severe liver degeneration and lack of nf kb activation in...

Severe liver degenerationand lack of NF-kB activationin NEMO/IKKg-deficient miceDorothea Rudolph,1 Wen-Chen Yeh,1 Andrew Wakeham,1 Bettina Rudolph,2 Dhani Nallainathan,1

Julia Potter,1 Andrew J. Elia,1 and Tak W. Mak1,3

1The Amgen Institute, Ontario Cancer Institute, and Departments of Medical Biophysics and Immunology,University of Toronto, Toronto, Ontario M5G 2C1, Canada; 2Imperial Cancer Research Fund, WC2A 3PX London, UK

Phosphorylation of IkB, an inhibitor of NF-kB, is an important step in the activation of the transcriptionfactor NF-kB. Phosphorylation is mediated by the IkB kinase (IKK) complex, known to contain two catalyticsubunits: IKKa and IKKb. A novel, noncatalytic component of this kinase complex called NEMO (NF-kBessential modulator)/IKKg was identified recently. We have generated NEMO/IKKg-deficient mice by genetargeting. Mutant embryos die at E12.5–E13.0 from severe liver damage due to apoptosis. NEMO/IKKg-deficient primary murine embryonic fibroblasts (MEFs) lack detectable NF-kB DNA-binding activity inresponse to TNFa, IL-1, LPS, and Poly(IC) and do not show stimulus-dependent IkB kinase activity, whichcorrelates with a lack of phosphorylation and degradation of IkBa. Consistent with these data, mutant MEFsshow increased sensitivity to TNFa-induced apoptosis. Our data provide in vivo evidence that NEMO/IKKg isthe first essential, noncatalytic component of the IKK complex.

[Key Words: NEMO/IKKg; IKK; NF-kB; liver apoptosis]

Received November 16, 1999; revised version accepted February 23, 2000.

The transcription factors of the Rel/NF-kB family arecritical regulators of genes involved in inflammatory,immune and acute phase responses, proliferation, andapoptosis (Baeuerle and Henkel 1994). NF-kB activationis tightly controlled by IkB proteins. In resting cells, NF-kB is bound by IkB proteins, which mask the nuclearlocalization signal of NF-kB and prevent its nucleartranslocation. Stimulation of the cell by a variety ofstimuli, including proinflammatory cytokines such astumor necrosis factor (TNFa) and interleukin-1 (IL-1),bacterial lipopolysaccharide (LPS) or viral infection,leads to the phosphorylation of IkB proteins on two con-served serine residues (Ser-32 and Ser-36 on IkBa andSer-19 and Ser-23 on IkBb) (DiDonato et al. 1996). Thephosphorylated IkB proteins become targets for ubiqui-tination and proteasome-mediated degradation, conse-quently releasing NF-kB and allowing its translocationinto the nucleus (Verma et al. 1995).

Recently, an IkB kinase activity was identified as alarge complex, which contains two catalytic subunits,IKKa and IKKb (DiDonato et al. 1997; Mercurio et al.1997; Regnier et al. 1997; Woronicz et al. 1997; Zandi etal. 1997). These kinases are 52% identical and share sev-eral structural domains, including an amino-terminal ki-nase domain, a helix–loop–helix (HLH) domain and a

leucine zipper (LZ) domain (Mercurio et al. 1997; Zandiet al. 1997). IKKa and IKKa form homo- and het-erodimers in vitro, although only heterodimers are foundin vivo (Woronicz et al. 1997). In vitro phosphorylationassays have shown that both kinases can phosphorylateIkBa on Ser-32 and Ser-36, but IKKb seems to be moreactive in this regard (Woronicz et al. 1997). In addition,whereas IKKb phosphorylates both critical serine resi-dues of IkBa and IkBb with equal efficiency (Woronicz etal. 1997), IKKa preferentially phosphorylates IkBb onSer-23 (Regnier et al. 1997). Catalytically inactive formsof both kinases inhibited TNFa and IL-1-induced NF-kBactivation (Mercurio et al. 1997; Regnier et al. 1997;Woronicz et al. 1997; Zandi et al. 1997), suggesting thatthe kinases would have similar physiological functions.Surprisingly, recent gene targeting experiments haveshown that, even though IKKa and IKKb are part of thesame IKK complex, share sequence homology and seemto have similar activities in vitro, IKKa-deficient miceand IKKb-deficient mice have completely different phe-notypes and the kinases seem to respond to differentbiological inducers. Although IKKb mediates the re-sponse to proinflammatory cytokines such as TNFa andIL-1 (Li et al. 1999b; Z. Li et al. 1999; Tanaka et al. 1999),IKKa seems to respond to an unknown morphogeneticsignal (Hu et al. 1999; Li et al. 1999a; Takeda et al. 1999).IKKb−/− mice die at E12.5 to E14.5 from liver degenera-tion due to apoptosis (Li et al. 1999b; Z. Li et al. 1999;

3Corresponding author.E-MAIL [email protected]; FAX (416) 204-2278.

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Tanaka et al. 1999), a phenotype similar to, but moresevere than, that of RelA−/− mice (Beg et al. 1995). Incontrast, IKKa−/− mice survive to term but die shortlyafter birth (Hu et al. 1999; Li et al. 1999a; Takeda et al.1999). IKKa−/− mice display rudimentary limbs and a tailas well as severe craniofacial deformities and skeletalabnormalities. Lack of IKKa also affects skin develop-ment, because the mutant epidermis shows increasedthickness due to hyperproliferation of cells at the basallayer and a block in keratinocyte differentiation.

Two additional subunits of the IkB kinase complexhave now been identified, IKAP (IKK-complex-associ-ated protein) (Cohen et al. 1998), a potential scaffold pro-tein, and NEMO (NF-kB essential modulator) (Yamaokaet al. 1998), also called IKKg (Rothwarf et al. 1998) orIKKAP1 (IkB kinase-associated protein 1) (Mercurio et al.1999). NEMO/IKKg is a glutamine-rich protein of 48 kD,which lacks a catalytic domain but contains two coiled–coil motifs and a LZ. NEMO/IKKg seems to be essentialfor NF-kB activation. Introduction of a cDNA encodingNEMO/IKKg was able to restore NF-kB DNA-bindingactivity in two cell lines with defective NF-kB activation(Yamaoka et al. 1998). Considering that both catalyticsubunits of the IKK complex, IKKa and IKKb, showsimilar in vitro activities but turned out to have verydifferent physiological functions in vivo, we have gener-ated NEMO/IKKg-deficient mice to analyze the physi-ological relevance of this noncatalytic subunit of the IKKcomplex. In particular, we were curious to find outwhether the phenotype of NEMO/IKKg-deficient micewould be similar to the phenotype observed in IKKa- orIKKb-deficient mice.

NEMO/IKKg-deficient mice die at E12.5–E13.0 fromsevere liver degeneration due to apoptosis. NEMO/IKKg-deficient primary murine embryonic fibroblasts (MEFs)show no detectable NF-kB DNA-binding activity in re-sponse to stimulation with TNFa, IL-1, LPS, or Poly(IC).Stimulus-dependent phosphorylation and degradation ofIkBa is absent in mutant MEFs and IkB kinase activity isnot detectable. Mutant MEFs are highly sensitive toTNFa and display reduced viability in response to TNFatreatment even in the absence of cycloheximide.

Results

Generation of NEMO/IKKg-deficient mice

A targeting vector was designed to replace exon 1, en-coding the translation start site, and exon 2 with a PGK–neo cassette (Fig. 1A). Correctly targeted ES cell cloneswere identified by Southern blot analysis and three ESclones were injected into C57BL/6 blastocysts to gener-ate chimeric mice. Chimeric mice from all three clonestransmitted the targeted NEMO/IKKg-allele to theirprogeny. Genotypes were confirmed by Southern blot(Fig. 1B) and PCR analysis (Fig. 1C). NEMO/IKKg hasbeen mapped to the X chromosome (Jin and Jeang 1999;data not shown). Therefore, we could only generate het-erozygous females from intercrosses of chimeric maleswith C57BL/6 females, which were then mated with

C57BL/6 males to generate homozygous mutant mice.Although NEMO/IKKg+/− females appeared normal andwere fertile, viable homozygous mutant mice could notbe generated from matings with C57BL/6 males, sug-gesting that a lack of NEMO/IKKg results in embryoniclethality.

Western blot analysis of protein extracts from NEMO/IKKg-deficient MEFs confirmed the absence of NEMO/IKKg protein in mutant cells (Fig. 1D), indicating thatwe have generated a NEMO/IKKg null mutation. Wealso did not detect changes in protein levels of IKKa,IKKb, IkBa, or p65 (RelA) in mutant cells (data notshown).

Liver degeneration in NEMO/IKKg-deficient embryos

Extensive timed matings were performed (Table 1).

Figure 1. Generation of NEMO/IKKg−/− mice. (A) Wild-typeNEMO/IKKg locus (top). Exon 1 and 2 are shown as open boxesand the ATG translation start site is indicated. (P) Externalflanking probe, (B) BamHI, (RV) EcoRV, (S) SpeI. The targetingvector (middle) was designed to replace exon 1 and exon 2 of thewild-type locus with a neomycin cassette in antisense orienta-tion. Flanking probe P detects a 6-kb BamHI fragment corre-sponding to the wild-type allele and a 3-kb BamHI fragment forthe targeted allele (bottom). (B) Genomic Southern blot ofBamHI-digested DNA from E12.5 embryos of the indicatedgenotypes, showing the 6-kb wild-type band and the 3-kb mu-tant band detected by probe P. (C) Genotyping by PCR of DNAfrom E12.5 embryos using the primers described in Material andMethods. (D) Western blot analysis of NEMO/IKKg protein ex-pression in cytoplasmic extracts of MEFs of the indicated geno-types.

Targeted disruption of NEMO/IKKg

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NEMO/IKKg−/− embryos were normal at E11.5. On sec-tions of E12.5−/− embryos, areas of hemorrhaging andtissue loss were apparent in parts of the liver (Fig. 2A,B;data not shown). At E13.0–E13.5, mutant embryos weredead and anemic in appearance. Mutant embryos atthese stages showed massive liver hemorrhages, which

are most likely due to the breakdown of the liver archi-tecture at this developmental stage (Fig. 2D). To deter-mine whether the observed cell death in the liver wasdue to apoptosis, TUNEL assays were performed on sec-tions of E11.5 and E12.5 wild-type and mutant embryos.Only a few isolated TUNEL-positive cells could be de-tected on sections from wild-type embryos at both stages(Fig. 2G; data not shown). Mutant embryos at E11.5showed only a slightly increased number of TUNEL-positive cells in small areas of the liver (data not shown).However, by E12.5, greatly increased numbers ofTUNEL-positive cells in large areas throughout the liverwere observed in mutant embryos (Fig. 2H), suggestingthat apoptosis in the liver is a major cause of death of themutant embryos. Enhanced apoptosis in the mutant em-bryos appeared to be restricted to the liver, as other partsof the NEMO/IKKg−/− embryos did not show an increasein TUNEL-positive cells. Cell proliferation in E12.5 mu-tant embryos as detected by BrdU incorporation was gen-erally comparable with wild-type embryos (data notshown) except in the liver (Fig. 2E,F). Of note, sections ofdorsal skin at E12.5, which at this stage consists of asingle layer of cells with underlying mesenchyme,showed similar proliferation in wild-type and mutantskin (data not shown).

Lack of NF-kB activation in NEMO/IKKg-deficientMEFs

The above-mentioned phenotype of NEMO/IKKg−/−

mice is similar to, albeit more severe than, that of RelA-deficient mice (Beg et al. 1995). RelA is a subunit of themost common form of NF-kB, the p50–RelA heterodi-mer. We therefore assessed whether the activation of NF-kB DNA-binding activity was impaired in NEMO/IKKg-deficient MEFs. Wild-type and NEMO/IKKg-deficientMEFs were treated with TNFa, IL-1, LPS, or poly(IC) toinduce NF-kB DNA-binding activity and nuclear ex-tracts were examined by gel shift assay. In contrast towild-type MEFs, mutant MEFs showed no detectable NF-kB DNA-binding activity in response to any of thesestimuli (Fig. 3; data not shown). Interestingly, IKKa−/−

MEFs show normal NF-kB DNA-binding activity in re-sponse to these stimuli (Hu et al. 1999; Li et al. 1999a;Takeda et al. 1999), whereas NF-kB activation in IKKb−/−

MEFs is impaired, although it still occurred with delayedkinetics and reduced magnitude (Li et al. 1999b; Tanakaet al. 1999).

Lack of IkBa phosphorylation/degradationin NEMO/IKKg-deficient MEFs

Next, we analyzed whether stimulus-dependent phos-phorylation and degradation of IkBa was affected by theabsence of NEMO/IKKg. Wild-type and NEMO/IKKg-deficient MEFs were stimulated with TNFa for 0, 6, and10 min. Cytoplasmic extracts were prepared and used forWestern blot analysis with an anti-phospho IkBa-spe-cific antibody. Phosphorylation of IkBa could be de-

Table 1. Genotypes of embryos derived from matingsof NEMO/IKKg+/− females with C57BL/6 males

Stage +/+ +/− −/−a Total

E11.5 12 8 2 22E12.5 54 29 20 103E13.0 3 1 3 7E13.5 13 5 2* 20E14.5 6 2 1* 9

a(*) In resorption.

Figure 2. Massive liver apoptosis in the absence of NEMO/IKKg. (A–D) Histological analysis of liver sections from E12.5(A,B) and E13.0 (C,D) wild-type (A,C) and NEMO/IKKg-defi-cient (B,D) embryos. Hematoxylin and eosin staining; high-power view. (D) The inset shows a low power view of the sameregion shown in D. The arrow points to a large area of tissue lossin which erythrocytes have accumulated. (E,F) Detection of cellproliferation as determined by BrdU incorporation. Pregnant fe-males were injected IP with BrdU 60-min prior to sacrifice. Sec-tions of E12.5 wild-type and mutant embryos were prepared andimmunostained with anti-BrdU antibody. A high power view ofthe liver is shown. (G,H) Enhanced apoptosis in livers of E12.5NEMO/IKKg-deficient embryos. TUNEL assays were per-formed on sections of wild-type and mutant E12.5 embryos. Ahigh power view of the liver is shown in both cases.

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tected in wild-type cells as soon as 6 min after TNFastimulation (Fig. 4A, top left). However, no phosphory-lation of IkBa could be detected in extracts of NEMO/IKKg-deficient MEFs even after 10 min stimulation withTNFa (Fig. 4A, top right). An anti-IkBa specific antibodywas used as a control to detect IkBa protein expression in−/− extracts. (Fig. 4A, bottom). As shown in Figure 4B,IkBa is degraded in wild-type cells and reappears 30–60min after stimulation (Fig. 4B, top), whereas degradationof IkBa is completely absent in mutant cells (Fig. 4B,bottom).

Impaired IkB kinase activity in NEMO/IKKg-deficientMEFs

The failure to phosphorylate IkBa in the absence ofNEMO/IKKg prompted us to examine the in vitro kinase

activity of the IKK complex. Wild-type and NEMO/IKKg-deficient MEFs were treated for 7 min with TNFa

Figure 4. Defective TNFa-induced IkBa phosphorylation anddegradation and IkB kinase activity in the absence of NEMO/IKKg. (A) IkBa phosphorylation. (Top) Wild-type and NEMO/IKKg-deficient MEFs were treated with TNFa (10 ng/ml) for theindicated times followed by Western blot analysis of cytoplas-mic extracts with anti-phospho-IkBa-specific antibody. (Bot-tom) Western blot of the same extracts with anti-IkBa antibodyas a control to show IkBa protein expression in mutant cells. (B)IkBa degradation. Wild-type (top) and NEMO/IKKg-deficient(bottom) MEFs were treated with TNFa (10 ng/ml) for the in-dicated times followed by Western blot analysis with anti-IkBa

specific antibody. (C) IkB kinase activity. Wild-type and NEMO/IKKg-deficient MEFs were either left in medium alone ortreated with TNFa (10 ng/ml) for 7 min. Whole cell extractswere immunoprecipitated with anti-IKKa antibody and in vitrokinase activity was determined as described in Materials andMethods using GST–IkBa (1–72) (left) or GST–IkBa (1–72) S/A(right) as a substrate. One representative experiment of four isshown.

Figure 3. Impaired NF-kB activation in NEMO/IKKg-deficientcells. Wild-type or NEMO/IKKg−/− MEFs were incubated withmouse recombinant TNFa (10 ng/ml) (A), LPS (15 µg/ml) (B), orIL-1 (10 ng/ml) (C) for the indicated times. NF-kB activation in10 µg of nuclear extract was determined by gel shift assay asdescribed in Materials and Methods.






to induce kinase activity. Whole cell extracts were im-munoprecipitated with anti-IKKa antibody and kinaseactivity was assessed in vitro using GST–IkBa (1–72) orGST–IkBa (1–72) S/A, containing a Ser-32/Ser-36 to Ala-32/Ala-36 mutation as a substrate. Extracts of wild-typecells were able to phosphorylate GST–IkBa (1–72) invitro, whereas stimulus-dependent kinase activity in ex-tracts of NEMO/IKKg-deficient MEFs was severely im-paired (Fig. 4C), indicating that NEMO/IKKg is essentialfor IkB kinase activity. Similar results were obtainedwith a longer substrate [GST–IkBa (1–317)] (data notshown).

Expression of NF-kB-dependent target genesis impaired in NEMO/IKKg-deficient MEFs

The above data showed that NF-kB activation is defec-tive in the absence of NEMO/IKKg. We therefore exam-ined the expression of known NF-kB-dependent targetgenes. IkBa is one of the first genes to be up-regulatedfollowing NF-kB activation, establishing a feedback loop,which will eventually shut down the NF-kB response.This induction, but not basal IkBa mRNA expression, isregulated by RelA (Scott et al. 1993). To analyze IkBamRNA expression in the absence of NEMO/IKKg, RNAfrom wild-type and NEMO/IKKg-deficient MEFs treatedwith TNFa for 0, 30, and 60 min was analyzed by North-ern blot. As shown in Figure 5A, an IkBa transcript in-duced within 60 min of TNFa stimulation in wild-typecells is absent in NEMO/IKKg-deficient MEFs. Interest-ingly, IKKb−/− MEFs are still able to express the TNFa-induced IkBa transcript, although the level of transcrip-tion is reduced compared with wild-type cells (Li et al.1999b).

Stimulation of cells with IL-1 leads to expression ofvarious genes, including interleukin-6 (IL-6), an impor-tant mediator of acute phase reactions. IL-1 induces IL-6expression through the coordinated action of NF-kB andthe transcription factor NF-IL6 (Akira and Kishimoto1992). We therefore treated wild-type and NEMO/IKKg-deficient MEFs with IL-1 for 16 hr and measured thelevels of IL-6 in the culture supernatant using an ELISA.Whereas wild-type MEFs showed stimulus-dependentproduction of IL-6, IL-1 treatment of mutant MEFs failedto up-regulate IL-6 expression (Fig. 5B). Levels of IL-6 inthe stimulated NEMO/IKKg-deficient cultures wereequivalent to those in wild-type cultures treated withmedium alone. These data demonstrate impaired induc-tion of acute phase response proteins in the absence ofNEMO/IKKg.

Cells lacking NF-kB activity have been shown to un-dergo apoptosis in response to TNFa stimulation (Begand Baltimore 1996; Van Antwerp et al. 1996; Wang et al.1996), suggesting that NF-kB activation provides protec-tion against TNFa-induced apoptosis. To determinewhether sensitivity to TNFa-induced apoptosis was in-creased in the absence of NEMO/IKKg, the viability ofwild-type and NEMO/IKKg-deficient MEFs after treat-ment with various concentrations of TNFa for 18 hr wasassessed. Wild-type MEFs were resistant to TNFa-medi-

ated cytotoxicity, but NEMO/IKKg deficient MEFsshowed a dramatic reduction in viability even in the ab-sence of cycloheximide (Fig. 6A). TNFa-sensitivity ofNEMO/IKKg-deficient MEFs could be further increasedby addition of very low concentrations of cycloheximide(30–100 ng/ml). Significantly higher concentrations ofcycloheximide in combination with TNFa were requiredto affect viability of wild-type MEFs (Fig. 6B).

Discussion

In this study, we have shown that NEMO/IKKg, a com-ponent of the IKK complex, is required for the phos-phorylation and degradation of IkBa. Lack of NEMO/IKKg affects IkB kinase activity, in particular also IKKakinase activity (Fig. 4C). We have demonstrated thatNEMO/IKKg is crucial for normal stimulus-dependentNF-kB activation and target gene expression. NEMO/

Figure 5. Impaired expression of NF-kB target genes in NEMO/IKKg-deficient MEFs. (A) TNFa-induced IkBa mRNA synthe-sis. Wild-type and NEMO/IKKg-deficient MEFs were stimu-lated with TNFa (10 ng/ml) for the indicated times and 20 µg ofRNA was analyzed by Northern blot analysis using a 32P-labeledrandom priming probe corresponding to the full-length IkBa

cDNA. b-actin, loading control. (B) IL-1 induced IL-6 produc-tion. Wild-type and NEMO/IKKg-deficient MEFs (3 × 104) wereleft untreated or stimulated with IL-1 (10 ng/ml) for 16 hr. Lev-els of IL-6 (pg/ml) in culture supernatants were determined byELISA. Values shown are the mean and standard deviation oftriplicate samples.

Rudolph et al.





IKKg-deficient MEFs show increased sensitivity toTNFa treatment, implying that the liver apoptosis ob-served in NEMO/IKKg-deficient mice could be due to alack of NF-kB-mediated protection from TNFa cytotox-icity. RelA−/− and IKKb−/− fibroblasts are also highly sen-sitized to TNFa in vitro (Beg et al. 1995; Li et al. 1999b;Tanaka et al. 1999). Interestingly, RelA/TNFa double-deficient mice (Doi et al. 1999) as well as IKKb/TNFR1double-deficient mice (Li et al. 1999b) are born alive,consistent with the hypothesis that enhanced sensitivityto endogenous TNFa is responsible for the liver apopto-sis observed in these mice.

NEMO/IKKg is a noncatalytic component of the IkBkinase complex. Precisely how it functions within thecomplex remains to be clarified. Analysis of a NEMO/IKKg-deletion mutant series showed that the carboxylterminus of the protein (amino acids 300–419) is requiredfor TNFa-stimulated IkB kinase activity but not basalkinase activity (Rothwarf et al. 1998). Furthermore, thismutation did not interfere with the binding of NEMO/IKKg to IKKa/b in cells, suggesting that the carboxyl

terminus might be required to connect the IKK complexto upstream activators. Gene-targeting experiments haveshown that IKKb rather than IKKa mediates the re-sponse to proinflammatory cytokines and that IKKaseems to respond to an unknown morphogenetic signal(Hu et al. 1999; Li et al. 1999a,b; Z. Li et al. 1999; Takedaet al. 1999; Tanaka et al. 1999). At E18.5, IKKa−/− micedisplay rudimentary limbs and a tail as well as severecraniofacial deformities and skeletal abnormalities. Ta-keda et al. (1999) and Li et al. (1999a) show that IKKa−/−

embryos at E12.5 already have mild alterations in themorphology of the limb buds (Li et al. 1999a; Takeda etal. 1999), whereas Hu et al. (1999) state that at E14.5, theforelimbs and hindlimbs of IKKa−/− embryos are not sig-nificantly shorter than those of wild-type littermates. AtE12.5, limbbuds of NEMO/IKKg-deficient mice are in-distinguishable from those of wild-type littermates (datanot shown), even though kinase assays (Fig. 4C) showedthat IKKa activity is severely impaired in extracts ofNEMO/IKKg-deficient MEFs. However, this does not ex-clude the possibility that NEMO/IKKg-deficient micewould show a phenotype similar to that observed inIKKa-deficient mice at E18.5, if they survived until thisdevelopmental stage. Lack of IKKa also affects skin de-velopment because the mutant epidermis at E18.5 showsincreased thickness due to hyperproliferation of cells atthe basal layer and a block in keratinocyte differentia-tion (Hu et al. 1999; Li et al. 1999a; Takeda et al. 1999).However, at E12.5, proliferation of the skin as detectedby BrdU incorporation is comparable in wild-type andNEMO/IKKg-deficient embryos (data not shown). AtE12.5, the skin consists of a single layer of cells withunderlying mesenchyme. Epidermal differentiation doesnot occur until later in development. Therefore, we can-not exclude the possibility that NEMO/IKKg-deficientmice would show defects in epidermal differentiationsimilar to those observed in IKKa-deficient mice atE18.5, if they survived until this developmental stage,especially as IKKa kinase activity is severely impaired inNEMO/IKKg-deficient MEFs (Fig. 4C).

Overall, the phenotype observed in NEMO/IKKg-defi-cient mice is more severe than that of IKKb−/− mice. Inparticular, the onset of the liver phenotype occurs atleast 12 hr earlier in NEMO/IKKg-deficient embryosthan in IKKb−/− embryos. Even though IKKb−/− embryosshow hemorrhages in the liver on E13.0, the overall liverarchitecture is still intact (Li et al. 1999b). In contrast,NEMO/IKKg-deficient embryos show large areas of tis-sue loss at this stage and a massive accumulation oferythrocytes. Moreover, IKKb−/− MEFs still show re-sidual NF-kB DNA-binding activity in response tostimulation with TNFa or IL-1 (Z. Li et al. 1999; Tanakaet al. 1999), whereas NF-kB DNA-binding activity in re-sponse to these stimuli is completely abolished inNEMO/IKKg-deficient MEFs. This suggests that the re-sidual NF-kB DNA-binding activity observed in IKKb−/−

mice might provide limited protection to liver cellsagainst apoptosis induced by endogenous TNFa. The ear-lier onset of the phenotype in NEMO/IKKg-deficientmice might then be explained by the complete absence

Figure 6. TNFa-induced cytotoxicity. (A) TNFa-induced cyto-toxicity. Wild-type and NEMO/IKKg-deficient (2 × 105) MEFswere left untreated or stimulated with the indicated concentra-tions of TNFa for 18 hr. Cells were then harvested and stainedwith PI/AnnexinV-FITC. Values shown are the percentage ofviable cells after treatment relative to untreated controls. (B)Cytotoxicity induced by TNFa plus cycloheximide. Wild-typeand NEMO/IKKg-deficient MEFs (2 × 105) were left untreatedor stimulated with TNFa (10 ng/ml) in combination with theindicated concentrations of cycloheximide for 18 hr. Cells wereharvested and stained as described for A, and values shown weredetermined as for A.






of NF-kB DNA-binding activity. As shown by Li et al.(1999b) the residual NF-kB DNA-binding activity ob-served in IKKb−/− MEFs is still sufficient to induce a lowlevel of expression of NF-kB-dependent target genes suchas IkBa (Li et al. 1999b). Sixty minutes after TNFastimulation of IKKb−/− MEFs, about half the amount ofIkBa mRNA observed in stimulated wild-type cells isdetected in IKKb mutant cells. However, IkBa mRNA isnot induced at all in NEMO/IKKg-deficient MEFs,which is consistent with the lack of detectable NF-kBDNA-binding activity in these cells.

In summary, the effects of the targeted disruption ofthe NEMO/IKKg gene on NF-kB activation by a varietyof stimuli are more dramatic than the deletion of eithercatalytic component of the IKK complex. We thereforespeculate that the phenotype of IKKa/IKKb double-de-ficient mice will be similar to that of NEMO/IKKg-de-ficient mice. In any case, our data provide strong in vivoevidence that NEMO/IKKg is essential for activation ofNF-kB by various stimuli. Our study combined with pre-vious reports clearly demonstrates that NEMO/IKKg isthe first essential, non-kinase component of the IKKcomplex.

Material and methods

Generation of NEMO/IKKg-deficient mice

Phage clones containing the murine NEMO/IKKg gene wereisolated by screening a 129/J genomic library with a probe cor-responding to the 58 end of the NEMO cDNA (Yamaoka et al.1998). A targeting vector was designed to replace exon 1 andexon 2 with a PGK–neo cassette in antisense orientation, lead-ing to the replacement of the first 131 amino acids of theNEMO/IKKg protein and a downstream frameshift. A total of30 µg of the linearized targeting vector were used to electropor-ate 5 × 106 E14K ES cells (Bio-Rad Gene Pulser, 0.34 kV, 0.25mF), which were subsequently cultured for 10 days in DMEMsupplemented with 15% FBS, glutamine, pyruvate, b-mercap-toethanol, and leukemia inhibitory factor, containing 300 µg/ml G418 (Sigma). Recombinants were identified by PCR andconfirmed by Southern blot analysis using BamHI-digested ge-nomic DNA hybridized to an external flanking probe, whichdetects a 6-kb band for the wild-type locus and a 3-kb band forthe mutant allele. Single integration was confirmed using aprobe corresponding to the neo gene. Three correctly targetedES clones were injected into C57BL/6 blastocysts and all threesuccessfully generated germ-line chimeras. Genotyping of micewas performed by PCR using tail genomic DNA with the fol-lowing primers: 58-ACTTAAAGGTGTGTGGAGGAGGTAG-G-38, 58-TGCATGTGACTCTTCACAGAGAACCC-38, 58-GG-GTGGGATTAGATAAATGCCTGCTC-38. These primers giverise to a 621-bp PCR product for the wild-type locus and a 354-bp PCR product for the mutant allele. The following PCR con-ditions were used: 1 min at 95°C, 30 sec at 64°C, 1.5 min at72°C, for 35 cycles.

Animal husbandry

Mice were maintained in the animal facility of the OntarioCancer Institute in accordance with its ethical guidelines.

Histology

Embryos were fixed with 4% buffered formalin at 4°C for 12 hr

and embedded in paraffin. Sections were stained with hema-toxylin and eosin according to standard protocols. For the de-tection of BrdU incorporation, immunostaining with anti-BrdUantibody was performed on paraffin sections according to stan-dard procedures. Pregnant females were injected with 300 µl ofBrdU (10 mg/ml) 60 min prior to sacrifice. For determination ofapoptosis, TUNEL assays were performed using an in situ celldeath detection kit (Boehringer Mannheim) according to themanufacturer’s instructions.

Generation of primary MEFs

Primary MEFs were derived from E12.5 embryos. The head andliver were removed and the remainder of the embryo was cutinto small pieces and trypsinized. Cells were washed in mediumand plated onto 6-well plates. Embryonic fibroblasts were ex-panded in DMEM supplemented with 10% FBS and b-mercap-toethanol and frozen.

Western blot analysis

Cytoplasmic extracts were prepared from primary MEFs as de-scribed previously (Yamaoka et al. 1998). Briefly, cells werestimulated, washed with cold PBS, resuspended at 106/20 µl inhypotonic solution [10 mM HEPES at pH 7.8, 10 mM KCl, 2 mM

MgCl2, 1 mM DTT, and 0.1 mM EDTA supplemented with aprotease inhibitor cocktail (Boehringer Mannheim)] and kept onice for 10 min. NP-40 was added to a final concentration of 1%and the cells were centrifuged at 14,000 rpm for 1 min. Thesupernatant, containing the cytoplasmic fraction, was recov-ered and the protein concentration was determined using a Bio-Rad protein assay. Protein samples were fractionated on a 10%SDS–polyacrylamide gel and blotted onto a PVDF membrane.Western blots were developed using an enhanced chemilumi-nescence system according to the manufacturer’s instructions(Amersham). Anti-phospho-IkBa and anti-IkBa were from NewEngland Biolabs. Anti-NEMO antiserum was kindly provided byAlain Israel (Institute Pasteur, Paris, France).

Gel mobility shift assay

MEFs (2 × 106) were lysed in 400 µl of hypotonic solution asdescribed above and nuclear extracts were prepared for gel mo-bility shift assays as described previously (Yeh et al. 1997). Atotal of 10 µg of nuclear extract was incubated with an end-labeled, double-stranded oligo containing two NF-kB bindingsites (58-ATCAGGGACTTTCCGCTGGGGACTTTCCG-38 and58-CGGAAAGTCCCCAGCGGAAAGTCCTGAT-38). The re-action was performed in 20 µl of binding buffer (5 mM HEPES atpH 7.8, 50 mM KCl, 0.5 mM DTT, 1 µg of [poly d(I-C)], 10%glycerol] for 20 min at room temperature. Samples were frac-tionated on a 5% polyacrylamide gel and visualized by autora-diography.

Kinase assay

MEFs (5 × 106) were lysed as described previously (Tanaka et al.1999). Briefly, cells were lysed with lysis buffer containing 50mM HEPES (pH 7.6), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA,10% glycerol, 1% Triton X-100, protease inhibitor cocktail(Boehringer Mannheim), 20 mM b-glycerophosphate, 1 mM so-dium orthovanadate, and 10 mM NaF. After incubation on icefor 30 min, lysates were centrifuged for 10 min at 14,000 rpmand the protein concentration of the supernatant was deter-mined using the Bio-Rad protein assay. A total of 500 µg ofprotein extract was precleared using protein G–Sepharose (Phar-

Rudolph et al.





macia) and immunoprecipitated with anti-IKKa (Santa Cruz).The immunoprecipitates were washed twice with lysis bufferand twice with kinase buffer [20 mM HEPES at pH 7.9, 10 mM

MgCl2, 100 mM NaCl, protease inhibitor cocktail (BoehringerMannheim), 20 mM b-glycerophospate, 1 mM sodium ortho-vanadate, 2 mM DTT] and assayed for kinase activity as de-scribed previously (Tanaka et al. 1999) using [g32P]ATP and 3 µgof GST–IkBa (1–72) or GST–IkBa (1–72) S/A as a substrate. Re-action products were fractionated by 10% SDS-PAGE and phos-phorylated IkBa was visualized by autoradiograhy.

Measurement of IL-6 production

MEFs (3 × 104) were plated onto 24-well plates in DMEMsupplemented with 10% FBS. After 24 hr, cells were treatedwith IL-1 (10 ng/ml) (R&D Systems) for 16 hr. The concentra-tion of IL-6 in the culture supernatants was determined byELISA (R&D Systems).

Northern blot analysis

Total RNA was prepared from MEFs using Trizol (GIBCO BRL).RNA (20 µg) was fractionated on a 1% formaldehyde gel andtransferred onto a Hybond N membrane (Amersham). Filterswere hybridized at 65°C in 50% formamide, 5× SSC, 25 mM

Na2HPO4 (pH 6.5), 8× Denhardt’s solution, 0.5 mg/ml salmonsperm DNA, and 1% SDS with a random priming probe corre-sponding to the full-length murine IkBa cDNA or a b-actincDNA as a control.

MEF cell death assay (PI/AnnexinV-FITC staining)

MEFs (2 × 105) were plated onto six-well plates 24 hr prior toinduction with various concentrations of TNFa (R&D Systems)alone or TNFa (10 ng/ml) in combination with various concen-trations of cycloheximide (Sigma). Cells were harvested and vi-ability was determined by FACS analysis of PI/AnnexinV-FITCstaining using an apoptosis detection kit (R&D Systems) accord-ing to the manufacturer’s instructions.

Acknowledgments

We thank Alain Israel for providing the NEMO antiserum aswell as GST–IkBa (1–72) and GST–IkBa (1–72) S/A expressionplasmids, Mary Saunders for scientific editing, Malte Peters andScott Pownall for critically reading the manuscript, DenisBouchard for technical expertise, and Irene Ng for expert admin-istrative assistance. D.R. was supported by a fellowship of theDeutsche Forschungsgemeinschaft.

The publication costs of this article were defrayed in part bypayment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 USC section1734 solely to indicate this fact.

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