Abstract
We previously reported that IL-2 deprivation induced acid sphingomyelinase-mediated (ASM-mediated) ceramide elevation and apoptosis in an NK/T lymphoma cell line KHYG-1. However, the molecular mechanism of ASM–ceramide-mediated apoptosis during IL-2 deprivation is poorly understood. Here, we showed that IL-2 deprivation induces caspase-dependent apoptosis characterized by phosphatidylserine externalization, caspase-8, -9, and -3 cleavage, and degradation of X-linked inhibitor of apoptosis protein (XIAP). IL-2 re-supplementation rescued apoptosis via inhibition of XIAP degradation without affecting caspase cleavage. However, IL-2 deprivation induced ceramide elevation via ASM in lysosomes and activated lysosomal cathepsin B (CTSB) but not cathepsin D. A CTSB inhibitor CA-074 Me and knockdown of CTSB inhibited ceramide-mediated XIAP degradation and apoptosis. Inhibition of ceramide accumulation in lysosomes using an ASM inhibitor, desipramine, decreased cytosolic activation of CTSB by inhibiting its transfer into cytosol from the lysosome. Knockdown of ASM also inhibited XIAP degradation and apoptosis. Furthermore, cell permeable N-acetyl sphingosine (C2-ceramide), which increases mainly endogenous d18:1/16:0 and d18:1/24:1 ceramide-like IL-2 deprivation, induced caspase-dependent apoptosis with XIAP degradation through CTSB. These findings suggest that lysosomal ceramide produced by ASM mediates XIAP degradation by activation of cytosolic CTSB and caspase-dependent apoptosis. The ASM–ceramide–CTSB signaling axis is a novel pathway of ceramide-mediated apoptosis in IL-2-deprived NK/T lymphoma cells.
Highlights
Apoptosis uses two major signaling pathways to induce programmed cell death; extrinsic and intrinsic pathways.[4]
We demonstrated that IL-2( − ) activated the acid sphingomyelinase (ASM)–ceramide pathway in lysosomes and that generation of d18:1/16:0 and d18:1/24:1 lysosomal ceramides caused the release of cathepsin B (CTSB), but not cathepsin D (CTSD), into the cytosol
CTSB-mediated degradation of X-linked inhibitor of apoptosis protein (XIAP) subsequently induced activation of caspase-3 and its nuclear entry to execute apoptosis. These results suggest the ASM/ceramide/CTSB axis is a novel pathway for the degradation of XIAP in IL-2deprived natural killer T (NK/T) lymphoma cell apoptosis
Summary
Apoptosis uses two major signaling pathways to induce programmed cell death; extrinsic and intrinsic pathways.[4]. The intrinsic pathway mediates the disruption of mitochondria and induced the formation of an apoptosome complex composed of cytochrome C secreted from mitochondria, apoptotic protease-activating factor 1 (Apaf-1), and caspase-9.4 Both extrinsic and intrinsic pathways lead to the activation of caspase-3/-7, which trigger various apoptotic phenomena such as phosphatidylserine (PS) externalization or DNA fragmentation. In addition to these mechanisms that induce apoptosis, it is important to clarify the role of lysosomal proteases in the regulation of antiapoptotic proteins such. The activation of CTSB through transforming growth factor-β signaling was reported to increase the proliferation of melanoma cells and short hairpin RNA (shRNA) of CTSB had an apoptotic effect mediated through the degradation of X-linked inhibitor of apoptosis protein (XIAP) in invasive meningioma cells, suggesting the positive effect of CTSB in cell proliferation.[8,9] In contrast, it was reported that CTSB induced apoptosis by activating caspase[3] and -9 in dengue virus-infected HepG2 hepatocytes.[10]
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