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.
Highlights
Cleavage of Bid Is a Caspase-mediated Event That Occurs Downstream of apoptotic protease activating factor-1 (Apaf-1)—Previously, we reported that effector caspases cleaved Bid to truncated Bid (tBid) during etoposide-induced apoptosis and that tBid functioned as part of a feed-forward amplification loop to induce Bak oligomerization and cytochrome c release [20]
Caspase-2 was suggested to be the apical caspase activated in response to hyperthermia, in part because its activation was reported to occur even in Bcl-2- or Bcl-xL-overexpressing cells and because caspase-2-deficient mouse splenocytes were partially resistant to heat-induced apoptosis [17]
A different study that was published around the same time reported that caspase-2-deficient mouse embryonic fibroblasts were sensitive to heat-induced apoptosis and, instead, suggested that an unknown Z-VAD-inhibitable protease was the apical protease activated in response to hyperthermia [13]
Summary
The extrinsic pathway is activated upon binding of a death ligand to its cognate receptor (e.g. Fas binding to the Fas receptor), which causes the receptors to move within close proximity to one another and recruit the adaptor protein Fas-associated protein with death domain (FADD), followed by the recruitment of initiator procaspase-8 or -10 [8, 9] This protein complex is termed the death-inducing signaling complex (DISC) and serves as the activating platform for initiator caspase-8 and -10 during receptor-mediated apoptosis. Heat-induced Apoptosis Requires Caspase-9 cleaves and activates downstream effector caspases, which cleave various target substrates, resulting in the biochemical and morphological characteristics associated with apoptotic cell death. Bid was observed to play a role in this form of apoptosis as a regulator of MOMP, its cleavage to tBid occurred downstream of Apaf-1, suggesting that tBid likely plays an amplification role in this process
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