Abstract
In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C(16)- ceramide predominated, was observed 6 h after BcR triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BcR triggering. The formation of these very long-chain ceramide species, in which C(24)-ceramide predominated, required the activation of effector caspases. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which long-chain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta-lactone, an irreversible inhibitor of the proteasome, prevented BcR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BcR cross-linking-induced activation of caspases. The formation of C(16)-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C(24)-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and important insights into the significance of specific ceramide species in defined stages of apoptosis.
Highlights
In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis
The early increase at 6 h after BcR cross-linking of C16-ceramide is seen both by Diacylglycerol Kinase (DGK) analysis as an increase in the lower ceramide-1-P spot upon separation by TLC (Fig. 1B) and by tandem MS analysis (Fig. 1D)
We have previously shown that the increase in ceramide after BcR cross-linking results from activation of the de novo pathway of sphingolipid biosynthesis [5]
Summary
We describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which longchain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide Both ISP-1 and clasto-lactacystin -lactone, an irreversible inhibitor of the proteasome, prevented BcR crosslinking-induced XIAP degradation. The formation of C16-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C24-ceramide formation was completely abolished These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. CH2F; APCI, atmospheric pressure chemical ionization; MS, mass spectrometry; XIAPwt, wild type XIAP; XIAP⌬R, RING-less XIAP; EGFP, enhanced green fluorescent protein; CLC, clasto-lactacystin -lactone; XIAP, X-linked inhibitor of apoptosis; PARP, poly (ADPribose)polymerase; IRES, internal ribosome entry site; BIR, Baculovirus IAP repeat; sn, sphingomyelin
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