Phospholipase A2 Is Necessary for Tumor Necrosis Factor α-induced Ceramide Generation in L929 Cells

Abstract

The role of cytosolic phospholipase A 2 (cPLA 2 ) in the regulation of ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor ␣ (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 Mg 2؉ -dependent, neutral Mg 2؉ -independent, and acidic enzymes). The variant cell line C12, which lacks cPLA 2 , 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. C 6 -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 cPLA 2 gene into C12 cells resulted in partial restoration of TNF␣-induced arachidonate release, ceramide accumulation, and cytotoxicity. This study suggests that cPLA 2 is a necessary component in the pathways leading to ceramide accumulation and cell death.The sphingomyelin (SM) 1 cycle, first described by Okazaki et al.(1), has gained recognition over the past few years as a key mechanism for regulating anti-mitogenic signals. Activation of this cycle through the regulation of a signal-induced sphingomyelinase (SMase) results in generation of the lipid second messenger ceramide. Ceramide then modulates a number of biological fates, including growth inhibition (1-3), differentiation (2), apoptosis (4 -6), and cell cycle arrest (7). Although recent studies have begun to catalogue inducers such as TNF␣, interleukin-1␤, nerve growth factor, and Fas that are capable of signaling through the SM cycle (see Refs. 5, 6, and 8 for reviews), the mechanisms by which these inducers stimulate SMase activity remain poorly understood.TNF␣, through interaction with either a 55-or 75-kDa TNF receptor (9, 10), impacts upon a myriad of intracellular signaling cascades, including protein phosphorylation cascades, transcription factors, and lipid messengers (11). Two classes of lipid mediators have been implicated in TNF␣ signaling, glycerophospholipid metabolites and sphingolipid metabolites (11,12), and recent evidence suggests that these two classes of lipids may interact (13). In HL-60 cells, a linear correlation was established among TNF␣ stimulation, AA generation, and SM cycle activation: TNF␣-stimulated AA liberation preceded ceramide generation, and AA reproduced the effects of TNF␣ on the SM cycle (13). Although these studies suggested that AA and/or its metabolites may be involved in activation of SMase, the physiologic role of the PLA 2 /AA pathway in regulating SMase activity has not been determined.In this study, we examined the role of PLA 2 in SMase activation in the L929 murine fibroblast cell l...

Crm-A, bcl-2 and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8 processing

“…In contrast, we find that CD95L did not induce the formation of ceramide in LN-18 or LN-229 either in the absence or presence of CHX ( Figure 4A). As a positive control, we confirm a significant increase of ceramide in L929 fibrosarcoma cells exposed to TNF-a (Jayadev et al, 1997). Consistent with this negative finding in glioma cells, we observed that inhibitors of acid sphingomyelinase such as desipramine (Hurwitz et al, 1994) or of ceramide synthase such as fumonisin B 1 (Bose et al, 1995), that is, of the key enzymes involved in ceramide generation, had no effect on CD95L-induced apoptosis.…”

Anti-CD3 and Concanavalin A-induced Human T Cell Proliferation Is Associated with an Increased Rate of Arachidonate-Phospholipid Remodeling

“…The sensitivity of HL-60 cells, which rapidly remodel arachidonate, to CoA-IT inhibition was suggested to be due to the rapid accumulation of the pathway intermediate free AA and its activation of a sphingomyelinase leading to ceramide generation (22). A similar cell death mechanism where the cell's inability to control free AA levels leads to increased ceramide concentration has been suggested to occur after cPLA 2 activation in TNF-sensitive cells (46,47) and in cells treated with cyclooxygenase inhibitors (23). In resting cells where arachidonate-PL remodeling proceeds slowly, pathway intermediates like free AA would accumulate to a lesser extent after CoA-IT inhibition, likely allowing the cell to cope with any released free AA via reacylation into phospholipids or triglycerides.…”

Intracellular unesterified arachidonic acid signals apoptosis

“…The cell-killing effect of TNF-␣ has been shown to signal through ceramide, and AA stimulated this pathway under different conditions (17,28). To evaluate the involvement of ceramide, we measured the cellular ceramide level after exposure of the cells to exogenous AA (300 M) for 1, 4, and 24 h. At 1 h there was a small increase (less than 2-fold) and at the other times there was no change compared with control cells (not shown).…”

“…TNF-␣ exerts a cytotoxic effect against certain tumor cells, and release of AA by cPLA 2 has been implicated in this process (13,28). In the cells that overexpressed FACL4 or COX-2, the cell survival after TNF-␣ treatment was increased moderately (not shown), and it was significantly improved in the FACL4͞COX-2 stable cells: the surviving fraction increased from 43.5 to 70.5% (n ϭ 3, P Ͻ 0.001) (Fig.…”