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 factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta)-receptor internalization in the sphingomyelin pathway signal transduction, we investigated receptor signaling under conditions in which receptor internalization is blocked. We demonstrate that human recombinant TNF-alpha and IL-1 beta both induced sphingomyelin and phosphatidylcholine hydrolysis at either 4, 14, or 37 degrees C in human skin fibroblasts and U937 monocytic cells. Cytokine-induced sphingomyelin degradation also occurred when endocytosis was inhibited by incubating the cells in hypertonic medium. While internalization was not required for the production of ceramide, activation of the transcription factor NF-kappa B was strongly 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 exogenous sphingomyelinase or by phorbol myristate acetate was also inhibited. These results suggest that low temperature and hypertonicity, two inhibitors of receptor internalization: (i) do not affect the TNF-alpha- or IL-1 beta-induced sphingomyelin hydrolysis, but (ii) do inhibit a step distal to ceramide of the intracellular signaling pathway leading to NF-kappa B activation.