THE TUMOR NECROSIS FACTOR-SENSITIVE POOL OF SPHINGOMYELIN IS RESYNTHESIZED IN A DISTINCT COMPARTMENT OF THE PLASMA-MEMBRANE

Sphingomyelin (SM) biosynthesis is believed to occur in the early Golg i apparatus, plasma membrane and recycling endosomes. In the present s tudy, the localization of the SM synthesis that follows its hydrolysis upon activation of the SM signal-transduction pathway was investigate d in human skin fibroblasts treated with tumour necrosis factor (TNF) alpha. After TNF alpha-induced degradation, the intracellular SM level s returned to baseline levels within 30-60 min in cells treated at 37 degrees C. Pretreatment or co-incubation of cells with bacterial sphin gomyelinase or phospholipase C, decreasing the SM and phosphatidylchol ine content in the external leaflet of the plasma membrane respectivel y, did not inhibit SM resynthesis. However, SM resynthesis was not obs erved when TNF alpha-treated cells were continuously exposed to exogen ous sphingomyelinase, suggesting that under these particular condition s the resynthesized SM becomes accessible to the enzyme. Furthermore, whereas inhibition of vesicular traffic/endocytosis at 4 degrees C blo cked exoplasmic SM resynthesis, it did not alter SM resynthesis in TNF alpha-treated fibroblasts, negating the role of endosomes and the Gol gi apparatus. This was further evidenced by the finding that after SM resynthesis, TNF alpha was again able to promote SM turnover, even at 4 degrees C. In addition, when the exoplasmic leaflet SM was hydrolyse d by treating fibroblasts with bacterial sphingomyelinase, resynthesis of SM occurred at 37 degrees C much more slowly than after TNF alpha treatment. These findings support strongly the conclusion that the SM, which is resynthesized after TNF alpha-induced hydrolysis, resides in the cytosolic leaflet of the plasma membrane, and that the process in volved in this resynthesis displays characteristics different from tho se of the previously described SM synthases.