Inhibitor of kappaB kinase is required to activate a subset of interferon gamma-stimulated genes

Abstract

IkappaB kinase (IKK), discovered as the major activator of NF-kappaB, plays additional roles in signaling. By using mouse embryo fibroblasts (MEFs) lacking both the alpha and beta subunits of IKK, we find that these proteins are required for induction of a major subset of IFNgamma-stimulated genes and that this requirement is independent of NF-kappaB activation. Furthermore, there is no defect in IFNgamma-stimulated signal transducer and activator of transcription 1 (Stat1) activation or function in the IKKalpha/beta-null MEFs. Therefore, although activated Stat1 dimers are necessary for the activation of these genes in response to IFNgamma, they are not sufficient. These results reveal an important additional pathway for IFNgamma-stimulated gene expression in which an NF-kappaB-independent function of IKK is required.

Fig. 1.

IKK-dependent responses to IFNγ. WT or IKKα/β-null cells were treated with IFNγ (γ) for 6 h or were untreated (C), and total RNA was analyzed by the Northern procedure.

Fig. 2.

Normal activation of Stat1 by IFNγ in IKKα/β-null cells. WT or IKKα/β-null cells were treated for 15 or 30 min with IFNγ or were untreated. Western analysis was performed with antibodies against phosphoserine-727 Stat1, phosphotyrosine-701 Stat1, and total Stat1.

Fig. 3.

Responses of GAS and NF-κB elements to IFNγ.(a) Normal activation of a GAS-dependent reporter by IFNγ in IKKα/β-null cells. WT or IKKα/β-null cells were transfected transiently with p7XmycGAS-luciferase and pSV2βgal and treated with IFNγ as shown. (b) IFNγ fails to activate an NF-κB-dependent promoter. WT or IKKα/β-null cells were transfected transiently with p5XIP10κB luciferase and pSV2βgal. After 8 h, the cells were divided into different plates and incubated for 16 h more. The cells were treated as indicated with IFNγ, IL-1, or TNF for 6 h or were untreated, and the luciferase activity was compared with β-galactosidase activity to normalize for transfection efficiency.

Fig. 4.

The requirement of IKKα/β for IFNγ-stimulated IP10 gene expression is independent of NF-κB activation. (a) The response of IP10 to IFNγ does not require p65. WT or p65NFκB-null cells were treated with IL-1, TNF, or IFNγ for 6 h or were untreated, and total RNA was analyzed by the Northern procedure. (b) The response of IP10 to IFNγ is not inhibited by the IκBα super repressor. WT cells or cells expressing the IκBαSR were treated with IFNβ, IFNγ, IL-1, or TNF for 6 h or were untreated, and total RNA was analyzed by the Northern procedure.

Fig. 5.

Reexpression of IKKα and IKKβ in the IKKα/β-null cells restores IFNγ-stimulated IP10 expression. Three plates each of WT or IKKα/β-null cells were infected with adenoviruses capable of expressing GFP, IκBSR, or IKKα and IKKβ together. After 18 h, the cells were treated with IFNγ for 6 h or were untreated. (a) Western analysis, performed on equal amounts of total protein from each infection, for GFP, the hemagglutinin (HA) tag of IκBSR, IKKα/IKKβ, and β-actin (loading control). (b) Northern analysis, performed with an equal amount of total RNA from each sample.

Fig. 6.

Reexpression of kinase-active IKKβ, but not IKKα or KI IKKβ, in IKKα/β-null cells restores IFNγ-stimulated IP10 expression. Three plates each of the cells were infected with adenoviruses expressing IKKα (α), IKKβ (β), GFP, or kinase-inactive IKKβ (KIβ). After 18 h, the cells were treated with IFNγ for 6 h or were untreated. (a) Western analysis, performed on an equal amount of total protein from each infection for GFP, IKKα, IKKβ, and β-actin (loading control). (b) Northern analysis, performed with an equal amount of total RNA from each sample.