Tumor necrosis factor-alpha and ceramide induce cell death through different mechanisms in rat mesangial cells

It has been proposed that ceramide acts as a cellular messenger to mediate tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Based on this hypothesis, it was postulated that resistance of some cells to TNF-alpha cytotoxicity was due to an insufficient production of ceramide on stimulation by TNF-alpha. The present study was initiated to investigate whether this was the case in mesangial cells, which normally are insensitive to TNF-alpha-induced apoptosis. Our results indicate that although C2 ceramide was toxic to mesangial cells, the cell death it induced differed both morphologically and biochemically from that induced by TNF-alpha in the presence of cycloheximide (CHX). The most apparent effect of C2 ceramide was to cause cells to swell, followed by disruption of the cell membrane. It is evident that C2 ceramide caused cell death by necrosis, whereas TNF-alpha in the presence of CHX killed the cells by apoptosis. C2 ceramide did not mimic the effects of TNF-alpha on the activation of c-Jun NH2-terminal protein kinase and nuclear factor-kappaB transcription factor. Although mitogen-activated protein kinase [extracellular signal-related kinase (ERK)] was activated by both C2 ceramide and TNF-alpha, such activation appeared to be mediated by different mechanisms as judged from the kinetics of ERK activation. Furthermore, the cleavage of cytosolic phospholipase A2 during cell death induced by C2 ceramide and by TNF-alpha in the presence of CHX showed distinctive patterns. The present study provides evidence that apoptosis and necrosis use distinctive signaling machinery to cause cell death.