Tumor Necrosis Factor-Alfa Stimulates Sphingomyelin Turnover in Human Skin Fibroblasts by Two Different Pathways

The effect of tumor necrosis factor-α (TNF-α) on sphingomyelin (SM) turnover in human skin fibroblasts (HFS) was investigated. The activation of SM hydrolysis was observed to follow two different pathways depending on the concentration of TNF-α. Treatment of cells with 2 nM TNF-α induced the activation of phospholipase A 2 with subsequent stimulation of neutral sphingomyelinase. Pre-treatment of cells with NH 4 Cl or monensin did not inhibit this activation. At a higher concentration of TNF-α (4 nM), the exposed cells showed activation of SM turnover in an acidic cellular compartment, since both NH 4 C1 and monensin completely blocked SM hydrolysis induced by the cytokine. The activation of SM turnover in the acid cellular compartment was mediated by activation of phosphatidylcholine-dependent phospholipase C. In addition, TNF-α was shown also to stimulate ceramidase activity in this cell line since SM hydrolysis increased ceramide levels in the cells only when ceramidase was inhibited with genistein. In the absence of genistein SM hydrolysis led to increased sphingosine formation. Without TNF-α stimulation, ceramidase activities in the HSF cells were low, and marked generation of ceramide could be observed only after hydrolysis of SM using bacterial sphingomyelinase. Thus, our results implied that TNF-α activates sphingomyelin turnover in human skin fibroblast by two different mechanisms which operate in two different cellular compartments and respond to different TNF-α concentrations.