EVIDENCE FOR THE LACK OF INVOLVEMENT OF SPHINGOMYELIN HYDROLYSIS IN THE TUMOR NECROSIS FACTOR-INDUCED SECRETION OF NERVE GROWTH-FACTOR IN PRIMARY ASTROCYTE CULTURES

The signal mechanism underlying tumor necrosis factor alpha (TNF alpha ) up-regulation of nerve growth factor (NGF) production was studied in primary rat astrocyte cultures. Because ceramide is also able to indu ce NGF secretion and because TNF alpha is a known agonist of the sphin gomyelin (SPM)-ceramide pathway, we investigated whether the TNF alpha -induced NGF secretion by primary astrocytes is mediated by ceramide. TNF alpha stimulation of NGF secretion was shown to be independent of protein kinase C, abrogated by the tyrosine phosphoprotein phosphatase inhibitor phenylarsine oxide (PAO), and independent of the activation of the mitogen-activated protein kinase (MAPK) cascade. In marked con trast, inhibition of MAPK counteracted the NGF secretion induced by ce ramide, TNF alpha stimulation of the nuclear transcription factor NF-k appa B was prevented by cell pretreatment with PAO, whereas ceramide a nd sphingomyelinase had a marginal effect on NF-kappa B activation. Mo reover, TNF alpha failed to activate the SPM pathway, as indicated by the lack of SPM degradation and the absence of ceramide generation. To clarify further the role of NF-kappa B in NGF synthesis, electrophore tic mobility shift assays were performed with an NF-kappa B site from the NGF promoter. The absence of significant binding of NF-kappa B to the NGF gene promoter indicates the existence of an indirect role of N F-kappa B in the regulation of NGF synthesis. Altogether, our data str ongly suggest that TNF alpha-mediated up-regulation of NGF occurs inde pendently of ceramide generation.