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Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and
R K Srivastava1, S J Sollott, L Khan
1Laboratory of Immunology, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825, USA.
Abstract:
The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) 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 N(G)-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 Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) 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-X(L) 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 Ca(2+) 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, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.
Insights
Bcl-2 and Bcl-X(L) proteins prevent apoptosis by regulating calcium (Ca2+) and nitric oxide (NO) production. These proteins protect Jurkat T cells against thapsigargin-induced apoptosis by inhibiting NO synthase activity.
Area of Science:
- Cellular Biology
- Molecular Biology
- Immunology
Background:
- The anti-apoptotic proteins Bcl-2 and Bcl-X(L) are crucial regulators of programmed cell death, but their precise molecular mechanisms remain incompletely understood.
- Disruption of calcium (Ca2+) homeostasis is implicated in apoptosis, and its interplay with signaling pathways like nitric oxide (NO) production and c-Jun NH2-terminal kinase (JNK) activation is a key area of investigation.
Purpose of the Study:
- To elucidate the role of Bcl-2 and Bcl-X(L) in regulating Ca2+ levels, NO production, JNK activation, and apoptosis in Jurkat T cells.
- To investigate the signaling cascade initiated by Ca2+ dysregulation and its downstream effects on apoptosis, particularly in the context of Bcl-2 and Bcl-X(L) expression.
Main Methods:
- Jurkat T cells overexpressing Bcl-2 or Bcl-X(L) (JT/Bcl-2, JT/Bcl-X(L)) or vector control (JT/Neo) were treated with thapsigargin (TG) to disrupt Ca2+ homeostasis.
- Measurements included intracellular and mitochondrial Ca2+ levels, NO production, JNK activation, mitochondrial membrane potential, cytochrome c release, caspase-3 activation, and apoptosis.
- Pharmacological inhibitors (BAPTA-AM, L-NAME) and dominant-negative mutants (SEK1, c-Jun) were used to dissect signaling pathways.
Main Results:
- Thapsigargin induced Ca2+ release, NO production, and apoptosis in JT/Neo cells, which were blocked by BAPTA-AM or L-NAME.
- Bcl-2 and Bcl-X(L) overexpression significantly reduced TG-induced NO production, late Ca2+ accumulation, and apoptosis, despite comparable early Ca2+ changes.
- Bcl-2 and Bcl-X(L) inhibited TG-induced mitochondrial dysfunction, cytochrome c release, caspase-3 activation, and JNK activation, with JNK activation downstream of caspase-3.
Conclusions:
- Thapsigargin-induced apoptosis in Jurkat T cells is mediated by Ca2+-dependent NO production, leading to mitochondrial damage, caspase-3 activation, and subsequent JNK pathway activation.
- Bcl-2 and Bcl-X(L) protect against TG-induced apoptosis by negatively regulating Ca2+-sensitive NO synthase activity or expression, thereby preventing the downstream apoptotic cascade.
- These findings highlight a novel mechanism by which Bcl-2 family proteins modulate cellular apoptosis through the regulation of Ca2+-NO signaling.