To elucidate the intracellular mechanism of NF-κB activation, we performed the involvement of IκBα of NF-κB in the expression of inducible NO synthase (iNOS) and chemokine (CINC) following pretreatment with bacterial endotoxin (LPS) or IL-1β, respectively, using rat C6 glioma cells. We found that herbimycin A, a tyrosine protein kinase inhibitor, blocked: 1) LPS/IFNγ-induced iNOS expression, 2) LPS-induced intranuclear translocation of activated NF-κB (p50 · p65) and 3) IFNγ-induced autophosphorylation and activation of Jak 2 and Stat 1 as well as intranuclear translocation of phosphorylated Stat 1. Furthermore, transfection of a dominant negative form of IκBα(SS → AA) suppressed LPS/IFNγ-induced iNOS expression, suggesting that NF-κB, in particular, IκBα molecules could play important roles in the iNOS expression. We also found in IL-Iβ-induced CINC expression using cultured C6 glioma cells, the transient translocation of NF-κB in response to IL- 1β is partly dependent on transient proteasome activation. Thus we suggest that the formation of heterodimer p50·p65 from inactive trimer p50·p65·IκBα, particularly, proteolytic degradation and dissociation of IκBα from p50·p65 are a critical phase in NF-κB activation during LPS- induced iNOS and IL- 1β induced CINC expression in astroglial cells.
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