Loss of Caspase-9 Provides Genetic Evidence for the Type I/II Concept of CD95-mediated Apoptosis

Abstract

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.CD95 (APO-1/Fas) is the prototype member of the death receptor family (1). Stimulation of CD95 with its cognate ligand or agonistic antibodies results in receptor oligomerization and recruitment of signaling proteins in a death-inducing signaling complex (DISC) 3 (2, 3). Essential for apoptosis signaling through death receptors are the DISC components Fas-associated death domain and caspase-8. Recruitment of caspase-8 into the DISC leads to its dimerization-induced activation and the autoproteolytic release of its active subunits into the cytosol. The active caspase-8 activates downstream caspases, such as caspase-3, -6 and-7, that are responsible for most of the morphological manifestations of cell death (1). This death receptor-mediated pathway is also called the extrinsic pathway and is distinguished from the intrinsic mitochondrial death pathway. Apoptosis induction via mitochondria is regulated by the Bcl-2 family and involves early loss of mitochondrial membrane potential (⌬⌿ M ), release of cytochrome c, and other apoptogenic factors (4). In the cytosol, cytochrome c binds to Apaf-1 leading to caspase-9 recruitment. At this apoptosome complex, caspase-9 is activated and initiates the caspase cascade and subsequent apoptosis.An involvement of mitochondria has also been demonstrated in CD95 signaling, suggesting a functional role of this organelle in death receptor-mediated apoptosis of certain cell types (5, 6). Two types of CD95 signaling pathways have been proposed (7). CD95 type I cells show efficient DISC formation and strong activation of caspase-8 at the receptor level, which directly triggers the caspase cascade. Type II cells, in contrast, form a weak DISC and produce very little active caspase-8 at the receptor level, which is insufficient to initiate the apoptotic process. The low amounts of active caspase-8 can cleave Bid, a proapoptotic Bcl-2 protein that tra...

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