TNFα‐induced glutathione depletion lies downstream of cPLA₂ in L929 cells

Abstract

Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor K K (TNFK K)-induced cell death. In addition, both have hypothesized direct roles in activation of N-sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N-SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with Lbuthionin-(S,R)-sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF-induced SM hydrolysis as well as TNF-induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A 2 (cPLA 2 ) mRNA and protein, in order to determine the relationship of cPLA 2 and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA 2 . Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA 2 was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wildtype L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA 2 activity, and suggest a pathway in which activation of cPLA 2 is required for the oxidation and reduction of GSH levels followed by activation of SMases. ß 2000 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Ceramide Enhances Acrosomal Exocytosis Triggered by Calcium and the Calcium Ionophore A23187 in Boar Spermatozoa

“…Arachidonic acid generated by PLA2 action has been shown to be a mediator of sphingomyelin hydrolysis induced by TNF-α in human leukemia-derived HL-60 cells [33,34]. It has also been demonstrated that, in L929 cells stimulated with TNF-α, arachidonic acid generated by the action of cytosolic PLA 2 causes glutathione depletion followed by sphingomyelinase activation [18]. Although in boar spermatozoa, no decrease in the levels of sphingomyelin during capacitating processes was found [35], further studies will be r e q u i r e d t o e x a m i n e t h e h y d r o l y s i s o f sphingomyelin after stimulation for acrosomal exocytosis.…”

“…The sphingomyelin cycle has been recognized as an important signalling pathway involved in many cell types and, upon activation, s p h i n g o m y e l i n i s h y d r o l y z e d i n t o phosphorylcholine and ceramide, the latter playing an activator role [15,16]. A messenger role for ceramide in cells activated by tumour necrosis factor (TNF)-α [17,18], transforming growth factor-β [19], and 1α,25-dihydroxyvitamin D 3 [19] has been demonstrated. In human spermatozoa, the involvement of a sphingomyelin/ceramide pathway has been suggested as playing a role in the capacitating processes [21], and a neutral sphingomyelinase has been identified in ram spermatozoa [22].…”

PKC-dependent Activation of Sphingosine Kinase 1 and Translocation to the Plasma Membrane

“…Several studies suggest that sphingomyelinases and ceramidases are activated in response to the same inducers that stimulate SK (11,(25)(26)(27)(28)(29), thus raising the possibility that sphingosine could be generated in or close to the plasma membrane concomitant with SK activation. These studies are consistent with S1P being generated in a location conducive for swift secretion out of the cell.…”

Oxidation of Ferulic Acid by Momordica charantia Peroxidase and Related Anti-inflammation Activity Changes

“…18) They were incubated with different concentrations of FA, FA-1, and FA-2 for 96 h. The supernatants were used for the TNF-a assay. A 100 ml of supernatant was added to 96-well microplates on which L929 cells were incubated; after 24 h, the number of cells were measured by MTT method.…”