Orai1 and STIM1 mediate SOCE and contribute to apoptotic resistance of pancreatic adenocarcinoma

The store-operated calcium channels (SOCs) represent one of the major calcium-entry pathways in non-excitable cells. SOCs and in particular their major components ORAI1 and STIM1 have been shown to be implicated in a number of physiological and pathological processes such as apoptosis, proliferation and invasion. Here we demonstrate that ORAI1 and STIM1 mediate store-operated calcium entry (SOCE) in pancreatic adenocarcinoma cell lines. We show that both ORAI1 and STIM1 play pro-survival anti-apoptotic role in pancreatic adenocarcinoma cell lines, as siRNA-mediated knockdown of ORAI1 and/or STIM1 increases apoptosis induced by chemotherapy drugs 5-fluorouracil (5-FU) or gemcitabine. We also demonstrate that both 5-FU and gemcitabine treatments increase SOCE in Panc1 pancreatic adenocarcinoma cell line via upregulation of ORAI1 and STIM1. Altogether our results reveal the novel calcium-dependent mechanism of action of the chemotherapy drugs 5-FU and gemcitabine and emphasize the anti-apoptotic role of ORAI1 and STIM1 in pancreatic adenocarcinoma cells. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Autophagy of cytoplasmic bulk cargo does not require LC3

To investigate the role of LC3 in bulk autophagy we compared its autophagic-lysosomal processing (using an improved quantitative immunoblotting method) with autophagic-lysosomal bulk cargo flux (measured by our established LDH [lactate dehydrogenase] sequestration assay) in amino acid-starved rat hepatocytes treated with cycloheximide to prevent new LC3 influx. Block-release experiments with the reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin (TG) showed that while only 3MA suppressed phagophoric LC3 attachment (lipidation), both inhibitors prevented phagophore closure (cargo sequestration). Upon release from closure blockade, some autophagic-lysosomal LC3 flux was resumed even in the presence of 3MA, i.e., without an accompanying bulk cargo flux. Conversely, whereas the autophagic-lysosomal flux of LC3 halted within ～100 min of cycloheximide treatment, the bulk cargo flux continued at a high rate. siRNA-mediated knockdown of LC3 family proteins in LNCaP prostate carcinoma cells confirmed that autophagy of cytoplasmic bulk cargo was completely LC3 independent also in these cells, and in the absence of cycloheximide. However, a strong requirement for GABARAP family proteins was evident. Since bulk autophagy of cytoplasm (macroautophagy) and autophagic-lysosomal LC3 processing may apparently be mutually independent, LC3 would seem to be unsuitable as a general indicator of autophagy.     

Contributions of intracellular compartments to calcium dynamics: implicating an acidic store

Many cells show a plateau of elevated cytosolic Ca(2+) after a long depolarization, suggesting delayed Ca(2+) release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic beta-cells show a thapsigargin-sensitive plateau ('hump') of Ca(2+) after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na(+)-Ca(2+) exchange and by only 18% upon blocking ryanodine or IP(3) receptors together. Further, the time course of ER Ca(2+) measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine beta-napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca(2+) into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in beta-cells. We conclude that the dipeptide compromises dense-core secretory granules and that these granules comprise an acidic calcium store in beta-cells whose loading and/or release is sensitive to thapsigargin and which releases Ca(2+) after cytosolic Ca(2+) elevation.     

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl⁻ channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl⁻ channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl⁻ channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively. These authors contributed equally to this work.     

Effects of thapsigargicin on Ca2+ movements in L1210 cells permeabilized with digitonin

The effect of thapsigargicin (TGC), a non-phorbol ester type tumor promoter, on Ca²⁺ movements has been investigated using L1210 mouse lymphoma cells. Ca²⁺ release from intact and digitonin permeabilized cells was evaluated using Fura-2 and Fura-3. TGC like Thapsigargin (TG) has the ability to discharge the intracellular Ca²⁺ stores and to increase intracellular free Ca²⁺ concentrations. TGC in a concentration dependent manner (0.16–16 nM) also inhibited cell growth and this effect was at least partially reversed by arachidonate. The article is published in the original.     

Correlation between inhibition of calcium—dependent apoptosis by cyclosporin A and calcium transportation in HL—60 cells

Both calcium ionophore A23187 and endoplasmic reticulum Ca^2 -ATPase inhibitor thapsigargin (Tg) could increse intracellular free calcium concentration and induce apoptosis in some cell lines.In the present study,we found that HL-60 cells treated with A23187 (1μg/ml) for 4h or with Tg(0.5μg/ml) for 2h showed typical characteristics of apoptosis.Pretreatment with nontoxic concentration of cyclosporin A (CsA) (1μg/ml) could block these effects.Flow cytometric analysis of intracellular Ca^2 after staining with fluo-3 AM showed that CsA did not prevent the increase of intracellular calcium induced by A23187 or Tg,but it could maintain the high level of intracellular Ca^2 for a long time.These results suggest that CsA may prevent calcium-induced apoptosis by blocking the transportation of Ca^2 in HL-60 cells.     

Erratum

Allatotropin (AT) is a 13 amino acid neuropeptide isolated from the tobacco hornworm, Manduca sexta, which stimulates the biosynthesis of juvenile hormone (JH) in adult females of this insect. In this paper we report that AT stimulates inositol phosphate formation in both female and male corpora allata (CA) of adult Manduca sexta. Furthermore, thapsigargin, a potent Ca²⁺-ATPase inhibitor, staurosporine, a potent inhibitor of protein kinases A and C, and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca²⁺ sequestration, all stimulate JH biosynthesis without changing the basal level of inositol phosphates. These data suggest that AT activates an inositol 1,4,5-trisphosphate (IP₃) pathway. Furthermore, increases in the free intracellular Ca²⁺ concentration in the CA stimulate JH biosynthesis. © 1992 Wiley-Liss, Inc.     

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

Pancreatic β-cell dysfunction is an early hallmark of type 1 diabetes mellitus. Among the potentially critical factors that cause β-cell dysfunction are cytokine attack, glucotoxicity, induction of endoplasmic reticulum (ER) or mitochondria stress. However, the exact molecular mechanism underlying β-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 β-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 β-cell dysfunction.