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Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells.

ABSTRACT: Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH(2)F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop.

SUBMITTER: Shelton SN

PROVIDER: S-EPMC3003351 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells.

Shelton Shary N SN Dillard Cindy D CD Robertson John D JD

The Journal of biological chemistry 20101026 52

Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-per ...[more]

PMID: 20978129

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Project description:Endothelial injury is an initial mechanism mediating cardiovascular disease.Here, we investigated the effect of hyperhomocysteinemia on programed cell death in endothelial cells (EC).We established a novel flow-cytometric gating method to define pyrotosis (Annexin V(-)/Propidium iodide(+)). In cultured human EC, we found that: (1) homocysteine and lipopolysaccharide individually and synergistically induced inflammatory pyroptotic and noninflammatory apoptotic cell death; (2) homocysteine/lipopolysaccharide induced caspase-1 activation before caspase-8, caspase-9, and caspase-3 activations; (3) caspase-1/caspase-3 inhibitors rescued homocysteine/lipopolysaccharide-induced pyroptosis/apoptosis, but caspase-8/caspase-9 inhibitors had differential rescue effect; (4) homocysteine/lipopolysaccharide-induced nucleotide-binding oligomerization domain, and leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) protein caused NLRP3-containing inflammasome assembly, caspase-1 activation, and interleukin (IL)-1? cleavage/activation; (5) homocysteine/lipopolysaccharide elevated intracellular reactive oxygen species, (6) intracellular oxidative gradient determined cell death destiny as intermediate intracellular reactive oxygen species levels are associated with pyroptosis, whereas high reactive oxygen species corresponded to apoptosis; (7) homocysteine/lipopolysaccharide induced mitochondrial membrane potential collapse and cytochrome-c release, and increased B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 ratio which were attenuated by antioxidants and caspase-1 inhibitor; and (8) antioxidants extracellular superoxide dismutase and catalase prevented homocysteine/lipopolysaccharide -induced caspase-1 activation, mitochondrial dysfunction, and pyroptosis/apoptosis. In cystathionine ?-synthase-deficient (Cbs(-/-)) mice, severe hyperhomocysteinemia-induced caspase-1 activation in isolated lung EC and caspase-1 expression in aortic endothelium, and elevated aortic caspase-1, caspase-9 protein/activity and B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 ratio in Cbs(-/-) aorta and human umbilical vein endothelial cells. Finally, homocysteine-induced DNA fragmentation was reversed in caspase-1(-/-) EC. Hyperhomocysteinemia-induced aortic endothelial dysfunction was rescued in caspase-1(-/-) and NLRP3(-/-) mice.Hyperhomocysteinemia preferentially induces EC pyroptosis via caspase-1-dependent inflammasome activation leading to endothelial dysfunction. We termed caspase-1 responsive pyroptosis and apoptosis as pyrop-apoptosis.

Dataset Information

Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells.

Publications

Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells.

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