The IKKbeta Subunit of Ikappa B Kinase (IKK) is Essential for Nuclear Factor kappa B Activation and Prevention of Apoptosis

By Zhi-Wei Li,* Wenming Chu,* Yinling Hu,* Mireille Delhase,* Tom Deerinck,§ Mark Ellisman,§ Randall Johnson,Dagger and Michael Karin*

From the * Department of Pharmacology,  Laboratory of Gene Regulation and Signal Transduction, Dagger  Department of Biology, and § Department of Neuroscience, University of California, San Diego, La Jolla, California 92093-0636

    Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

The Ikappa B 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 Ikappa B 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)-kappa B 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-kappa B1 (p50/p105) subunits of NF-kappa B. Accordingly, IKKbeta -deficient cells are defective in activation of IKK and NF-kappa B 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-kappa B activity. In the absence of IKKbeta , IKKalpha is unresponsive to IKK activators.

Key words: inflammation;  tumor necrosis factor alpha ;  interleukin 1;  knockout mice;  signal transduction
    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The nuclear factor (NF)-kappa B1 transcription factor plays a key role in activation of inflammatory and innate immune responses (1, 2). In nonstimulated cells, NF-kappa B dimers are kept as cytoplasmic latent complexes through binding of specific inhibitors, the Ikappa Bs, which mask their nuclear localization signal (NLS). Upon exposure to proinflammatory stimuli, such as bacterial LPS, TNF-alpha , or IL-1, the Ikappa Bs are rapidly phosphorylated at two conserved NH2-terminal serines, a posttranslational modification that is rapidly followed by their polyubiquitination and proteasomal degradation (3). This results in unmasking of the NLS of NF-kappa B dimers followed by their translocation to the nucleus, binding to specific DNA sites (kappa B sites), and target gene activation. NF-kappa B target genes include many of the cytokine and chemokine genes, as well as genes coding for adhesion molecules, cell surface receptors, and enzymes that produce secondary inflammatory mediators (7, 8).

The protein kinase that phosphorylates Ikappa Bs in response to proinflammatory stimuli has been identified biochemically and molecularly (9). Named IKK, this protein kinase is a complex composed of at least three subunits: IKKalpha , IKKbeta and IKKgamma (for a review, see reference 12). IKKalpha and IKKbeta are highly similar protein kinases that act as the catalytic subunits of the complex (9, 11, 13, 14). In vitro, both IKKalpha and IKKbeta form homo- and heterodimers that can phosphorylate Ikappa B proteins at their NH2-terminal regulatory serines (15). In mammalian cells, IKKalpha and IKKbeta form a stable heterodimer that is tightly associated with the IKKgamma (NEMO) subunit (16, 17). As cell lines that fail to express IKKgamma (NEMO) exhibit a major defect in Ikappa B degradation and NF-kappa B activation in response to proinflammatory stimuli and double-stranded RNA, this regulatory subunit plays an essential function (at least in the examined cell lines) in IKK and NF-kappa B activation (17). The physiological function of the two catalytic subunits has been less clear. Initially, overexpression of catalytically inactive forms of IKKalpha and IKKbeta that blocked IKK and NF-kappa B activation suggested that both subunits play similar and possibly redundant roles in Ikappa B phosphorylation and NF-kappa B activation (13, 14). This hypothesis was fostered by finding that in vitro IKKalpha and IKKbeta can directly phosphorylate Ikappa Balpha and Ikappa Bbeta at the serines that trigger their degradation in vivo (15). However, it was also suggested that IKKalpha rather than IKKbeta is responsible for activation of the entire complex in response to certain stimuli, such as the NF-kappa B inducing kinase, NIK (18). Recently, we found that in addition to an IKKgamma subunit with an intact COOH terminus (16), IKK activation requires the phosphorylation of IKKbeta at two serines within its activation loop (19). Replacement of these serines, whose phosphorylation is stimulated by proinflammatory stimuli or NIK, with alanines abolishes IKK activation. Interestingly, although the entire activation loop is identical in sequence between IKKalpha and IKKbeta , replacement of the same two serines in IKKalpha with alanines has no effect on IKK activation (19). These results were further substantiated by gene targeting (knockout) experiments. Cells and tissues from mice that no longer express IKKalpha (Ikkalpha -/- mice) exhibit normal IKK activation in response to TNF, IL-1, or LPS (20). Although NF-kappa B is fully inducible, for an unknown reason, IKKalpha -deficient fibroblasts exhibit approximately twofold reduction in both basal and induced NF-kappa B binding activity (20). Thus, IKKalpha may somehow stimulate NF-kappa B DNA binding despite not being required for Ikappa B phosphorylation and degradation in most cell types. The gene targeting experiments reveal that, although not involved in activation of IKK by proinflammatory stimuli, IKKalpha plays an instrumental role in morphogenesis (20). The most important function of IKKalpha appears to be in the control of keratinocyte differentiation and formation of the epidermis (20). It is not yet clear whether these morphogenetic functions of IKKalpha are exerted through localized NF-kappa B activation in response to developmental cues.

To determine the physiological function(s) of IKKbeta , we have used gene targeting to create Ikkbeta knockout mice. We now show that the loss of IKKbeta results in embryonic lethality at mid-gestation due to extensive apoptosis of the developing liver. This phenotype is similar to that of mice deficient in the RelA (p65) subunit of NF-kappa B (21). It was recently shown that the lethality of RelA-/- mice is completely suppressed by the loss of TNF-alpha (22). As NF-kappa B is required for protection of cells from TNF-alpha -induced apoptosis (23), the apoptotic phenotype of Ikkbeta -/- mice strongly suggests that the absence of IKKbeta results in a severe defect in NF-kappa B activation. Indeed, neither IKK nor NF-kappa B can be activated by TNF-alpha or IL-1 in IKKbeta -deficient cells. Furthermore, we show that in the absence of IKKbeta , the IKKalpha subunit is not responsive to NIK even though it can still associate with the IKKgamma subunit.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Generation of IKKbeta -deficient Mice.

Using a 0.2-kb BstEII-Bsu36I restriction fragment from the 5' end of human IKKbeta cDNA as a probe, three murine IKKbeta genomic fragments were isolated from a 129/SvJ mouse genomic library (Stratagene, Inc.). One of the clones contained at least the first three coding exons and was used to construct the targeting vector IKKbeta KO. A 1.4-kb SacI restriction fragment harboring part of the second exon was used as the short homology arm, and the long arm was a 5.5-kb EcoRV-XhoI restriction fragment containing part of the third intron. The two arms were inserted into the XmnI and SmaI sites, respectively, of pGNA, which contains the G418 resistance gene (Neor) and LacZ (26). As a negative selection marker, a diphtheria toxin gene cassette (DT) was inserted into the KpnI site of pGNA. After cutting with PmeI, 20 µg of the linearized targeting vector was electroporated into 107 mouse embryonic stem (ES) cells (line GS from Genome Systems). After selection with G418 at 0.4 mg/ml, G418-resistant colonies were picked and screened by PCR. The genotype of the PCR-positive clones was confirmed by Southern blotting analysis. Homologous recombinants were karyotyped and analyzed for mycoplasma. Two homologous recombinant ES clones were injected into C57BL/6 blastocysts. Resulting male chimeras were crossed with C57BL/6 females, and germline transmission was scored by coat color. Heterozygous mice were identified by PCR and Southern analysis of mouse tail DNA. Embryos from intercrosses of heterozygous (Ikkbeta +/-) mice, as well as mouse embryonic fibroblasts (EFs), were genotyped by PCR and Southern analysis using DNA isolated from a piece of each embryo or a cell pellet, respectively.

PCR and Southern Blotting Analysis.

PCR was performed in the presence of 10% DMSO with Taq DNA polymerase using a Perkin-Elmer 9600 thermocycler programmed for denaturation at 95°C for 5 min, amplification for 35 cycles (94°C for 30 s, 55°C for 30 s, 65°C for 2 min), and elongation at 72°C for 10 min. Primers used were: P1 (5'-AGTCCAACTGGCAGCGAATA-3') located outside of the homology arm and P2 (5'-CAACATTAAATGTGAGCGAG-3') located within the LacZ gene. Southern blotting analysis was performed according to a standard protocol (27) except that hybridization was performed in phosphate-SDS buffer (28).

Kinase Assay, Immunoprecipitation, Immunoblotting, and Electrophoretic Mobility Shift Assays.

Ikkbeta -/-, Ikkbeta +/-, and Ikkbeta +/+ ES and EF cells were treated with TNF-alpha or IL-1 at 20 ng/ml. Kinase assays and immunoprecipitations were performed as described (9). Immunoblotting was performed as described (14, 16). Electrophoretic mobility shift assays (EMSAs) using the consensus kappa B and NF-1 sequences were performed as described (16, 29).

Histology, In Situ TUNEL Assay, and Transmission Electron Microscopy.

Mouse embryos or embryo livers were fixed in 10% buffered formalin and embedded in paraffin. After routine processing, the sections (5-µm thick) were stained with hematoxylin and eosin (H&E) for histological analysis. In situ TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay was done using the in situ cell death detection kit according to the manufacturer's instructions (Boehringer Mannheim). For electron microscopy, embryonic day 13 (E13) embryos were removed and the livers were dissected out and fixed for 1 h in 2% formaldehyde and 2% glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.4) at 4°C. The remainder of the embryos were placed in PBS for subsequent PCR and Southern analysis. After washing in cacodylate buffer, the livers were postfixed in 1% osmium tetroxide in cacodylate buffer for an additional 1 h. After postfixation, the samples were rinsed in double distilled water, dehydrated in a graded ethanol series, and infiltrated and polymerized in Durcupan ACM resin (Electron Microscopy Sciences). Sections 80-nm thick were stained with Sato lead and examined at 80 keV with either a JEOL 100CX or 2000EX transmission electron microscope.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Generation of Ikkbeta Knockout Mice.

To create a strain of IKKbeta -deficient mice, we used gene targeting technology (30). Mouse genomic Ikkbeta DNA was cloned from a 129 strain library and, after mapping and sequencing, was used to construct the targeting vector (Fig. 1 A). To eliminate IKKbeta kinase activity, part of the second and the entire third coding exon that specifies an essential part of the kinase domain were replaced with a DNA fragment encoding beta -galactosidase (LacZ) and neomycin resistance (Neor). Because the Neor gene contains transcription termination and polyadenylation signals, the COOH-terminal three quarters of IKKbeta including its protein interaction motifs are unlikely to be expressed from the targeted allele.


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Fig. 1.   Generation of IKKbeta -deficient mice. (A) The mouse Ikkbeta locus and the targeting vector. Map of the Ikkbeta 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 Ikkbeta ). After homologous recombination, the 9.7-kb EcoRI fragment of wild-type Ikkbeta 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 IKKbeta . Location of the IKKbeta band is indicated. The lower band is nonspecific (ns). The same blot was also probed with antibodies to IKKalpha , IKKgamma , p65(RelA), and p50(NF-kappa B1). The genotypes are as indicated.

After selection and screening by Southern blotting, six ES cell clones with homologous integration of the targeting vector into the Ikkbeta locus were isolated, and two of them were used to generate chimeric mice. Chimeric mice derived from these clones transmitted the targeted Ikkbeta allele to their progeny (Fig. 1 B). Although Ikkbeta +/- male and female mice appeared normal and were fertile, upon intercrossing they did not give rise to live Ikkbeta -/- progeny.

Analysis of protein extracts of Ikkbeta +/+, Ikkbeta +/-, and Ikkbeta -/- cells revealed that, as expected, no IKKbeta protein was expressed from the targeted allele (Fig. 1 C). In addition, Ikkbeta +/- cells expressed approximately half the dose of IKKbeta present in wild-type cells. No compensatory increases in IKKalpha , IKKgamma , p65(RelA), or p50(NF-kappa B1) expression were observed.

Phenotype of Ikkbeta -/- Mice.

Given the expected importance of IKKbeta for NF-kappa B activation and the embryonic lethality of RelA-/- mice (21), we suspected that the loss of IKKbeta would result in a similar phenotype. Therefore, we analyzed embryos from timed pregnancies of Ikkbeta +/- intercrosses. Although Ikkbeta -/- embryos isolated at E11.5 were alive and had perfectly normal appearance (data not shown), Ikkbeta -/- embryos isolated at E13.5 were no longer alive and were rather anemic in appearance (Fig. 2). Even external examination suggested that the liver of E13.5 Ikkbeta -/- embryos had degenerated. Notably, however, the limbs and head of Ikkbeta -/- embryos were normally developed, unlike those of Ikkalpha -/- E13.5 embryos (20). Histochemical examination of transverse sections of normal and mutant E13.5 mouse embryos stained with H&E revealed massive cell death in livers of Ikkbeta -/- embryos (Fig. 3 A). Essentially, no viable hepatocytes could be detected, and the numbers of dead cells with highly condensed and fragmented nuclei were markedly increased. However, hematopoietic precursors retained their normal appearance in Ikkbeta -/- livers. TUNEL staining revealed that the observed cell death is most likely due to apoptosis, whose rate was increased manyfold (Fig. 3 B). Examination of E13 Ikkbeta -/- embryos revealed close to normal external appearance (data not shown), but electronmicroscopic examination of ultrathin sections from their livers revealed massive numbers of dead hepatocytes with highly condensed nuclei characteristic of apoptotic cell death (Fig. 4). The livers of Ikkbeta +/+ or Ikkbeta +/- littermates had perfectly normal appearance.


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Fig. 2.   Appearance of an Ikkbeta -/- E13.5 embryo and a normal littermate. Wild-type (Ikkbeta +/+, WT) and mutant (Ikkbeta -/-, M) embryos were isolated at E13.5 and photographed. The genotypes of the embryos were later determined by PCR and Southern blot analysis.


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Fig. 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.


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Fig. 4.   Electron microscopic analysis of livers from E13 Ikkbeta +/+, Ikkbeta +/-, and Ikkbeta -/- embryos. Both E13 Ikkbeta +/+ (A) and Ikkbeta +/- (B) livers exhibited normal morphology. The Ikkbeta -/- liver (C) exhibited varying degrees of apoptosis characterized by collapsed and condensed nuclei and general cellular degeneration. Bars = 5 µm.

Defective NF-kappa B Activation in Ikkbeta -/- Cells.

We used two different approaches to determine the consequences of the loss of IKKbeta expression on IKK and NF-kappa B activation. First, we prepared Ikkbeta -/- ES cell lines by subjecting Ikkbeta +/- ES cells to selection at higher G418 concentration. One Ikkbeta -/- cell line was identified. As shown in Fig. 5 A, stimulation of these cells with either TNF-alpha or IL-1 did not result in IKK activation, whereas a normal activation response was observed in Ikkbeta +/- cells. Note, however, that Ikkbeta +/- cells had ~50% of the IKK activity of wild-type (Ikkbeta +/+) ES cells, consistent with the reduced amount of IKKbeta protein (data not shown). In addition to the defect in IKK activation, hardly any induction of NF-kappa B DNA binding activity was observed in Ikkbeta -/- cells after stimulation with either IL-1 or TNF-alpha (Fig. 5 B). Even the basal level of NF-kappa B DNA binding activity was considerably reduced in Ikkbeta -/- cells, despite no detectable changes in p65(RelA) or p50(NF-kappa B1) abundance (data not shown). The second approach to evaluate the function of IKKbeta was to prepare cultures of EFs from E11.5 mouse embryos of all three genotypes. As shown in Fig. 6, essentially no induction of IKK or NF-kappa B activity could be detected in Ikkbeta -/- EF cells treated with either IL-1 or TNF-alpha . Interestingly, Ikkbeta +/- EF cells exhibited an ~50% reduction in IKK activity (consistent with the reduction in IKKbeta expression) but a much larger decrease in NF-kappa B DNA binding activity.


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Fig. 5.   Defective IKK and NF-kappa B activation in IKKbeta - deficient ES cells. (A) IKK activity. Lysates of TNF-alpha - or IL-1-treated Ikkbeta +/- and Ikkbeta -/- cells were prepared at the indicated time points (in min) after stimulation and immunoprecipitated with antibody M280 to IKKalpha . IKK activity (KA) was measured by an immunecomplex kinase assay using GST-Ikappa Balpha (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: IKKalpha ) for loading control. (B) NF-kappa B binding activity. Nuclear extracts of Ikkbeta +/- and Ikkbeta -/- cells stimulated with IL-1 or TNF-alpha for the indicated times (in min) were incubated with 32P-labeled kappa B oligonucleotide probe and subjected to EMSA. Binding to an NF-1 probe was used to control the quality and amount of nuclear protein extracts.


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Fig. 6.   Defective IKK and NF-kappa B activation in IKKbeta -deficient EF cells. Second passage EFs from E11.5 Ikkbeta +/+, Ikkbeta +/-, and Ikkbeta -/- embryos were stimulated with TNF-alpha or IL-1. At the indicated times, whole cell extracts were prepared and used to measure (A) IKK activity (KA), and (B) NF-kappa B DNA binding activity. IB, immunoblotting.

IKKalpha Cannot Be Activated by NIK in the Absence of IKKbeta .

The results described above indicate that IKKalpha , which is expressed in normal levels in Ikkbeta -/- cells, cannot be activated by either TNF-alpha or IL-1. To further examine this point, we cotransfected an HA epitope-tagged IKKalpha expression vector into Ikkbeta -/- ES cells in the absence or presence of an NIK expression vector. NIK is the most potent IKK activator identified to date (31) and was suggested to be a direct IKKalpha kinase (18). Recently, however, we obtained results that suggested that NIK-induced IKKalpha phosphorylation is not direct and is likely to be dependent on IKKbeta (19). Consistent with this hypothesis, we found no increase in IKK activity towards Ikappa Balpha (1-54) substrate upon coexpression of HA-IKKalpha with NIK in Ikkbeta -/- cells (Fig. 7 A). Yet, when an IKKbeta expression vector was included in these transfections, NIK elicited a clear increase in IKK activity. As shown previously, NIK coexpression efficiently stimulates IKKalpha -associated IKK activity in IKKbeta -expressing cells (19).


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Fig. 7.   IKKalpha is refractory to activation in Ikkbeta -/- cells despite its association with IKKgamma . (A) Ikkbeta -/- ES cells were transiently transfected by electroporation with an HA-IKKalpha expression vector alone or together with XpressNIK or HA-IKKbeta 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-Ikappa Balpha (1-54) as a substrate. Protein expression levels were determined by immunoblotting (IB) with anti-HA. (B) Lysates of Ikkbeta +/+, Ikkbeta +/-, and Ikkbeta -/- cells were immunoprecipitated (IP) with either anti-IKKalpha or anti-IKKgamma 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-IKKalpha antibody. A lysate of 3T3 cells was used as a control (Cont).

One reason for the inability of IKKalpha to respond to proinflammatory stimuli or NIK in the absence of IKKbeta could be its inability to directly associate with IKKgamma , the regulatory subunit of the IKK complex. Previous experiments indicate that IKKgamma is essential for recruitment of upstream activators to IKK (16). In addition, using recombinant proteins, it was found that IKKbeta directly interacts with IKKgamma much more efficiently than does IKKalpha (16, 17). Having available IKKbeta -deficient cells, we reexamined the ability of IKKalpha to interact with IKKgamma . In contrast to the results obtained with recombinant proteins, very efficient coprecipitation of IKKalpha by anti-IKKgamma antibodies was observed using lysates of Ikkbeta -/- cells as a starting material (Fig. 7 B). Therefore, the refractoriness of IKKalpha to IKK activators in IKKbeta -deficient cells is not due to its inability to associate with IKKgamma .

    Discussion
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

The enzymatic activity of the IKK complex, composed of two catalytic subunits, IKKalpha and IKKbeta , and one regulatory subunit, IKKgamma , is rapidly stimulated by proinflammatory cytokines and LPS (for a review, see reference 12). Activated IKK phosphorylates the different Ikappa Bs at the two NH2-terminal serines that trigger their polyubiquitination and proteasome-mediated degradation. Once the Ikappa Bs are degraded, the freed NF-kappa B dimers migrate to the nucleus and activate target gene transcription. Based on their similar primary structures (11, 13, 14) and substrate specificities (15), IKKalpha and IKKbeta were expected to play redundant and interchangeable roles in proinflammatory signaling to NF-kappa B. Therefore, it was rather surprising that only IKKbeta was found to be involved in IKK activation. Alanine substitutions of the two serines in the activation loop of IKKbeta , whose phosphorylation is stimulated by either TNF-alpha treatment or NIK overexpression, prevented IKK activation. Yet, the same mutations introduced into the activation loop of the IKKalpha subunit had no effect on the response of IKK to TNF-alpha or NIK (19). These results were confirmed by the analysis of IKKalpha -deficient cells and tissues which revealed no defect in IKK activation and Ikappa Balpha degradation in response to TNF-alpha , IL-1, or LPS (20). However, it remained possible that the function of IKKalpha in Ikappa B phosphorylation in response to proinflammatory stimuli can be fully replaced by IKKbeta . The results described here indicate that IKKbeta and IKKalpha have different physiological functions and that IKKalpha cannot substitute for IKKbeta .

To determine the physiological function of IKKbeta , we generated Ikkbeta -/- knockout mice and cell lines. The loss of IKKbeta results in embryonic death at mid-gestation due to massive hepatocyte apoptosis. This phenotype is remarkably similar to that of RelA knockout mice (21), with one exception: while Ikkbeta -/- embryos die around E13, RelA-/- embryos die around E15. The earlier death of Ikkbeta -/- embryos is likely to be due to a more extensive reduction in NF-kappa B activity, as embryos that are deficient in both the p65 (RelA) and the p50 (NF-kappa B1) subunits of NF-kappa B die at E12.5, the same time as IKKbeta -deficient embryos, from massive hepatocyte apoptosis (32). Thus, IKKbeta and RelA are genetically proven to be components of the same pathway. Accordingly, cells that lack IKKbeta are completely defective in IKK and NF-kappa B activation in response to either TNF-alpha or IL-1. Therefore, the IKKbeta subunit is absolutely essential for mounting a response to proinflammatory stimuli. This function is not replaced by IKKalpha , whose expression is not diminished in the absence of IKKbeta . In addition, as indicated by the normal morphology of the head and limbs of E13.5 Ikkbeta -/- embryos, IKKalpha can carry out its developmental function (20) in the complete absence of IKKbeta . Interestingly, a 50% reduction in IKKbeta expression, as in Ikkbeta +/- cells, results in a similar decrease in IKK activity but a much more severe defect in NF-kappa B activation. These results underscore the importance of the IKKbeta subunit and indicate that the NF-kappa B activation response does not follow a simple linear relationship to the magnitude of IKK activation. It also appears from these results that a low level of NF-kappa B activity may be sufficient for protecting the liver from TNF-alpha -induced apoptosis.

One possible cause for the inability of IKKalpha to substitute for IKKbeta was its relatively lower affinity to IKKgamma , the regulatory subunit that is absolutely required for IKK activation (17). Using recombinant proteins, it was observed that IKKalpha does not form a stable complex with IKKgamma in vitro, whereas IKKbeta readily associates with IKKgamma (16, 17). However, immunoprecipitation experiments indicate that a similar amount of IKKalpha is precipitated by IKKgamma antibodies from Ikkbeta -/- cells as from Ikkbeta +/+ cells. Despite its ability to associate with IKKgamma in the absence of IKKbeta , IKKalpha is refractory to upstream activators involved in proinflammatory signaling, including the most potent IKK activator identified so far, NIK, in IKKbeta -deficient cells. These results underscore the differences in regulation of IKKalpha and IKKbeta activities.

In summary, together with the previous analysis of IKKalpha -deficient mice, the analysis of IKKbeta -deficient mice, described here, indicates that the two catalytic subunits of the IKK complex, although similar in structure, have very different functions. Although IKKbeta is responsible both for activation of the entire complex in response to proinflammatory stimuli, through phosphorylation at its activation loop, and for activation of NF-kappa B, through Ikappa B phosphorylation, IKKalpha is assigned the control of epidermal and skeletal morphogenesis. Although the stimuli that activate IKKbeta and the substrates that mediate its biological activity are known, the stimuli and the relevant substrates for IKKalpha remain to be identified.

    Footnotes

Address correspondence to Michael Karin, Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0636. Phone: 619-534-1361; Fax: 619-534-8158; E-mail: karinoffice{at}ucsd.edu

Received for publication 7 April 1999.

   Y. Hu was supported by a postdoctoral fellowship from the Arthritis Foundation. This work was supported by grants from the National Institutes of Health (AI43477, ES04151, AG05131, and RR04050) and the Department of Energy (DE-FG03-86ER60429). M. Karin is the Frank and Else Schilling-American Cancer Society Research Professor.

We thank G. Hageman for the gift of mouse TNF-alpha , D.M. Rothwarf for advice and technical assistance, and B. Thompson for assistance with manuscript preparation.

Abbreviations used in this paper EF, embryonic fibroblast; EMSA, electrophoretic mobility shift assay; ES, embryonic stem; H&E, hematoxylin and eosin; IKK, Ikappa B kinase; NF, nuclear factor; NIK, NF-kappa B inducing kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

    References
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

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