Glutathione Regulation of Neutral Sphingomyelinase in Tumor Necrosis Factor-α-induced Cell Death

Abstract

Tumor necrosis factor-␣ (TNF␣)-induced cell death involves a diverse array of mediators and regulators including proteases, reactive oxygen species, the sphingolipid ceramide, and Bcl-2. It is not known, however, if and how these components are connected. We have previously reported that GSH inhibits, in vitro, the neutral magnesium-dependent sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this study, GSH was found to reversibly inhibit the N-SMase from human mammary carcinoma MCF7 cells. Treatment of MCF7 cells with TNF␣ induced a marked decrease in the level of cellular GSH, which was accompanied by hydrolysis of sphingomyelin and generation of ceramide. Pretreatment of cells with GSH, GSH-methylester, or N-acetylcysteine, a precursor of GSH biosynthesis, inhibited the TNF␣-induced sphingomyelin hydrolysis and ceramide generation as well as cell death. Furthermore, no significant changes in GSH levels were observed in MCF7 cells treated with either bacterial SMase or ceramide, and GSH did not protect cells from death induced by ceramide. Taken together, these results show that GSH depletion occurs upstream of activation of N-SMase in the TNF␣ signaling pathway.TNF␣ has been shown to activate at least two groups of caspases involved in the initiation and "execution" phases of apoptosis. Therefore, additional studies were conducted to determine the relationship of GSH and the death proteases. Evidence is provided to demonstrate that depletion of GSH is dependent on activity of interleukin-1␤-converting enzyme-like proteases but is upstream 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 TNF␣ action and in connecting major components in the pathways leading to cell death.

Stress signals for apoptosis: ceramide and c-Jun kinase

“…These investigators showed that physiologic concentrations of glutathione, which neutralizes free radicals, inhibits NSMase (Liu and Hannun, 1997). A depletion in glutathione, which occurs during oxidative stress, may result in NSMase activation (Liu et al, 1998). During TNF-mediated cell death glutathione depletion is progressive, and NSMase activation and ceramide generation occur after 6 ± 12 h. This contrasts with the rapid ceramide generation that occurs in response to environmental stress in some cells (Figure 1), an event likely mediated via ASMase.…”

Oxidative stress and regulation of glutathione in lung inflammation

“…Downregulation of VEGF and its receptor may be associated with apoptosis, which may be linked in the pathogenesis of inflammatory lung diseases such as emphysema and COPD. GSH and other thiols such as NAC inhibit TNFa-induced sphingomyelin hydrolysis, ceramide generation and programmed cell death (apoptosis), suggesting that GSH has antiapoptotic properties through its ability to detoxify oxidants and free radicals [214].…”

Ceramide: A common pathway for atherosclerosis?

“…TNF-α initiates the pathway through TNFR1 (55-kDa receptor) leading to phospholipase A2 activation, generation of arachidonic acid, and subsequent activation of SMase [27]. TNF-α-stimulated cells are redox-sensitive [28]. Some studies showed that ROS participate in TNF-α-mediated ceramide formation, suggesting that the ROS-sensitive pathway may be located upstream of ceramide formation [29].…”