GLUTATHIONE REGULATION OF NEUTRAL SPHINGOMYELINASE IN TUMOR NECROSIS FACTOR-ALPHA-INDUCED CELL-DEATH

Tumor necrosis factor-alpha (TNF alpha)-induced cell death involves a diverse array of mediators and regulators including proteases, reactiv e oxygen species, the sphingolipid ceramide, and Bcl-2. It is not know n, however, if and how these components are connected. We have previou sly reported that GSH inhibits, in vitro, the neutral magnesium-depend ent sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this stu dy, GSH was found to reversibly inhibit the N-SMase from human mammary carcinoma MCF7 cells. Treatment of MCF7 cells with TNF alpha induced a marked decrease in the level of cellular GSH, which was accompanied by hydrolysis of sphingomyelin and generation of ceramide. Pretreatmen t of cells with GSH, GSH-methylester, or N-acetylcysteine, a precursor of GSH biosynthesis, inhibited the TNF alpha-induced sphingomyelin hy drolysis and ceramide generation as well as cell death. Furthermore, n o significant changes in GSH levels were observed in MCF7 cells treate d with either bacterial SMase or ceramide, and GSH did not protect cel ls from death induced by ceramide. Taken together, these results show that GSH depletion occurs upstream of activation of N-SMase in the TNF alpha signaling pathway. TNF alpha has been shown to activate at leas t two groups of caspases involved in the initiation and ''execution'' phases of apoptosis. Therefore, additional studies were conducted to d etermine the relationship of GSH and the death proteases. Evidence is provided to demonstrate that depletion of GSH is dependent on activity of interleukin-1 beta-converting enzyme-like proteases but is upstrea m of the site of action of Bcl-2 and of the execution phase caspases. Taken together, these studies demonstrate a critical role for GSH in T NF alpha action and in connecting major components in the pathways lea ding to cell death.