| Activation of NFkappaB requires the inhibitor of kappaB (IkappaB) kinase (IKK) complex, which comprises two functional kinases, IKKalpha and IKKbeta, and the scaffolding protein IKKgamma. We previously identified several interferon (IFN)-gamma-stimulated genes (ISGs) that are not induced in mouse embryonic fibroblast cells (MEFs) doubly-null for the expression of IKKalpha and IKKbeta, and we show here that IFN-gamma-induced transcription of IKK-dependent ISGs requires IKKbeta but not IKKalpha. Through microarray studies, we identified IKKbeta-dependent ISGs in both MEFs and RAW 264.7 macrophages. Many have both kappaB and ISRE elements, the latter of which bind IRF proteins, in their promoters. Although the IFN-gamma-induced expression of the IKKbeta-dependent gene ip-10 is sensitive to the super-repressor of NFkappaB and requires the p65 subunit of NFkappaB, IFN-gamma does not activate NFkappaB. Thus, a distinct subset of IRF-activated IFN-gamma-dependent genes requires some components of a normal NFkappaB activation pathway but not actual activation of NFkappaB in response to IFN-gamma. IFN-gamma activates IRF1 to bind the ISRE of ip-10 . In IKKbeta-null cells, IRF1 activation and binding to DNA are slightly weaker and the ability of IRF1 to transactivate a synthetic promoter composed of tandem copies of the ip-10 ISRE element is impaired. IFN-gamma and IL-1beta strongly synergize to induce ip-10. However, except at 1 h, the binding of IRF1 to the ip-10 ISRE in response to IFN-gamma is not enhanced by IL-1beta, and NFkappaB activation in response to IL-1beta is not affected by IFN-gamma. IRF1-null cells show impaired ip-10 induction in response to IFN-gamma compared to wild-type cells. However, synergy between IFN-gamma and IL-1beta is unaffected. The results presented provide new evidence of how NFkB components function in the induction of a specific subset of ISGs, and they provide mechanistic information about how IRF1 and NFkB cooperate to activate these promoters. |
