Modulation of Mitochondrial Ca2+ Homeostasis by Bcl-2

doi:10.1074/jbc.274.47.33267

Modulation of Mitochondrial Ca²⁺ Homeostasis by Bcl-2*

From the ^‡Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201 and the ^§Institute for Molecular Virology, St. Louis University Medical Center, St. Louis, Missouri 63110

Abstract

We have investigated the role of mitochondrial Ca²⁺ (Ca_m) homeostasis in cell survival. Disruption of Ca_m homeostasis via depletion of the mitochondrial Ca²⁺ store was the earliest event that occurred during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca_m preceded activation of the caspase cascade and DNA fragmentation. Overexpression of the anti-apoptosis protein Bcl-2 led to increased Ca_m load, increased mitochondrial membrane potential (ΔΨ_m), and inhibition of staurosporine-induced apoptosis. On the other hand, ectopic expression of the pro-apoptotic protein Bik led to decreased Ca_m load and decreased ΔΨ_m. Inhibition of calcium uptake into mitochondria by ruthenium red induced a dose-dependent apoptosis as determined by nuclear staining and DNA ladder assay. Similarly, reducing the Ca_m load by lowering the extracellular calcium concentration also led to apoptosis. We suggest that the anti-apoptotic effect of Bcl-2 is related to its ability to maintain a threshold level of Ca_mand ΔΨ_m while the pro-apoptotic protein Bik has the opposite effect. Furthermore, both ER and mitochondrial Ca²⁺ stores are important, and the depletion of either one will result in apoptosis. Thus, our results, for the first time, provide evidence that the maintenance of Ca_mhomeostasis is essential for cell survival.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL-39481 (to T. H. K.) and CA73803 and CA33616 (to G. C.) and by a grant-in-aid from the American Heart Association of Michigan (to T. H. K).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: Dept. of Pathology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. Tel.: 313-577-1131; Fax: 313-577-0057; E-mail: tkuo@med.wayne.edu.
↵2 L. Zhu, S. Ling, X.-D. Yu, and T. H. Kuo, unpublished results.
Abbreviations:

ER

endoplasmic reticulum

Ca_m

mitochondrial Ca²⁺

Ca_c

cytoplasmic Ca²⁺

Ca_o

extracellular Ca²⁺

FCCP

carbonyl cyanidep-trifluoromethoxyphenylhydrazone

Tg

thapsigargin

SR

sarcoplasmic reticulum

HA

hemagglutinin

DMEM

Dulbecco's modified Eagle's medium

STS

staurosporine

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

ΔΨ_m

mitochondrial membrane potential

MPT

mitochondrial permeability transition

TMRM

tetramethylrhodamine methyl ester
- Received July 14, 1999.
- Revision received September 9, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.