) stress, leading to apoptosis. In this study, we tested the hypothesis that β-AR stimulation induces ER stress, and induction of ER stress plays a pro-apoptotic role in cardiac myocytes. Using thapsigargin and brefeldin A, we demonstrate that ER stress induces apoptosis in adult rat ventricular myocytes (ARVMs). β-AR-stimulation (isoproterenol; 3h) significantly increased expression of ER stress proteins, such as GRP-78, Gadd-153, and Gadd-34, while activating caspase-12 in ARVMs. In most parts, these effects were mimicked by thapsigargin. β-AR stimulation for 15 min increased PERK and eIF-2α phosphorylation. PERK phosphorylation remained higher, while eIF-2α phosphorylation declined thereafter, reaching to ~50% below basal levels at 3 h after β-AR stimulation. This decline in eIF-2α phosphorylation was prevented by β1-AR, not by β2-AR antagonist. Forskolin, adenylyl cyclase activator, simulated the effects of ISO on eIF-2α phosphorylation. Salubrinal (SAL), an ER stress inhibitor, maintained eIF-2α phosphorylation and inhibited β-AR-stimulated apoptosis. Furthermore, inhibition of caspase-12 using z-ATAD inhibited β-AR-stimulated and thapsigargin-induced apoptosis. In vivo, β-AR stimulation induced ER stress in the mouse heart as evidenced by increased expression of GRP-78 and Gadd-153, activation of caspase-12, and dephosphorylation of eIF-2α. SAL maintained phosphorylation of eIF-2α, inhibited activation of caspase-12, and decreased β-AR-stimulated apoptosis in the heart. Thus, β-AR stimulation induces ER stress in cardiac myocytes and in the heart, and induction of ER stress plays a pro-apoptotic role.

Topics: Adenylyl Cyclases; Adrenergic beta-1 Receptor Antagonists; Adrenergic beta-2 Receptor Antagonists; Animals; Antigens, Differentiation; Apoptosis; Brefeldin A; Caspase 12; Caspase Inhibitors; Cells, Cultured; Cinnamates; Colforsin; eIF-2 Kinase; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Heat-Shock Proteins; Isoproterenol; Male; Mice; Myocytes, Cardiac; Phosphorylation; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Signal Transduction; Thapsigargin; Thiourea; Transcription Factor CHOP

Fatty acids such as palmitic acid at high levels are known to induce endoplasmic reticulum (ER) stress and lipotoxicity in numerous cell types and thereby contribute to cellular dysfunctions in obesity. To understand the impact of high fatty acids on oocytes, ER stress and lipotoxicity were induced in mouse cumulus-oocyte complexes during in vitro maturation using the ER Ca(2+) channel blocker thapsigargin or high physiological levels of palmitic acid; both of which significantly induced ER stress marker genes (Atf4, Atf6, Xbp1s, and Hspa5) and inositol-requiring protein-1α phosphorylation, demonstrating an ER stress response that was reversible with the ER stress inhibitor salubrinal. Assessment of pentraxin-3, an extracellular matrix protein essential for fertilization, by immunocytochemistry and Western blotting showed dramatically impaired secretion concurrent with ER stress. Mitochondrial activity in oocytes was assessed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining of inner mitochondrial membrane potential, and oocytes matured in thapsigargin or high-dose palmitic acid had significantly reduced mitochondrial activity, reduced in vitro fertilization rates, and were slower to develop to blastocysts. The deficiencies in protein secretion, mitochondrial activity, and oocyte developmental competence were each normalized by salubrinal, demonstrating that ER stress is a key mechanism mediating fatty acid-induced defects in oocyte developmental potential.

Topics: Animals; C-Reactive Protein; Cells, Cultured; Cinnamates; Cumulus Cells; Embryo Culture Techniques; Embryonic Development; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Female; Fertilization in Vitro; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Oocytes; Palmitic Acid; Serum Amyloid P-Component; Thapsigargin; Thiourea

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

Topics: Carcinogens; Cell Line; Cinnamates; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Epstein-Barr Virus Infections; Eukaryotic Initiation Factor-2; Gene Expression Profiling; Gene Expression Regulation, Viral; Genes, Immediate-Early; Germinal Center; Herpesvirus 4, Human; Humans; Immediate-Early Proteins; Lymphocytes; Lymphoma; Membrane Glycoproteins; Plasma Cells; Tetradecanoylphorbol Acetate; Thapsigargin; Thiourea; Trans-Activators; Viral Matrix Proteins; Virus Replication

T cells alter their functional phenotype during the evolution of an immune response (intra-lineage differentiation), but the driving forces to this plastic intra-lineage differentiation are poorly understood. The endoplasmic reticulum (ER) stress response is a possible critical event for the initial T cell differentiation upon antigen recognition. Here we studied the relationship between ER and Il-10 transcription in human Treg clones. The induction of ER stress with a canonical stressor, thapsigargin, enhances Il-10 transcription. Salubrinal, a small molecule inhibitor of the eukaryotic translation initiation factor 2α (eIF2α) dephosphporylation, dramatically inhibits it. Il-10 transcription is also enhanced by exogenous TNFα. These results disclose a role for ER stress in driving T cell plasticity.

Topics: Antigens, Differentiation; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Cycle Proteins; Cell Differentiation; Cinnamates; Clone Cells; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Eukaryotic Initiation Factor-2; Forkhead Transcription Factors; Gene Expression; Heat-Shock Proteins; Humans; Interferon-gamma; Interleukin-10; Interleukin-23 Subunit p19; Mucocutaneous Lymph Node Syndrome; Protein Phosphatase 1; Stress, Physiological; T-Lymphocytes, Regulatory; Thapsigargin; Thiourea; Transcription Factor CHOP; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Cells adapt to endoplasmic reticulum (ER)-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD), however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.. Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.. Although PP1 activity does not play a major role in regulating the ER stress response in leukemic cells, phosphatase signaling nevertheless significantly limits proteasome inhibitor-mediated ER-stress and apoptosis. Inclusion of specific phosphatase inhibitors might therefore represent an option to improve current proteasome inhibitor-based treatment modalities for hematological cancers.

Topics: Activating Transcription Factor 4; Apoptosis; Cell Cycle; Cell Line, Tumor; Cinnamates; DNA-Binding Proteins; Drug Synergism; Endoplasmic Reticulum; Enzyme Inhibitors; Genes, abl; Humans; K562 Cells; Leukemia; Phosphoric Monoester Hydrolases; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Phosphatase 2; Regulatory Factor X Transcription Factors; RNA, Messenger; Thapsigargin; Thiourea; Transcription Factor CHOP; Transcription Factors; X-Box Binding Protein 1

The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), enhances cisplatin [cis-diammine dichloroplatinum (II)] (CDDP)-induced apoptosis in the oral squamous cell carcinoma (OSCC) cell line by complex, multifunctional mechanisms. We investigated the role of endoplasmic reticulum (ER) stress in the enhancing effect of SAHA on CDDP, compared with the ER stressor thapsigargin.. We chose OSCC cell line HSC-3 to ascertain the mechanism of SAHA-enhanced cytotoxicity among various cell lines. HSC-3 cells were incubated with CDDP/SAHA for 48 h, followed by the assessment of cell chemosensitivity to CDDP with MTT and TUNEL assays. Western blot analysis was used to detect the expressions of ER-related molecules, and flow cytometry was used to monitor caspase activity.. Treatment with CDDP/SAHA potently induced apoptosis in HSC-3 cells with a significant increase in caspase-4 and -12 functions. For example, 60% of cells became apoptotic after 48 h of treatment with CDDP/SAHA. In addition, SAHA alone rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)alpha, which is up-regulated during ER stress. Inhibition of ER stress by salubrinal, an inhibitor of eIF2alpha dephosphorylation, abrogated SAHA's enhancement of CDDP cytotoxicity. Levels of phospho-Akt are decreased in SAHA-treated cells, and this is in turn associated with increased activity of protein phosphatase 1 (PP1) by SAHA, the phosphatase upstream of Akt.. These data indicate that up-regulation of specific-ER stress-associated events is an integral part of the mechanism by which SAHA enhances CDDP-induced apoptosis, and PP1 up-regulation followed by Akt dephosphorylation plays an important role in SAHA-enhanced CDDP apoptosis.

Topics: Apoptosis; Carcinoma, Squamous Cell; Caspase 12; Caspase Inhibitors; Caspases, Initiator; Cell Line, Tumor; Cinnamates; Cisplatin; Drug Synergism; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Female; Heat-Shock Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; In Situ Nick-End Labeling; Membrane Glycoproteins; Mouth Neoplasms; Phosphorylation; Protein Phosphatase 1; Proto-Oncogene Proteins c-akt; Thapsigargin; Thiourea; Tunicamycin; Valproic Acid; Vorinostat

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-selective ion channel expressed in human lung cells. We show that activation of the intracellular subpopulation of TRPV1 causes endoplasmic reticulum (ER) stress and cell death in human bronchial epithelial and alveolar cells. TRPV1 agonist (nonivamide) treatment caused calcium release from the ER and altered the transcription of growth arrest- and DNA damage-inducible transcript 3 (GADD153), GADD45alpha, GRP78/BiP, ATF3, CCND1, and CCNG2) in a manner comparable with prototypical ER stress-inducing agents. The TRPV1 antagonist N-(4-tert-butylbenzyl)-N'-(1-[3-fluoro-4-(methylsulfonylamino)-phenyl]ethyl)thiourea (LJO-328) inhibited mRNA responses and cytotoxicity. EGTA and ruthenium red inhibited cell surface TRPV1 activity, but they did not prevent ER stress gene responses or cytotoxicity. Cytotoxicity paralleled eukaryotic translation initiation factor 2, subunit 1 (EIF2alpha) phosphorylation and the induction of GADD153 mRNA and protein. Transient overexpression of GADD153 caused cell death independent of agonist treatment, and cells selected for stable overexpression of a GADD153 dominant-negative mutant exhibited reduced sensitivity. Salubrinal, an inhibitor of ER stress-induced cytotoxicity via the EIF2alphaK3/EIF2alpha pathway, or stable overexpression of the EIF2alpha-S52A dominant-negative mutant also inhibited cell death. Treatment of the TRPV1-null human embryonic kidney 293 cell line with TRPV1 agonists did not initiate ER stress responses. Likewise, n-benzylnonanamide, an inactive analog of nonivamide, failed to cause ER calcium release, an increase in GADD153 expression, and cytotoxicity. We conclude that activation of ER-bound TRPV1 and stimulation of GADD153 expression via the EIF2alphaK3/EIF2alpha pathway represents a common mechanism for cytotoxicity by cell-permeable TRPV1 agonists. These findings are significant within the context of lung inflammatory diseases where elevated concentrations of endogenous TRPV1 agonists are probably produced in sufficient quantities to cause TRPV1 activation and lung cell death.

Topics: Activating Transcription Factor 3; Arachidonic Acids; Calcium; Capsaicin; Cell Line; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cinnamates; Cyclin D1; Cyclin G2; Cyclins; Diterpenes; Dithiothreitol; Endocannabinoids; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Epithelial Cells; Eukaryotic Initiation Factor-2; Gene Expression; Humans; Lung; Phosphorylation; Polyunsaturated Alkamides; Thapsigargin; Thiourea; Transcription Factor CHOP; Transfection; TRPV Cation Channels