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Caspase-3 and -9 are activated in human myeloid HL-60 cells by calcium signal

  • D. González
  • J. Espino
  • I. Bejarano
  • J. J. López
  • A. B. Rodríguez
  • J. A. Pariente
Article

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.

Keywords

Apoptosis Caspases Calcium signal Thapsigargin HL-60 cells 

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

Notes

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.

References

  1. 1.
    Fadeel B, Orrenius S (2005) Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med 258:479–517CrossRefPubMedGoogle Scholar
  2. 2.
    Groenendyk J, Michalak M (2005) Endoplasmic reticulum quality control and apoptosis. Acta Biochim Pol 52:381–395PubMedGoogle Scholar
  3. 3.
    Stennicke HT, Salvesen GS (1997) Biochemical characteristics of caspases-3, -6, -7, and -8. J Biol Chem 272:25123–25719CrossRefGoogle Scholar
  4. 4.
    Shi Y (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9:459–470CrossRefPubMedGoogle Scholar
  5. 5.
    Hajnóczky G, Davies E, Madesh M (2003) Calcium signaling and apoptosis. Biochem Biophys Res Commun 304:445–454CrossRefPubMedGoogle Scholar
  6. 6.
    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 274:31185–31188CrossRefPubMedGoogle Scholar
  7. 7.
    Starkov AA, Polster BM, Fiskum G (2002) Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax. J Neurochem 83:220–228CrossRefPubMedGoogle Scholar
  8. 8.
    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 271:15597–15601CrossRefPubMedGoogle Scholar
  9. 9.
    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 58:491–497PubMedGoogle Scholar
  10. 10.
    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 131:489–497CrossRefPubMedGoogle Scholar
  11. 11.
    Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Bio 4:517–529CrossRefGoogle Scholar
  12. 12.
    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 296:77–84CrossRefPubMedGoogle Scholar
  13. 13.
    Grynkiewicz C, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450PubMedGoogle Scholar
  14. 14.
    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 97:847–856CrossRefPubMedGoogle Scholar
  15. 15.
    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 46:392–400CrossRefPubMedGoogle Scholar
  16. 16.
    Demaurex N, Distelhorst C (2003) Cell biology. Apoptosis—the calcium connection. Science 300:65–67CrossRefPubMedGoogle Scholar
  17. 17.
    Rao RV, Ellerby HM, Bredesen DE (2004) Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ 11:372–380CrossRefPubMedGoogle Scholar
  18. 18.
    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 91:479–489CrossRefPubMedGoogle Scholar
  19. 19.
    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 115:371–388CrossRefPubMedGoogle Scholar
  20. 20.
    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 88:2909–2918CrossRefPubMedGoogle Scholar
  21. 21.
    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 225:39–49CrossRefPubMedGoogle Scholar
  22. 22.
    Naziroğlu M (2007) New molecular mechanisms on the activation of TRPM2 channels by oxidative stress and ADP-ribose. Neurochem Res 32:1990–2001CrossRefPubMedGoogle Scholar
  23. 23.
    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 Google Scholar
  24. 24.
    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 279:22964–22972CrossRefPubMedGoogle Scholar
  25. 25.
    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 90:1340–1347CrossRefPubMedGoogle Scholar
  26. 26.
    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 209:142–152CrossRefPubMedGoogle Scholar
  27. 27.
    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 283:34819–34832CrossRefPubMedGoogle Scholar
  28. 28.
    Meldolesi J, Pozzan T (1998) The endoplasmic reticulum Ca2+ store: a view from the lumen. Trends Biochem Sci 23:10–14CrossRefPubMedGoogle Scholar
  29. 29.
    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 36:421–430CrossRefPubMedGoogle Scholar
  30. 30.
    Putney JW Jr, Ribeiro CM (2000) Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores. Cell Mol Life Sci 57:1272–1286CrossRefPubMedGoogle Scholar
  31. 31.
    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 40:553–560CrossRefPubMedGoogle Scholar
  32. 32.
    Csordas G, Thomas AP, Hajnozcky G (1999) Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria. EMBO J 18:96–108CrossRefPubMedGoogle Scholar
  33. 33.
    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 280:1763–1766CrossRefPubMedGoogle Scholar
  34. 34.
    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 63:283–292CrossRefPubMedGoogle Scholar
  35. 35.
    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 278:39224–39234CrossRefPubMedGoogle Scholar
  36. 36.
    Hajnoczky G, Csordas G, Madesh M, Pacher P (2000) The machinery of local Ca2+ signalling between sarco-endoplasmic reticulum and mitochondria. J Physiol 529:69–81CrossRefPubMedGoogle Scholar
  37. 37.
    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 580:39–49CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • D. González
    • 1
  • J. Espino
    • 1
  • I. Bejarano
    • 1
  • J. J. López
    • 1
  • A. B. Rodríguez
    • 1
  • J. A. Pariente
    • 1
  1. 1.Department of Physiology, Faculty of ScienceUniversity of ExtremaduraBadajozSpain

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