Synovial fibroblasts and the sphingomyelinase pathway: sphingomyelin turnover and ceramide generation are not signaling mechanisms for the actions 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)-induced nuclear factor (NF)-kappaB 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 pannus 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 fibroblasts (HSF) and the potential role of ceramide in HSF proliferation and apoptosis. Cultured human synovial fibroblasts were stimulated with exogenous TNF-alpha, sphingomyelinase, and ceramide. Apoptosis was assessed by cell morphology and annexin V labeling. NF-kappaB and stress kinase pathway activation were determined by immunoblotting techniques. Sphingomyelinase activation was determined by quantitation of sphingomyelin and ceramide radioactivity in [14C]serine-prelabeled HSF cells. The addition of TNF-alpha (50 ng/ml) to HSF did not elicit detectable sphingomyelinase activation. TNF-alpha was shown to activate NF-kappaB (p65 translocation and degradation of IkappaBalpha) and the stress kinase pathway (phosphorylation of ATF-2, p38, and c-jun). In contrast, exogenous ceramide had no effect on these signaling pathways nor did ceramide stimulate the generation of interleukin-6 or interleukin-8. High concentrations of ceramide (> or =25 micromol/L) were cytotoxic, whereas lower concentrations of ceramide inhibited cell cycle progression. Thus, although TNF-alpha stimulates the NF-kappaB and stress kinase pathways in HSF, these effects of TNF-alpha are not associated with sphingomyelinase turnover or induction of apoptosis.