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

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-γand TNF-αon iNOS gene activation in NB-39-nu cells.IFN-γprimed NB-39-nu cells induced TNF-αand TNF type2 receptor(TNF-R2)as well as iNOS expressions.On the other hand，TNF type 1 receptor(TNF 0R1)was continuously expressed in untreated cells，and its expression level did not change during the further treatment with IFN-γ。The involvement of TNF-αin the induction of iNOS mRNA expression by IFN-γwas evidenced by the fact that anti-TNF-αbut not anti-TNF-βneutralizing 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-αalone induces iNOS mRNA expression in the cells stably overexpressing TNF-R2(NB-39-nu/TNF-R2)but not in control cells stably expressingβ-GAL(NB-39-nu/β-GAL)，and2)although iNOS mRNA firstly detected in NB-39-nu cells at 12hr after-γand TNF-αtreatment，NB-39-nu cells at 12hr after-IFN-γand TNF-αtreatment，NB-39-nu cells。The addition of NF-I D2k ii D2B inhibitor significantly suppressed TNF-αstimulated iNOS mRNA expression in NB-39-nu/TNF-R2cells.Thus，IFN-γmay involved in the activation of TNF signal transduction machinery，and the NF-I D2k ii D2B activation by TNF-αthrough TNF-R2 signaling might play an important role for induction of iNOS expression in NB-39-nu cells upon stimulation with IFN-γand TNF-α。These findings may provide a new intracelluar signal transduction mechanism for the iNOS gene regulation in human neuronal cells.

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.