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.
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 (Lys
Arg), 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.
This article has been cited by other articles:
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|