 |
Introduction |
The nuclear factor (NF)-
B1 transcription factor plays a
key role in activation of inflammatory and innate immune responses (1, 2). In nonstimulated cells, NF-
B
dimers are kept as cytoplasmic latent complexes through
binding of specific inhibitors, the I
Bs, which mask their
nuclear localization signal (NLS). Upon exposure to proinflammatory stimuli, such as bacterial LPS, TNF-
, or IL-1,
the I
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-
B dimers followed by their translocation to the nucleus, binding to specific DNA sites (
B sites), and target
gene activation. NF-
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 I
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: IKK
, IKK
and IKK
(for a review, see reference 12). IKK
and IKK
are highly similar protein kinases that act as the catalytic subunits of the complex (9, 11, 13, 14). In vitro, both IKK
and
IKK
form homo- and heterodimers that can phosphorylate
I
B proteins at their NH2-terminal regulatory serines (15).
In mammalian cells, IKK
and IKK
form a stable heterodimer that is tightly associated with the IKK
(NEMO) subunit (16, 17). As cell lines that fail to express IKK
(NEMO)
exhibit a major defect in I
B degradation and NF-
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-
B activation (17). The physiological function of the
two catalytic subunits has been less clear. Initially, overexpression of catalytically inactive forms of IKK
and IKK
that blocked IKK and NF-
B activation suggested that both
subunits play similar and possibly redundant roles in I
B
phosphorylation and NF-
B activation (13, 14). This hypothesis was fostered by finding that in vitro IKK
and
IKK
can directly phosphorylate I
B
and I
B
at the
serines that trigger their degradation in vivo (15). However,
it was also suggested that IKK
rather than IKK
is responsible for activation of the entire complex in response to certain
stimuli, such as the NF-
B inducing kinase, NIK (18). Recently, we found that in addition to an IKK
subunit with
an intact COOH terminus (16), IKK activation requires the
phosphorylation of IKK
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 IKK
and IKK
, replacement of the same two serines in IKK
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 IKK
(Ikk
/
mice) exhibit normal IKK activation in
response to TNF, IL-1, or LPS (20). Although NF-
B is
fully inducible, for an unknown reason, IKK
-deficient
fibroblasts exhibit approximately twofold reduction in both
basal and induced NF-
B binding activity (20). Thus, IKK
may somehow stimulate NF-
B DNA binding despite not being required for I
B phosphorylation and degradation in
most cell types. The gene targeting experiments reveal that,
although not involved in activation of IKK by proinflammatory stimuli, IKK
plays an instrumental role in morphogenesis (20). The most important function of IKK
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 IKK
are exerted through localized NF-
B activation in response to developmental cues.
To determine the physiological function(s) of IKK
, we
have used gene targeting to create Ikk
knockout mice.
We now show that the loss of IKK
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-
B (21). It was
recently shown that the lethality of RelA
/
mice is completely suppressed by the loss of TNF-
(22). As NF-
B is
required for protection of cells from TNF-
-induced apoptosis (23), the apoptotic phenotype of Ikk
/
mice
strongly suggests that the absence of IKK
results in a severe defect in NF-
B activation. Indeed, neither IKK nor
NF-
B can be activated by TNF-
or IL-1 in IKK
-deficient cells. Furthermore, we show that in the absence of
IKK
, the IKK
subunit is not responsive to NIK even
though it can still associate with the IKK
subunit.
 |
Materials and Methods |
Generation of IKK
-deficient Mice.
Using a 0.2-kb BstEII-Bsu36I
restriction fragment from the 5' end of human IKK
cDNA as a
probe, three murine IKK
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 IKK
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
(Ikk
+/
) 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.
Ikk
/
, Ikk
+/
, and Ikk
+/+ ES
and EF cells were treated with TNF-
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
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 |
Generation of Ikk
Knockout Mice.
To create a strain of
IKK
-deficient mice, we used gene targeting technology
(30). Mouse genomic Ikk
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
IKK
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
-galactosidase (LacZ) and neomycin resistance (Neor).
Because the Neor gene contains transcription termination
and polyadenylation signals, the COOH-terminal three
quarters of IKK
including its protein interaction motifs
are unlikely to be expressed from the targeted allele.

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Fig. 1.
Generation of IKK -deficient mice. (A) The mouse Ikk locus and the targeting vector. Map of
the Ikk 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 Ikk ). After homologous recombination, the 9.7-kb EcoRI fragment of wild-type Ikk 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 IKK . Location of the IKK band is indicated. The lower band is nonspecific (ns). The
same blot was also probed with antibodies to IKK , IKK , p65(RelA), and p50(NF- 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 Ikk
locus were isolated, and two of them
were used to generate chimeric mice. Chimeric mice derived from these clones transmitted the targeted Ikk
allele
to their progeny (Fig. 1 B). Although Ikk
+/
male and female mice appeared normal and were fertile, upon intercrossing they did not give rise to live Ikk
/
progeny.
Analysis of protein extracts of Ikk
+/+, Ikk
+/
, and
Ikk
/
cells revealed that, as expected, no IKK
protein
was expressed from the targeted allele (Fig. 1 C). In addition,
Ikk
+/
cells expressed approximately half the dose of IKK
present in wild-type cells. No compensatory increases in
IKK
, IKK
, p65(RelA), or p50(NF-
B1) expression were observed.
Phenotype of Ikk
/
Mice.
Given the expected importance of IKK
for NF-
B activation and the embryonic lethality of RelA
/
mice (21), we suspected that the loss of
IKK
would result in a similar phenotype. Therefore, we
analyzed embryos from timed pregnancies of Ikk
+/
intercrosses. Although Ikk
/
embryos isolated at E11.5 were
alive and had perfectly normal appearance (data not shown),
Ikk
/
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 Ikk
/
embryos had degenerated. Notably, however, the limbs
and head of Ikk
/
embryos were normally developed,
unlike those of Ikk
/
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 Ikk
/
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 Ikk
/
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 Ikk
/
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 Ikk
+/+ or
Ikk
+/
littermates had perfectly normal appearance.

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Fig. 2.
Appearance of an Ikk / E13.5 embryo and a normal littermate. Wild-type (Ikk +/+, WT) and mutant (Ikk / , 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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|

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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 Ikk +/+,
Ikk +/ , and Ikk / embryos. Both E13 Ikk +/+ (A) and Ikk +/ (B)
livers exhibited normal morphology. The Ikk / liver (C) exhibited
varying degrees of apoptosis characterized by collapsed and condensed
nuclei and general cellular degeneration. Bars = 5 µm.
|
|
Defective NF-
B Activation in Ikk
/
Cells.
We used two
different approaches to determine the consequences of the
loss of IKK
expression on IKK and NF-
B activation. First, we prepared Ikk
/
ES cell lines by subjecting
Ikk
+/
ES cells to selection at higher G418 concentration.
One Ikk
/
cell line was identified. As shown in Fig. 5 A,
stimulation of these cells with either TNF-
or IL-1 did
not result in IKK activation, whereas a normal activation
response was observed in Ikk
+/
cells. Note, however, that
Ikk
+/
cells had ~50% of the IKK activity of wild-type
(Ikk
+/+) ES cells, consistent with the reduced amount of
IKK
protein (data not shown). In addition to the defect
in IKK activation, hardly any induction of NF-
B DNA
binding activity was observed in Ikk
/
cells after stimulation with either IL-1 or TNF-
(Fig. 5 B). Even the basal
level of NF-
B DNA binding activity was considerably reduced in Ikk
/
cells, despite no detectable changes in
p65(RelA) or p50(NF-
B1) abundance (data not shown).
The second approach to evaluate the function of IKK
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-
B activity could be detected in Ikk
/
EF cells treated with either IL-1 or TNF-
. Interestingly,
Ikk
+/
EF cells exhibited an ~50% reduction in IKK activity (consistent with the reduction in IKK
expression) but a
much larger decrease in NF-
B DNA binding activity.

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Fig. 5.
Defective IKK and
NF- B activation in IKK -
deficient ES cells. (A) IKK activity. Lysates of TNF- - or
IL-1-treated Ikk +/ and Ikk /
cells were prepared at the indicated time points (in min) after
stimulation and immunoprecipitated with antibody M280 to
IKK . IKK activity (KA) was
measured by an immunecomplex kinase assay using GST-I B (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: IKK ) for loading control. (B)
NF- B binding activity. Nuclear
extracts of Ikk +/ and Ikk /
cells stimulated with IL-1 or
TNF- for the indicated times
(in min) were incubated with
32P-labeled 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- B activation in IKK -deficient EF
cells. Second passage EFs from E11.5 Ikk +/+, Ikk +/ , and Ikk / embryos were stimulated with TNF- or IL-1. At the indicated times,
whole cell extracts were prepared and used to measure (A) IKK activity
(KA), and (B) NF- B DNA binding activity. IB, immunoblotting.
|
|
IKK
Cannot Be Activated by NIK in the Absence of
IKK
.
The results described above indicate that IKK
,
which is expressed in normal levels in Ikk
/
cells, cannot
be activated by either TNF-
or IL-1. To further examine
this point, we cotransfected an HA epitope-tagged IKK
expression vector into Ikk
/
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 IKK
kinase (18). Recently, however, we obtained
results that suggested that NIK-induced IKK
phosphorylation is not direct and is likely to be dependent on IKK
(19).
Consistent with this hypothesis, we found no increase in IKK
activity towards I
B
(1-54) substrate upon coexpression of
HA-IKK
with NIK in Ikk
/
cells (Fig. 7 A). Yet, when
an IKK
expression vector was included in these transfections,
NIK elicited a clear increase in IKK activity. As shown previously, NIK coexpression efficiently stimulates IKK
-associated IKK activity in IKK
-expressing cells (19).

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Fig. 7.
IKK is refractory to activation
in Ikk / cells despite its association with
IKK . (A) Ikk / ES cells were transiently
transfected by electroporation with an
HA-IKK expression vector alone or together with XpressNIK or HA-IKK 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-I B (1-54) as a substrate. Protein expression levels were determined by immunoblotting (IB) with anti-HA. (B) Lysates of
Ikk +/+, Ikk +/ , and Ikk / cells were immunoprecipitated (IP) with either anti-IKK or anti-IKK 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-IKK antibody. A lysate of 3T3 cells was used as a control (Cont).
|
|
One reason for the inability of IKK
to respond to
proinflammatory stimuli or NIK in the absence of IKK
could be its inability to directly associate with IKK
, the
regulatory subunit of the IKK complex. Previous experiments indicate that IKK
is essential for recruitment of upstream activators to IKK (16). In addition, using recombinant proteins, it was found that IKK
directly interacts with IKK
much more efficiently than does IKK
(16,
17). Having available IKK
-deficient cells, we reexamined
the ability of IKK
to interact with IKK
. In contrast to
the results obtained with recombinant proteins, very efficient coprecipitation of IKK
by anti-IKK
antibodies was
observed using lysates of Ikk
/
cells as a starting material
(Fig. 7 B). Therefore, the refractoriness of IKK
to IKK
activators in IKK
-deficient cells is not due to its inability
to associate with IKK
.
 |
Discussion |
The enzymatic activity of the IKK complex, composed
of two catalytic subunits, IKK
and IKK
, and one regulatory subunit, IKK
, is rapidly stimulated by proinflammatory cytokines and LPS (for a review, see reference 12).
Activated IKK phosphorylates the different I
Bs at the two
NH2-terminal serines that trigger their polyubiquitination
and proteasome-mediated degradation. Once the I
Bs are
degraded, the freed NF-
B dimers migrate to the nucleus
and activate target gene transcription. Based on their similar
primary structures (11, 13, 14) and substrate specificities (15), IKK
and IKK
were expected to play redundant
and interchangeable roles in proinflammatory signaling to
NF-
B. Therefore, it was rather surprising that only IKK
was found to be involved in IKK activation. Alanine substitutions of the two serines in the activation loop of IKK
,
whose phosphorylation is stimulated by either TNF-
treatment or NIK overexpression, prevented IKK activation. Yet, the same mutations introduced into the activation loop of the IKK
subunit had no effect on the response of IKK to TNF-
or NIK (19). These results were
confirmed by the analysis of IKK
-deficient cells and tissues which revealed no defect in IKK activation and I
B
degradation in response to TNF-
, IL-1, or LPS (20). However, it remained possible that the function of IKK
in I
B
phosphorylation in response to proinflammatory stimuli can
be fully replaced by IKK
. The results described here indicate that IKK
and IKK
have different physiological functions and that IKK
cannot substitute for IKK
.
To determine the physiological function of IKK
, we
generated Ikk
/
knockout mice and cell lines. The loss
of IKK
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 Ikk
/
embryos die around E13, RelA
/
embryos die around E15. The earlier death of Ikk
/
embryos is likely to be due to a more extensive reduction in NF-
B activity, as embryos that are deficient in both the
p65 (RelA) and the p50 (NF-
B1) subunits of NF-
B die
at E12.5, the same time as IKK
-deficient embryos, from
massive hepatocyte apoptosis (32). Thus, IKK
and RelA
are genetically proven to be components of the same pathway. Accordingly, cells that lack IKK
are completely defective in IKK and NF-
B activation in response to either
TNF-
or IL-1. Therefore, the IKK
subunit is absolutely
essential for mounting a response to proinflammatory stimuli. This function is not replaced by IKK
, whose expression is not diminished in the absence of IKK
. In addition,
as indicated by the normal morphology of the head and
limbs of E13.5 Ikk
/
embryos, IKK
can carry out its
developmental function (20) in the complete absence of
IKK
. Interestingly, a 50% reduction in IKK
expression,
as in Ikk
+/
cells, results in a similar decrease in IKK activity but a much more severe defect in NF-
B activation.
These results underscore the importance of the IKK
subunit and indicate that the NF-
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-
B activity may be sufficient for protecting the
liver from TNF-
-induced apoptosis.
One possible cause for the inability of IKK
to substitute
for IKK
was its relatively lower affinity to IKK
, the regulatory subunit that is absolutely required for IKK activation
(17). Using recombinant proteins, it was observed that IKK
does not form a stable complex with IKK
in vitro, whereas
IKK
readily associates with IKK
(16, 17). However,
immunoprecipitation experiments indicate that a similar
amount of IKK
is precipitated by IKK
antibodies from
Ikk
/
cells as from Ikk
+/+ cells. Despite its ability to
associate with IKK
in the absence of IKK
, IKK
is refractory to upstream activators involved in proinflammatory signaling, including the most potent IKK activator identified so
far, NIK, in IKK
-deficient cells. These results underscore
the differences in regulation of IKK
and IKK
activities.
In summary, together with the previous analysis of IKK
-deficient mice, the analysis of IKK
-deficient mice, described
here, indicates that the two catalytic subunits of the IKK
complex, although similar in structure, have very different
functions. Although IKK
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-
B, through I
B phosphorylation, IKK
is assigned the control of epidermal and skeletal morphogenesis.
Although the stimuli that activate IKK
and the substrates
that mediate its biological activity are known, the stimuli and
the relevant substrates for IKK
remain to be identified.
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
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