LOW-TEMPERATURES AND HYPERTONICITY DO NOT BLOCK CYTOKINE-INDUCED STIMULATION OF THE SPHINGOMYELIN PATHWAY BUT INHIBIT NUCLEAR FACTOR-KAPPA-B ACTIVATION

In order to better understand the significance of tumor necrosis facto r-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta)-receptor intern alization in the sphingomyelin pathway signal transduction, we investi gated receptor signaling under conditions in which receptor internaliz ation is blocked. We demonstrate that human recombinant TNF-alpha and IL-1 beta both induced sphingomyelin and phosphatidylcholine hydrolysi s at either 4, 14, or 37 degrees C in human skin fibroblasts and U937 monocytic cells. Cytokine-induced sphingomyelin degradation also occur red when endocytosis was inhibited by incubating the cells in hyperton ic medium. While internalization was not required for the production o f ceramide, activation of the transcription factor NF-kappa B was stro ngly reduced when cells were stimulated with TNF at low temperature or in hypertonic medium. Under these conditions, activation of NF-kappa B by the cell-permeant C-2-ceramide (N-acetylsphingosine), by exogenou s sphingomyelinase or by phorbol myristate acetate was also inhibited. These results suggest that low temperature and hypertonicity, two inh ibitors of receptor internalization: (i) do not affect the TNF-alpha- or IL-1 beta-induced sphingomyelin hydrolysis, but (ii) do inhibit a s tep distal to ceramide of the intracellular signaling pathway leading to NF-kappa B activation.