thapsigargin and Cell-Transformation--Viral

Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Activating transcription factor 4 (ATF4) is induced by various stressors, including endoplasmic reticulum (ER) and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Transcriptional regulation of the ATF4 gene under oxidative stress was investigated in human papillomavirus 16 (HPV-16)-transformed retinal pigment epithelial ARPE-19/HPV-16 cells.. Retinal pigment epithelial cells, trabecular meshwork cells, and corneal endothelial cells were treated with anoxia and thapsigargin (TG). Gene expression of ATF4 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and transcription factors was investigated by Western blot analysis, reporter assays, chromatin immunoprecipitation (ChIP) assays, and small interfering (si)RNA strategies. Cellular sensitivity to oxidative stress was determined.. The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by anoxia and TG. The Nrf2 regulator Keap1 was downregulated by anoxia. Downregulation of Nrf2 abolished ATF4 expression. On the other hand, downregulation of Keap1 enhanced the expression of both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA. The ChIP assays demonstrated that Nrf2 bound to the promoter of the ATF4 gene. Nrf2 downregulation nearly abolished the ATF4 induction by anoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by treatment with TG, HCA, H(2)O(2), and anoxia. However, stress induction of ATF4 promoter activity was observed, even when a mutation was introduced into the antioxidant-responsive elements site. Furthermore, stress induction of the ATF4 promoter was completely abolished when the 5' untranslated region of the ATF4 gene was deleted. Downregulation of ATF4 rendered ARPE-19/HPV-16 cells sensitive to oxidative stress.. These results suggest that the stress induction of ATF4 is significantly regulated transcriptionally through a Nrf2-dependent mechanism and may be a double-edged sword in the pathogenesis of various retinopathies.

Topics: Activating Transcription Factor 4; Blotting, Western; Cell Line, Transformed; Cell Transformation, Viral; Chromatin Immunoprecipitation; Endothelium, Corneal; Enzyme Inhibitors; Gene Expression Regulation; Human papillomavirus 16; Humans; Hydrogen Peroxide; Hypoxia; NF-E2-Related Factor 2; Oxidative Stress; Plasmids; Retinal Pigment Epithelium; Thapsigargin; Trabecular Meshwork

Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.

Topics: B-Lymphocytes; Calcium; Calcium Channels; Cell Line, Tumor; Cell Transformation, Viral; Endoplasmic Reticulum; Enzyme Inhibitors; Herpesvirus 4, Human; Humans; ORAI1 Protein; Thapsigargin; Viral Matrix Proteins

Transient phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2alpha phosphorylation-dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2alpha phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress-inducible eIF2alpha kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2alpha kinase, Fv2E-PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E-PERK activation enhanced the expression of numerous stress-induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2alpha phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress-induced signals and that activation of this pathway can single-handedly promote a stress-resistant preconditioned state.

Topics: Activating Transcription Factor 4; Animals; Cell Line, Transformed; Cell Survival; Cell Transformation, Viral; Cytoprotection; Dimerization; eIF-2 Kinase; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Glutamates; Mice; Oxidative Stress; Pancreas; Phosphorylation; Protein Biosynthesis; Protein Structure, Tertiary; Reactive Oxygen Species; Recombinant Fusion Proteins; Retroviridae; Signal Transduction; Tacrolimus; Thapsigargin; Trans-Activators; Tunicamycin

Elevated basal intracellular calcium (Ca(2+)) levels ([Ca(2+)](B)) in B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) patients implicate altered Ca(2+) homeostasis in this illness. Chronic lithium treatment affects key proteins modulating intracellular Ca(2+) signaling. Thus, we sought to determine if chronic exposure to therapeutic lithium concentrations also modifies intracellular Ca(2+) homeostasis in this surrogate cellular model of signal transduction disturbances in BD. BLCLs from BD-I (N=26) and healthy subjects (N=17) were regrown from frozen stock and incubated with 0.75 mM lithium or vehicle for 24 h (acute) or 7 days (chronic). [Ca(2+)](B), lysophosphatidic acid (LPA)-stimulated Ca(2+) mobilization ([Ca(2+)](S)), and thapsigargin-induced store-operated Ca(2+) entry (SOCE) were determined using ratiometric fluorometry with Fura-2. Compared with vehicle, chronic lithium exposure resulted in significantly higher [Ca(2+)](B) (F=8.47; p=0.006) in BLCLs from BD-I and healthy subjects. However, peak LPA-stimulated [Ca(2+)](S) and SOCE were significantly reduced (F=11.1, p=0.002 and F=8.36, p=0.007, respectively). Acute lithium exposure did not significantly affect measured parameters. In summary, the effect of chronic lithium to elevate [Ca(2+)](B) in BLCLs while attenuating both receptor-stimulated and SOCE components of intracellular Ca(2+) mobilization in BLCLs suggests that modulation of intracellular Ca(2+) homeostasis may be important to the therapeutic action of lithium.

Topics: Adult; B-Lymphocytes; Bipolar Disorder; Calcium; Case-Control Studies; Cell Count; Cell Line; Cell Transformation, Viral; Chi-Square Distribution; Drug Administration Schedule; Enzyme Inhibitors; Female; Fura-2; Herpesvirus 4, Human; Homeostasis; Humans; Intracellular Space; Lithium; Lysophospholipids; Male; Multivariate Analysis; Thapsigargin; Time Factors

The transverse redistribution of plasma membrane phosphatidylserine is one of the hallmarks of cells undergoing apoptosis and also occurs in cells fulfilling a more specialized function, such as platelets after appropriate activation. Although an increase in intracellular Ca2+ is required to trigger the remodeling of the plasma membrane, little information regarding intracellular signals leading to phosphatidylserine externalization has been provided. Scott syndrome is an extremely rare inherited disorder of the migration of phosphatidylserine toward the exoplasmic leaflet of the plasma membrane of stimulated blood cells. We have studied here the intracellular Ca2+ mobilization and Ca2+ entry involved in tyrosine phosphorylation in Epstein Barr virus (EBV)-infected B cells derived from a patient with Scott syndrome, her daughter, and control subjects. An alteration of Ca2+ entry through the plasma membrane and subsequent tyrosine phosphorylation induced by Ca2+ were observed in Scott EBV-B cells, but the release of Ca2+ from intracellular stores was normal. Furthermore, phosphatidylserine externalization at the surface of stimulated cells does not depend on tyrosine kinases. These results suggest that the defect of phosphatidylserine exposure in Scott syndrome cells is related to the alteration of a particular way of Ca2+ entry, referred to as capacitative Ca2+ entry, although some differences may be related to the cell type. Hence, this genetic mutant testifies to the prime significance of Ca2+ signaling in the regulation of phosphatidylserine expression at the surface of stimulated cells.

Topics: Aged; B-Lymphocytes; Blood Coagulation Disorders; Calcimycin; Calcium Channels; Cell Membrane; Cell Transformation, Viral; Cells, Cultured; Enzyme Inhibitors; Female; Genistein; Humans; Lymphocyte Activation; Phosphatidylserines; Phospholipids; Phosphorylation; Protein-Tyrosine Kinases; Syndrome; Thapsigargin; Tyrosine

In the present study, the bombesin-induced changes in cytosolic free Ca2+ ([Ca2+]i) were investigated in single Fura-2 loaded SV-40 transformed hamster beta-cells (HIT). Bombesin (50-500 pM) caused frequency-modulated repetitive Ca2+ transients. The average frequency of the Ca2+ transients induced by bombesin (200 pM) was 0.58 +/- 0.02 min-1 (n = 121 cells). High concentrations of bombesin (> or = 2 nM) triggered a large initial Ca2+ transient followed by a sustained plateau or by a decrease to basal levels. In Ca(2+)- free medium, bombesin caused only one or two Ca2+ transients and withdrawal of extracellular Ca2+ abolished the Ca2+ transients. The voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (50 microM) and nifedipine (10 microM), reduced amplitude and frequency of the Ca2+ transients and stopped the Ca2+ transients in some cells. Thapsigargin caused a sustained rise in [Ca2+]i in the presence of extracellular Ca2+ while in its absence the rise in [Ca2+]i was transient. Verapamil (50 microM) inhibited the thapsigargin-induced increase in [Ca2+]i by about 50%. Depletion of intracellular Ca2+ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca(2+)-free medium caused an agonist-independent increase in [Ca2+]i when extracellular Ca2+ was restored, which was larger than in control cells that had been incubated in Ca(2+)-free medium for the same period of time. This rise in [Ca2+]i and the thapsigargin-induced increase in [Ca2+]i were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca2+ pool enhances Ca2+ influx through VDCC and voltage-independent Ca2+ channels (VICC). In conclusion, the bombesin-induced Ca2+ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca2+ transients. Intracellular Ca2+ is mobilized during each Ca2+ transient, but Ca2+ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca2+ response. Ca2+ influx in whole or part is activated by a capacitative Ca2+ entry mechanism.

Topics: Animals; Bombesin; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Line, Transformed; Cell Transformation, Viral; Chelating Agents; Cricetinae; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Insulin; Insulin Secretion; Intracellular Fluid; Islets of Langerhans; Nifedipine; Thapsigargin; Verapamil

Calcium homeostasis has long been thought to be altered in transformed cells but mechanisms have not been established. In this study, the photoprotein, aequorin, was used to examine calcium regulation in 3T3 and SV40-transformed 3T3 cells. It was found that calcium transients induced by bradykinin or serum in serum-starved cells are lower and delayed in the transformed cells and decay kinetics are altered. These changes are not related to differences in cell cycle distribution. Though the serum transient is insensitive to nifedipine, verapamil, or lanthanum, removal of extracellular calcium accelerates transient decay in both cell types. Treatment of unstimulated cells with the ER Ca(2+)-ATPase inhibitor, thapsigargin, causes a 4-5-fold greater increase in [Ca2+]i in the transformed than in the nontransformed cells. Following serum stimulation, transformed cells still exhibit a large thapsigargin-induced increase in [Ca2+]i whereas the response in nontransformed cells is nearly abolished. When the 3T3 or SV3T3 cells are exposed to serum or thapsigargin in the absence of extracellular calcium and subsequently exposed to 11.8 mM Ca2+, a much greater influx of calcium again occurs in the SV3T3 cells. The observed changes in SV3T3 cells are most likely due to an alteration in a capacitative mechanism which regulates influx of calcium through the plasma membrane.

Topics: 3T3 Cells; Animals; Bradykinin; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Cell Compartmentation; Cell Cycle; Cell Line, Transformed; Cell Transformation, Viral; Ion Channel Gating; Kinetics; Mice; Simian virus 40; Terpenes; Thapsigargin