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J. Biol. Chem., Vol. 278, Issue 26, 23278-23284, June 27, 2003
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and Lymphotoxin-
Receptor Activation
¶
From the
Laboratoire Oncogenèse,
Différenciation et Transduction du Signal, CNRS UPR 9079, Institut
André Lwoff, 7 rue Guy Moquet, 94801 Villejuif, France and
Division of Cellular Immunology, La Jolla
Institute for Allergy and Immunology, San Diego, California 92121
Received for publication, January 6, 2003 , and in revised form, April 17, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(TNF-) and lymphotoxin-
receptor
(LTR) signaling both play important roles in inflammatory and immune
responses through activation of NF-
B. Using various deficient mouse
embryonic fibroblast cells, we have compared the signaling pathways leading to
NF-B induction in response to TNF- and LTR activation. We
demonstrate that LTR ligation induces not only RelA/p50 dimers but also
RelB/p50 dimers, whereas TNF- induces only RelA/p50 dimers.
LTR-induced binding of RelB/p50 requires processing of p100 that is
mediated by IKK but is independent of IKK, NEMO/IKK
, and
RelA. Moreover, we show that RelB, p50, and p100 can associate in the same
complex and that TNF- but not LT signaling increases the
association of p100 with RelB/p50 dimers in the nucleus, leading to the
specific inhibition of RelB DNA binding. These results suggest that the
alternative NF-B pathway based on p100 processing may account not only
for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and
that p100 regulates the binding activity of RelB/p50 dimers via at least two
distinct mechanisms depending on the signaling pathway involved. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B transcription factors are key regulators of transcription of a
variety of genes involved in inflammatory and immune responses and in the
control of cell proliferation, differentiation, and apoptosis
(1–7).
In mammalian cells, the NF-B family is composed of five members, RelA
(p65), RelB, c-Rel (Rel), NF-B1 (p50 and its precursor p105), and
NF-B2 (p52 and its precursor p100), and exists as a heterogeneous
collection of homodimers and heterodimers
(3,
8).
In resting cells, NF-B activity is tightly controlled by IB
family members, which include IB, IB,
IB
, Bcl-3, p100, and p105
(9,
10). Phosphorylation of a
NF-B inhibitor protein at specific serine residues by the
IKK1 complex targets
it for ubiquitination and subsequent degradation by the proteasome, thus
enabling NF-B dimers to translocate into the nucleus
(11). The IKK complex is
composed of two catalytic subunits, IKK and IKK, and a regulatory
subunit, IKK (also known as NEMO)
(11). The disruption of genes
encoding individual subunits have demonstrated that IKK and IKK
are required for mediating the canonical NF-B activity (i.e.
RelA/p50 dimers) induced by inflammatory signals
(12–15),
whereas IKK participates in other physiological processes. In
particular, IKK has been shown to be essential for the regulation of
keratinocyte differentiation
(16,
17), receptor activator of
nuclear factor B ligand (RANKL) induced IB degradation in
mammary epithelial cells (18),
and appropriate basal and inducible processing of NF-B2 p100 precursor
to p52 in B cells and lymphotoxin- receptor (LTR)-expressing cells
(19–21).
RelB is the only NF-B member that cannot homodimerize and only
triggers potent transcriptional activation when coupled to p50 or p52
(22–25).
Analyses of RelB-deficient mice have shown that RelB is essential to the
development of medullary epithelium, mature dendritic cell function, and
secondary lymphoid tissue organization
(26–29).
Relb–/– mice
also spontaneously develop a generalized persistent non-infectious multi-organ
inflammatory syndrome
(30).
Until recently, the canonical NF-B (RelA/p50) was considered to be
the predominant inducible B DNA binding activity in most cell types in
response to a broad range of stimuli, whereas RelB represented the major
constitutive B activity in lymphoid cells
(31,
32). However, in the past few
months, an alternative mechanism for inducing NF-B activity has emerged
based on the observation that inducible IKK-dependent p100 processing
allows the resultant p52 to function as transcriptional activator
(20,
21,
33,
34). Remarkably, a
pathway-dependent specificity in p52 binding partner was demonstrated. Whereas
RelA/p52 dimers are the targets of the canonical pathway, nuclear
translocation of RelB/p52 is regulated via the alternative NF-B pathway
and leads to the transcription of a specific pool of genes
(21). Most importantly, all of
these studies point to a crucial role for the alternative NF-B pathway
in controlling the development, organization, and function of lymphoid
tissue.
The participation of RelB in non-lymphoid function is much less well
documented. Although RelB was initially identified as an immediate-early gene
in fibroblasts (24), it has
now been shown to play an essential role in limiting the expression of
proinflammatory mediators in lipopolysaccharide-induced fibroblasts
(35,
36), thereby playing an
important role in the resolution of acute inflammation. Interestingly, in the
non-lymphoid cells examined so far (e.g. NIH 3T3, smooth muscle
cells), RelB was found mainly in association with p50 but not p52 in the
inducible B DNA binding complexes
(24,
37,
38). In contrast to recent
progress in understanding the regulation of RelB/p52 dimers, the mechanisms
controlling the inducible RelB/p50 DNA binding activity are still poorly
understood.
In this study, we have investigated the regulation of RelB/p50 dimers in
fibroblasts in response to ligation of TNFR and LTR, two members of the
TNFR superfamily involved in the regulation of inflammatory and immune
responses
(39–43).
We demonstrate that RelB/p50 activation triggered by LTR ligation
requires processing of p100 that is mediated through IKK but not
IKK, IKK, or RelA. Moreover, we show that RelB, p50, and p100 can
associate in the same complex and that TNF- signaling leads to the
inhibition of RelB DNA binding via an increased association of p100 with
RelB/p50 dimers in the nucleus.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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was
purchased from Sigma, and agonistic anti-LTR mAb AC.H6 was a kind gift
from J. Browning. J. Hiscott and N. Rice generously provided anti-p52/p100 and
anti-p50/p105 polyclonal antibodies. The remaining antibodies were purchased
from Santa Cruz Biotechnology (IKK, RelA, RelB, p105/p50, cRel, and
phospholipase C-1), Upstate Biotechnology (IKK, p100/p52, and
p105/p50), and BD Biosciences (IKK).
Cell Culture and Cell Lines—IKK-, IKK-,
NEMO/IKK-deficient mouse embryonic fibroblasts (MEFs) were described
previously (12,
14,
16). RelA-, RelB-, and
NF-B2-deficient MEFs were a kind gift from A. Beg, F. Weih, and J.
Caamano, respectively. MEFs were grown in Dulbecco's modified Eagle medium
(Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 2
mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml
penicillin, and 100 µg/ml streptavidin. HT29 (ATCC) were cultured in
McCoy's 5A medium with the same supplements.
Cell Extract Preparation—Whole cell extracts were prepared
as reported previously (44).
For cytosolic and nuclear proteins, cells were lysed for 5 min on ice in
hypotonic buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 0.5
mM dithiothreitol, 0.1% Nonidet P-40, 10% glycerol, 1
µM leupeptin, 1 µM aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). The cytosolic fraction was harvested after
centrifuging the lysate for 5 min at 4500 x g. The nuclear
pellet was washed once with hypotonic buffer and lysed for 30 min on ice in
extraction buffer (20 mM Hepes, 500 mM NaCl, 1.5
mM MgCl2, 0.5 mM EDTA, 0.5 mM
dithiothreitol, 25% glycerol, 1 µM leupeptin, 1 µM
aprotinin, and 1 mM phenylmethylsulfonyl fluoride).
Anti-phospholipase C- was used as control for cytoplasmic contamination
in nuclear fractions.
Coimmunoprecipitation and Immunoblotting—For coimmunoprecipitation experiments, 500 µg of nuclear or whole cell extracts were immunoprecipitated for 2 h or overnight at 4 °C using specific antibodies, after which protein A/G-agarose beads (Amersham Biosciences) were added and incubation continued for 90 min at 4 °C. After four washes in lysis buffer, the beads were heat-denatured to release the proteins. Immunoprecipitated proteins were resolved on 8% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore). Immunoblotting was performed with specific antibodies and visualized using the ECL Western blotting detection kit (Amersham Biosciences). For double immunoprecipitation, nuclear or whole cell extracts were incubated with anti-p50 antibody and protein A/G-agarose beads. After five washes in lysis buffer, the antigen-antibody complexes were eluted with a 15-fold excess (w/w) of the specific peptide (Santa Cruz Biotechnology) overnight at 4 °C. The resulting supernatants were immunoprecipitated with anti-RelB antibody and protein A/G-agarose beads. The immune complexes obtained were separated on 8% SDS-polyacrylamide gel and detected by immunoblotting with anti-p100 antibody.
Electrophoretic Mobility Shift Assays (EMSA)—Nuclear
extracts were prepared and analyzed as previously described using the human
immunodeficiency virus long terminal repeat tandem B oligonucleotide as
B probe (45). For
supershift assays, nuclear extracts were incubated with specific antibodies
for 30 min on ice before incubation with the labeled probe.
RT-PCR—RT-PCR were performed as described previously (46). Linear response ranges were determined for each gene to semiquantify their expression levels. Primer sequences are available upon request.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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R but Not TNFR Activates RelB/p50
DNA Binding—We used EMSA to evaluate the nuclear NF-B DNA
binding activity induced by ligation of LTR and TNFR. As shown in
Fig. 1A, whereas
nuclear extracts from untreated WT MEFs contained only low levels of
NF-B DNA binding activity, treatment with either TNF- or
agonistic anti-LTR mAb AC.H6 both resulted in two phases of NF-B
activation. TNF--induced NF-B binding activity was detected
after 30 min of treatment (complex I), decreased to basal levels after 60 min,
returned to near maximal levels after 4 h of treatment, and persisted for at
least 8 h. Complex I was also induced after 30 min of treatment with
anti-LTR antibody, but a faster migrating B complex (complex II)
was detected after 4–8 h of treatment.
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The subunit composition of the NF-B DNA binding complexes was then
examined by supershift assays (Fig.
1B). Incubation of the agonistic LTR Ab-stimulated
protein extracts with anti-RelA and anti-p50 antibodies supershifted complex I
almost completely, whereas complex II was effectively supershifted with
anti-RelB and anti-p50 antibodies. Antibodies to p52
(Fig. 1B) and c-Rel
(data not shown) had very little effect on either complex.
LTR-induced Binding of RelB/p50 Dimers Requires
IKK but Not IKK nor
IKK—To determine which subunit of the IKK complex
controls the binding of RelB/p50 dimers in response to LTR ligation, we
analyzed the DNA binding activity of the nuclear NF-B complexes in
IKK-, IKK-, and IKK-deficient fibroblasts
(Fig. 2A) and found
that a weak constitutive binding of RelB/p50 (complex II) was present only in
MEFs lacking IKK. Most importantly, we found that IKK is
absolutely required for the induction of RelB/p50 DNA binding (complex II),
whereas IKK and IKK are not. In contrast, RelA/p50 binding
(complex I) was strongly reduced in IKK-deficient MEFs and abolished in
IKK- and IKK-deficient MEFs. During the preparation of this
paper, LTR-ligation-induced RelB/p50 activation was also reported by
others to be independent of IKK
(47). Because RelB
transcription has been reported to be regulated by RelA
(48), we also analyzed
LTR-mediated NF-B DNA binding activity in MEFs lacking RelA.
Complex II was clearly induced in the absence of RelA, albeit at somewhat
lower levels. A strong constitutive binding of a third complex (complex III)
was also observed in these cells. Super-shift assays revealed that complex III
corresponds to a p50-containing complex (data not shown).
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Given that IKK exerts a specific function that is not controlled by
the two other subunits of the IKK complex, it is possible that some of the
IKK present in cells is not incorporated into the large
IKK/IKK/IKK-containing complex
(49). To test this
possibility, we carried out immunodepletion experiments on whole cell extracts
from fibroblasts. Five rounds of depletion were performed using an
anti-IKK antibody, and the IKK subunit content was analyzed after each
round by immunoblotting for IKK, IKK, and IKK. IKK
was almost entirely depleted from the protein extracts after one round of
IKK depletion (Fig.
2B), showing that most of the IKK binds to
IKK. In contrast, a considerable fraction of IKK was still
detectable after five rounds of IKK depletion. This observation
suggests the existence of an IKK-containing complex independent of
IKK and IKK. Whether this alternative complex represents
IKK homodimers or IKK associated with a different protein(s)
remains to be determined.
IKK but Not IKK and IKK
Regulates LTR-induced p100 Processing and RelB Nuclear
Translocation in Mouse Embryonic Fibroblasts—We have previously
reported that LTR-induced processing of p100 generates RelB/p52 dimers
that translocate to the nucleus to activate a set of specific target genes
(21). To determine whether a
control mechanism based on p100 processing could also apply to RelB/p50
dimers, we examined p100 and p52 protein levels in WT, IKK-,
IKK-, and IKK-deficient fibroblasts
(Fig. 3A). Treatment
with anti-LTR antibody strongly enhanced processing of p100 to p52 in WT
fibroblasts but failed to do so in IKK-deficient MEFs
(Fig. 3A).
Importantly, LTR ligation led to p100 processing with kinetics parallel
to those of RelB/p50 binding. Although the steady state level of expression of
p100 is low in IKK- and even lower in IKK-deficient fibroblasts
compared with WT fibroblasts, p100 processing still occurred in these cells in
response to treatment with the agonistic LTR Ab. Thus, coincident with
the induction of RelB/p50 DNA binding (Fig.
2A), IKK but not IKK and IKK is also
absolutely required for LTR-induced processing of p100
(Fig. 3A). These
results strongly suggest that the IKK-dependent p100 processing plays a
critical role in the regulation of LTR-induced activation of RelB/p50
dimers. To further investigate underlying mechanisms, we compared
intracellular distributions of RelB in LTR-stimulated IKK-,
IKK-, and IKK-deficient MEFs
(Fig. 3B). We observed
that LTR ligation-induced RelB nuclear localization was abolished in
IKK- but not - or -deficient MEFs
(Fig. 3B). In
addition, although similar constitutive RelB protein levels were observed in
the cytoplasm of the three IKK-deficient cell lines, constitutive RelB levels
in the nucleus were markedly greater in IKK-deficient cells, which may
explain the constitutive RelB/p50 DNA binding activity observed in these cells
(Fig. 2A). Taken
together, our data demonstrate that IKK is required for the
LTR-induced activation of RelB/p50 dimers. Most probably, the processing
of p100 and thus the removal of this main inhibitory partner of RelB allows
RelB nuclear translocation and DNA binding.
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Nuclear p100 Inhibits RelB/p50 DNA Binding in Response to
TNF-—To determine whether the failure of
TNF- to induce RelB DNA binding is the result of a lack of nuclear
translocation of RelB, we compared RelB protein levels and cellular
distributions in response to TNF- versus agonistic LTR
activation in WT MEFs (Fig. 4).
TNF- induction resulted in a strong increase of RelB in both cytoplasm
and nucleus, whereas LTR stimulation markedly increased only nuclear
RelB. Importantly, the levels of induced nuclear RelB were similar in response
to TNFR and LTR ligation. Thus, the increased nuclear RelB expression
level observed in TNF--induced fibroblasts does not lead to an
increased binding activity, suggesting a nuclear control of RelB/p50
activity.
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The absence of TNF--induced RelB DNA binding, despite its
accumulation in the nucleus, could also be attributed to a lack of production
of its heterodimerization partners p50 and p52 and/or to the absence of p100
degradation. Therefore, we also compared the protein levels and cellular
distributions of p105/p50 and p100/p52 in TNF-- and
anti-LTR-treated WT MEFs (Fig.
4). TNFR and LTR ligation both had very little effect on
p105 and p50 protein levels and cellular distribution. Although no nuclear
p105 was detected, a fraction of p50 was constitutively present in the
nucleus. Within 4 h after TNFR ligation, p100 levels increased slightly in the
cytoplasm but strongly in the nucleus. In contrast, LTR ligation led to
p100 processing accompanied by nuclear accumulation of p52
(Fig. 4) with kinetics parallel
to those of RelB/p50 binding. Because the availability of RelB for its DNA
binding heterodimerization partner p50 is similar in TNF- and
anti-LTR mAb AC.H6-stimulated fibroblasts, we hypothesized that the
TNF--induced increase of nuclear p100 might block RelB/p50 DNA
binding.
To test this hypothesis, we first examined whether RelB associates with
p100 in the nucleus of TNF--treated fibroblasts in vivo. p100
was immunoprecipitated using antibody directed against its C-terminal domain
to avoid immunoprecipitation of p52. As shown in
Fig. 5A, endogenous
RelB coimmunoprecipitates with p100 in the nucleus of TNF--activated WT
fibroblasts. Importantly, the increase of RelB protein levels parallels the
increase of p100 protein levels. In contrast, using whole cell extracts as
well as nuclear fractions, no association was detected between RelB and p105
(data not shown). We next investigated whether p100 was able to sequester
RelB/p50 dimers within the nucleus of TNF--treated cells. To first
confirm the existence of such an association, we performed double
immunoprecipitation using whole cell extracts from untreated fibroblasts. A
first immunoprecipitation was performed with an anti-peptide antibody directed
against p50 followed by incubation with the corresponding p50 peptide to elute
the precipitated proteins. The eluate was then immunoprecipitated with an
anti-RelB antibody. An analysis of this second eluate by immunoblotting with
an anti-p100 antibody revealed that p100 participates in a multi-protein
complex that contains both p50 and RelB
(Fig. 5B). The
existence of such a p100/RelB/p50 complex is not restricted to fibroblasts.
Indeed, we have also observed the association of p100 with RelB/p50 dimers in
HT29 cells (see below). To further explore underlying mechanisms, we then used
nuclear fractions instead of whole cell extracts in double immunoprecipitation
as described above. Most importantly, 8 h of TNF- stimulation resulted
in a strong increase in the association of nuclear RelB/p50 dimers with p100,
whereas such an association was not detected in the nucleus of
LTR-activated cells (Fig.
5C).
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To further elucidate the inhibitory role of p100 on RelB/p50 DNA binding
activity downstream of TNFR, we analyzed B DNA binding activity in
NF-B2-deficient fibroblasts, i.e. lacking p100
(Fig. 6). A constitutive
binding of RelB/p50 dimers (complex II) was observed in these cells.
Importantly, TNF- treatment resulted in dramatic increase of RelB/p50
DNA binding (complex II) in the absence of p100, whereas only RelA-containing
dimers (complex I) were induced by TNF- in WT fibroblasts
(Fig. 6). Together, these
results demonstrate that p100 inhibits TNF--induced RelB/p50 DNA
binding, most probably via the "trapping" of nuclear RelB/p50
dimers by p100.
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Ligation of LTR and TNFR Differentially Regulate Gene
Expression—We have shown that LTR ligation induces both the
canonical NF-B pathway, leading to a rapid and transient activation of
RelA/p50 dimers, and the alternative NF-B pathway, leading to a more
delayed and sustained activation of RelB-containing dimers (Ref.
21 and this report). In
contrast, TNF- only induces the canonical NF-B pathway, leading
primarily to the activation of RelA-containing dimers. To address the
physiological relevance of the alternative pathway in the activation of gene
expression, WT MEFs were either left untreated or treated with agonistic
LTR mAb or TNF- for8h and expression of several
NF-B-responsive genes with roles in inflammation was monitored by
semiquantitative RT-PCR (Fig.
7). LTR mAb and TNF- both induced the genes encoding
monocytic chemoattractant protein-1 (MCP-1) and p100, but expression of other
target genes including those for the chemokines RANTES (regulated on
activation normal T cell expressed and secreted) and interferon-inducible
protein-10 was clearly specifically induced by TNF-
(Fig. 7). None of the genes
tested thus far was specifically induced in response to LTR ligation.
Nevertheless, our results indicate that there is only partial overlap in the
set of genes induced by LTR mAb and TNF-, suggesting that the
LTR-induced activation of p100 processing may control a set of specific
target genes that remain to be identified.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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R-mediated signaling events that control NF-B activity in
fibroblasts. We observe that different IKK subunits are required for RelA and
RelB regulation. Consistent with previous observations, the data also show
that TNF--induced RelA/p50 activation requires IKK and
IKK, whereas IKK is dispensable
(12,
15,
17,
50). In contrast, no RelB/p50
DNA binding is induced by TNFR ligation, whereas LTR ligation activates
both RelA/p50 and RelB/p50 complexes. In addition, we demonstrate that
LTR-induced binding of RelA/p50 requires IKK, IKK, and
IKK, whereas LTR-induced binding of RelB/p50 absolutely requires
IKK but not IKK and IKK. Recently, it has been reported
that IKK may function as an essential component of the classical IKK
complex, being specifically required for RANKL-mediated activation of this
complex in mammary epithelial cells
(18). The data presented here
thus provide a second body of evidence for a crucial role of IKK in the
induction of canonical NF-B DNA binding activity.
In contrast to the canonical NF-B (RelA/p50), we and others have
observed that p100 is the only IB family member that strongly inhibits
RelB activity (48,
51,
52).2
Recently, we have shown that LTR-induced IKK-dependent p100
processing controls RelB/p52 dimer nuclear translocation and gene regulation
(21). In this report, we show
that the control of p100 processing also plays a critical role in the
regulation of LTR-induced activation of RelB/p50 dimers. Therefore, the
newly discovered alternative NF-B pathway based on p100 processing
seems to account not only for the regulation of RelB/p52 dimers but also for
that of RelB/p50 dimers. Although RelB/p52 dimers might be expected to result
from the processing of RelB/p100 dimers, it was less clear a priori
how p100 processing could control RelB/p50 binding activity. Interestingly, we
have found that endogenous p100, p50, and RelB can associate in a single
multi-protein complex in fibroblasts as well as in HT29 cells. Thus, our data
suggest that LTR ligation releases RelB/p50 dimers from their
interaction with full-length p100, allowing RelB nuclear translocation and
subsequent DNA binding. Endogenous complexes containing p100 together with
RelA/p50 (53,
54) or RelB/p50
(55) have also been found in
human breast and lymphoid cancer cells, suggesting that the release of
NF-B dimers from p100 inhibition could represent a more general
mechanism for regulation of NF-B activity.
In IKK-deficient fibroblasts, we have observed a clear reduction of
LTR-induced binding of RelB/p50 dimers that does not correlate with an
impaired processing of p100 or decreased RelB nuclear translocation. Most
probably, the diminished RelB/p50 activity is related to the markedly reduced
RelB protein expression in these cells
(Fig. 3B).
Interestingly, a weak constitutive RelB/p50 DNA binding was detected in MEFs
lacking IKK, correlating with a high constitutive level of nuclear RelB
and a very low level of p100 expression in these cells
(Fig. 3). A constitutive
RelB/p50 DNA binding was also detected in NF-B2-deficient fibroblasts
(Fig. 6). These observations
suggest that there are at least two levels of complexity in the regulation of
RelB/p50 activity: 1) the overall expression level of RelB and p100 proteins;
and 2) the control of p100 processing.
Although TNF- signaling did not induce RelB/p50 DNA binding in WT
fibroblasts, a marked increase of RelB protein level was observed in the
nucleus of these cells. This absence of a direct correlation between the
nuclear localization of RelB and its DNA binding activity clearly suggested
that an additional negative control of RelB activity existed in the nucleus of
TNF--treated fibroblasts. Here again, p100 was a good candidate,
because the level of nuclear p100 was also strongly increased in response to
TNF-. Indeed, we observe that TNF- signaling strongly induces
RelB/p50 activity in NF-B2-deficient cells, suggesting that it is not
the processing of p100 per se but rather the "removal" of
p100 that allows RelB/p50 dimers to bind to the DNA. In addition, we
demonstrate that the association of p100 with RelB/p50 dimers is dramatically
increased in the nucleus of TNF--treated cells. In conclusion,
TNF--induced assembly of the p100/RelB/p50 multimeric complex in the
nucleus seems to account for the inhibition of RelB/p50 DNA binding, implying
that p100 controls RelB/p50 dimers not only in the cytoplasm but also in the
nucleus.
How might full-length p100 interact with NF-B dimers? NF-B
members all contain an N-terminal Rel homology domain responsible for DNA
binding, dimerization, and association with the IBs
(3,
56). The C-terminal domain of
p100, like the other IBs, is characterized by an ankyrin-rich domain
that interacts with NF-B via the Rel homology domain. Structures of
co-crystals of NF-B proteins in association with IB and
IB have been determined previously
(56), and it emerges that the
dimerization domain of the NF-B dimers is the primary region of
interaction with IBs. It seems plausible that p100 might self-associate
through its dimerization domain and that its C-terminal ankyrin domain could
then serve as a platform for the binding of RelB/p50 dimers.
Analyses of NF-B knock-out mice have revealed that mice lacking
p100/p52 have marked defects in splenic microarchitecture very similar to
those observed in LTR-, NIK-, and RelB-deficient mice
(29,
57,
58). Interestingly, during the
revisions of this paper, mice lacking RelB were also reported to be deficient
in Peyer's patch organogenesis
(59), a phenotype also
observed in NF-B2-, NIK-, and LTR-deficient animals. Animals
lacking p50 do not show those dramatic developmental defects. Nevertheless,
their Peyer's patches are reduced in number and size
(60). Therefore, although
p50-containing dimers are not absolutely required, they seem to contribute to
the Peyer's patch developmental program. Therefore, it is tempting to conclude
that the processing of p100 downstream of LTR is critically involved in
the functions of stromal cells during secondary lymphoid organ development,
most probably through the control of RelB/p52 and, perhaps to a lesser extent,
RelB/p50-responsive genes.
In addition to the lymphoid organ defects, RelB-deficient mice display a
multi-organ inflammatory syndrome that contributes significantly to premature
mortality in these mice (28).
In an effort to better elucidate the physiological relevance of the
LTR-induced alternative NF-B pathway, we have performed RT-PCR on
several known NF-B target genes with roles in inflammation. We have
observed that in WT fibroblasts, p100 and monocytic chemoattractant protein-1
are induced by ligation of both TNFR and LTR. Interestingly, MCP-1 was
previously found to be specifically regulated by NIK in response to LTR
but not TNFR activation (61).
Because NIK is required for LTR-induced p100 processing
(21), the loss of MCP-1
induction observed in NIK-deficient cells could reflect the lack of activation
of RelB-containing dimers. These findings suggest that RelB/p50 dimers control
the transcription of inflammatory genes downstream of LTR. Chromatin
immunoprecipitation experiments and microarray analyses designed to determine
which genes are specifically regulated by RelB heterodimers will provide a
direct test of this hypothesis.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 33-1-49-58-33-96; Fax: 33-1-49-58-33-07; E-mail: m.korner{at}vjf.cnrs.fr.
1 The abbreviations used are: IKK, IB kinase; LTR,
lymphotoxin- receptor; TNF-, tumor necrosis factor-; TNFR,
TNF receptor; mAb, monoclonal antibody; Ab, antibody; MEF, mouse embryonic
fibroblasts; EMSA, electrophoretic mobility shift assays; RT, reverse
transcription; WT, wild type; NIK, NF-B-inducing kinase; MCP-1,
monocytic chemoattractant protein-1.
2 E. Derudder and M. Körner, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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