INTRACELLULAR SIGNAL TRANSDUCTION OF INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION IN THE CENTRAL NERVOUS SYSTEM

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We previously showed that the expression of functional inducible nitric oxide synthase (iNOS) was induced by interferon-γ (IFN-γ) in a human neuroblastoma cell line NB-39-nu, and its mRNA level was synergistically induced by simultaneous treatment of TNF-α. In the present study, we examined the signal transduction mechanism of the synergistic effect of IFN-γ and TNF-α on iNOS gene activation in NB-39 -nu cells. IFN-γ primed NB-39-nu cells induced TNF-α and TNF type 2 receptor (TNF-R2) as well as iNOS expressions. On the other hand, TNF type 1 receptor (TNF0R1) was continuously expressed in untreated cells, and its expression level did not change during the further treatment with IFN-γ. The involvement of TNF-α in the induction of iNOS mRNA expression by IFN-γ was evidenced by the fact that anti-TNF-α but not anti-TNF-β neutralizing antibody inhibits the induction of iNOS mRNA in IFN-γ-treated NB-39-nu cells. The involvement of TNF-R2 in the signal transduction for iNOS gene activation was also evidenced by followings: 1) TNF-α alone induces iNOS mRNA expression in the cells stably overexpressing TNF-R2 (NB-39-nu/TNF-R2) but not in control cells stably expressing β-GAL (NB-39-nu/β-GAL), and 2) although iNOS mRNA firstly detected in NB-39-nu cells at 12 hr after IFN-γ and TNF-α treatment, NB-39-nu/TNF-R2 cells could express iNOS mRNA at 6 hr after TNF-α alone treatment. The addition of NF-ィイD2kィエD2B inhibitor significantly suppressed TNF-α stimulated iNOS mRNA expression in NB-39-nu/TNF-R2 cells. Thus, IFN-γ may involved in the activation of TNF signal transduction machinery, and the NF-ィイD2kィエD2B activation by TNF-α through TNF-R2 signaling might play an important role for induction of iNOS expression in NB-39-nu cells upon stimulation with IFN-γ and TNF-α. These findings may provide a new intracelluar signal transduction mechanism for the iNOS gene regulation in human neuronal cells.