Cell Surface Ceramide Generation Precedes and Controls FcγRII Clustering and Phosphorylation in Rafts

Abstract

Despite the role of sphingolipid/cholesterol rafts as signaling platforms for Fc␥ receptor II (Fc␥RII), the mechanism governing translocation of an activated receptor toward the rafts is unknown. We show that at the onset of Fc␥RII cross-linking acid sphingomyelinase is rapidly activated. This enzyme is extruded from intracellular compartments to the cell surface, and concomitantly, exofacially oriented ceramide is produced. Both non-raft and, to a lesser extent, raft sphingomyelin pools were hydrolyzed at the onset of Fc␥RII cross-linking. The time course of ceramide production preceded the recruitment of Fc␥RII to rafts and the receptor phosphorylation. Exogenous C 16 -ceramide facilitated clustering of Fc␥RII and its association with rafts. In contrast, inhibition of acid sphingomyelinase diminished both the ceramide generation and clustering of crosslinked Fc␥RII. Under these conditions, tyrosine phosphorylation of Fc␥RII and receptor-accompanying proteins was also reduced. All the inhibitory effects were bypassed by treatment of cells with exogenous ceramide. These data provide evidence that the generation of cell surface ceramide is a prerequisite for fusion of cross-linked Fc␥RII and rafts, which triggers the receptor tyrosine phosphorylation and signaling.

Inhibition of acid sphingomyelinase by tricyclic antidepressants and analogons

“…In 2001, Grassmé and coworkers demonstrated for the first time that ASM activation is linked to translocation of the enzyme to the plasma membrane upon CD95 stimulation (Grassmé et al, 2001a). Since then, a large number of studies have analyzed the regulation of lysosomal ASM with respect to ASM translocation in combination with ASM activation in response to different stimuli (e.g., Grassmé et al, 2002; Abdel Shakor et al, 2004; Lacour et al, 2004; Rotolo et al, 2005; Dumitru and Gulbins, 2006; Zeidan and Hannun, 2007; Zhang et al, 2007; Perrotta et al, 2010; Edelmann et al, 2011; Li et al, 2012, 2013), but only a few studies have addressed the secreted form of the enzyme (Schissel et al, 1996, 1998; Wong et al, 2000; Jenkins et al, 2009) and these studies have been limited to cell models, plasma, or serum (Mühle et al, 2013). …”

The Role of Sphingolipids and Ceramide in Pulmonary Inflammation in Cystic Fibrosis

“…Ceramide molecules self-associate to small ceramide-enriched membrane microdomains, which have the tendency to spontaneously fuse to large ceramide-enriched membrane domains [36-38]. The formation of ceramide-enriched membrane domains was shown to occur in cells after stimulation via a variety of stimuli including CD95 [39-41], CD40 [42], DR5 [43], FcγRII [44], the PAF-receptor [45], but also after infection with P. aeruginosa [19], Neisseriae gonorrhoeae [46] , Rhinovirus [47], application of stress stimuli such as UV-light [48], cisplatin [49] or Cu 2+ -treatment [50]. The great variety of stimuli that trigger the formation of ceramide-enriched membrane platforms suggests that these membrane domains facilitate signal transduction, but are very likely not part of the specific signal transduction pathway elicited by the these stimuli.…”

Lysenin-His, a sphingomyelin-recognizing toxin, requires tryptophan 20 for cation-selective channel assembly but not for membrane binding

“…The cholesterol effect can be attributed to the changes of sphingomyelin topology in the membrane yielding local concentration of sphingomyelin into discrete microdomains [14,16]. Accordingly, lysenin was applied for identification of sphingomyelin-rich domains in the plasma membrane [17].…”