Role of caspases in CD95-induced biphasic activation of acid sphingomyelinase

Abstract

Acid sphingomyelinase (A-SMase) plays an important role in the initiation of CD95 signaling by forming ceramide-enriched membrane domains that enable clustering and activation of the death receptors. In TNF-R1 and TRAIL-R1/R2 signaling, A-SMase also contributes to the lysosomal apoptosis pathway triggered by receptor internalization. Here, we investigated the molecular mechanism of CD95-mediated A-SMase activation, demonstrating that A-SMase is located in internalized CD95-receptosomes and is activated by the CD95/CD95L complex in a biphasic manner.Since several caspases have been described to be involved in the activation of A-SMase, we evaluated expression levels of caspase-8, caspase-7 and caspase-3 in CD95-receptosomes. The occurrence of cleaved caspase-8 correlated with the first peak of A-SMase activity and translocation of the A-SMase to the cell surface which could be blocked by the caspase-8 inhibitor IETD.Inhibition of CD95-internalization selectively reduced the second phase of A-SMase activity, suggesting a fusion between internalized CD95-receptosomes and an intracellular vesicular pool of A-SMase. Further analysis demonstrated that caspase-7 activity correlates with the second phase of the A-SMase activity, whereas active caspase-3 is present at early and late internalization time points. Blocking caspases-7/ -3 by DEVD reduced the second phase of A-SMase activation in CD95-receptosomes suggesting the potential role of caspase-7 or -3 for late A-SMase activation.In summary, we describe a biphasic A-SMase activation in CD95-receptosomes indicating (I.) a caspase-8 dependent translocation of A-SMase to plasma membrane and (II.) a caspase-7 and/or -3 dependent fusion of internalized CD95-receptosomes with intracellular A-SMase-containing vesicles.

Synergistic interaction of the cannabinoid and death receptor systems – a potential target for future cancer therapies?

“…It is widely accepted that sphingomyelinases (SMases) become activated by cannabinoid receptors and stress stimuli . In addition to extracellular stimuli, caspases have also been shown to be involved in the activation and translocation of SMases . Mechanistically, there are several theories to delineate the mechanism of action of ceramide‐induced cell death.…”

A toolbox for the immunomagnetic purification of signaling organelles

“…In addition, we applied the method to isolate CD95-receptosomes to analyze the role of A-SMase in CD95L induced signaling. 16 The biological importance of the compartmentalized signal transduction of these members of the TNF-receptor superfamily has meanwhile been validated in various settings, while especially for TRAIL receptors cell line-dependent differences have been observed. [35][36][37][38][39][40][41][42][43][44][45][46][47] The experimental designs for all described protocols here are provided as schematic workflows.…”

“…In all cases, the blockade of internalization also inhibits apoptosis induction by prohibiting the formation of the “death induced signalling complex” (DISC) and subsequent activation of caspases and the lysosomal enzymes A‐SMase and cathepsin D which activate the mitochondrial apoptosis amplification (for review, see Reference ). In addition, we applied the method to isolate CD95‐receptosomes to analyze the role of A‐SMase in CD95L induced signaling …”

“…Identification of the underlying molecular mechanisms has been facilitated by the protocol we present here. The method allows the selective purification of biological materials from cellular lysates, in particular morphologically and functionally intact endosomes containing activated receptors and receptor-associated proteins, [16][17][18][19][20][21][22][23][24][25] as well as phagosomes from bacteria-infected cells described elsewhere. 26,27 Here, we provide a detailed protocol for the isolation of TNF-and CD95-receptosomes ( Figure 1).…”

“…26,27 Here, we provide a detailed protocol for the isolation of TNF-and CD95-receptosomes ( Figure 1). 16,[18][19][20][21][22][23]25,28,29 This basic protocol allows to simultaneously isolate plasma membranes (PM) from the same pool of cells, allowing to discriminate signaling emanating from PM-resident vs endosome-resident receptors.…”