Abstract
Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine, whereas overexpression of Bcl-x(L) had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable C(2)-ceramide, induced cytochrome c release from mitochondria in a caspase-independent fashion leading to activation of caspase-9 and executioner caspases and, surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely abolished by Bcl-x(L) overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced cell death in type II cells.
Highlights
Fas induces apoptosis when cross-linked with either agonistic antibodies or its natural ligand via formation of the deathinducing signal complex (DISC)1 by recruiting Fas-associated death domain protein adapter proteins and caspase-8 proenzymes, leading to activation of caspase-8 by autocatalytic cleavage [1]
Transient Increases in Ceramide and Sphingosine during Fas-induced Apoptosis Precede Executioner Caspases Activation and Cell Death and Are Followed by a Sustained Accumulation of Ceramide—As previously reported [10, 14, 20, 21], we found that treatment of Jurkat cells with 50 ng/ml anti-Fas antibody resulted in acute changes in ceramide levels within 30 min from a basal level of ϳ10 pmol/nmol total phospholipids (Fig. 1A)
These data are consistent with the reports in which a rapid rise in ceramide has been implicated as an early event in the Fas-signaling pathway in Jurkat cells [10, 14, 20, 21], as well as association solely with a late increase in ceramide [15, 27]
Summary
Fas induces apoptosis when cross-linked with either agonistic antibodies or its natural ligand via formation of the deathinducing signal complex (DISC)1 by recruiting Fas-associated death domain protein adapter proteins and caspase-8 (caspase-8) proenzymes, leading to activation of caspase-8 by autocatalytic cleavage [1]. We report that treatment of Jurkat cells with antiFas antibody induces rapid generation of ceramide immediately followed by transient production of sphingosine, both preceding cyt c release and activation of executioner caspase-3, -6, and -7 as well as caspase-8 and Bid activation.
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