Abstract
D609 is known to modulate death receptor-induced ceramide generation and cell death. We show that in Jurkat cells, non-toxic D609 concentrations inhibit sphingomyelin synthase and, to a lesser extent, glucosylceramide synthase, and transiently increase the intracellular ceramide level. D609 significantly enhanced FasL-induced caspase activation and apoptosis. D609 stimulated FasL-induced cell death in caspase-8-deficient Jurkat cells, indicating that D609 acts downstream of caspase-8. At high FasL concentration (500 ng/mL), cell death was significantly, but not completely, inhibited by zVAD-fmk, a broad-spectrum caspase inhibitor, indicating that FasL can activate both caspase-dependent and -independent cell death signaling pathways. FasL-induced caspase activation was abolished by zVAD-fmk, whereas ceramide production was only partially impaired. D609 enhanced caspase-independent ceramide increase and cell death in response to FasL. Also, D609 overcame zVAD-fmk-conferred resistance to a FasL concentration as low as 50 ng/mL and bypassed RIP deficiency. It is likely that mitochondrial events were involved, since Bcl-xL over-expression impaired D609 effects. In PHA-activated human T lymphocytes, D609 enhanced FasL-induced cell death in the presence or absence of zVAD-fmk. Altogether, our data strongly indicate that the inhibition of ceramide conversion to complex sphingolipids by D609 is accompanied by an enhancement of FasL-induced caspase-dependent and -independent cell death in T lymphocytes.
Highlights
Treatment with 50 μg/mL (i.e., 187.5 μM) D609 resulted in a transient elevation of endogenous ceramide levels up to 200% of the basal value at 2 h, followed by a decline to basal level by 4 h (Figure 1C) with no significant toxicity induction (Figure 1D). 100 μg/mL (i.e., 375 μM) D609 triggered cell death (Figure 1D) that was not totally inhibited by the broad-spectrum caspase inhibitor zVAD-fmk (Figure 1E), under conditions that abolished caspase-3 and PARP cleavage
Whereas D609 alone triggered apoptosis, as evidenced by nuclear fragmentation, D609-induced cell death in the presence of zVAD-fmk was associated with marginal chromatin condensation and necrotic features (Figure 1F)
Cell death was strongly impaired by Bcl-xL-over-expressing E6 cells (Bcl-xL) over-expression, suggesting that mitochondrial events are involved in D609-induced toxicity (Figure 1G)
Summary
D609 has been widely used as an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC) [2,6,7,8,9] and, indirectly, acidic sphingomyelinase (SMase) [2,4,10,11,12,13,14]. D609 impairs TNF- and anti-Fas-induced activation of a PC-PLC (the molecular identity of which remains unknown) and acidic SMase [4,10,11]. In this context, PC-PLC stimulation leads to the rise of diacylglycerol (DAG), which enhances acidic SMase activity and ceramide levels [10,12]. D609 has been shown to be being capable of inhibiting sphingomyelin synthase (SMS) [15,16,17,18], an enzyme that regulates ceramide and DAG levels in the opposite direction [17].
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