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-induced Ceramide Generation in L929 Cells
(Received for publication, January 21, 1997, and in revised form, March 21, 1997)
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From the The role of cytosolic phospholipase
A2 (cPLA2) in the regulation of ceramide
formation was examined in a cell line (L929) responsive to the
cytotoxic action of tumor necrosis factor 
Departments of Medicine and Cell Biology,
Duke University Medical Center, Durham, North Carolina 27710, the
§ Laboratoire de Biochimie, "Maladies Metaboliques,"
Institut Louis Bugnard, 31054 Toulouse, France, and the
¶ Department of Biochemistry, Setsunan University, Hirakata,
Osaka 573-01, Japan
(TNF). In L929 cells,
the addition of TNF resulted in the release of arachidonate, which
was followed by a prolonged accumulation of ceramide occurring over
5-12 h and reaching 250% over base line. The formation of ceramide
was accompanied by the hydrolysis of sphingomyelin and the activation
of three distinct sphingomyelinases (neutral
Mg2+-dependent, neutral
Mg2+-independent, and acidic enzymes). The variant cell
line C12, which lacks cPLA2, is resistant to the cytotoxic
action of TNF. TNF was able to activate nuclear factor 
B in
both the wild-type L929 cells and the C12 cells. However, TNF was
unable to cause the release of arachidonate or the accumulation of
ceramide in C12 cells. C6-ceramide overcame the resistance
to TNF and caused cell death in C12 cells to a level similar to that
in L929 cells. The introduction of the cPLA2 gene into C12
cells resulted in partial restoration of TNF-induced arachidonate
release, ceramide accumulation, and cytotoxicity. This study suggests
that cPLA2 is a necessary component in the pathways leading
to ceramide accumulation and cell death.
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