SPHINGOSINE SYNERGISTICALLY STIMULATES TUMOR NECROSIS FACTOR-ALPHA-INDUCED PROSTAGLANDIN-E2 PRODUCTION IN HUMAN FIBROBLASTS

Sphingosine is a biologically active derivative of sphingomyelin. It affects diverse cellular functions and its mechanism(s) of action is poorly defined. Tumor necrosis factor-alpha (TNF-alpha) has recently been shown to rapidly induce sphingomyelin turnover, implicating this metabolic pathway in TNF-alpha signal transduction. Because TNF-alpha is known to induce prostaglandin E2 (PGE2) production in human fibroblasts, we tested the effect of sphingosine on TNF-alpha-induced PGE2 production. We found that sphingosine enhanced TNF-alpha-induced PGE2 production by as much as 18-fold over TNF-alpha alone. Sphingosine appeared to stimulate TNF-alpha-induced PGE2 production independent of TNF-alpha-mediated interleukin 1 (IL-1) production, because anti-IL-1 antibodies and IL-1 receptor antagonist protein (IRAP) did not inhibit TNF-alpha-induced PGE2 production or the stimulatory effect of sphingosine. TNF-alpha stimulated PGE2 production to the same degree in normal and protein kinase C (PKC) downregulated cells in the presence and absence of sphingosine, indicating that neither TNF-alpha nor sphingosine require active PKC to elicit their respective effects. The sphingosine analogues stearylamine and stearoyl-D-sphingosine had little or no effect on TNF-alpha-mediated PGE2 production, supporting a specific role for sphingosine in the activation process. Short-term (1 min) exposure of cells to sphingosine dramatically increased TNF-alpha-induced PGE2 production. A potential mechanism by which sphingosine could increase TNF-aLpha-induced PGE2 production involves enhancement of phospholipase A2 (PLA2) and/or cyclooxygenase (Cox) activity, the rate-limiting enzymes in PGE2 production. We found that both TNF-alpha and sphingosine alone enhanced these enzymatic activities, and that sphingosine additively increased the effect of TNF-alpha on phospholipase A2 activity. It appears that sphingosine affects TNF-alpha-induced PGE2 production via a mechanism that is independent of PKC involvement, and that sphingosine may function as an endogenous second messenger capable of modulating the responsiveness of the cell to external stimuli.