Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH2-terminal kinase activity, and apoptosis

Rakesh K. Srivastava, Steven J. Sollott, Leila Khan, Richard Hansford, Edward Lakatta, Dan L. Longo

Research output: Contribution to journalArticle

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 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 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 Ca2+ 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) Ca2+ 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 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-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 Ca2+-sensitive NO synthase activity or expression.

Original languageEnglish (US)
Pages (from-to)5659-5674
Number of pages16
JournalMolecular and Cellular Biology
Volume19
Issue number8
StatePublished - Aug 1999
Externally publishedYes

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ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

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