Document Detail

TNFalpha-induced glutathione depletion lies downstream of cPLA(2) in L929 cells.
MedLine Citation:
PMID:  11684089     Owner:  NLM     Status:  MEDLINE    
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 alpha (TNFalpha)-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 L-buthionin-(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 wild-type 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.
H L Hayter; B J Pettus; F Ito; L M Obeid; Y A Hannun
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  FEBS letters     Volume:  507     ISSN:  0014-5793     ISO Abbreviation:  FEBS Lett.     Publication Date:  2001 Oct 
Date Detail:
Created Date:  2001-10-30     Completed Date:  2001-12-11     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0155157     Medline TA:  FEBS Lett     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  151-6     Citation Subset:  IM    
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
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MeSH Terms
Buthionine Sulfoximine / metabolism
Cell Line
Cell Survival
Ceramides / metabolism
Cytosol / enzymology
Glutathione / metabolism*
Phospholipases A / genetics,  metabolism*
Sphingomyelin Phosphodiesterase / metabolism
Tumor Necrosis Factor-alpha / pharmacology*
Grant Support
Reg. No./Substance:
0/Ceramides; 0/Tumor Necrosis Factor-alpha; 5072-26-4/Buthionine Sulfoximine; 70-18-8/Glutathione; EC 3.1.1.-/Phospholipases A; EC Phosphodiesterase

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