Abstract
Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-x(L) were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-x(L)-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells.
Highlights
Apoptosis can occur through two distinct signaling pathways
Concluding Remarks— the type I/II cell distinction was first described in studies involving tumor cell lines (e.g. Jurkat cells are type II, and SKW6.4 cells are type I) [5, 16], subsequent evidence has shown that the model applies to primary cells (46 – 48)
Type II cells are distinguished from type I cells by their reported heavy reliance on the intrinsic apoptotic pathway for apoptosis induced by a death receptor ligand (e.g. TNF␣, TRAIL, and FasL) or agonistic antibody
Summary
Apoptosis can occur through two distinct signaling pathways. One is the extrinsic (receptor-mediated) pathway in which binding of a death receptor (e.g. tumor necrosis factor (TNF) receptor-1, TNF-related apoptosis-inducing ligand (TRAIL) receptor-1, TRAIL-R2, and Fas) by a cognate ligand (e.g. TNF␣, TRAIL, and FasL) or an agonistic antibody (e.g. CH-11) causes the recruitment of adaptor proteins (e.g. TRADD and FADD) and initiator procaspase-8 molecules to the cytosolic side of the receptor to form the death-inducing signaling complex (DISC) [2]. TBid can activate a multidomain Bcl-2 family protein (i.e. Bax and Bak) that stimulates mitochondrial outer membrane permeabilization (MOMP) and the release of intermembrane space proteins into the cytosol [9, 10] In this regard, it is widely accepted that the mitochondrial apoptotic pathway plays an essential role during receptor-mediated apoptosis in type II cells [5, 11,12,13,14,15,16]; conflicting results exist regarding the precise molecular signaling requirements. A different study using a reconstituted in vitro cell-free system showed that recombinant Smac was sufficient to activate caspase-3 activity by inhibiting XIAP [13] These findings suggested that Smac-dependent neutralization of XIAP-mediated caspase-3 inhibition might be the most important consequence of MOMP during Fas-mediated apoptosis in type II cells, neither study examined this possibility in an experimental system in which a component of the apoptosome was absent. Our findings suggested that the inhibition of XIAP was a better determinant of the susceptibility of type II cells to anti-Fasinduced apoptosis
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