FcγRII Activation Induces Cell Surface Ceramide Production which Participates in the Assembly of the Receptor Signaling Complex

Abstract

We studied an involvement of various cellular ceramide pools in signaling of immunoreceptor FcγII (FcγRII). The cell surface ceramide level was assessed by a technique based on binding of ceramide probes to intact cells. Total cellular ceramide was estimated by radioactive measurements. The activity of sphingomyelinases was measured by NBD-ceramide release while immunoprecipitation and immunoblotting were applied to analyze protein tyrosine phosphorylation. A complex pattern of protein phosphorylation was found to accompany FcγRII activation and the phosphorylation was either diminished by imipramine or increased by B13, modulators of acid sphingomyelinase and acid ceramidase activity. The effects of the drugs on the phosphorylation of FcγRII and NTAL were prominent and correlated with a reduction of the cell surface ceramide production by imipramine and an augmentation of the ceramide generation by B13. The ceramide generation followed activation of acid sphingomyelinase and preceded that of neutral sphingomyelinase. The level of cell surface ceramide was additionally elevated by exogenous bacterial sphingomyelinase, but only at later stages of the receptor activation. The total mass of ceramide was diminished in the course of receptor activation pointing to an engagement of enzymes metabolizing ceramide. The data indicate that FcγRII activates enzymes of the sphingomyelin cycle which affect various sphingomyelin/ceramide pools in a cell.

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

“…Subsequent studies involving the imaging of immunolabeled ceramide show that many stimuli, including the activation of other immune cell receptors, induce the acid sphingomyelinase-mediated formation of ceramide-rich domains (Grassmé et al, 2002; Abdel Shakor et al, 2004; Korzeniowski et al, 2007). The activation of cluster of differentiation 40 (CD40), a member of the TNF-receptor superfamily found on antigen presenting cells, induced the formation of ceramide patches that largely colocalized with clustered CD40 and acid sphingomyelinase (Grassmé et al, 2002).…”

“…Similar to CD95, CD40 clustering, and subsequent signaling was inhibited by a loss of acid sphingomyelinase activity, neutralization of cell surface ceramide, and cholesterol depletion (Grassmé et al, 2002). Likewise, immunofluorescence imaging of ceramide showed the activation of Fc gamma receptor II (FcγRII), an immune cell receptor for IgG, induced acid sphingomyelinase activity at the cell surface and the formation of ceramide-rich membrane patches (Abdel Shakor et al, 2004; Korzeniowski et al, 2007). This ceramide production was required for the clustering of the crosslinked FcγRII, subsequent receptor phosphorylation, and signaling.…”

“…Cellular processes are sensitive to sphingolipid catabolism and inhibitors of sphingolipid biosynthesis (Wiegmann et al, 1994; Tepper et al, 1995; Grassmé et al, 1997, 2001a,b, 2003a; Brenner et al, 1998; Junge et al, 1999; Grullich et al, 2000; Cremesti et al, 2001; Paris et al, 2001; Grassmé et al, 2003b; Abdel Shakor et al, 2004, 2012; Grassmé, 2005; Korzeniowski et al, 2007; Verdurmen et al, 2010; Serrano et al, 2012). …”

Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

“…Monolayers of ECs were fixed (1% PFA in TBS), then incubated with 2µg/mL anti-ceramide Ab (45-60 min, RT) and washed 3x with TBS as in [46]. Cell-bound Ab was released by 30 sec treatment with ice-cold 100 mM glycine, pH3.5, neutralized with 1M NaOH, and cell lysates spotted onto NC membrane using a dot-blot apparatus followed by incubation with anti-goat IgM HRP-conjugated.…”

One lipid, multiple functions: how various pools of PI(4,5)P2 are created in the plasma membrane

“…Further ultrastructural studies performed on sheets of plasma membrane obtained by mechanical cleavage of cells revealed that PI(4,5)P 2 accumulated at the margins of raft conglomerates [43]. Such structures form during Fcc receptor II activation and accommodate the receptor and proteins of its signaling cascade [175,176]. At these particular locations, PI(4,5)P 2 may function in tethering the actin cytoskeleton to the plasma membrane (as proposed for T cell receptor) and govern internalization of particles.…”