Role of an acidic compartment in tumor-necrosis-factor-α−induced production of ceramide, activation of caspase-3 and apoptosis

Authors


  • Correspondence to C. Borner, Institute of Biochemistry, Rue du Musee 5, CH-1700 Fribourg, Switzerland

  • Fax: +41 26 300 97 35.

  • E-mail:christoph.borner@unifr.ch

  • Abbreviations. TNF-α, tumor necrosis factor-α; TRADD, TNF-receptor-associated death-domain-containing protein; FADD, Fas-associated death-domain-containing protein; Z-Val-Ala-Asp-CH2F, benzyloxycarbonylvalylalanylaspartylfluormethane; Ac-Asp-Glu-Val-Asp-CHO, acetylaspartylglutamylvalylaspartyl aldehyde; PARP, poly(ADP ribose) polymerase; CrmA, cowpox virus cytokine-response modifier A; PLA2, phospholipase A2.

  • Enzymes. Neutral sphingomyelinase ( EC3.1.4.12); phospholipase A2 ( EC3.1.1.4), poly(ADP ribose) synthase (2.4.2.30).

Abstract

Tumor necrosis factor-α (TNF-α) apoptosis by recruiting a complex of cytosolic proteins at its plasma membrane receptor. Among them is caspase-8, an interleukin-1β−converting enzyme (ICE)-like protease that initiates an amplified protease cascade to activate the cell-death machinery. The latter comprises at least caspase-3 and caspase-7, which execute cell death by cleaving numerous protein substrates, including poly(ADP-ribose) polymerase. In addition, TNF-α stimulates the production of ceramide, which also activates the death machinery. Whether the signaling pathways elicited by caspase-8 and ceramide proceed independently or intersect at a specific subcellular site is unknown. Using the lysosomotropic agent NH4Cl and the vesicularization inhibitor brefeldin A, we show here the convergence of TNF-α-induced death signaling on an acidic, subcellular compartment reminiscent of lysosomes. This compartment generates at least two signaling pathways that account for the caspase-3 activation and apoptosis induced by TNFα, one involving ceramide and caspase-unrelated adapter molecules and another involving yet unknown lysosomal mediators. The apoptosis inhibitor Bcl-2 specifically acts on the ceramide-activated pathway to block caspase-3 activation and apoptosis. The latter result explains why Bcl-2 only partially blocks TNF-α-induced apoptosis.

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