The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis

Abstract

The IkappaB kinase (IKK) complex is composed of three subunits, IKKalpha, IKKbeta, and IKKgamma (NEMO). While IKKalpha and IKKbeta are highly similar catalytic subunits, both capable of IkappaB phosphorylation in vitro, IKKgamma is a regulatory subunit. Previous biochemical and genetic analyses have indicated that despite their similar structures and in vitro kinase activities, IKKalpha and IKKbeta have distinct functions. Surprisingly, disruption of the Ikkalpha locus did not abolish activation of IKK by proinflammatory stimuli and resulted in only a small decrease in nuclear factor (NF)-kappaB activation. Now we describe the pathophysiological consequence of disruption of the Ikkbeta locus. IKKbeta-deficient mice die at mid-gestation from uncontrolled liver apoptosis, a phenotype that is remarkably similar to that of mice deficient in both the RelA (p65) and NF-kappaB1 (p50/p105) subunits of NF-kappaB. Accordingly, IKKbeta-deficient cells are defective in activation of IKK and NF-kappaB in response to either tumor necrosis factor alpha or interleukin 1. Thus IKKbeta, but not IKKalpha, plays the major role in IKK activation and induction of NF-kappaB activity. In the absence of IKKbeta, IKKalpha is unresponsive to IKK activators.

Figure 1

Generation of IKKβ-deficient mice. (A) The mouse Ikkβ locus and the targeting vector. Map of the Ikkβ genomic fragment used for gene targeting is shown. The exons are indicated by solid black boxes, the introns are indicated by bold lines, and the selection markers, lengths of restriction fragments, restriction enzyme sites, the probes used for Southern analysis, and the location of primers used in PCR screening are also shown. RI, EcoRI; P, PstI; S, SacI; RV, EcoRV; Xh, XhoI. (B) Southern blot analysis of mouse genomic DNA. Mouse genomic DNA was digested with EcoRI and probed with probe A (1.2-kb HindIII-PstI fragment of Ikkβ). After homologous recombination, the 9.7-kb EcoRI fragment of wild-type Ikkβ is replaced by a 7.2-kb EcoRI fragment, as indicated in panel A. (C) Western blot analysis of mouse proteins using antibody H470 specific for IKKβ. Location of the IKKβ band is indicated. The lower band is nonspecific (ns). The same blot was also probed with antibodies to IKKα, IKKγ, p65(RelA), and p50(NF-κB1). The genotypes are as indicated.

Figure 1

Generation of IKKβ-deficient mice. (A) The mouse Ikkβ locus and the targeting vector. Map of the Ikkβ genomic fragment used for gene targeting is shown. The exons are indicated by solid black boxes, the introns are indicated by bold lines, and the selection markers, lengths of restriction fragments, restriction enzyme sites, the probes used for Southern analysis, and the location of primers used in PCR screening are also shown. RI, EcoRI; P, PstI; S, SacI; RV, EcoRV; Xh, XhoI. (B) Southern blot analysis of mouse genomic DNA. Mouse genomic DNA was digested with EcoRI and probed with probe A (1.2-kb HindIII-PstI fragment of Ikkβ). After homologous recombination, the 9.7-kb EcoRI fragment of wild-type Ikkβ is replaced by a 7.2-kb EcoRI fragment, as indicated in panel A. (C) Western blot analysis of mouse proteins using antibody H470 specific for IKKβ. Location of the IKKβ band is indicated. The lower band is nonspecific (ns). The same blot was also probed with antibodies to IKKα, IKKγ, p65(RelA), and p50(NF-κB1). The genotypes are as indicated.

Figure 1

Generation of IKKβ-deficient mice. (A) The mouse Ikkβ locus and the targeting vector. Map of the Ikkβ genomic fragment used for gene targeting is shown. The exons are indicated by solid black boxes, the introns are indicated by bold lines, and the selection markers, lengths of restriction fragments, restriction enzyme sites, the probes used for Southern analysis, and the location of primers used in PCR screening are also shown. RI, EcoRI; P, PstI; S, SacI; RV, EcoRV; Xh, XhoI. (B) Southern blot analysis of mouse genomic DNA. Mouse genomic DNA was digested with EcoRI and probed with probe A (1.2-kb HindIII-PstI fragment of Ikkβ). After homologous recombination, the 9.7-kb EcoRI fragment of wild-type Ikkβ is replaced by a 7.2-kb EcoRI fragment, as indicated in panel A. (C) Western blot analysis of mouse proteins using antibody H470 specific for IKKβ. Location of the IKKβ band is indicated. The lower band is nonspecific (ns). The same blot was also probed with antibodies to IKKα, IKKγ, p65(RelA), and p50(NF-κB1). The genotypes are as indicated.

Figure 2

Appearance of an Ikkβ^−/− E13.5 embryo and a normal littermate. Wild-type (Ikkβ^+/+, WT) and mutant (Ikkβ^−/−, M) embryos were isolated at E13.5 and photographed. The genotypes of the embryos were later determined by PCR and Southern blot analysis.

Figure 3

Analysis of wild-type (WT) and mutant (M) livers. E13.5 embryos were fixed and sectioned. Paraffin-embedded transverse sections at the area of the liver were subjected to H&E (top; original magnification: 400×) or TUNEL (bottom; original magnification: 600×) staining. The stained sections were photographed.

Figure 4

Electron microscopic analysis of livers from E13 Ikkβ^+/+, Ikkβ^+/−, and Ikkβ^−/− embryos. Both E13 Ikkβ^+/+ (A) and Ikkβ^+/− (B) livers exhibited normal morphology. The Ikkβ^−/− liver (C) exhibited varying degrees of apoptosis characterized by collapsed and condensed nuclei and general cellular degeneration. Bars = 5 μm.

Figure 5

Defective IKK and NF-κB activation in IKKβ- deficient ES cells. (A) IKK activity. Lysates of TNF-α– or IL-1–treated Ikkβ^+/− and Ikkβ^−/− cells were prepared at the indicated time points (in min) after stimulation and immunoprecipitated with antibody M280 to IKKα. IKK activity (KA) was measured by an immunecomplex kinase assay using GST-IκBα(1-54) as a substrate. The kinase assay products were separated by SDS-PAGE, transferred to nitrocellulose membrane, and autoradiographed. The membrane was reprobed with antibody M280 (IB: IKKα) for loading control. (B) NF-κB binding activity. Nuclear extracts of Ikkβ^+/− and Ikkβ^−/− cells stimulated with IL-1 or TNF-α for the indicated times (in min) were incubated with ³²P-labeled κB oligonucleotide probe and subjected to EMSA. Binding to an NF-1 probe was used to control the quality and amount of nuclear protein extracts.

Figure 5

Defective IKK and NF-κB activation in IKKβ- deficient ES cells. (A) IKK activity. Lysates of TNF-α– or IL-1–treated Ikkβ^+/− and Ikkβ^−/− cells were prepared at the indicated time points (in min) after stimulation and immunoprecipitated with antibody M280 to IKKα. IKK activity (KA) was measured by an immunecomplex kinase assay using GST-IκBα(1-54) as a substrate. The kinase assay products were separated by SDS-PAGE, transferred to nitrocellulose membrane, and autoradiographed. The membrane was reprobed with antibody M280 (IB: IKKα) for loading control. (B) NF-κB binding activity. Nuclear extracts of Ikkβ^+/− and Ikkβ^−/− cells stimulated with IL-1 or TNF-α for the indicated times (in min) were incubated with ³²P-labeled κB oligonucleotide probe and subjected to EMSA. Binding to an NF-1 probe was used to control the quality and amount of nuclear protein extracts.

Figure 6

Defective IKK and NF-κB activation in IKKβ-deficient EF cells. Second passage EFs from E11.5 Ikkβ^+/+, Ikkβ^+/−, and Ikkβ^−/− embryos were stimulated with TNF-α or IL-1. At the indicated times, whole cell extracts were prepared and used to measure (A) IKK activity (KA), and (B) NF-κB DNA binding activity. IB, immunoblotting.

Figure 6

Defective IKK and NF-κB activation in IKKβ-deficient EF cells. Second passage EFs from E11.5 Ikkβ^+/+, Ikkβ^+/−, and Ikkβ^−/− embryos were stimulated with TNF-α or IL-1. At the indicated times, whole cell extracts were prepared and used to measure (A) IKK activity (KA), and (B) NF-κB DNA binding activity. IB, immunoblotting.

Figure 7

IKKα is refractory to activation in Ikkβ^−/− cells despite its association with IKKγ. (A) Ikkβ^−/− ES cells were transiently transfected by electroporation with an HA-IKKα expression vector alone or together with XpressNIK or HA-IKKβ and XpressNIK expression vectors. 24 h after transfection, HA-IKK proteins were immunoprecipitated (IP) with anti-HA antibody and their associated IKK activity (KA) was determined using GST-IκBα(1-54) as a substrate. Protein expression levels were determined by immunoblotting (IB) with anti-HA. (B) Lysates of Ikkβ^+/+, Ikkβ^+/−, and Ikkβ^−/− cells were immunoprecipitated (IP) with either anti-IKKα or anti-IKKγ antibodies as indicated. The immunecomplexes were dissolved in SDS loading buffer and separated by SDS-PAGE. After transfer to an Immobilon membrane, the proteins were analyzed by immunoblotting (IB) with anti-IKKα antibody. A lysate of 3T3 cells was used as a control (Cont).

Figure 7

IKKα is refractory to activation in Ikkβ^−/− cells despite its association with IKKγ. (A) Ikkβ^−/− ES cells were transiently transfected by electroporation with an HA-IKKα expression vector alone or together with XpressNIK or HA-IKKβ and XpressNIK expression vectors. 24 h after transfection, HA-IKK proteins were immunoprecipitated (IP) with anti-HA antibody and their associated IKK activity (KA) was determined using GST-IκBα(1-54) as a substrate. Protein expression levels were determined by immunoblotting (IB) with anti-HA. (B) Lysates of Ikkβ^+/+, Ikkβ^+/−, and Ikkβ^−/− cells were immunoprecipitated (IP) with either anti-IKKα or anti-IKKγ antibodies as indicated. The immunecomplexes were dissolved in SDS loading buffer and separated by SDS-PAGE. After transfer to an Immobilon membrane, the proteins were analyzed by immunoblotting (IB) with anti-IKKα antibody. A lysate of 3T3 cells was used as a control (Cont).