Tumor necrosis factor activation of the sphingomyelin pathway signals nuclear factor kappa B translocation in intact HL-60 cells.
Recent investigations suggest that tumor necrosis factor (TNF)-alpha may utilize the sphingomyelin pathway for signal transduction. Signaling in this system involves hydrolysis of sphingomyelin to ceramide by action of a neutral sphingomyelinase and stimulation of a ceramide-activated protein kinase (Dressler, K. A., Mathias, S., and Kolesnick, R. N. (1992) Science 255, 1715-1718). To clarify the role of this pathway in TNF action, the present studies assessed the effect of the sphingomyelin pathway on activation of nuclear factor kappa B (NF-kappa B), an event considered integral to the transfer of the TNF message to the cell nucleus. As shown previously, TNF (1 nM) induced a marked increase in nuclear NF-kappa B binding in human leukemia (HL-60) cells within 5 min, and elevated binding was detected for as long as 1 h. Addition of a maximally effective concentration of sphingomyelinase, 0.1 units.ml-1, induced a 50% reduction in sphingomyelin content by 5 min from a basal level of 560 pmol.10(6) cells-1 and a quantitative increase in ceramide levels from 89 pmol.10(6) cells-1. Sphingomyelinase 0.1 units.ml-1 also induced an increase in nuclear NF-kappa B binding within 5 min, an effect measurable for as long as 1 h. As little as 1 x 10(-5) units.ml-1 sphingomyelinase was effective and a maximal effect occurred with 1 x 10(-3) units.ml-1. A cell-permeable ceramide analog, C8-ceramide, which mimics biologic effects of TNF-alpha, also enhanced nuclear NF-kappa B activation within minutes. In contrast, addition of a phospholipase C or a synthetic diacylglycerol (DG) analog, 1,2-dioctanoylglycerol, failed to enhance nuclear NF-kappa B binding despite large increases in cellular DG content. Further, TNF-alpha induced elevation in ceramide content by 2 min to 185% of control but did not affect DG levels. These studies provide evidence that stimulation of the sphingomyelin pathway leads to NF-kappa B activation in HL-60 cells.
Laboratory of Signal Transduction, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
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Yang
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Costanzo
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Golde
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Kolesnick
D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010741:Phospholipases A / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000494:pharmacology
D002518:Ceramides; D004075:Diglycerides; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D013109:Sphingomyelins; D014409:Tumor Necrosis Factor-alpha; C044295:1,2-dioctanoylglycerol; D016718:Arachidonic Acid; D010741:Phospholipases A; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase