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Evidence against an early signalling role for ceramide in Fas-mediated apoptosis.

ABSTRACT: We have investigated whether the increases in ceramide levels that occur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depend on the activation of caspases. Using cells prelabelled to equilibrium with [14C]acetate, it was shown that the amount of ceramide approximately doubled after 24 h incubation with anti-Fas, but the time course of ceramide changes was slower than that of anti-Fas-induced cell death. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added together with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a structurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, but in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentation. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence of processes downstream of the activation of caspases and increases in cytosolic calcium concentration.

SUBMITTER: Sillence DJ

PROVIDER: S-EPMC1218397 | biostudies-other | 1997 May

REPOSITORIES: biostudies-other

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Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis.

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Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis.

Project description:Systemic infusion of bone marrow mesenchymal stem cells (BMMSCs) yields therapeutic benefit for a variety of autoimmune diseases, but the underlying mechanisms are poorly understood. Here we show that in mice systemic infusion of BMMSCs induced transient T cell apoptosis via the FAS ligand (FASL)-dependent FAS pathway and could ameliorate disease phenotypes in fibrillin-1 mutated systemic sclerosis (SS) and dextran-sulfate-sodium-induced experimental colitis. FASL(-/-) BMMSCs did not induce T cell apoptosis in recipients, and could not ameliorate SS and colitis. Mechanistic analysis revealed that FAS-regulated monocyte chemotactic protein 1 (MCP-1) secretion by BMMSCs recruited T cells for FASL-mediated apoptosis. The apoptotic T cells subsequently triggered macrophages to produce high levels of TGFβ, which in turn led to the upregulation of CD4(+)CD25(+)Foxp3(+) regulatory T cells and, ultimately, immune tolerance. These data therefore demonstrate a previously unrecognized mechanism underlying BMMSC-based immunotherapy involving coupling via FAS/FASL to induce T cell apoptosis.

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Project description:Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, -3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

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Project description:Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

| S-EPMC3674671 | biostudies-literature

Fas-activated serine/threonine kinase (FAST) phosphorylates TIA-1 during Fas-mediated apoptosis.

Project description:We have identified a serine/threonine kinase that is rapidly activated during Fas-mediated apoptosis. Fas-activated serine/threonine kinase (FAST) is phosphorylated on serine and threonine residues in Jurkat cells. In response to Fas ligation, it is rapidly dephosphorylated and concomitantly activated to phosphorylate TIA-1, a nuclear RNA-binding protein that has been implicated as an effector of apoptosis. Phosphorylation of TIA-1 precedes the onset of DNA fragmentation, suggesting a role in signaling downstream events in the apoptotic program. Our results introduce Fast and TIA-1 as components of a molecular cascade involved in signaling Fas-mediated apoptosis.

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