Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis

Abstract

The accumulation of Ca2+ in the mitochondrial matrix can stimulate oxidative phosphorylation, but can also, at high Ca2+ concentrations, transmit and amplify an apoptotic signal. Here, we characterized the capacity of physiological stimuli (for example, histamine and inositol-1,4,5-triphosphate) and inducers of endoplasmic reticulum (ER) stress (for example, A23187, thapsigargin and tunicamycin) to release Ca2+ from ER stores, induce mitochondrial Ca2+ accumulation, and trigger cell death in human cervix and colon carcinoma cell lines. Sustained Ca2+ accumulation in the mitochondrial matrix induced by ER stress triggered signs of proapoptotic mitochondrial alteration, namely permeability transition, dissipation of the electrochemical potential, matrix swelling, relocalization of Bax to mitochondria and the release of cytochrome c and apoptosis-inducing factor from mitochondria. In contrast, rapid and transient accumulation of Ca2+ induced by physiological stimuli failed to promote mitochondrial permeability transition and to affect cell viability. The specificity of this apoptosis pathway was validated in cells using a panel of pharmacological agents that chelate Ca2+ (BAPTA-AM) or inhibit inositol-1,4,5-trisphosphate receptor (IP3R; 2-aminoethoxydiphenyl borate), voltage-dependent anion channel (VDAC) (4,4′-diisothiocyanatostilbene-2,2′-disulfonate, NADH), the permeability transition pore (cyclosporin A and bongkrekic acid), caspases (z-VAD-fmk) and protein synthesis (cycloheximide). Finally, we designed an original cell-free system in which we confronted purified mitochondria and ER vesicles, and identified IP3R, VDAC and the permeability transition pore as key proteins in the ER-triggered proapoptotic mitochondrial membrane permeabilization process.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

Abbreviations

2-APB:

2-aminoethoxydiphenyl borate

AIF:

apoptosis inducing factor

ANT:

adenine nucleotide translocase

BA:

bongkrekic acid

BFA:

brefeldin A

CHX:

cycloheximide

CsA:

cyclosporin A

DIDS:

4,4′-diisothiocyanatostilbene-2,2′-disulfonate

ΔΨm:

mitochondrial transmembrane potential

ER:

endoplasmic reticulum

Hist:

histamine

IP3:

inositol-1,4,5-triphosphate

IP3R:

inositol-1,4,5-trisphosphate receptor

CCCP:

carbonylcyanide m-chlorophenylhydrazone

MMP:

mitochondrial membrane permeabilization

IM:

inner membrane

OM:

outer membrane

PI:

propidium iodide

ROS:

reactive oxygen species

PT:

permeability transition

PTPC:

permeability transition pore complex

TG:

thapsigargin

TN:

tunicamycin

VDAC:

voltage-dependent anion channel

References

  • Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA et al. (2005). Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434: 658–662.

    Article  CAS  PubMed  Google Scholar 

  • Bathori G, Csordas G, Garcia-Perez C, Davies E, Hajnoczky G . (2006). Ca2+-dependent control of the permeability properties of the mitochondrial outer membrane and voltage-dependent anion-selective channel (VDAC). J Biol Chem 281: 17347–17358.

    Article  CAS  PubMed  Google Scholar 

  • Belzacq A, Vieira H, Verrier F, Vandecasteele G, Cohen I, Prevost M et al. (2003). Bcl-2 and bax modulate adenine nucleotide translocase activity. Cancer Res 63: 541–546.

    CAS  PubMed  Google Scholar 

  • Belzacq AS, El Hamel C, Vieira HL, Cohen I, Haouzi D, Metivier D et al. (2001). Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437. Oncogene 20: 7579–7587.

    Article  CAS  PubMed  Google Scholar 

  • Beutner G, Ruck A, Riede B, Welte W, Brdiczka D . (1996). Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. FEBS Lett 396: 189–195.

    Article  CAS  PubMed  Google Scholar 

  • Bhanumathy C, Nakao S, Joseph S . (2006). Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells. J Biol Chem 281: 3722–3730.

    Article  CAS  PubMed  Google Scholar 

  • Boehning D, Patterson RL, Sedaghat L, Glebova NO, Kurosaki T, Snyder SH . (2003). Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nat Cell Biol 5: 1051–1061.

    Article  CAS  PubMed  Google Scholar 

  • Brdiczka D . (1991). Contact sites between mitochondrial envelope membranes. Structure and function in energy- and protein-transfer. Biochim Biophys Acta 1071: 291–312.

    Article  CAS  PubMed  Google Scholar 

  • Breckenridge D, Germain M, Mathai J, Nguyen M, Shore G . (2003). Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene 22: 8608–8618.

    Article  CAS  PubMed  Google Scholar 

  • Cianchi F, Cortesini C, Schiavone N, Perna F, Magnelli L, Fanti E et al. (2005). The role of cyclooxygenase-2 in mediating the effects of histamine on cell proliferation and vascular endothelial growth factor production in colorectal cancer. Clin Cancer Res 11: 6807–6815.

    Article  CAS  PubMed  Google Scholar 

  • Costantini P, Belzacq AS, Vieira HL, Larochette N, de Pablo MA, Zamzami N et al. (2000). Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2-independent permeability transition pore opening and apoptosis. Oncogene 19: 307–314.

    Article  CAS  PubMed  Google Scholar 

  • Crompton M, Barksby E, Johnson N, Capano M . (2002). Mitochondrial intermembrane junctional complexes and their involvement in cell death. Biochimie 84: 143–152.

    Article  CAS  PubMed  Google Scholar 

  • Csordas G, Madesh M, Antonsson B, Hajnoczky G . (2002). tcBid promotes Ca(2+) signal propagation to the mitochondria: control of Ca(2+) permeation through the outer mitochondrial membrane. EMBO J 21: 2198–2206.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Csordas G, Renken C, Varnai P, Walter L, Weaver D, Buttle K et al. (2006). Structural and functional features and significance of the physical linkage between ER and mitochondria. J Cell Biol 174: 915–921.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dolce V, Scarcia P, Iacopetta D, Palmieri F . (2005). A fourth ADP/ATP carrier isoform in man: identification, bacterial expression, functional characterization and tissue distribution. FEBS Lett 579: 633–637.

    Article  CAS  PubMed  Google Scholar 

  • Gincel D, Zaid H, Shoshan-Barmatz V . (2001). Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory role in mitochondrial function. Biochem J 351: 147–155.

    Article  Google Scholar 

  • Green DR, Kroemer G . (2004). The pathophysiology of mitochondrial cell death. Science 305: 626–629.

    Article  CAS  PubMed  Google Scholar 

  • Hajnoczky G, Csordas G, Krishnamurthy R, Szalai G . (2000a). Mitochondrial calcium signaling driven by the IP3 receptor. J Bioenerg Biomembr 32: 15–25.

    Article  CAS  PubMed  Google Scholar 

  • Hajnoczky G, Csordas G, Madesh M, Pacher P . (2000b). Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals. Cell Calcium 28: 349–363.

    Article  CAS  PubMed  Google Scholar 

  • Halestrap AP, Brenner C . (2003). The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr Med Chem 10: 1507–1525.

    Article  CAS  PubMed  Google Scholar 

  • Haouzi D, Lekehal M, Tinel M, Vadrot N, Caussanel L, Letteron P et al. (2001). Prolonged, but not acute, glutathione depletion promotes Fas-mediated mitochondrial permeability transition and apoptosis in mice. Hepatology 33: 1181–1188.

    Article  CAS  PubMed  Google Scholar 

  • Ichas F, Jouaville L, Mazat J . (1997). Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell 89: 1145–1153.

    Article  CAS  PubMed  Google Scholar 

  • Inesi G, Sagara Y . (1992). Thapsigargin, a high affinity and global inhibitor of intracellular Ca2+ transport ATPases. Arch Biochem Biophys 298: 313–317.

    Article  CAS  PubMed  Google Scholar 

  • Iwasaki H, Mori Y, Hara Y, Uchida K, Zhou H, Mikoshiba K . (2001). 2-Aminoethoxydiphenyl borate (2-APB) inhibits capacitative calcium entry independently of the function of inositol 1,4,5-trisphosphate receptors. Receptors Channels 7: 429–439.

    CAS  PubMed  Google Scholar 

  • Kokoszka JE, Waymire KG, Levy SE, Sligh JE, Cai J, Jones DP et al. (2004). The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore. Nature 427: 461–465.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Korge P, Weiss JN . (1999). Thapsigargin directly induces the mitochondrial permeability transition. Eur J Biochem 265: 273–280.

    Article  CAS  PubMed  Google Scholar 

  • Lauquin GJ, Vignais PV . (1976). Interaction of (3H) bongkrekic acid with the mitochondrial adenine nucleotide translocator. Biochemistry 15: 2316–2322.

    Article  CAS  PubMed  Google Scholar 

  • Le Bras M, Borgne-Sanchez A, Touat Z, Deniaud A, Maillier E, Lecellier G et al. (2006). Chemosensitization by knock-down of adenine nucleotide translocase-2. Cancer Res 66: 9143–9152.

    Article  CAS  PubMed  Google Scholar 

  • Le Bras M, Clement MV, Pervaiz S, Brenner C . (2005). Reactive oxygen species and the mitochondrial signaling pathway of cell death. Histol Histopathol 20: 205–220.

    CAS  PubMed  Google Scholar 

  • Lee A, Zizi M, Colombini M . (1994). Beta-NADH decreases the permeability of the mitochondrial outer membrane to ADP by a factor of 6. J Biol Chem 269: 30974–30980.

    CAS  PubMed  Google Scholar 

  • Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y et al. (1998). The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366: 177–196.

    Article  CAS  PubMed  Google Scholar 

  • Luo X, He Q, Huang Y, Sheikh M . (2005). Transcriptional upregulation of PUMA modulates endoplasmic reticulum calcium pool depletion-induced apoptosis via Bax activation. Cell Death Differ 12: 1310–1318.

    Article  CAS  PubMed  Google Scholar 

  • Madesh M, Hajnoczky G . (2001). VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release. J Cell Biol 155: 1003–1015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Madesh M, Hawkins BJ, Milovanova T, Bhanumathy CD, Joseph SK, Ramachandrarao SP et al. (2005). Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis. J Cell Biol 170: 1079–1090.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL et al. (1998b). Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281: 2027–2031.

    Article  CAS  PubMed  Google Scholar 

  • Marzo I, Brenner C, Zamzami N, Susin SA, Beutner G, Brdiczka D et al. (1998a). The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med 187: 1261–1271.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McDowell W, Schwarz RT . (1988). Dissecting glycoprotein biosynthesis by the use of specific inhibitors. Biochimie 70: 1535–1549.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Misumi Y, Miki K, Takatsuki A, Tamura G, Ikehara Y . (1986). Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J Biol Chem 261: 11398–11403.

    CAS  PubMed  Google Scholar 

  • Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H et al. (2005). Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 434: 652–658.

    Article  CAS  PubMed  Google Scholar 

  • Nieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ . (1995). Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. Biochem J 307: 99–106.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oakes SA, Scorrano L, Opferman JT, Bassik MC, Nishino M, Pozzan T et al. (2005). Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum. Proc Natl Acad Sci USA 102: 105–110.

    Article  CAS  PubMed  Google Scholar 

  • Paulik M, Nowack D, Morre D . (1988). Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver. J Biol Chem 263: 17738–17748.

    CAS  PubMed  Google Scholar 

  • Peppiatt C, Collins T, Mackenzie L, Conway S, Holmes A, Bootman M et al. (2003). 2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels. Cell Calcium 34: 97–108.

    Article  CAS  PubMed  Google Scholar 

  • Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P et al. (1999). Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 76: 725–734.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Petrosillo G, Ruggiero FM, Pistolese M, Paradies G . (2004). Ca2+-induced reactive oxygen species production promotes cytochrome c release from rat liver mitochondria via mitochondrial permeability transition (MPT)-dependent and MPT-independent mechanisms: role of cardiolipin. J Biol Chem 279: 53103–53108.

    Article  CAS  PubMed  Google Scholar 

  • Poncet D, Boya P, Metivier D, Zamzami N, Kroemer G . (2003). Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization. Apoptosis 8: 521–530.

    Article  CAS  PubMed  Google Scholar 

  • Reddy R, Mao C, Baumeister P, Austin R, Kaufman R, Lee A . (2003). Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP binding site in suppression of caspase-7 activation. J Biol Chem 278: 20915–20924.

    Article  CAS  PubMed  Google Scholar 

  • Risso A, Braidot E, Sordano MC, Vianello A, Macri F, Skerlavaj B et al. (2002). BMAP-28, an antibiotic peptide of innate immunity, induces cell death through opening of the mitochondrial permeability transition pore. Mol Cell Biol 22: 1926–1935.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rizzuto R, Pozzan T . (2006). Microdomains of intracellular Ca2+: molecular determinants and functional consequences. Physiol Rev 86: 369–408.

    Article  CAS  PubMed  Google Scholar 

  • Schroder M, Kaufman RJ . (2005). The mammalian unfolded protein response. Annu Rev Biochem 74: 739–789.

    Article  PubMed  Google Scholar 

  • Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T et al. (2003). BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 300: 135–139.

    Article  CAS  PubMed  Google Scholar 

  • Szabadkai G, Rizzuto R . (2004). Participation of endoplasmic reticulum and mitochondrial calcium handling in apoptosis: more than just neighborhood? FEBS Lett 567: 111–115.

    Article  CAS  PubMed  Google Scholar 

  • Thinnes FP, Florke H, Winkelbach H, Stadtmuller U, Heiden M, Karabinos A et al. (1994). Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes or bovine skeletal muscle, reversibly binds the stilbene-disulfonate group of the chloride channel blocker DIDS. Biol Chem Hoppe Seyler 375: 315–322.

    Article  CAS  PubMed  Google Scholar 

  • Trollinger D, Cascio W, Lemasters J . (1997). Selective loading of Rhod 2 into mitochondria shows mitochondrial Ca2+ transients during the contractile cycle in adult rabbit cardiac myocytes. Biochem Biophys Res Commun 236: 738–742.

    Article  CAS  PubMed  Google Scholar 

  • Verrier F, Deniaud A, LeBras M, Metivier D, Kroemer G, Mignotte B et al. (2004). Dynamic evolution of the adenine nucleotide translocase interactome during chemotherapy-induced apoptosis. Oncogene 23: 8049–8064.

    Article  CAS  PubMed  Google Scholar 

  • Vieira HL, Boya P, Cohen I, El Hamel C, Haouzi D, Druillenec S et al. (2002). Cell permeable BH3-peptides overcome the cytoprotective effect of Bcl-2 and Bcl-X(L). Oncogene 21: 1963–1977.

    Article  CAS  PubMed  Google Scholar 

  • Weaver JGR, Tarze A, Moffat TC, Le Bras M, Deniaud A, Brenner C et al. (2005). Inhibition of adenine nucleotide translocator pore function and protection against apoptosis in vivo by an HIV protease inhibitor. J Clin Inves 115: 1828–1838.

    Article  CAS  Google Scholar 

  • White C, Li C, Yang J, Petrenko N, Madesh M, Thompson C et al. (2005). The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP(3)R. Nat Cell Biol 7: 1021–1028.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zoratti M, Szabo I . (1994). Electrophysiology of the inner mitochondrial membrane. J Bioenerg Biomembr 26: 543–553.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work is supported by grants funded by ARC, CNRS and by Agence Nationale pour la Valorisation de la Recherche (ANVAR) to CB, n°A0505001, INCa (n°0610–3D1616–123/PL-2006) as well by a grant from European Union (RIGHT to GK). A Deniaud receives a PhD fellowship of Ligue Nationale Contre le Cancer.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to C Brenner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Deniaud, A., Sharaf el dein, O., Maillier, E. et al. Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene 27, 285–299 (2008). https://doi.org/10.1038/sj.onc.1210638

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.onc.1210638

Keywords

This article is cited by

Search

Quick links