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Molecular and Cellular Biology, August 1999, p. 5659-5674, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Bcl-2 and Bcl-XL Block Thapsigargin-Induced Nitric Oxide Generation, c-Jun NH2-Terminal Kinase Activity, and Apoptosis

Rakesh K. Srivastava,1,* Steven J. Sollott,2 Leila Khan,1 Richard Hansford,3 Edward G. Lakatta,2 and Dan L. Longo1

Laboratory of Immunology,1 Laboratory of Cardiovascular Sciences,2 and Laboratory of Molecular Genetics,3 Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825

Received 31 August 1998/Returned for modification 27 October 1998/Accepted 29 April 1999

The proteins Bcl-2 and Bcl-XL prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-XL in the regulation of cytosolic Ca2+, nitric oxide production (NO), c-Jun NH2-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca2+ ATPase, was used to disrupt Ca2+ homeostasis. TG acutely elevated intracellular free Ca2+ and mitochondrial Ca2+ levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca2+ response with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca2+ level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-XL (JT/Bcl-2 or JT/Bcl-XL), NO production, late (36-h) Ca2+ accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-XL cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lysright-arrowArg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-XL inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca2+ release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca2+-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-XL cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-XL protect the cells against TG-induced apoptosis by negatively regulating Ca2+-sensitive NO synthase activity or expression.

* Corresponding author. Mailing address: Laboratory of Immunology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dr., Box 9, Baltimore, MD 21224-6825. Phone: (410) 558-8110. Fax: (410) 558-8137. E-mail: longod{at}vax.grc.nia.nih.gov.

Molecular and Cellular Biology, August 1999, p. 5659-5674, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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