Fas, a member of the tumor necrosis factor receptor family, can upon ligation by its ligand or agonistic antibodies trigger signaling cascades leading to cell death in lymphocytes and other cell types. Such signaling cascades are initiated through the formation of a membrane death-inducing signaling complex (DISC) that includes Fas, the Fas-associated death domain protein (FADD) and caspase-8. We report here that a considerable fraction of Fas is constitutively partitioned into sphingolipid-and cholesterol-rich membrane rafts in mouse thymocytes as well as the L12.10-Fas T cells, and Fas ligation promotes a rapid and specific recruitment of FADD and caspase-8 to the rafts. Raft disruption by cholesterol depletion abolishes Fas-triggered recruitment of FADD and caspase-8 to the membrane, DISC formation and cell death. Taken together, our results provide the first demonstration for an essential role of membrane rafts in the initiation of Fas-mediated cell death signaling.

Apoptosis is a highly regulated process of cell suicide that occurs during development, host defense, and pathophysiology. The transcription factor IFN regulatory factor 5 (IRF5), known to be involved in the activation of innate immune responses, recently has been shown to be critical for DNA damage-induced apoptosis and tumor suppression. Here, we report on a cell-type-specific role of IRF5 in promoting apoptosis upon signaling through the death receptor Fas (CD95/APO-1/TNFRSF6). In particular, we show that mice deficient in the Irf5 gene are resistant to hepatic apoptosis and lethality in response to the in vivo administration of a Fasactivating monoclonal antibody, and that IRF5 is involved in a stage of Fas signaling that precedes the activation of caspase 8 and c-Jun N-terminal kinase (JNK). In addition to hepatocytes, IRF5 is also required for apoptosis in dendritic cells activated by hypomethylated CpG but not in thymocytes and embryonic fibroblasts in vitro. Thus, these findings reveal a cell-type-specific function for IRF5 in the complex regulatory mechanism of death-receptor-induced apoptosis.death receptor ͉ programmed cell death ͉ transcription factor ͉ liver ͉ dendritic cell I FN regulatory factor 5 (IRF5) plays a critical role in activating innate immune responses by transmitting pathogen-derived danger signals (i.e., pathogen-associated molecular patterns, or PAMPs) sensed by pattern recognition receptors to induce transcription of various cytokine genes (1-3). More recently, its tumor-suppressive function has been revealed by showing that IRF5 is required for DNA damage-induced apoptosis in c-HaRas-expressing mouse embryonic fibroblasts (MEFs) and that MEFs undergo transformation by this single oncogene in the absence of IRF5 (3).IRF5 is constitutively expressed in a variety of cell types and is further induced transcriptionally by treatment with type I IFNs or DNA damage (3). Upon stimulation with PAMPs or upon DNA damage, IRF5 is activated, presumably by phosphorylation, and then translocates from the cytoplasm to the nucleus where it promotes gene transcription by binding to target DNA sequences such as the IFN-stimulated response element in the cis-regulatory region of target genes (1,3,4). This finding is exemplified by the induction by IRF5 of the Il12b gene (1). DNA damage increases Irf5 transcript levels in a p53-dependent manner (3, 5). However, the induction of p53 target genes occurred normally in Irf5 Ϫ/Ϫ cells (3), and overexpression of IRF5 enhanced the cellular susceptibility to anticancer druginduced apoptosis even in p53-deficient cancer cell lines (6, 7), which suggests that IRF5 probably acts on a pathway that is distinct from that for p53. Nevertheless, how IRF5 participates in the apoptotic signaling pathway is not well understood.Fas (CD95/APO-1/TNFRSF6) belongs to the death receptor family of the tumor necrosis factor (TNF) receptor superfamily, whose engagement induces apoptosis in a cell-type-specific and, typically, p53-independent manner (8). Ligation of Fas by its liga...

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Fas-induced death of motoneurons in vitro has been shown to involve two signaling cascades that act together to execute the death program: a Fas-Daxx-ASK-1-p38 kinase-nNOS branch, which controls transcriptional and post-translational events, and the second classical Fas-FADD-caspase-8 branch. To analyze the role of Daxx in the developmental motoneuron cell death, we studied Fas-dependent cell death in motoneurons from transgenic mice that overexpress a dominant-negative form of Daxx. Motoneurons purified from these transgenic mice are resistant to Fas-induced death. This protective effect is specific to Fas because ultraviolet irradiation-triggered death is not affected by the transgene. The Daxx and the FADD pathways work in parallel because only Daxx, but not FADD, is involved in the transcriptional control of neuronal nitric oxide synthase and nitric oxide production. Nevertheless, we do not observe involvement of Daxx in developmental motoneuronal cell death, as the pattern of naturally occurring programmed cell death in vivo is normal in transgenic mice overexpressing the dominant negative form of Daxx, suggesting that Daxx-independent pathways are used during development.