Neutral sphingomyelinase action stimulates signal transduction of tumor necrosis factor-alpha in the synthesis of cholesteryl esters in human fibroblasts.

We have investigated biochemical mechanisms of tumor necrosis factor (TNF)-alpha signaling in cultured human skin fibroblasts. We found that TNF-alpha signaling may involve activation of a cell membrane neutral sphingomyelinase (N-SMase) in that within 2.5-5 min of treatment of cells with TNF-alpha there was a 2-fold increase in the activity of N-SMase compared to control. This reaction led to the hydrolysis of sphingomyelin as evidenced by a decrease in sphingomyelin mass and in the radioactivity associated with [14C]choline-labeled sphingomyelin. This was accompanied by a 4-fold increase in the formation of cholesteryl [14C]oleate within 2.5 min of incubation with TNF-alpha. This reaction also stimulated the mobilization of cell surface-associated [3H]cholesterol and its utilization in the synthesis of [3H]cholesteryl esters via acyl coenzyme-A cholesterol acyltransferase (ACAT). Gas chromatographic analysis revealed that the cellular level of cholesteryl esters increased about 2.5-3-fold following treatment with TNF-alpha compared to control. Cholesteryl ester synthesis was compromised upon incubation of cells with antibody against N-SMase and remained unaltered with TNF-beta and fibroblast growth factor. Furthermore, TNF-alpha-mediated stimulation of cholesteryl ester synthesis was compromised by incubation of cells with an inhibitor of ACAT. These findings suggest a possible biological role of N-SMase in the signal transduction of TNF-alpha in the synthesis of cholesteryl esters in human fibroblasts.