SYNOVIAL FIBROBLASTS AND THE SPHINGOMYELINASE PATHWAY - SPHINGOMYELINTURNOVER AND CERAMIDE GENERATION ARE NOT SIGNALING MECHANISMS FOR THEACTIONS OF TUMOR-NECROSIS-FACTOR-ALPHA

The activation of sphingomyelinase and the generation of ceramide has been proposed to mediate tumor necrosis factor-alpha (TNF-alpha)-induc ed nuclear factor (NF)-kappa B activation through its second messenger ceramide. Ceramide may also be an important regulator of cell growth, senescence, and apoptosis. Aberrant cell proliferation and apoptosis have been implicated in the rampant fibroblast proliferation and pannu s formation characteristic of rheumatoid arthritis. However, the role of TNF-alpha and the sphingomyelinase pathway in the process have not been determined, The objective of this study was to determine whether TNF-alpha activates the sphingomyelin pathway in human synovial fibrob lasts (HSF) and the potential role of ceramide in HSF proliferation an d apoptosis. Cultured human synovial fibroblasts were stimulated with exogenous TNF-alpha, sphingomyelinase, and ceramide. Apoptosis was ass essed by cell morphology and annexin V labeling. NF-kappa B and stress kinase pathway activation were determined by immunoblotting technique s. Sphingomyelinase activation was determined by quantitation of sphin gomyelin and ceramide radioactivity in [C-14]serine-prelabeled HSF cel ls. The addition of TNF-alpha (50 ng/ml) to HSF did not elicit detecta ble sphingomyelinase activation, TNF-alpha was shown to activate NF-ka ppa B (p65 translocation and degradation of I kappa B alpha) and the s tress kinase pathway (phosphorylation of ATF-2, p38, and c-jun). In co ntrast, exogenous ceramide had no effect on these signaling pathways n or did ceramide stimulate the generation of interleukin-6 or interleuk in-8. High concentrations of ceramide (greater than or equal to 25 mu mol/L) were cytotoxic, whereas lower concentrations of ceramide inhibi ted cell cycle progression. Thus, although TNF-alpha stimulates the NF -kappa B and stress kinase pathways in HSF, these effects of TNF-alpha are not associated with sphingomyelinase turnover or induction of apo ptosis.