Abstract
This study is aimed to determine the role of calcium signaling evoked by the calcium-mobilizing agonist uridine-5′-triphosphate (UTP) and by the specific inhibitor of the endoplasmic reticulum calcium reuptake thapsigargin on caspase activation in human leukemia cell line HL-60. We have analyzed cytosolic free calcium concentration ([Ca2+]c) determination, mitochondrial membrane potential and caspase-3 and -9 activity by fluorimetric methods, using the fluorescent ratiometric calcium indicator Fura-2, the dye JC-1, and specific fluorogenic substrate, respectively. Our results indicated that treatment of HL-60 cells with 10 μM UTP or 1 μM thapsigargin induced a transient increase in [Ca2+]c due to calcium release from internal stores. The stimulatory effect of UTP and thapsigargin on calcium signal was followed by a mitochondrial membrane depolarization. Our results also indicated that UTP and thapsigargin were able to increase the caspase-3 and -9 activities. The effect of UTP and thapsigargin on caspase activation was time dependent, reaching a maximal caspase activity after 60 min of stimulation. Loading of cells with 10 μM dimethyl BAPTA, an intracellular calcium chelator, for 30 min significantly reduced both UTP- or thapsigargin-induced mitochondrial depolarization and caspase activation. Similar results were obtained when the cells were pretreated with 10 μM Ru360 for 30 min, a specific blocker of calcium uptake into mitochondria. The findings suggest that UTP- and thapsigargin-induced caspase-3 and -9 activation and mitochondrial membrane depolarization is dependent on rises in [Ca2+]c in human myeloid HL-60 cells.
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Abbreviations
- BAPTA-AM:
-
1,2-bis(o-aminophenoxy)ethane-N,N,N′,N-tetraacetic acid-acetoxymethyl ester
- [Ca2+]c :
-
Cytosolic free calcium concentration
- CHAPS:
-
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate
- DMSO:
-
Dimethylsulfoxide
- DTT:
-
Dithiothreitol
- EGTA:
-
Ethylene glycol-bis (2-aminoethyleter)-N,N,N′,N′-tetraacetic acid
- ER/SR:
-
Sarco-endoplasmic reticulum
- FCCP:
-
Carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone
- HEPES:
-
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- IP3 :
-
Inositol 1,4,5-triphosphate
- PLC:
-
Phospholipase C
- ROS:
-
Reactive oxygen species
- SERCA:
-
Sarco-endoplasmic reticulum Ca2+-ATPase
- TG:
-
Thapsigargin
- UTP:
-
Uridine-5′-triphosphate
References
Fadeel B, Orrenius S (2005) Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med 258:479–517
Groenendyk J, Michalak M (2005) Endoplasmic reticulum quality control and apoptosis. Acta Biochim Pol 52:381–395
Stennicke HT, Salvesen GS (1997) Biochemical characteristics of caspases-3, -6, -7, and -8. J Biol Chem 272:25123–25719
Shi Y (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9:459–470
Hajnóczky G, Davies E, Madesh M (2003) Calcium signaling and apoptosis. Biochem Biophys Res Commun 304:445–454
Ghafourifar P, Schenk U, Klein SD, Richter C (1999) Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation. J Biol Chem 274:31185–31188
Starkov AA, Polster BM, Fiskum G (2002) Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax. J Neurochem 83:220–228
Verghese MW, Kneisler TB, Boucheron JA (1996) P2U agonists induce chemotaxis and actin polymerization in human neutrophils and differentiated HL-60 cells. J Biol Chem 271:15597–15601
Alemany R, Sichelschmidt B, zu Heringdorf DM, Lass H, van Koppen CJ, Jakobs KH (2000) Stimulation of sphingosine-1-phosphate formation by the P2Y2 receptor in HL-60 cells: Ca2+ requirement and implication in receptor-mediated Ca2+ mobilization, but not MAP kinase activation. Mol Pharmacol 58:491–497
Suh BC, Kim TD, Lee IS, Kim KT (2000) Differential regulation of P2Y11 receptor-mediated signalling to phospholipase C and adenylyl cyclase by protein kinase C in HL-60 promyelocytes. Br J Pharmacol 131:489–497
Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Bio 4:517–529
Bejarano I, Terrón MP, Paredes SD, Barriga C, Rodríguez AB, Pariente JA (2007) Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation. Mol Cell Biochem 296:77–84
Grynkiewicz C, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Granados MP, Salido GM, Pariente JA, González A (2005) Effect of H2O2 on CCK-8-evoked changes in mitochondrial activity in isolated mouse pancreatic acinar cells. Biol Cell 97:847–856
Bejarano I, Redondo PC, Espino J, Rosado JA, Paredes SD, Barriga C, Reiter RJ, Pariente JA, Rodríguez AB (2009) Melatonin induces mitochondrial-mediated apoptosis in human myeloid HL-60 cells. J Pineal Res 46:392–400
Demaurex N, Distelhorst C (2003) Cell biology. Apoptosis—the calcium connection. Science 300:65–67
Rao RV, Ellerby HM, Bredesen DE (2004) Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ 11:372–380
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489
Colegrove SL, Albrecht MA, Friel DD (2000) Quantitative analysis of mitochondrial Ca2+ uptake and release pathways in sympathetic neurons. Reconstruction of the recovery after depolarization-evoked [Ca2+]i elevations. J Gen Physiol 115:371–388
Kovács R, Schuchmann S, Gabriel S, Kann O, Kardos J, Heinemann U (2002) Free radical-mediated cell damage after experimental status epilepticus in hippocampal slice cultures. J Neurophysiol 88:2909–2918
Naziroğlu M, Kutluhan S, Yilmaz M (2008) Selenium and topiramate modulates brain microsomal oxidative stress values, Ca2+-ATPase activity, and EEG records in pentylentetrazol-induced seizures in rats. J Membr Biol 225:39–49
Naziroğlu M (2007) New molecular mechanisms on the activation of TRPM2 channels by oxidative stress and ADP-ribose. Neurochem Res 32:1990–2001
Naziroğlu M (2009) Role of selenium on calcium signaling and oxidative stress-induced molecular pathways in epilepsy. Neurochem Res. doi:10.1007/s11064-009-0015-8
White JA, Blackmore PF, Schoenbach KH, Beebe SJ (2004) Stimulation of capacitative calcium entry in HL-60 cells by nanosecond pulsed electric fields. J Biol Chem 279:22964–22972
Bejarano I, Lozano GM, Ortiz A, García JF, Paredes SD, Rodríguez AB, Pariente JA (2008) Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone. Fertil Steril 90:1340–1347
Rosado JA, López JJ, Gómez-Arteta E, Redondo PC, Salido GM, Pariente JA (2006) Early caspase-3 activation independent of apoptosis is required for cellular function. J Cell Physiol 209:142–152
Lei X, Zhang S, Bohrer A, Ramanadham S (2008) Calcium-independent Phospholipase A2 (iPLA2{beta})-mediated ceramide generation plays a key role in the cross-talk between the endoplasmic reticulum (ER) and mitochondria during ER stress-induced insulin-secreting cell apoptosis. J Biol Chem 283:34819–34832
Meldolesi J, Pozzan T (1998) The endoplasmic reticulum Ca2+ store: a view from the lumen. Trends Biochem Sci 23:10–14
Pigozzi D, Tombal B, Ducret T, Vacher P, Gaily P (2004) Role of store-dependent influx of Ca2+ and efflux of K+ in apoptosis of CHO cells. Cell Calcium 36:421–430
Putney JW Jr, Ribeiro CM (2000) Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores. Cell Mol Life Sci 57:1272–1286
Hajnóczky G, Csordas G, Das S, Garcia-Perez C, Saotome M, Roy SS, Yi M (2006) Mitochondrial calcium signaling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40:553–560
Csordas G, Thomas AP, Hajnozcky G (1999) Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria. EMBO J 18:96–108
Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz LM, Tuft RA, Pozzan T (1998) Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280:1763–1766
Camello-Almaraz C, Salido GM, Pariente JA, Camello PJ (2002) Role of mitochondria in Ca2+ oscillations and shape of Ca2+ signals in pancreatic acinar cells. Biochem Pharmacol 63:283–292
Filippin L, Magalhaes PJ, Di Benedetto G, Colella M, Pozzan T (2003) Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria. J Biol Chem 278:39224–39234
Hajnoczky G, Csordas G, Madesh M, Pacher P (2000) The machinery of local Ca2+ signalling between sarco-endoplasmic reticulum and mitochondria. J Physiol 529:69–81
Vay L, Hernández-SanMiguel E, Santo-Domingo J, Lobatón CD, Moreno A, Montero M, Alvarez J (2007) Modulation of Ca2+ release and Ca2+ oscillations in HeLa cells and fibroblasts by mitochondrial Ca2+ uniporter stimulation. J Physiol 580:39–49
Acknowledgments
This work was supported by MEC-DGI and Junta de Extremadura grants BFU2007-60091 and PRI07-A024, respectively. I. Bejarano was beneficiary of grant by Junta de Extremadura PRE06070.
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González, D., Espino, J., Bejarano, I. et al. Caspase-3 and -9 are activated in human myeloid HL-60 cells by calcium signal. Mol Cell Biochem 333, 151–157 (2010). https://doi.org/10.1007/s11010-009-0215-1
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DOI: https://doi.org/10.1007/s11010-009-0215-1