Lysosomal ceramide generated by acid sphingomyelinase triggers cytosolic cathepsin B-mediated degradation of X-linked inhibitor of apoptosis protein in natural killer/T lymphoma cell apoptosis

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.

Ceramide activates lysosomal cathepsin B and cathepsin D to attenuate autophagy and induces ER stress to suppress myeloid-derived suppressor cells

“…However, simultaneous inhibition of both cathepsin B and cathepsin D significantly decreased LCL521-induced cell death (Figure 8A). Western blotting analysis indicated that LCL521 does not alter the levels of cathepsin B and cathepsin D proteins (Figure 8B), suggesting that LCL521 activates the enzymatic activity but not the protein levels of cathepsin B and cathepsin D. Previous studies have shown that ceramide induces xIAP degradation to mediate cell death [44, 46, 48]. Western blotting analysis indicates that although LCL521 treatment decreased xIAP protein level in J774M cells, xIAP protein level is essentially low in J774M cells (Figure 8B), suggesting that xIAP may not play a significant role in MDSCs.…”

Inhibitors of the sphingomyelin cycle: Sphingomyelin synthases and sphingomyelinases

“…In another study, aSMase is also proposed as target for cancer therapy since its inhibition induced lysosomal cell death (Aits and Jaattela, 2013). Taniguchi M et al showed that IL-2 deprivation resulted in LaSMase-dependent ceramide generation and caspase-mediated cell death in NK/T lymphoma cells (Taniguchi et al, 2015). Inhibition of aSMase using desipramine, knock-down of aSMase, or inhibition of cathepsin B rescued cells from apoptotic cell death induced by IL-2 deprivation, and proving the role of aSMase in this kind of cell death.…”

Staphylococcus aureus α-Toxin Induces Inflammatory Cytokines via Lysosomal Acid Sphingomyelinase and Ceramides

“…Cathepsin D has been linked to the induction of endoplasmatic reticulum stress [50], suppression of autophagy [51], degradation of XIAP [52] and induction of apoptosis [53]. …”