SummaryRecent investigations provided evidence that the sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor c~ (TNF-o 0 in several hematopoietic and nonhematopoietic ceils. In this pathway, TNF-receptor interaction initiates sphingomyelin hydrolysis to ceramide by a sphingomyelinase. Ceramide acts as a second messenger stimulating a ceramide-activated serine/threonine protein kinase. The present studies show that ionizing radiation, like TNF, induces rapid sphingomyelin hydrolysis to ceramide and apoptosis in bovine aortic endothelial cells. Elevation of ceramide with exogenous ceramide analogues was su~cient for induction of apoptosis. Protein kinase C activation blocked both radiation-induced sphingomyelin hydrolysis and apoptosis, and apoptosis was restored by ceramide analogues added exogenously. Ionizing radiation acted directly on membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis enzymatically through a neutral sphingomyelinase. These studies provide the first conclusive evidence that apoptotic signaling can be generated by interaction of ionizing radiation with cellular membranes and suggest an alternative to the hypothesis that direct DNA damage mediates radiation-induced cell kill.T he biochemical regulation of programmed cell death (apoptosis) has recently attracted a great deal of attention because of its role as a physiological mechanism of cell death. Apoptosis represents a major regulatory mechanism in embryonal development, growth and differentiation, and in the wear and tear maintenance of adult mammalian tissues (1-3). In addition, programmed cell death serves as one of the pleiotropic mechanisms of cell kill by cytokines (4, 5), chemical agents (6, 7), radiation (8, 9), and heat (10). Apoptosis is conceptualized as a pre-programmed pathway of sequential biochemical events that are only partially known, eventually leading to activation of a calcium-magnesium-dependent endonuclease that cleaves the nuclear chromatin at selective internucleosomal linker sites (1). Signals generated at the membrane of the affected cell activate neighboring intact cells and infiltrating macrophages to phagocytize the dying cell and its disintegrating nucleus (11). Presently, there is little information on the signaling mechanisms that initiate the apoptotic response. Recently, however, the sphingomyelin pathway has been shown to constitute the early events in the apoptotic cascade of TNF-ol-induced programmed cell death (12, 13).The sphingomyelin pathway is a signal transduction pathway (14) that mediates the signaling of several cytokines, such as 13,15) and IL-1B (16). In these systems, stimulation of cell surface receptors activates a plasma membrane neutral sphingomyelinase that hydrolyzes sphingomyelin to generate ceramide and phosphocholine. Ceramide then serves as a second messenger, activating a proline-directed serine/ threonine kinase, termed ceramide-activated protein kinase (17, 18). The further downstream signaling events are only partially k...

The mechanism of interleukin-1 (IL-1) signaling is unknown. Tumor necrosis factor-alpha uses a signal transduction pathway that involves sphingomyelin hydrolysis to ceramide and stimulation of a ceramide-activated protein kinase. In intact EL4 thymoma cells, IL-1 beta similarly stimulated a rapid decrease of sphingomyelin and an elevation of ceramide, and enhanced ceramide-activated protein kinase activity. This cascade was also activated by IL-1 beta in a cell-free system, demonstrating tight coupling to the receptor. Exogenous sphingomyelinase, but not phospholipases A2, C, or D, in combination with phorbol ester replaced IL-1 beta to stimulate IL-2 secretion. Thus, IL-1 beta signals through the sphingomyelin pathway.

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