Involvement of ERK MAP kinase in endoplasmic reticulum stress in SH-SY5Y human neuroblastoma cells

Endoplasmic reticulum (ER) stress has increasingly come into focus as a factor contributing to neuronal injury. Although caspase-dependent mechanisms have been implicated in ER stress, the signaling pathways involved remain unclear. In this study, we examined the role of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase pathway that is highly conserved in many systems for balancing cell survival and death. Prolonged treatment of the human neuroblastoma cell line SH-SY5Y with thapsigargin, an inducer of ER stress, increased cell death over 24-48 h, as measured by LDH release. Caspases were involved; increased levels of active caspase-3 and cleaved caspase substrate PARP were detected, and treatment with Z-VAD-FMK reduced thapsigargin-induced cytotoxicity. In contrast, inhibition of calpain was not protective, although calpain was activated following thapsigargin treatment. An early and transient phosphorylation of ERK1/2 occurred after thapsigargin-induced ER stress, and targeting this pathway with the MEK inhibitors U0126 or PD98059 significantly reduced cell death. Similar cytoprotection was obtained against brefeldin A, another ER stress agent. However, protection against ER stress via ERK inhibition was not accompanied by amelioration of caspase-3 activation, PARP cleavage, or DNA laddering. These data indicate that ERK may contribute to non-caspase-dependent pathways of injury after ER stress.     

Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-alpha or thapsigargin

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. The proliferation of the cells was significantly suppressed in transfectants cultured for 72 h, as shown previously (Tsurusaki and Yamaguchi [2003]: J Cell Biochem 90:619-626). After culture for 72 h, cells were further cultured for 24-72 h in medium without FBS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The number of wild-type cells was significantly decreased by culture for 42 or 72 h in the presence of TNF-alpha (0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The effect of TNF-alpha (0.1 or 1 ng/ml) or thapsigargin (10(-7) or 10(-6) M) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. The presence of TNF-alpha (10 ng/ml) or thapsigargin (10(-5) M) caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity in wild-type cells was significantly increased by culture with TNF-alpha (10 ng/ml) for 48 or 72 h. This increase was significantly prevented in transfectants. Culture with thapsigargin (10(-5) M) caused a significant increase in Ca(2+)/calmodulin-dependent NO synthase activity in wild-type cells or transfectants. TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with N omega-nitro-L-arginine (10(-4) M), an inhibitor of caspase. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with thapsigargin (10(-6) M), and this DNA fragmentation was not suppressed by culture with caspase inhibitor. Thapsigargin-induced DNA fragmentation was significantly suppressed in transfectants cultured with or without caspase inhibitor. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by TNF-alpha or thapsigargin.     

Intracellular calcium puffs in osteoclasts

We studied intracellular calcium ([Ca(2+)](i)) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. Acid secretion and [Ca(2+)](i) were followed using H(+)-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was approximately 100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca(2+)](i) transients to 500-1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca(2+)](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca(2+)](i) transients in osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H(+)-ATPase activity was insensitive to calcium in the 100 nM-1 microM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.     

Thapsigargin increases apoptotic cell death in human hepatoma BEL—7404 cells

Effects of thapsigargin,an inhibitor of Ca^2 -ATPase in surface of endoplasmic reticulum,on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron microscopy.Propidium iodide staining and flow cytometry revealed that in the serum-free condition,thapsigargin increased the rate of apoptosis of BEL-7404 cells in a dose-dependent manner.Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment.Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation,apoptotic bodies existed in TG-treated cells,supporting that thapsigargin is a potent activator of apoptosis in the cells.     

Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts

Summary Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30–50 m) of indo-1 and with high concentrations (3.5–4.5mm) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca²⁺] _i ) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases, aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca²⁺] _i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.     

Deformation of the Bowditch staircase in Ca2+-overloaded mammalian cardiac tissue — a calcium phenomenon?

Concentrations of 1–4 mol l^–1 isoproterenol cause both in right ventricular papillary muscles and in enzymatically isolated myocytes of the guinea-pig a Ca^2– overload-induced state which is functionally characterized by biphasic (multiphasic) twitches and biphasic (multiphasic) intracellular calcium transients, respectively, during excitation-contraction coupling. This state was stabilized in the in vitro experiments for some hours by a co-ordination of the interstimulus interval, the temperature of the superfasion fluid and the addition of calcium agonists. The functional stability is the precondition for the reproducibility of the experimental results particularly after the application of long-lasting stimulation programmes. Changes in the shape of biphasic contractions were compared with changes in the time course of biphasic intracellular calcium transients using three manipulations of a different kind: (1) the interruption of the steady pacing rhythm, (2) the variation of the interstimulus interval, (3) the addition of ryanodine. It was shown that: (1) The BOWDITCH staircase in calcium overloaded multicellular preparations is changed in that each individual component of the twitch passes through its own staircase. A homologous behaviour can be observed in the configuration of the phasic and tonic component of biphasic intracellular calcium transients. (2) At different driving frequencies the relative proportion of the two components of a biphasic twitch corresponds to the time integrals of the two components of biphasic intracellular calcium transients. (3) Ryanodine suppresses both the first component of the biphasic twitch and the phasic component of the biphasic intracellular calcium transient.The SR Ca²⁺-ATPase inhibitor thapsigargin increases the second component of the biphasic calcium transient. This supports the hypothesis that the size of the tonic component is in part determined by intracellular calcium reuptake. The results of both kinds of experiments would be compatible with the assumption that in calcium overloaded mammalian cardiac cells calcium reaches the contractile system directly as well as via two intracellular stores (extended two-Ca-store concept).     

Effect of thapsigargin on inhibitory synaptic transmission between cultured neurons of the rat hippocampus

We carried out electrophysiological experiments on cultured neurons of the rat hippocampus. The voltage-clamp technique and extracellular stimulation of single presynaptic axons were used for measurements of the evoked inhibitory postsynaptic currents (eIPSCs). It was found that 1 μM thapsigargin is capable of modulating inhibitory synaptic transmission, and the effects were ambivalent. Among 21 examined cells, eIPSCs decreased in 15 neurons and were augmented in 6 units; the kinetic parameters of these currents underwent no changes. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 374–376, July–October, 2007.     

Resveratrol derivatives increase cytosolic calcium by inhibiting plasma membrane ATPase and inducing calcium release from the endoplasmic reticulum in prostate cancer cells

Resveratrol (RES) is a putative chemotherapeutic naturally found in grapes, peanuts, and Japanese knotweed. Previous studies demonstrate that RES modulates calcium signaling as part of its chemotherapeutic activity. In this study, we determined the chemotherapeutic activity of three RES esters that have been modified at the 4’ hydroxyl by the addition of pivalate, butyrate, and isobutyrate. All of the RES derivatives disrupted the calcium signaling in prostate cancer cells more than the parent compound, RES. Further, we demonstrate that the RES derivatives may disrupt the calcium homeostasis by activating calcium release from the endoplasmic reticulum and inhibiting plasma membrane Ca²⁺-ATPase. The pivalated and butyrated RES derivatives decreased cell viability significantly more than RES. Because pivalated and butyrated RES are more effective than RES at targeting calcium signaling pathways, pivalated and butyrated RES may serve as more effective chemotherapeutics.     

Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

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Substrate-induced conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase probed by surface modification using diethylpyrocarbonate with mass spectrometry

We have identified 15 residues from the surface of sarcoplasmic reticulum Ca²⁺-pump ATPase, by mass spectrometry using diethylpyrocarbonate modification. The reactivity of 9 residues remained high under all the conditions. The reactivity of Lys-515 at the nucleotide site was severely inhibited by ATP, whereas that of Lys-158 in the A-domain decreased by one-half and increased by five-fold in the presence of Ca²⁺ and MgF₄, respectively. These are well explained by solvent accessibility, pK_a and nearby hydrophobicity of the reactive atom on the basis of the atomic structure. However, the reactivity of 4 residues near the interface among A-, N- and P-domain suggested larger conformational changes of these domains in membrane upon binding of Ca²⁺ (Lys-436), ATP (Lys-158) and MgF₄ (His-5, -190, Lys-436).