CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway

The possibility that the sphingomyelin (SM)-ceramide pathway is activated b y CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of i mmune responses has been investigated. We demonstrate that incubation of Ep stein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acti ng as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition , SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as w ell as the soluble CD40 ligand induced a decrease in thymidine incorporatio n and morphological features of apoptosis, which were mimicked by cell-perm eant or bacterial sphingomyelinase-produced ceramides, Stable expression of a dominant-negative form of the FAN protein (factor associated with neutra l sphingomyelinase activation), which has been reported to mediate tumor ne crosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis o f transformed human fibroblasts, Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to coimmunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating inter action between CD40 and FAN. Altogether, these results strongly suggest tha t CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ce ramide pathway that is involved in the cell growth inhibitory effects of CD 40.