COMPARATIVE-STUDY OF THE METABOLIC POOLS OF SPHINGOMYELIN AND PHOSPHATIDYLCHOLINE SENSITIVE TO TUMOR-NECROSIS-FACTOR

The metabolism and localization of the pools of sphingomyelin and phos phatidylcholine (PtdCho) which are hydrolyzed upon activation of the s phingomyelin signal transduction pathway were studied in human skin fi broblasts treated with tumor necrosis factor alpha (TNF-alpha). In a f irst series of experiments, cellular phospholipids were labeled with [ H-3]choline under conditions that inhibit the vesicular traffic to the plasma membrane. Thus, in human fibroblasts metabolically labeled in the presence of brefeldin A, monensin or at 20 degrees C, the arrival of newly synthesized sphingomyelin to the cell surface was prevented, supporting previous conclusions for a vesicular mechanism of sphingomy elin transport to the plasma membrane. Under these conditions, TNF-alp ha induced the hydrolysis of PtdCho but did not promote the hydrolysis of H-3-labeled sphingomyelin, suggesting that the sphingomyelin signa ling pool resides in a compartment distal to the Golgi apparatus, and possibly in the plasma membrane. TNF was also unable to trigger the br eakdown of a radioactive sphingomyelin, [ceramide-H-3] sphingomyelin, exogenously added to the cells to label the exoplasmic side of the cel l surface. However, TNF caused PtdCho and sphingomyelin degradation in fibroblasts that had been treated with bacterial sphingomyelinase to degrade the sphingomyelin pool of the external leaflet of the plasma m embrane. A similar result was obtained at 4 degrees C, i.e. under cond itions which inhibit endocytosis, thereby excluding the endosomes as a potential site for TNF-induced sphingomyelin hydrolysis. Altogether, these results strongly argue for a localization of the sphingomyelin s ignaling pool at the inner leaflet of the plasma membrane, but neither in the endolysosomal nor the Golgi compartments. In addition, when [H -3]choline-labeled fibroblasts were treated under non-lytic conditions with bacterial phospholipase C to degrade the external pool of PtdCho , TNF was still able to stimulate the hydrolysis of PtdCho. This demon strates that the pool of PtdCho involved in TNF-alpha signaling (and w hich is hydrolyzed concurrently with sphingomyelin to,generate diacylg lycerol), is not located in the outer leaflet of the plasma membrane.