By
From The Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000
The nfkb2 gene encodes the p100 precursor which produces the p52 protein after proteolytic
cleavage of its COOH-terminal domain. Although the p52 product can act as an alternative
subunit of NF-B, the p100 precursor is believed to function as an inhibitor of Rel/NF-
B
activity by cytoplasmic retention of Rel/NF-
B complexes, like other members of the I
B
family. However, the physiological relevance of the p100 precursor as an I
B molecule has not
been understood. To assess the role of the precursor in vivo, we generated, by gene targeting,
mice lacking p100 but still containing a functional p52 protein. Mice with a homozygous deletion of the COOH-terminal ankyrin repeats of NF-
B2 (p100
/
) had marked gastric hyperplasia, resulting in early postnatal death. p100
/
animals also presented histopathological alterations of hematopoietic tissues, enlarged lymph nodes, increased lymphocyte proliferation in
response to several stimuli, and enhanced cytokine production in activated T cells. Dramatic
induction of nuclear
B-binding activity composed of p52-containing complexes was found in
all tissues examined and also in stimulated lymphocytes. Thus, the p100 precursor is essential
for the proper regulation of p52-containing Rel/NF-
B complexes in various cell types and its
absence cannot be efficiently compensated for by other I
B proteins.
In most cell types, Rel/NF- Activation of the Rel/NF- The nfkb2 gene was initially isolated as a subunit of NF- Although genetic evidence suggests that abnormal NF- Targeting Vector.
A genomic library (cloned in Generation of Mutant Mice.
CJ7 ES cells were electroporated
with 25 µg of the NotI-linealized pPNT/I
Histology, In Situ Hybridization, and Flow Cytometry.
Mouse tissues were immersion fixed in 10% buffered formalin and embedded in paraffin blocks. Sections were stained with hematoxylin and eosin. In situ hybridization of a wild-type newborn mouse using nfkb2 cDNA (25) as a probe was performed as previously described (12). Flow cytometry analysis with single cell suspension from 7-10-d-old mice were performed as previously described (19).
Immunoprecipitation Assay, Electrophoretic Mobility Shift Assay
(EMSA), and Western Blot Analysis.
Thymocytes from 10-d-old
animals isolated as previously described (32) were labeled with
800 µCi/ml of [35S]methionine (Amersham Corp., Arlington
Heights, IL) in the presence or absence of 20 ng/ml of PMA
(Sigma Chemical Co., St. Louis, MO) and 1 µg/ml of PHA
(Sigma Chemical Co.) for 6 or 8 h. Nuclear and cytoplasmic extracts from single cell suspensions with or without stimulation
isolated from several tissues of 10-d-old mice were prepared as
previously described (33). Cell were lysed directly in RIPA
buffer, followed by immunoprecipitation as previously described
(34). Western blot analysis using cytoplasmic extracts and EMSA
using nuclear extracts were carried out as previously described
(34). The full length murine I Reverse Transcriptase (RT)-PCR Analysis.
Total RNAs from
10-d-old mouse spleen, stomach, and thymus were prepared using RNAzol (Cinna/Biotecx Laboratories, Inc., Houston, TX).
RT-PCR was performed as previously described (36). The sequences of the primers were as follows: for MHC-I, 5 Proliferation Assays In Vitro.
Peripheral T cells were purified
from 10-d-old mouse splenocyte suspensions. Erythrocytes were
depleted by ammonium chloride lysis, and subsequently T cells
were purified by murine T cell enrichment columns (R & D Systems, Inc., Minneapolis, MN). Purified T cells were stimulated
with coated CD3 antibody (PharMingen, San Diego, CA),
coated CD3 plus CD28 antibodies (PharMingen), or 7 ng/ml of
PMA plus 1 µg/ml of PHA. Purified T cells (105) in 96-well
plates were incubated with or without the different stimuli in 200 µl medium for 48 h, and cell proliferation was measured by
[3H]thymidine incorporation after 12 h culture with 1 µCi
[3H]thymidine (Amersham Corp.) in each well.
ELISA.
Splenic T cells (5 × 105/ml) isolated from 10-d-old
mice were incubated with or without coated anti-CD3 and anti-CD28 antibodies for 72 h. Cytokine levels in supernatants were
determined by ELISA (R & D Systems, Inc.).
A targeted disruption of the COOH terminus of
NF- CJ7 ES cells were electroporated with the targeting vector pPNT/I Since expression of the nfkb2 gene has been shown to be
very low in lymphocytes but rapidly induced by mitogens
(25), thymocytes stimulated with PMA and PHA were
used for immunoprecipitation analysis to verify the absence
of the p100 protein. Protein extracts prepared from +/+
and Newborn p100
The most striking histopathological alterations of p100
Histopathological alterations in hematopoietic tissues were also observed
in p100
The hematopoietic populations were analyzed by flow
cytometry (Fig. 4 B). In contrast to wild-type mice, in 10-d-old p100-deficient mice CD4+CD8+ double positive
thymic cells were nearly absent and single positive CD4+
or CD8+ were the dominant cell populations (Fig. 4 B,
compare a and b). Despite the dramatic histopathological
alteration of the spleen, both B and T cell populations
(B220+ or Thy-1.2+ cells) were not severely altered in
p100 The
p100 precursor, like p105 (NF-
Since different tissues presented Since the increased The processing of p100 is believed to be controlled by external signals. Although the precise mechanisms have not
been elucidated, they have been assumed to resemble the
processing of p105 which might involve phosphorylation,
ubiquitination, and subsequent proteasome-mediated degradation (20, 44). EMSA was used to determine the effect of the deletion of the p100 inhibitor region on the kinetics and composition of Rel/NF-
Rel/NF- The consequences of the increased
The enlarged lymph nodes in p100
To further investigate the alteration of lymphocyte function in p100 Mice homozygous for the deletion of the COOH terminus of NF- A significant fraction of the p52-containing heterodimers
probably results from the processing of the p100 precursor-
containing heterodimers. The COOH-terminal ankyrin
domain of the precursor masks its NLS as well as the NLS
of the dimeric partner, resulting in cytoplasmic retention of
the precursor-containing dimers (1, 5, 7). The deletion of
the COOH-terminal half of NF- Hematopoietic organs of p100 The human NF The increased proliferation and cytokine secretion in activated p100 A surprising outcome obtained from mice lacking the ankyrin motif of
NF- Several regulated steps of cellular proliferation, migration, differentiation, and senescence are required for gastric
epithelial renewal, and disturbance of any processes may
cause development of gastric abnormalities. In humans,
there are hyperplastic and hypertrophic gastropathies, such
as Menetrier's disease, which is characterized by extensive
hyperplasia of the surface epithelium and associated with
gastric carcinoma (53). The gastric pathology in p100 It has been reported that TGF- Despite the increase in
understanding the mechanism of the Rel/NF- The expression of the nfkb1 and nfkb2 genes is induced
by some extracellular stimuli, and may be regulated by the
Rel/NF- The p105 inhibitor in ES cells is preferentially associated
with p50 (34). Although p105 can interact with RelA or
c-Rel, the significant levels of the p50-RelA or p50-c-Rel
dimers derived from the processing of the p105 precursor
fail to be induced after stimulation of thymocytes (see Fig.
6 A, lanes 5 and 6), implying that these may be inactivated
by I Since p52-containing heterodimers dramatically accumulate in the absence of the p100 precursor, the processing
of the precursor as a regulatory mechanism for controlling
Rel/NF- Mutant mice lacking different members of the
Rel/NF- Mice lacking I Table 1.
Comparison of the Phenotype between IB complexes are inactive in
the cytoplasm but can be rapidly induced by a variety of
stimuli leading to degradation of the inhibitory I
B molecules, allowing nuclear translocation of the different Rel/
NF-
B complexes (1). The majority of the genes regulated by these complexes are involved in immune, acute
phase, and inflammatory responses (8). The Rel/NF-
B transcription factors are homo- and heterodimeric
complexes composed of various combinations of structurally related subunits. In mammals, members of this family
include NF-
B1 (p50 and its precursor p105), NF-
B2
(p52 and its precursor p100), RelA, RelB, and c-Rel. They
share a conserved NH2-terminal region of 300 amino acids,
termed the Rel homology domain, responsible for DNA
binding, dimerization, and association with inhibitors of
the I
B family. In the case of RelA, RelB, and c-Rel, the
diverged COOH-terminal domains mediate transcriptional
activation. Interestingly, NF-
B1 and NF-
B2 are synthesized as cytoplasmic precursors p105 and p100, respectively, which in addition to the Rel homology domain, contain repeated ankyrin-like sequences in the COOH-terminal half. Removal of this ankyrin domain by proteolytic processing generates active p50 and p52 products
(1, 10, 11). The genes of the Rel/NF-
B family are differentially expressed in lymphoid tissues (12) and studies with mice lacking p50, RelB, RelA, or c-Rel demonstrate that individual members of this family have distinct functions in vivo (15).
B transcription factors is
regulated by posttranslational modification and degradation
of the I
B proteins that interact with the Rel/NF-
B
complexes and sequester them in the cytoplasm by masking
their nuclear localization signal (NLS)1. Posttranslational
modification and degradation of I
Bs do not require de
novo protein synthesis, resulting in a rapid NF-
B activation and expression of immune response genes after cell stimulation. In mammals, members of the I
B family include I
B
, I
B
, I
B
, Bcl-3, p105, and p100, which all
share the conserved ankyrin-like repeats responsible for the
interaction with the Rel/NF-
B complexes. I
B
, I
B
,
I
B
(the latter being identical to the COOH-terminal
half of NF-
B1), and Bcl-3 form ternary complexes with
Rel/NF-
B dimers, whereas the p105 and p100 precursors
form dimers with individual members of the Rel/NF-
B
family including their products p50 and p52 (1, 5, 7, 11).
In the case of I
B
, the phosphorylation and subsequent
degradation of the inhibitor release the active Rel/NF-
B
complexes, resulting in the nuclear translocation of Rel/
NF-
B. In addition, proteolytic cleavage and the presumably subsequent degradation of the COOH-terminal inhibitor region of the precursors also result in Rel/NF-
B activation (5, 7). Recent reports indicate that degradation of
both I
B
and the COOH terminus of p105 is mediated
by the ubiquitin-proteasome pathway, and that phosphorylation of I
B
also involves a ubiquitin-dependent protein kinase (20).
B, a candidate protooncogene, and a mitogen-inducible
gene (23). The function of NF-
B2 may be similar to
that of the better characterized NF-
B1 molecule because
of their structural similarity; however, the distinct expression pattern of the nfkb1 and nfkb2 transcripts in adult mice
indicates different roles for these molecules (14). Moreover,
in contrast to p50, the ubiquitous component of NF-
B, p52 barely contributes to the NF-
B activity in cells, presumably due to its lower abundance or inefficient
B-binding ability (27).
B2 can be involved in lymphomagenesis (23, 28, 29), little is known about NF-
B2 functions in vivo. To understand the physiological roles of NF-
B2, particularly those
of the p100 precursor, we generated, by gene targeting,
mutant mice lacking the precursor but still containing the
p52 product. Deletion of the COOH terminus of NF-
B2
in mice resulted in marked gastric hyperplasia, causing early
postnatal death. In addition, histopathological changes were
observed in hematopoietic tissues, such as enlargement of lymph nodes and granulocytosis in bone marrow. T cells
from p100-deficient mice displayed hyperproliferative responses to several stimuli and enhanced cytokine production in vitro. A significant increase in
B-binding complexes containing p52 were found in both lymphoid and
nonlymphoid tissues and in stimulated lymphocytes lacking p100. These findings demonstrate that the processing of the
p100 precursor to generate the p52 molecule is an important regulatory step, and indicate that overexpression/deregulation of p52-containing Rel/NF-
B complexes increases gastric and lymphoid cell proliferation.
DashII;
Stratagene Inc., La Jolla, CA) prepared from D3 embryonic stem
(ES) cell DNA was screened with the mouse nfkb2 cDNA probe
corresponding to nucleotides 1280-2290 of the human sequence
(25). Two overlapping phages were isolated and the genomic
nfkb2 DNA fragments were subcloned into pBluescript KS+
(Stratagene Inc.). A 5.6-kb HindIII-XbaI (XbaI is present in
exon 14 of murine nfkb2 gene) genomic DNA fragment was isolated and subcloned in pGEM-9Zf(
) (Promega Corp., Madison,
WI). A termination codon and KpnI and SalI sites were introduced by PCR mutagenesis at a position corresponding to amino
acid 451 using the murine nfkb2 genomic DNA as a template.
Codon 451 is between the glycine-rich region and the first
ankyrin motif. A XbaI-SalI (present in exon 14 and created by
mutagenesis, respectively) genomic DNA fragment containing
parts of exons 14 and 15, a 79-bp-long intronic fragment, a termination signal, and a KpnI site was subcloned into pBluescript KS+. The KpnI-SalI fragment of the 852-bp-long SV40 polyadenylation recognition sequence [p(A)] was amplified by PCR
using the pMSG vector (Pharmacia Biotech, Piscataway, NJ) as a
template and inserted downstream of the termination codon.
Subsequently, an XbaI-SalI fragment containing the nfkb2 genomic DNA and SV40 p(A), was ligated into the XbaI (exon 14 of nfkb2) and SalI sites of the pGEM-9Zf(
) containing the 5.7-kb-long upstream nfkb2 genomic DNA fragment, generating the
5
arm of the targeting vector. The 6.8-kb NotI-SalI DNA fragment spanning exon 1 and part of exon 15, the termination codon, and the SV40 p(A) was inserted at the 5
end of the phosphoglycerate kinase (PGK) promoter driving the neo gene (PGK-
neo cassette; reference 30) into the NotI and XhoI sites of the
pPNT vector (31). A 7.3-kb SpeI-KpnI DNA fragment containing exons 20-24 (the last exon of nfkb2) was inserted between the
PGK-neo cassette and the PGK promoter, driving the herpes simplex virus thymidine kinase gene (PGK-tk cassette) into the XbaI
and KpnI sites of pPNT containing the 5
arm, creating the targeting vector pPNT/I
B
. This resulted in the deletion of a 1.3-kb genomic DNA fragment that contains part of exons 15-19,
encoding the six repeats of ankyrin-like motif of NF-
B2.
B
/107 cells using a
gene pulser (Bio-Rad Labs., Hercules, CA) and grown under
double selection using G418 and fialuridine, and double-resistant clones were selected. Homologous recombination (3 out of 65 neo-containing clones) was screened by Southern blot analysis using a 5
external probe and additional random integrations were
excluded with a 5
internal probe (see Fig. 1 A). Targeted ES
clones were identified by the appearance of a 7.2-kb recombinant
band in addition to the 8.5-kb wild-type band in KpnI- and
SpeI-digested DNA. These clones were injected into C57BL/6
blastocysts, which were subsequently implanted into foster mothers. Resulting male chimeras were backcrossed to C57BL/6 females and heterozygous offspring were then interbred to generate
homozygous mutant animals.
Fig. 1.
Generation of mice deficient in the p100 precursor. (A) Targeting strategy of the ankyrin-encoding region of the nfkb2 gene. The relevant part of the mouse nfkb2 gene structure is shown at the top. Exons
13-19, encoding residues 332-690, are indicated by closed boxes. Targeting vector pPNT/IB
and the targeted allele are shown at the middle
and bottom, respectively. Open boxes indicate the SV40 polyadenylation
recognition sequences, PGK-neo and PGK-tk cassettes. The position of
KpnI and SpeI sites are indicated by K and S, respectively. The diagnostic
restriction fragments used for Southern blot analysis are indicated at the
top (wild-type allele) and bottom (targeted allele). The DNA fragments
used as 5
external (H) and internal (B) probes are indicated at the bottom. (B) Genotype analysis of mice generated from p100+/
heterozygote intercrosses. Tail DNAs were digested with KpnI and SpeI, and subjected
to Southern blot analysis using the 5
external probe H indicated in A. The 8.5-kb band indicates the wild-type allele, while the 7.2-kb band
represents the targeted allele. (C) Absence of p100 in homozygous mutant
mice. Protein extracts from control (+/+) and homozygous (
/
) mutant thymocytes labeled with [35S]methionine for 8 h in the presence of
PMA (20 ng/ml) and PHA (5 µg/ml) were immunoprecipitated with an
anti-p52 antiserum. p100 and p52 proteins are indicated by the arrows.
[View Larger Version of this Image (23K GIF file)]
B
protein was used to generate
polyclonal rabbit I
B
antiserum. Other antibodies used in this
study have been previously described (34, 35).
-TAC
CTG AAG AAC GGG AAC-3
and 5
-GAC TAA AGA GAA
CTG AGG GC-3
; for endothelial leukocyte adhesion molecule
1, 5
-CTT TGA CCC ACC CTG CCC ACG GTA TCA G-3
and 5
-GAA CTC ACA ACT GGA CCC ATT TTG GAA A-3
; for intercellular adhesion molecule 1, 5
-CCG CTT CCG CTA
CCA TCA CCG TGT ATT C-3
and 5
-GCC TTC CAG
GGA GCA AAA CAA CTT CTG C-3
; for vascular adhesion
molecule 1, 5
-AAC AGA CAG GAG TTT TC-3
and 5
-GTC
AAC AAT AAA TGG TT-3
; and for
-actin, 5
-CCA CCA
GAC AAC ACT GTG TTG GCA T-3
and 5
-AGA GGT
ATC CTG ACC CTG AAG TAC C-3
. The primers for TNF-
were obtained from Stratagene, Inc.
Generation of Mice Lacking the COOH Terminus of NF-B2.
B2 was generated by introducing a termination signal
at codon 451 of nfkb2 and replacing 4.5 exons encoding
residues 451 to 690 with the SV40 p(A) and the PGK-neo
cassette (Fig. 1 A). This would produce a p52 molecule of
450 amino acids, but not the full length p100 precursor. As
the correct length of the p52 molecule is unknown, we decided that the 450-amino acid protein was the most convenient as several of the previous studies characterizing the
activity of p52 were performed with a molecule of a very
similar size. These studies demonstrated that the p52 protein was a very weak transactivator but one that could interact with other NF-
B proteins and Bcl-3 (23, 37, 38).
B
and resistant clones were screened by
Southern blot analysis. Homologous recombination was
demonstrated by the detection of a new 7.2-kb DNA fragment (8.5-kb wild-type) in KpnI- and SpeI-digested DNA
from targeted ES cells using a 5
external probe (Fig. 1 A and data not shown). Injection of targeted ES cells into
C57BL/6 blastocysts and subsequent implantation into
pseudopregnant foster mothers generated chimeric mice
that transmitted the mutated nfkb2 allele to their offspring.
Homozygous mutant (p100
/
) mice were generated by
intercrossing between heterozygous (p100+/
) animals.
Genotyping analysis demonstrated that although homozygous mutants were born at the expected Mendelian ratios
(25%), at postnatal days (P) 5-7 they were already underrepresented (19.5%, n = 788), indicating that some p100
/
pups died neonatally. The results obtained from Southern
blot analysis using tail DNA of animals representing the
three different genotypes are presented in Fig. 1 B.
/
thymocytes labeled for 8 h with [35S]methionine
in the presence of PMA and PHA were immunoprecipitated with a p52 antiserum. The immunocomplexes were
then denatured, renatured by fourfold dilution, and precipitated again with p52 antiserum (Fig. 1 C). The results revealed the presence of both the precursor (p100) and the
processed p52 protein in control thymocytes (Fig. 1 C, lane
1). In contrast, p100
/
thymocytes lack the precursor but
express the processed and truncated p52 product (Fig. 1 C,
lane 2). The truncated NF-
B2 of 450 amino acids in
p100
/
mice showed a higher molecular weight than the
processed p52 in p100+/+ mice. However, previous in
vitro analyses have demonstrated that it lacked transcriptional activity but could interact with other NF-
B proteins and with Bcl-3 (23, 37, 38). It is important to
note that only a small fraction of the p100 precursor was
processed during the 8 h period to generate p52.
/
mice were grossly indistinguishable from their littermates,
but by P10-14 they could be recognized by their smaller size. After P10-14, p100
/
mice started losing body
weight, and presented a disheveled appearance and hunched
posture. Their body weights were reduced 25-33% compared to control littermates and by 4 wk of age 90% of
p100
/
mice had died (Fig. 2, A and B). None of the
p100
/
animals survived beyond 10 wk of age (Fig. 2 B).
Fig. 2.
Postnatal growth
and survival of p100-deficient
mice. (A) Changes in body
weight of p100/
mice (open triangles) and control littermates
(+/+ and +/
, closed circles).
(B) Survival of control (+/+
and +/
, closed circles, n = 290)
and p100
/
mice (open triangles,
n = 100). Survival is shown as a
percentage of the total initial
number of control (+/+ and
+/
) or p100
/
mice.
[View Larger Version of this Image (16K GIF file)]
/
mice were detected in the stomach. By 2 wk of age the
stomachs of p100
/
mice appeared smaller than those of
the control littermates, and contained little food or milk
(data not shown). The stomach showed a marked hyperplasia of the epithelial cell layer in the antrum with lymphocytic infiltration in the lamina propria and hyperkeratosis in
the cardiac portion. In 3-wk-old p100
/
mice, the gastric
abnormalities had increased in severity until the gastric lumen was mostly occluded, which most likely led to premature death of p100
/
mice (Fig. 3 A, compare a and c with
b and d, respectively). The strong expression of the nfkb2
transcript found in the epithelial cell layer of the wild-type
mouse stomach by in situ hybridization (Fig. 3 B, a and c)
supports a physiological role for NF-
B2 in this area. Although young heterozygous mutant mice exhibited unremarkable histopathology, mild gastric hyperplasia was also
observed in 10-mo-old p100+/
mice (data not shown).
Fig. 3.
Histopathology of a
p100/
mouse stomach. (A)
Stomach sections of 3-wk-old
wild-type (a and c) and p100
/
(b and d) animals stained with
hematoxylin and eosin (original magnification: 12.5-fold). As
shown in (b) the epithelial layer (EL) was markedly thick,
whereas the gastric lumen (Lu)
was narrow in p100
/
mice.
Also, hyperkeratosis in cardiac
portion was evident in the
p100
/
stomach (d). (B) A section of a wild-type newborn
mouse was probed with nfkb2
cDNA, stained with carmine
red (b and c), and photographed under dark (a and c) or light (b)
field illumination (original magnification: a, 4-fold; b and c, 25-fold). The nfkb2 transcript is expressed in thymus (Th) and in
the surface epithelium (c; arrows)
of the stomach (St).
[View Larger Version of this Image (62K GIF file)]
/
Mice.
/
mice. Spleen weight of p100
/
mice started to
decrease at P10-14, and both spleen and thymus became
very atrophic by 3 wk of age. Spleen of 3-wk-old p100
/
mice showed reduced cellularity and poorly demarcated
white and red pulp areas (Fig. 4 A, compare a and b). Thymus also presented structural alterations with poorly demarcated cortico-medullary junctions (Fig. 4 A, compare c
and d). Remarkably, in contrast to other hematopoietic organs, lymph nodes of p100
/
mice older than 2 wk were
clearly enlarged with increased paracortical areas but had
reduced number and cell density of lymphatic follicles (Fig.
4 A, compare e and f). Bone marrow of 3-wk-old p100
/
mice had an increased number of granulocytes but a reduced number of other hematopoietic cell populations
(Fig. 4 A, compare g and h), and granulocytosis was also
observed in peripheral blood of p100
/
mice (data not
shown). In addition, liver and spleen of some p100
/
pups
were anemic at 3 wk (data not shown). Analysis of other organs showed no evident histopathological alterations.
Fig. 4.
Alterations of hematopoietic tissues in p100/
mice. (A) Sections of 3-wk-old wild type (a, c, e, and g) and p100
/
(b, d, f, and h) spleen (a
and b), thymus (c and d), lymph node (e and f), and bone marrow (g and h) stained with hematoxylin and eosin (original magnification: a, b, e and f, 12.5-fold; c and d, 5-fold; g and h, 375-fold). Both spleen (b) and thymus (d) of p100
/
mice were atrophic, showing poorly demarcated white (WP, arrows) and red pulp, and cortico-medullary junctions, respectively. (f) Lymph nodes were clearly enlarged and the lymphatic follicle (LF, arrows) was not well
defined in p100
/
mice. (h) Granulocyte precursors and neutrophils (arrows) markedly accumulated in p100
/
bone marrow. WP, white pulp; RP, red pulp;
Co, cortex; Me, medulla; LF, lymphatic follicle; PA, paracortical area. (B) Flow cytometry of thymocytes (Th) stained for CD4 and CD8 (a and b), splenocytes (Sp) stained for B220 and Thy-1.2 (c and d), bone marrow cells (BM) stained for Gr-1 (e and f) or B220 (g and h), and lymph node cells (LN) stained for
CD25 (i and j) from 3-wk-old wild-type (+/+, a, c, e, g, and i) and p100
/
(
/
, b, d, f, h, and j) mice. Percentages of positive cells are indicated.
[View Larger Version of this Image (83K GIF file)]
/
mice (Fig. 4 B, compare c and d). The low percentage of T cells observed in spleen at this age is explained
by the fact that during the first 4 wk of age, peripheral T
cell accumulation is predominantly due to emigration from
thymus, whereas in adult mice peripheral expansion becomes the main mechanism maintaining a constant number of T cells (39). Analysis of bone marrow cell populations
from 3-wk-old p100
/
mice revealed increased number
of granulocytes defined by the Gr-1 surface marker (Fig. 4
B, compare e and f), while B cells defined by B220 were
dramatically reduced (Fig. 4 B, compare g and h). Despite
the slight decrease in the T to B cell ratio in p100
/
lymph nodes (data not shown), IL-2R
chain-expressing
T cells (CD25+ cells) were increased (Fig. 4 B, compare i
and j). These data suggest that lymphocyte development is
not impaired by the absence of the p100 precursor, although hematopoietic abnormalities increased in severity in
3-wk-old p100
/
mice (see Discussion).
B-Binding Activity in p100-deficient Mice.
B1), has been shown to
function as an I
B molecule since it retains Rel/NF-
B
subunits in the cytoplasm (40). To assess the consequence of p100 precursor elimination on NF-
B activity,
we examined the
B-binding activity in nuclear protein
extracts from wild-type and p100
/
mice by EMSAs using
a palindromic
B site (Fig. 5 A). To avoid alteration of
endogenous NF-
B activity in the different tissues from
mutant mice because of lymphocyte or granulocyte cell
infiltration, nuclear protein extracts were prepared from
10-d-old mice. Nuclear extracts from all tissues examined
revealed a dramatic increase in
B-binding activity in
p100
/
mice when compared with wild type animals.
Comparable levels of protein-nucleotide complexes in wild-type and p100
/
nuclear extracts were detected when an
octamer-specific probe was used in this assay (data not shown),
indicating similar amounts of nuclear extracts loaded. Longer
exposure of the film revealed the presence of
B-binding
activity in control thymus and stomach (data not shown).
These results indicate that the elimination of the ankyrin
domain of the p100 precursor dramatically increases the
constitutive
B-binding activity in lymphoid and nonlymphoid tissues.
Fig. 5.
Augmented B-binding activity in p100
/
mice. (A) Tissues from p100
/
mice present increased Rel/NF-
B activity. The
B-binding activity of nuclear extracts (2 µg) from several tissues was determined by EMSA using a palindromic
B site. (B) The accumulated
B-binding complexes
contain p52 in p100
/
mice. Antisera used for determining the composition of the Rel/NF-
B complexes are indicated at the top. p.i., preimmune serum. Different mobilities of
B-binding complexes are indicated by arrows. (C) The p100 precursor interacts with all members of the Rel/NF-
B family
in primary murine thymocytes. Whole cell lysates from wild-type thymocytes labeled with [35S]methionine for 8 h in the presence of PMA and PHA
were incubated with p100-COOH-terminus antiserum (p100-C). The resulting immune complexes were disrupted and reprecipitated with either p50,
p52, RelA, or RelB antiserum as indicated (2nd antiserum). Specific signals for the p105, p100, p50, RelA, and RelB proteins are indicated by arrows.
[View Larger Version of this Image (57K GIF file)]
B-binding complexes
with distinct mobilities that may reflect the presence of different Rel/NF-
B dimers, we further investigated the contribution of p52 to the
B-binding activity in nuclear extracts from p100
/
thymus and spleen by using specific
antibodies. Two major complexes with distinct mobilities
were present in both tissues (Fig. 5 B). Addition of p50 antiserum completely eliminated the faster migrating complex
and to a different degree the slower migrating complex in
thymus and spleen, while addition of a p52 antiserum removed slower migrating complex in the thymus and both
the faster and slower migrating complexes in the spleen.
These results indicated that the faster migrating complex
consisted of the p50 homodimers in the thymus, and of the
p50-p52 dimers in spleen, and that in both tissues the slower
migrating complex consisted mainly of the p52-containing
heterodimers.
B-binding detected in p100
/
tissues was mainly due to p52-containing heterodimeric
complexes, we further examined if the p100 precursor is
complexed with different Rel/NF-
B proteins in control
tissues. To address this, whole cell lysates of thymocytes
from 10-d-old wild-type mice labeled for 8 h with [35S]methionine in the presence of PMA and PHA were
first immunoprecipitated under nondenaturing conditions
with an antiserum raised against the COOH terminus of
p100 (p100-C, Fig. 5 C). The resulting immunocomplexes were denatured and sequentially reimmunoprecipitated
with p50, p52, RelA, RelB, or c-Rel antisera. RelA,
RelB, and p50, as well as the p105 precursor, were associated with p100 in wild-type thymocytes (Fig. 5 C, lanes 1,
3 and 4). Although p52 was not efficiently produced in
stimulated wild-type thymocytes, weak signals corresponding to p52 as well as c-Rel associated with the p100 precursor could be detected after long exposure (data not
shown). These results indicate that the p100 precursor can form dimers with all members of the Rel/NF-
B family
including the p105 precursor, consistent with previous observations (40). Thus, in murine thymocytes p100 can
be associated with any of the Rel/NF-
B subunits, and the
limited processing of the p100 precursor prevents the release of active p52-containing complexes.
B Activation after Stimulation of Thymocytes.
B activation after stimulation of thymocytes with PMA and PHA (Fig. 6 A).
In wild-type thymocytes, a slower migrating complex that
consisted of p50-RelA dimers was strongly induced during
the first 30 min of stimulation (Fig. 6 A, lane 2) and then
rapidly repressed (Fig. 6 A, lanes 3 and 4), while a faster
migrating complex composed of p50 dimers accumulated
after 4 and 8 h of stimulation (Fig. 6 A, lanes 5 and 6). In
p100
/
thymocytes, although similar kinetics of NF-
B
induction were observed, the level of
B-binding activities
are stronger than those of control thymocytes (Fig. 6 A,
lanes 7-10). The p50-RelA dimers were also induced after
30 min of stimulation in p100
/
thymocytes (Fig. 6 A,
lane 8). However, in contrast to control thymocytes, the
strong
B-binding activities induced after 4 and 8 h of
stimulation in p100-deficient cells (Fig. 6 A, lanes 11 and
12) consisted of p50-p52 and p50-p50 dimers (faster migrating bands) and mainly of the p52-RelB dimers (slower
migrating band). The identity of the complexes was determined by gel supershift using specific antibodies.
Fig. 6.
Induction of B-binding activity after stimulation of thymocytes. (A) Strong Rel/NF-
B activation in stimulated p100
/
thymocytes. Nuclear extracts (1.5 µg) from wild type (+/+, lanes 1-6) and p100
/
(
/
, lanes 7-12) thymocytes treated with PMA and PHA for the indicated periods were analyzed by EMSA. Two major bands are indicated by arrows. (Note: the exposure time of the autoradiogram is significantly shorter than that
of Fig. 5 A). (B) I
B
is responsible for the rapid activation of the p50-RelA complexes in thymocytes. The amounts of the I
B
and I
B
proteins in
wild-type thymocytes stimulated with PMA and PHA for different periods were determined by Western blot analysis using 30 µg of cytoplasmic extracts. (C) The processing of the p105 precursor is enhanced by stimulation of thymocytes. Whole cell lysates from wild-type thymocytes labeled with [35S]methionine for 6 h in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of PMA and PHA were incubated with a p52 antiserum (lanes 1 and 2) and the
resulting supernatants were immunoprecipitated with a p50 antiserum (lanes 3 and 4). The exposure time of the autoradiogram of the immunoprecipitation with p52 is 2.5 times longer than that of p50, so as to verify the p100 and p52 proteins. Specific signals for the p100, p52, p105, and p50 proteins are indicated by arrows. The numbers of methionine residues contained in human p100, p52, murine p105, and p50 are 16, 10, 20, and 9, respectively (25, 66).
[View Larger Version of this Image (25K GIF file)]
B activities are tightly regulated by the I
B
inhibitor proteins. The effect of PMA and PHA stimulation of thymocytes on I
B protein levels was examined by
Western blot or immunoprecipitation analysis (Fig. 6, B
and C). I
B
levels decreased after 30 min of stimulation
(Fig. 6 B, lane 2) and rapidly recovered (Fig. 6 B, lanes 3-6),
whereas I
B
started to be degraded after 1 h of stimulation (Fig. 6 B, lane 3) and recovered at 8 h (Fig. 6 B, lane
6) in both wild-type and p100
/
thymocytes (Fig. 6 B and
data not shown). These results suggest that I
B
-degradation is negligible compared to I
B
for the rapid activation
of NF-
B in primary thymocytes stimulated with PMA and PHA and that degradation of neither I
B
nor I
B
seems to contribute to the delayed activation of the p50
dimers. Interestingly, both I
B
and I
B
were expressed
at high levels for 8 h at a time when there was a dramatic
increase in
B-binding activity in p100-deficient cells, indicating that neither of the two is able to efficiently retain
p50 dimers and p52- or RelB-containing heterodimers in
the cytoplasm. As shown in Fig. 6 C, immunoprecipitation of wild-type thymocytes labeled with [35S]methionine in
the presence or absence of PMA and PHA for 6 h also
showed that the generation of both precursors was responsive to PMA and PHA stimulation (Fig. 6 C, compare lanes
1 and 3 with 2 and 4, respectively). In addition, these results demonstrated that significant amounts of p105 had
been processed, as indicated by the significant amount of
p50 accumulated during this period (Fig. 6 C, lane 4). In
contrast, only small amounts of p52 were detected after 6 h
of stimulation in wild-type thymocytes (Fig. 6 C, lane 2),
and the processed p52 molecule was stable for at least 5 h
under continuous stimulation with PMA (40), indicating that under these conditions the processing of the p100 precursor is significantly slower than that of p105. Similar results were obtained when wild-type splenocytes were treated
with LPS (data not shown). These findings indicate that in
addition to the degradation of I
B
responsible for the
rapid activation of the p50-RelA complex, the processing
of the p105 precursor is responsible for the delayed activation of the p50 dimers, because I
B
does not interact
with the p50 homodimer (47). It is also important to note
that extremely limited availability of p52 in control lymphocytes is due to the inefficient processing of the p100
precursor.
B-regulated Genes in
p100-deficient Mice.
B-binding activity in p100
/
tissues on the expression of
genes regulated by Rel/NF-
B were investigated by RT-PCR analysis of total RNA isolated from spleen (Fig. 7)
and thymus (not shown) of 10-d-old wild-type, heterozygous, and homozygous mutant animals. Semiquantitative
RT-PCR analysis showed that mRNA levels of class I
MHC, TNF-
, endothelial leukocyte adhesion molecule
1, intercellular adhesion molecule 1, and VCAM-1 were
elevated in both tissues from p100
/
mice (Fig. 7 and data
not shown). G-CSF expression was upregulated in thymus
from p100
/
mice (data not shown). The expression of
the nfkb2 gene was also augmented by the absence of p100
in both spleen and thymus (data not shown). This indicates
that the p52-containing heterodimers enhance the nfkb2
gene expression in an autoregulatory manner, agreeing
with the result obtained from in vitro experiments (48). In
contrast, expression of most cytokine genes, such as IFN-
, IL-2, M-CSF and TNF-
were not altered in the thymus
and spleen of p100
/
mice (data not shown). These results
suggest that the increase in p52-containing Rel/NF-
B
complexes are sufficient for regulating expression of some
target genes, while others are either not regulated by Rel/
NF-
B or are regulated by other complexes, such as p50- RelA dimers. Although the consequences of increased expression of
B responsive genes in p100
/
mice have not
been examined, the elevated G-CSF might contribute to
the increased granulopoiesis observed in p100
/
mice.
Fig. 7.
The expression of
Rel/NF-B-regulated genes is
upregulated in p100
/
mice.
Total RNA (0.25 µg) isolated
from the spleen of 10-d-old wild-type (+/+), heterozygous (+/
),
and homozygous (
/
) mutant mice was subjected to RT-PCR
analysis using the indicated specific primers.
[View Larger Version of this Image (79K GIF file)]
/
T cells.
/
mice aged 2-wk or older suggest that the increase in
B-binding activity promotes lymphocyte proliferation in vivo.
Therefore, we analyzed the proliferative responses of purified T cells from 10-d-old wild type and homozygous mutant mouse spleen stimulated with several mitogens by
[3H]thymidine incorporation. Anti-CD3, anti-CD3 plus
anti-CD28, or PMA plus PHA promoted proliferation of
p100
/
T cells two- to sixfold more efficiently than control T cells (Fig. 8 A). LPS treatment also increased proliferation of p100
/
B cells fivefold more efficiently than
control B cells (data not shown). These data indicate that
proliferative responses of p100
/
lymphocytes are also enhanced in vitro.
Fig. 8.
Accelerated proliferative responses
and cytokine production in activated T cells of
p100/
mice. (A) T cell proliferation in vitro.
Peripheral T cells isolated from spleen of 10-d-old wild-type (closed boxes) and p100
/
mice
(open boxes) were treated with either anti-CD3,
anti-CD3 plus anti-CD28, or PMA plus PHA,
followed by [3H]thymidine incorporation. Values of [3H]thymidine incorporation are shown
by mean ± S.D. (B) The cytokine production
from stimulated T cells of p100
/
mice is increased. Splenic T cells isolated from 10-d-old
wild-type (closed boxes) and p100
/
(open boxes)
mice were treated with (+) or without (
)
anti-CD3 and anti-CD28 antibodies for 72 h.
The cytokine levels in the supernatants were
determined by ELISA. Levels of IL-2, IL-4,
IL-10, GM-CSF, and TNF-
produced in
p100
/
T cells relative to control T cells are
represented by mean values ± S.D.
[View Larger Version of this Image (19K GIF file)]
/
mice, we analyzed cytokine production in
T cells isolated from 10-d-old mouse spleen. The levels of
cytokine released from T cells stimulated with anti-CD3
and anti-CD28 antibodies were detected by ELISA. The
levels of IL-2, IL-4, IL-10, GM-CSF, and TNF-
in the
culture supernatant from p100
/
T cells were increased
5-35-fold compared to control T cells isolated from wild-type littermates (Fig. 8 B). Enhanced IL-2 expression might
contribute to the vigorous proliferative responses of p100
/
T cells. Recent reports characterizing p50- and c-Rel-deficient mice show that p50 or c-Rel are required for proliferative responses of lymphocytes (17, 18). Mice deficient in
c-Rel also demonstrate that c-Rel is essential for expression
of some cytokines in activated T cells (17, 49). Thus, the
increased
B-binding activity in stimulated p100
/
lymphocytes might result in hyperproliferation of lymphocytes and overproduction of cytokines in activated T cells, further supporting the notion that Rel/NF-
B is important
for the regulation of those activities in lymphocytes.
B2 displayed extensive gastric hyperplasia and
enlarged lymph nodes at 2 wk of age. Granulocytosis in
bone marrow, atrophic spleen, and thymus appeared at 3 wk
of age. The proliferative in vitro responses of lymphocytes
and cytokine production in activated T cells were also increased. This mutation introduced in the nfkb2 gene resulted in a constitutive Rel/NF-
B activity containing the
p52 subunit in all tissues analyzed, and also dramatically increased the p52-containing Rel/NF-
B complexes in
stimulated thymocytes. Thus, elimination of the ankyrin
region of the p100 precursor ubiquitously affects the Rel/
NF-
B activity, and the activated Rel/NF-
B complexes
containing p52 promote proliferation of gastric mucosal
cells and peripheral lymphocytes. It is also important to
note that neither I
B
nor I
B
can compensate for the
lack of the p100 precursor in mice.
B2 has a dual effect as it
eliminates the p100 inhibitor and increases the mature p52
subunit. Abolition of the processing of the precursor, the
limiting step in generating p52, results in accumulation of active p52 molecule. Therefore, both increased p52 protein
and absence of the p100 inhibitor may contribute to activation of the p52-containing Rel/NF-
B complexes. Heterozygous animals have the increased p52 protein but still
retain half of the p100 precursor compared to wild-type
animals (see Fig. 1 C), whereas they behave similarly to
wild-type mice. According to the p100+/
phenotype, loss
of the p100 inhibitor may contribute to the abnormalities in homozygous mutant mice more than accumulation of
the p52 product. As heterozygous mice develop gastric hyperplasia at a much older age, the dominant effect of the
increased p52 protein under the presence of p100 may also
be considered.
/
Mice.
/
mice are almost normal by 2 wk of
age. However, they all are severely affected by 3 wk of age. Both spleen and thymus of p100
/
mice are very atrophic
with altered architecture. Granulocytosis is found in bone
marrow and peripheral blood of p100
/
mice without evidence of infection. In contrast to other atrophic hematopoietic organs, lymph nodes are clearly enlarged and contain an increased number of T cells expressing IL-2R
chain in p100
/
mice aged 2 wk or older. As 50% of
p100
/
pups die by 3 wk of age, the possibility that abnormal physiological conditions of 3-wk-old p100
/
mice
influence the viability of most hematopoietic cells can not
be excluded. Indeed, the serum glucocorticoid levels of
3-wk-old p100
/
mice were always elevated (1.4- to
10.2-fold, average 2-fold) relative to their control littermates (data not shown). Nevertheless, granulocytosis in
bone marrow, enlargement of lymph nodes, and lymphocyte infiltration in stomach of p100
/
mice are evident,
implying that the constitutive activation of Rel/NF-
B affects the hematopoietic cell functions in vivo.
B2 gene is located in chromosome
10q24, which is recurrently associated with lymphoid malignancies (23). Several cases of NF
B2 gene rearrangement, including a small deletion and chromosomal translocation, are found in primary human lymphomas and cell
lines (23, 28, 29, 50, 51). The rearranged NF
B2 genes encode either the truncated NF-
B2 or fusion products to
heterologous molecules that commonly lack some or all
ankyrin repeats at the COOH terminus but retain an intact
Rel homology domain. The abnormal NF-
B2 proteins
lack the inhibitory function and are directly converted into
the active subunit of the Rel/NF-
B transcription factors
(52). Thus, genetic evidence in humans also suggests that
the intact p100 precursor is important for lymphocytes.
/
T cells indicate that these cells are hyperresponsive to external stimuli. Interestingly, stimulation of
wild-type T cells clearly induces expression of the p100
precursor but not the generation of p52 (see Fig. 6 C), supporting a physiological role of the intact p100 precursor in
activated T cells. Although p100
/
splenocytes and thymocytes have constitutive
B-binding activities (see Fig. 5
A), T cells isolated from p100
/
spleen neither proliferate
nor produce cytokines without stimulation (see Fig. 8, A
and B). This suggests that although Rel/NF-
B is necessary to lymphocyte proliferation (17, 18) and cytokine production in T cells (17, 48), it is not sufficient per se to trigger those activities in lymphocytes.
/
Mice.
B2 was the marked gastric hyperplasia, providing novel evidence that activated Rel/NF-
B transcription
factors are involved in gastric cell proliferation. Accumulation of surface epithelial cells with infiltration of lymphocytes in the lamina propria and hyperkeratosis in the cardiac
portion of the stomach were constant observations in
p100
/
mice and, importantly, the nfkb2 gene is strongly
expressed in the surface epithelial layer of mouse stomach.
Pathological changes observed in the p100
/
stomachs
might cause alteration in homeostasis, advanced cachexia, and subsequent postnatal death.
/
mice may resemble that seen in human Menetrier's disease,
although p100
/
mice exhibit a much more severe phenotype. In contrast, heterozygous animals whose stomach is
histologically normal while young also develop gastric hyperplasia by 10 mo of age. This hyperplasia is pathologically
mild and seems to be more like human Menetrier's disease
(data not shown). Although genetic change(s) in Menetrier's disease has not been yet established, if a somatic mutation
of gene(s) causes this disease, it may occur in a single allele. Therefore, it may be possible to propose that p100+/
animals are useful for understanding the molecular basis of human Menetrier's disease.
is a potent mitogen of
gastric mucosal cells. Indeed, transgenic mice overexpressing TGF-
present gastric abnormalities reminiscent of
Menetrier's disease (54, 55). TGF-
and epidermal growth
factor (EGF) receptor but not EGF have been demonstrated to be expressed in gastric mucosal cells (56). Upon
ligand binding, phosphorylation of several proteins including autophosphorylation of EGFR initiates a number of intracellular responses, such as transient expression of the nuclear oncogene products c-myc and c-fos. However, no elevation of TGF-
, EGFR, c-myc, or c-fos mRNAs was
detected in the stomach of 10-d-old p100
/
mice compared with those of their control littermates by RT-PCR analysis (data not shown). It is likely that the activation of Rel/NF-
B is downstream of the EGFR signaling pathway, since the Ras-Raf pathway is proposed to participate
in the activation of NF-
B (57). Alternatively, there may
be a distinct pathway promoting gastric mucosal cell
growth through the Rel/NF-
B activation. Another possibility is that the infiltrating lymphocytes in the epithelial
layer and lamina propria of mutant mouse stomach may produce a different cytokine such as heparine-binding EGF
(58), and in that case gastric hyperplasia of mutant mice is
related to the activated lymphocyte functions.
B activation through modification of I
B inhibitors, the specificity
and physiological relevance of the different Rel/NF-
B and I
B proteins remain to be understood. It was initially
proposed that the rapid but transient activation of NF-
B is
mediated through I
B
, while the persistent activation of
NF-
B by some inducers, such as LPS or IL-1, is mediated
through I
B
, and that IkB
and IkB
exhibit a similar
preference among the Rel/NF-
B subunits (47). A more
recent report characterizing I
B
-deficient (I
B
/
)
mice (59) demonstrates that I
B
is also involved in the
rapid activation of NF-
B in fibroblasts, whereas I
B
plays an important role in hematopoietic tissues. It also
demonstrated that I
B
is required for the repression of
the NF-
B activity after stimulation of fibroblasts. According to these observations, the different properties of these
inhibitors are due to their different degradation and expression kinetics in the cell and their different expression patterns in mouse tissues. I
B
has been shown to be quite
stable in pre-B cells treated with PMA (47). However, our
results indicate that although PMA and PHA stimulation of
thymocytes leads to degradation of I
B
, it is unlikely to
be important for rapid activation of NF-
B in these cells.
As we have shown here, the expression and processing of
the p105 precursor is enhanced by extracellular stimuli in
primary murine lymphocytes, inducing the delayed activation of the p50 homodimers. In contrast, p100 expression is
increased but processing to p52 is not, showing that the
processing of the p105 and p100 precursors is differentially
regulated.
B transcription factors (25, 43, 60). Indeed, the
expression of the nfkb2 gene is increased in p100
/
mice
presumably due to the enhanced Rel/NF-
B activity.
Complexes containing p52 accumulate after 4- to 8-h stimulation of p100
/
thymocytes (see Fig. 6 A) because of induced de novo protein synthesis of p52. Therefore, the initial synthesis of the precursor form may have the advantage
to act as reservoirs for the Rel/NF-
B activity, avoiding
the rapid accumulation of the mature p50 or p52 molecules. RelB and c-Rel, unlike RelA, are also inducible gene products (61), and the newly synthesized RelB
and c-Rel may be sequestered immediately by p100 and
p105 to prevent the activation of the RelB- or c-Rel-containing complexes. As RelB is unable to dimerize with itself, RelA, or c-Rel, the precursors may be the important
inhibitors for RelB. Indeed, although the p100 precursor
efficiently interacts with all members of the Rel/NF-
B family (see Fig. 5 C), the p52-RelB dimer appears dominant in the
B-binding activity in the absence of the p100
precursor after thymocyte stimulation (see Fig. 6 A and
data not shown). This result agrees with the inefficient regulation of the p52-RelB complex by I
B
compared to
other heterodimers (35, 64). Taken together, we propose
that the COOH-terminal inhibitor of NF-
B2 controls the
activity of the p52-containing complexes, particularly the
p52-RelB and p52-p50 dimers.
B
or I
B
. Since the protein levels of p100 were
much lower than those of I
B
, I
B
, p105, and RelA in
lymphoid cells (Fig. 6 C and data not shown), I
B
and
I
B
would be the most important inhibitory molecules
regulating the ubiquitous components, RelA and c-Rel, of
the Rel/NF-
B complexes. After cell stimulation, the
RelA- or c-Rel-containing complexes are activated immediately, whereas the production of the p50 homodimers derived from the processing of p105 is delayed (see Fig. 6
A). Because of the absence of a potent transcriptional activation domain in p50 (65), the p50 homodimers may work
as transcriptional repressors of the Rel/NF-
B activity.
B activity is more important than we expected.
The differences observed in the processing efficiency, preference for dimeric partners, and tissue expression of p100
and p105 precursors suggest that these molecules have a
different biological role.
B Complexes.
B family present different phenotypes, suggesting distinct roles for the individual subunits (15).
B
exhibit skin defects, granulocytosis,
and activation of NF-
B activity composed of p50-RelA
dimers, resulting in neonatal mortality (59, 66). This suggests that I
B
is mainly involved in the regulation of the
p50-RelA dimer activity, and that the constitutively activated p50-RelA complex found in I
B
/
mice presumably causes abnormalities (59). Different Rel/NF-
B complexes are activated in I
B
/
and p100
/
mice and
therefore we found it of interest to compare the phenotype of these mutant animals (Table 1). Skin is severely affected in I
B
/
mice, whereas it seems to remain intact in
p100
/
mice. Increased granulopoiesis is found earlier in
I
B
/
mice than in p100
/
mice, which have no granulocytosis by 2 wk of age. Both gastric hyperplasia and enlargement of lymph nodes which are developed in p100
/
mice by 2 wk of age, are not noted in I
B
/
mice because
of either lack of the abnormalities or death earlier than 2 wk
of age. Although abnormalities in hematopoietic organs of
I
B
/
mice are hard to determine due to neonatal mortality, both spleen and thymus are atrophied. It is interesting that hyperplasia of different cell types (epidermal cells in
skin of I
B
/
mice and epithelial cells in stomach and
peripheral lymphocytes of p100
/
mice) and hyperkeratosis of different regions (skin in I
B
/
mice and
cardiac portion of stomach in p100
/
mice) are developed
in both mutant mice (reference 66 and Fig. 3 A), suggesting that the activation of different Rel/NF-
B complexes promotes cell proliferation although in vivo targets for the
p50-RelA and p52-containing heterodimers appear to be
different. In addition, expression of several genes, such as
G-CSF, TNF-
, and VCAM-1, assumed to be regulated
by Rel/NF-
B, is elevated without cell stimulation in both
mutant mice, implying that constitutive activation of different Rel/NF-
B complexes can activate in common
some, but not all, of the target genes. Hyperkeratosis in the
tail and delayed onset of granulocytosis and mortality that
resembles the p100
/
phenotype are observed in mice
lacking both p50 and I
B
(p50
/
I
B
/
). This is intriguing considering that the major
B-binding complex in
the tissues of these mutant mice, as in p100-deficient animals, is the p52-RelB dimer instead of the p50-RelA complex (59).
B
/
and p100
/
Mice
I
B
/
p100
/
Fate
Runting and lethal by P10
Runting and lethal by 4w
Activated Rel/NF-kB complexes
p50-RelA
p52-containing heterodimers
Pathological phenotype
Dermatitis
Hyperkeratosis in skin
Granulocytosis in bone marrow,
peripheral blood, and spleen
Atrophy of spleen and thymus
Gastric hyperplasia
Hyperkeratosis in cardiac portion of stomach
Enlargement of lymph nodes
Granulocytosis in bone marrow
and peripheral blood
Atrophy of spleen and thymus
Increased gene expression
G-CSF
TNF-
VCAM-1
MIP-2
G-CSF
TNF-
VCAM-1
ELAM-1
ICAM-1
nfkb2
MHC-I
*
Phenotype ovserved in I B
/
mice has been described previously (58, 65).
Thus, these findings suggest that different dimeric complexes of Rel/NF-B transcription factors play a distinct
role in vivo. Studies of I
B
, p105, and Bcl-3-deficient
mice will be helpful to clarify the specificity and physiological relevance of individual members of the I
B family and
of the different Rel/NF-
B complexes.
Address correspondence to Dr. Rodrigo Bravo, Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543. Phone: 609-252-5744; FAX: 609-252-6051.
Received for publication 31 December 1996 and in revised form 30 June 1997.
1 Abbreviations used in this paper: EGF, epidermal growth factor; EMSA, electrophoretic mobility shift assay; ES, embryonic stem; NLS, nuclear localization signal; P, postnatal day(s); PGK, phosphoglycerate kinase; RT, reverse transcriptase; VCAM-1, vascular adhesion molecule 1.We are grateful to S. Lira, M. Swerdel and A. Lee for generating mutant mice, C. Rizzo for tissue culture, A. Lewin for tissue sections, K. Class and C. Raventos-Suarez for FACS® analysis, N. Thomson and T. Nelson for oligo synthesis and DNA sequencing, T. Gridley for CJ7 ES cells, and the staff of Veterinary Sciences at Bristol-Myers Squibb for their excellent support. We also thank R. Attar, J. Caamano, J. Chen, V. Iotsova, H. Macdonald-Bravo, and F. Weih for their valuable comments.
1. |
Beg, A.A., and
A.S. Baldwin Jr..
1993.
The I![]() ![]() |
2. |
Gilmore, T.D., and
P.J. Morin.
1993.
The I![]() |
3. |
Liou, H.C., and
D. Baltimore.
1993.
Regulation of the NF-![]() ![]() |
4. |
Siebenlist, U.,
G. Franzoso, and
K. Brown.
1994.
Structure,
regulation and function of NF-![]() |
5. |
Finco, T.S., and
A.S. Baldwin.
1995.
Mechanistic aspects of
NF-![]() |
6. |
Thanos, D., and
T. Maniatis.
1995.
NF-![]() |
7. |
Verma, I.M.,
J.K. Stevenson,
E.M. Schwarz,
D. Van Antwerp, and
S. Miyamoto.
1995.
Rel/NF-![]() ![]() |
8. |
Grilli, M.,
J.-S. Chiu, and
M.J. Lenardo.
1993.
NF-![]() |
9. |
Baeuerle, P.A., and
T. Henkel.
1994.
Function and activation
of NF-![]() |
10. |
Kopp, E.B., and
S. Ghosh.
1995.
NF-![]() |
11. |
Miyamoto, S., and
I.M. Verma.
1995.
Rel/NF-![]() ![]() |
12. |
Carrasco, D.,
R.-P. Ryseck, and
R. Bravo.
1993.
Expression
of relB transcripts during lymphoid organ development: specific expression in dendritic antigen-presenting cells.
Development (Camb.).
118:
1221-1231
|
13. |
Carrasco, D.,
F. Weih, and
R. Bravo.
1994.
Developmental
expression of the c-rel protooncogene in hematopoietic organs.
Development (Camb.).
120:
2991-3004
|
14. |
Weih, F.,
D. Carrasco, and
R. Bravo.
1994.
Constitutive and
inducible Rel/NF-![]() |
15. |
Beg, A.A.,
W.C. Sha,
R.T. Bronson,
S. Ghosh, and
D. Baltimore.
1995.
Embryonic lethality and liver degeneration in
mice lacking the RelA components of NF-![]() |
16. | Burkly, L., C. Hesslon, L. Ogata, C. Reilly, L.A. Marconi, D. Olson, R. Tizard, R. Cate, and D. Lo. 1995. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature (Lond.). 373: 531-536 [Medline]. |
17. | Kontgen, F., R.J. Grumont, A. Strasser, D. Metcalf, R. Li, D. Tarlinton, and S. Gerondakis. 1995. Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Genes Dev. 9: 1965-1977 [Abstract]. |
18. |
Sha, W.C.,
H.-C. Liou,
E.I. Tuomanen, and
D. Baltimore.
1995.
Targeted disruption of the p50 subunit of NF-![]() |
19. |
Weih, F.,
D. Carrasco,
S.K. Durham,
D.S. Barton,
C.A. Rizzo,
R.-P. Ryseck,
S.A. Lira, and
R. Bravo.
1995.
Multiorgan inflammation and hemopoietic abnormalities in mice
with a targeted disruption of RelB, a member of the NF-![]() |
20. |
Palombella, V.J.,
O.J. Rando,
A.L. Goldberg, and
T. Maniatis.
1994.
The ubiquitin-proteasome pathway is required for
processing the NF-![]() ![]() |
21. |
Chen, Z.J.,
J. Hagler,
V.J. Palombella,
F. Melandri,
D. Scherer,
D. Ballard, and
T. Maniatis.
1995.
Signal-induced
site-specific phosphorylation targets I![]() ![]() |
22. |
Chen, Z.J.,
L. Parent, and
T. Maniatis.
1996.
Site-specific
phosphorylation of I![]() ![]() |
23. |
Neri, A.,
C.-C. Chang,
L. Lombardi,
M. Salina,
P. Corradini,
A.T. Maiolo,
R.S.K. Chaganti, and
R. Dalla-Favera.
1991.
B cell lymphoma-associated chromosomal translocation involves candidate oncogene lyt-10, homologous to NF-![]() |
24. |
Schmid, R.M.,
N.D. Perkins,
C.S. Duckett,
P.C. Andrews, and
G.J. Nabel.
1991.
Cloning of an NF-![]() |
25. |
Bours, V.,
P.R. Burd,
K. Brown,
J. Villalobos,
S. Park,
R.-P. Ryseck,
R. Bravo,
K. Kelly, and
U. Siebenlist.
1992.
A novel
mitogen-inducible gene product related to p50/p105-NF-![]() ![]() |
26. |
Mercurio, F.,
J. DiDonato,
C. Rosette, and
M. Karin.
1992.
Molecular cloning and characterization of a novel Rel/NF-![]() ![]() |
27. |
Duckett, C.S.,
N.D. Perkins,
T.F. Kowalik,
R.M. Schmid,
E.-S. Huang,
A.S. Baldwin Jr., and
G. Nabel.
1993.
Dimerization of NF-![]() ![]() ![]() |
28. |
Fracchiolia, N.S.,
L. Lombardi,
M. Slaina,
A. Migliazzi,
L. Baldini,
E. Berti,
L. Cro,
E. Polli,
A.T. Maiolo, and
A. Neri.
1993.
Structural alterations of the NF-![]() |
29. |
Migliazza, A.,
L. Lombardi,
M. Rocchi,
D. Trecca,
C.-C. Chang,
R. Antonacci,
N.S. Fracchiolla,
P. Ciana,
A.T. Maiolo, and
A. Neri.
1994.
Heterogeneous chromosomal aberrations generate 3![]() ![]() |
30. | McBurney, M.W., L.C. Sutherland, C.N. Adra, B. Leclair, M.A. Rudnicki, and K. Jardine. 1991. The mouse Pgk-1 gene promoter contains an upstream activator sequence. Nucleic Acids Res. 20: 5755-5761 . |
31. | Tybulewicz, V.L.J., C.E. Crawford, P.K. Jackson, R.T. Bronson, and R.C. Mulligen. 1991. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl protooncogene. Cell. 65: 1153-1163 [Medline]. |
32. | Coligan, J.E., A.M. Kruisbeek, D.H. Margulies, E.M. Schevach, and W. Strober. 1992. Isolation and fractionation of mononuclear cell populations. In Current Protocols in Immunology. Greene Publishing Associates & Wiley-Interscience, New York. Chapter 3. |
33. | Schreiber, E., P. Matthias, M.M. Muller, and W. Schaffner. 1989. Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells. Nucleic Acids Res. 17: 6419 [Medline]. |
34. |
Ishikawa, H.,
R.-P. Ryseck, and
R. Bravo.
1996.
Characterization of ES cells deficient for the p105 precursor (NF-![]() |
35. |
Dobrzanski, P.,
R.-P. Ryseck, and
R. Bravo.
1994.
Differential interactions of Rel/NF-![]() ![]() ![]() ![]() |
36. | Carrasco, D., C.A. Rizzo, K. Dorfman, and R. Bravo. 1996. The v-rel oncogene promotes malignant T-cell leukemia/ lymphoma in transgenic mice. EMBO (Eur. Mol. Biol. Organ.) J. 15: 3640-3650 [Abstract]. |
37. |
Bours, V.,
G. Franzoso,
V. Azarenko,
S. Park,
T. Kanno,
K. Brown, and
U. Siebenlist.
1993.
The oncoprotein Bcl-3 directly transactivates through ![]() |
38. |
Fujita, T.,
G.P. Nolan,
H.-C. Liou,
M.L. Scott, and
D. Baltimore.
1993.
The candidate proto-oncogene bcl-3 encodes a
transcriptional coactivator that activates through NF-![]() |
39. | Modigliani, Y., G. Coutinho, O. Burlen-Defranoux, A. Coutinho, and A. Bandeira. 1994. Differential contribution of thymic outputs and peripheral expansion in the development of peripheral T cell pools. Eur. J. Immunol. 24: 1223-1227 [Medline]. |
40. |
Mercurio, F.,
J. DiDonato,
C. Rosette, and
M. Karin.
1993.
p105 and p98 precursor proteins play an active role in NF-![]() |
41. |
Naumann, M.,
A. Nieters,
E.N. Hatada, and
C. Scheidereit.
1993.
NF-![]() ![]() |
42. |
Scheinman, R.I.,
A.A. Beg, and
A.S. Baldwin Jr..
1993.
NF-![]() ![]() |
43. |
Sun, S.-C.,
P.A. Ganchi,
C. Beraud,
D.W. Ballard, and
W.C. Greene.
1994.
Autoregulation of the NF-![]() |
44. |
Mellits, K.H.,
R.T. Hay, and
S. Goodbourn.
1993.
Proteolytic degradation of MAD3 (I![]() ![]() ![]() ![]() |
45. |
Naumann, M., and
C. Scheidereit.
1994.
Activation of NF-![]() |
46. |
MacKichan, M.L.,
F. Logeat, and
A. Israel.
1996.
Phosphorylation of p105 PEST sequences via a redox-insensitive pathway up-regulates processing to p50 NF-![]() |
47. |
Thompson, J.E.,
R.J. Phillips,
H. Erdjument-Bromage,
P. Tempst, and
S. Ghosh.
1995.
I![]() ![]() ![]() |
48. |
Liptay, S.,
R.M. Schmid,
E.G. Nabel, and
G.J. Nabel.
1994.
Transcriptional regulation of NF-![]() ![]() |
49. |
Gerondakis, S.,
A. Strasser,
D. Metcalf,
G. Grigoriadis,
J.-P.Y. Scheerlinck, and
R.J. Grumont.
1996.
Rel-deficient T cells
exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.
Proc. Natl. Acad.
Sci. USA.
93:
3405-3409
|
50. |
Thakur, S.,
J.-C. Lin,
W.-T. Tseng,
S. Kumar,
R. Bravo,
F. Foss,
C. Gelinas, and
A.B. Rabson.
1994.
Rearrangement
and altered expression of the NF![]() |
51. |
Zhang, J.,
C.-C. Chang,
L. Lombardi, and
R. Dalla-Favera.
1994.
Rearranged NF![]() |
52. |
Chang, C.-C.,
J. Zhang,
L. Lombardi,
A. Neri, and
R. Dalla-Favera.
1995.
Rearranged NF![]() |
53. | Menetrier, P.. 1888. Des polyadenomes gastriques et de leurs rapports avec le cancer de l'estomac. Arch. Physiol. Norm. Pathol. 1: 32-55 . |
54. |
Dempsey, P.J.,
J.R. Goldenring,
C.J. Soroka,
I.M. Modlin,
R.W. McClure,
C.D. Lind,
D.A. Ahlquist,
M.R. Pittelkow,
D.C. Lee,
E.P. Sandgren, et al
.
1992.
Possible role of transforming growth factor ![]() |
55. |
Takagi, H.,
C. Jhappan,
R. Sharp, and
G. Merlino.
1992.
Hypertrophic gastropathy resembling Menetrier's disease in
transgenic mice overexpressing transforming growth factor ![]() |
56. | Bennett, C., I.M. Paterson, C.M. Corbishley, and Y.A. Luqmani. 1989. Expression of growth factor and epidermal growth factor receptor encoded transcripts in human gastric tissues. Cancer Res. 49: 2104-2111 [Abstract]. |
57. |
Folgueira, L.,
A. Algecias,
W.S. MacMorran,
G.D. Bren, and
C.V. Paya.
1996.
The Ras-Raf pathway is activated in human immunodeficiency virus-infected monocytes and participates in the activation of NF-![]() |
58. | Higashiyama, S., J.A. Abbraham, J. Miller, J.C. Fiddes, and M. Klagsburn. 1991. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science (Wash. DC). 251: 936-939 [Medline]. |
59. |
Beg, A.A.,
W.C. Sha,
R.T. Bronson, and
D. Baltimore.
1995.
Constitutive NF-![]() ![]() ![]() |
60. |
Bours, V.,
J. Villalobos,
P.R. Burd,
K. Kelly, and
U. Siebenlist.
1990.
Cloning of a mitogen-inducible gene encoding a
![]() |
61. | Bull, P., T. Hunter, and I.M. Verma. 1989. Transcriptional induction of the murine c-rel gene with serum and phorbol 12-myristrate 13-acetate in fibroblasts. Mol. Cell. Biol. 9: 5239-5243 [Medline]. |
62. |
Molitor, J.A.,
W.H. Walker,
S. Doerre,
D.W. Ballard, and
W.C. Greene.
1990.
NF-![]() |
63. |
Ryseck, R.-P.,
P. Bull,
M. Takamiya,
V. Bours,
U. Siebenlist,
P. Dobrzanski, and
R. Bravo.
1992.
RelB, a new rel
family transcription activator that can interact with p50-NF-![]() |
64. | Dobrzanski, P., R.-P. Ryseck, and R. Bravo. 1995. Specific inhibition of RelB/p52 transcriptional activity by the COOH-terminal domain of p100. Oncogene. 10: 1003-1007 [Medline]. |
65. |
Schmitz, M.L., and
P.A. Baeuerle.
1991.
The p65 subunit is
responsible for the strong transactivating potential of NF-![]() |
66. |
Klement, J.F.,
N.R. Rice,
B.D. Car,
S.J. Abondanzo,
G.D. Powers,
H. Bhatt,
C.-H. Chen,
C.A. Rosen, and
C.L. Stewart.
1996.
I![]() ![]() ![]() |
67. |
Ghosh, S.,
A.M. Gifford,
L.R. Riviere,
P. Tempst,
G.P. Nolan, and
D. Baltimore.
1990.
Cloning of the p50 DNA binding subunit of NF-![]() |