 |
INTRODUCTION |
NF-
B1 transcription
factors are critical mediators in the fight of the host against
invading pathogens (reviewed in Refs. 1-5). These factors are integral
parts of the innate machinery that translates initial detection of
foreign pathogens, for example by epithelial cells, into activation of
these cells, including production of chemokines and cytokines to in
turn attract and activate professional immune cells. The innate system
further involves NF-
B factors to produce antipathogenic effectors as well as chemokines and cytokines to mediate evolving cell-cell communications needed to coordinate responses. Depending on the exact
nature of the initial innate response, NF-
B factors then help to
develop the appropriate adaptive responses by lymphocytes. In the final
phase of the immune response, NF-
B factors have important roles
during the expansion and differentiation of lymphocytes involved in the
adaptive response. Beyond the innate and adaptive antipathogenic
responses, NF-
B factors are also essential during development and
maintenance of lymphoid organ structures (6, 7), and they make
important contributions during the development of hematopoietic cells,
including B cells and osteoclasts (8, 9). To carry out its diverse
physiologic roles, NF-
B factors not only help to induce expression
of various factors and effectors, but depending on the cellular
context, they also transcriptionally induce proteins that function to
protect cells from apoptosis and that help to stimulate proliferation
(1, 2, 5, 10).
NF-
B is a collective term for a family of dimeric complexes
comprised of combinations of five polypeptides, RelA, c-Rel, RelB,
p50/NF-
B1, and p52/NF-
B2. p50 and p52 are the N-terminal parts of the longer p105/NF-
B1 and p100/NF-
B2 proteins,
respectively, and they are generated by proteolytic processing (1, 2, 4, 5). High levels of p50 are produced constitutively by a
cotranslational mechanism. In contrast, usually only small amounts of
p52 exist in cells, but higher amounts may be induced by select signals.
To activate NF-
B, appropriate environmental signals must bring about
the release of NF-
B dimers from their bound cytoplasmic inhibitors,
in particular from the prototypical inhibitor I
B
and its close
relatives, I
B
and I
B
(1, 2, 4, 5). NF-
B factors are in
addition subject to various direct and indirect mechanisms that
modulate their ability to stimulate transcription, dependent also on
promoter context (1, 2, 5), but the release from the inhibitors is a
first and necessary step in the activation process. Most of the NF-
B
activation signals, and in particular inflammatory cytokines, such as
TNF
and IL-1, induce the phosphorylation of the I
Bs followed by
the rapid ubiquitin- and proteasome-mediated degradation of the
inhibitors, thus freeing NF-
B dimers to migrate to the nucleus to
initiate gene transcription (1, 2, 4, 5). I
Bs are phosphorylated on
two conserved serines by the I
B kinase (IKK) complex. IKKs consist
of the catalytic subunits, IKK
and IKK
, and the regulatory
subunit IKK
(also known as Nemo).
Most signals have been shown to activate NF-
B by the
classical, IKK-dependent pathway and, in particular, to be
dependent on the IKK
catalytic and IKK
/Nemo regulatory subunit to
bring about the degradation of small I
B inhibitors (1, 2, 5, 11). In
addition to the small I
Bs, the long forms of the NF-
B1 and
NF-
B2 proteins, p105 and p100, can also act as cytoplasmic inhibitors of bound Rel proteins due to the presence of I
B-like inhibitory ankyrin domains in their C-terminal halves (1, 2, 4, 5).
p105 may be completely degraded in response to some signals in a manner
similar to that of small I
Bs, including IKK
/IKK
-induced phosphorylation of two serines embedded in a small I
B-like
phosphorylation motif (12). Recently, a second or alternative signaling
path has been reported to liberate NF-
B activity via induced
processing of p100 inhibitor (13, 14). Although physiologic signals for this pathway were not reported, processing was mediated by the NF-
B-inducing kinase (NIK) and IKK
.
In the present report, we demonstrate that physiologic signaling via
the lymphotoxin
receptor (LT
R) in stromal cells induced the
degradation of I
B
via the classical pathway, and it induced processing of p100 via an alternative pathway. p100 processing was
shown to be dependent on NIK and IKK
but independent of IKK
and
IKK
/Nemo. Therefore, the p100 processing pathway was entirely independent of the IKK complex, not just of the IKK
kinase subunit. We also demonstrate that transient activation of the
classical pathway caused the transient activation of p50-RelA dimers,
whereas the delayed and protein synthesis-dependent p100
processing led to the delayed and sustained liberation of p50-RelB and
p52-RelB complexes. We also provide an explanation and supporting
evidence for how p100 processing liberated p50-RelB complexes.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
IKK
/
and
IKK
/
mouse embryonic fibroblasts (MEFs) were kindly
provided by Drs. Q. Li and I. M. Verma, and
IKK
/
MEFs were kindly provided by Drs. M. Pasparakis
and K. Rajewsky. NF-
B1
/
and
NF-
B2
/
MEFs were kindly provided by Dr. E. Claudio.
To prepare embryonic fibroblasts from wild-type and aly/aly
mice, 12-day-old embryos were dissected, heads and inner organs were
removed, and remaining parts were minced, filtered, and
subjected to trypsin (0.25%) digestion for 10 min at 37 °C.
The resulting cells were filtered and washed in Dulbecco's modified
Eagle's medium (Invitrogen). Fibroblasts were grown in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated fetal
bovine serum and antibiotics.
Transfections and Western Analyses--
Fibroblasts were plated
into 6-well plates at 105 cells/well 24 h prior To
Whom It May Concern: stimulation. After treatment, cells were lysed in
100 mM Tris, pH 6.8, 4% SDS, 20% glycerol, sonicated, and
subjected to SDS-PAGE. MEFs were transfected with LipofectAMINE 2000 (Invitrogen). Cells were analyzed 24 h after transfection.
Expression vectors for IKK
, IKK
, and NIK were kindly provided by
Drs. R. Geleziunas and W. C. Greene (15). Human p100 was excised
from a previously described expression vector (16) and inserted into
pcRSV (Invitrogen).
Nuclear/Cytoplasmic Extracts and
Electrophoretic Mobility Shift Assays (EMSAs)--
For each
nuclear preparation, 5 × 105 cells were plated
24 h prior to stimulation. Stimulation was done under serum-free
conditions. Following stimulation, nuclear and cytoplasmic extracts
were prepared essentially as described (17). Briefly, fibroblasts were
mechanically removed, washed twice in phosphate-buffered saline, and
resuspended in 400 µl of low salt buffer (10 mM Hepes, pH
7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, supplemented with protease and phosphatase inhibitors).
After a 15-min incubation on ice, Triton-X-100 was added to a final
concentration of 0.6%, and the suspension was vigorously vortexed for
10 s. The nuclei were pelleted, and the supernatant served as
cytoplasmic extract. The pelleted nuclei were resuspended in 50 µl of
high salt buffer (20 mM Hepes, pH 7.9, 400 mM
NaCl, supplemented with protease and phosphatase inhibitors) and
incubated for an additional 15 min on ice. 2.5 µl of this preparation
were used in DNA binding reactions. An NF
B-binding site
from the
light chain enhancer was used as a probe:
5'-AGTTGAGGGGACTTTCCCAGGC-3' (Promega, Madison, WI). Oct1
oligonucleotides were used in controls: 5'-TGTCGAATGCAAATCACTAGAA-3'.
Complementary and annealed oligonucleotides were end-labeled with
[
- 32P]ATP. Approximately 20,000 cpm of probe were
used per assay. The binding reaction was carried out at room
temperature for 15 min in a total volume of 25 µl containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM
MgCl2, 0.5 mM dithiothreitol, 50 µg/ml
poly(dI-dC)·poly(dI-dC), 4% glycerol. For supershift analyses,
nuclear extracts were preincubated with antibodies for 30 min on ice
prior to adding the probe. For Western blot analyses, 20 µl of each
preparation were subjected to SDS-PAGE. The efficiency of the
nuclear-cytoplasmic fractionation was confirmed in several ways,
including by the fact that entry of factors was dependent on
stimulation and by the fact that RelA did not enter nuclei in
NEMO-deficient cells, whereas RelB did (see Fig.6, A and
C).
For immunoprecipitation, cytoplasmic and nuclear preparations were
generated from 1.5 × 106 cells/condition. These
extracts were adjusted to 150 mM NaCl and mixed with
anti-RelB antibodies (SC-848) or anti-NEMO antibodies (SC-8330) (Santa
Cruz Biotechnology, Santa Cruz, CA) as a negative control that had been
conjugated to agarose beads (Pierce). Following a 2-h incubation at
4 °C, the agarose beads were washed four times in 150 mM
NaCl, 25 mM Hepes, pH 7.3, 10% glycerol, 1% Triton-X-100, and the immunoprecipitated preparations were subjected to SDS-PAGE.
Antibodies and Reagents--
The agonistic monoclonal
anti-murine LT
R antibodies were kindly provided by Dr. J. Browning
and used at 10 µg/ml. For EMSA supershift experiments, the following
antibodies to detect murine proteins were used: anti-RelA (SA-171)
(Biomol, Plymouth Meeting, PA); anti-RelB (SC-226X), anti-c-Rel
(SC-71X), anti-NF-
B1 (SC-114X), anti-NF-
B2 (SC-848X) (Santa Cruz
Biotechnology). For Western analyses, the following antibodies
were used: anti-I
B
(SC-945), anti-I
B
(SC-371), anti-RelB
(SC-226), anti-c-Rel (SC-71) (Santa Cruz Biotechnology). Polyclonal
anti-murine RelA, anti-murine p105, and anti-human p100 antibodies
(also detects murine p100) were raised against the 13 C-terminal amino
acids (RelA), the 15 N-terminal amino acids (p105), and the 398 N-terminal amino acids (p100), respectively. TNF
was purchased from
PeproTech (Rocky Hill, NJ); Light and platelet-derived growth
factor (PDGF-BB) were purchased from R&D Systems (Minneapolis, MN).
Light was used at 20 ng/ml. The IKK
-specific inhibitor PS1145 was
kindly provided by Dr. J. Adams, Millennium Pharmaceuticals (Cambridge,
MA). To inhibit protein synthesis, cells were pretreated with 50 µM cycloheximide (Sigma) for 30 min prior to stimulation.
 |
RESULTS |
LT
R Induces Processing of p100 and Degradation of I
B
in
Mouse Embryo Fibroblasts--
NF-
B2-deficient mice are impaired in
their splenic microarchitecture, they lack Peyer's patches, and they
are severely impaired in lymph node formation, primarily due to defects
within the stromal compartment (6, 7, 18, 19). These deficiencies in
secondary lymphoid organs, which also include loss of follicular
dendritic cell networks, in turn contribute to defective immune
responses in these mutant mice. Similar deficiencies have been noted in aly/aly mice (20, 21) and in mice lacking
lymphotoxin
(LT
) receptor or its ligands (LT

and
Light) (20, 22-25), members of the TNF receptor/ligand family.
aly/aly mice are mutated in NIK (26).
Overexpression of the wild-type form of NIK induces processing of the
p100 protein of NF-
B2 to p52, but its mutant form (aly)
does not (13). Furthermore, splenocytes from
aly/aly mice contain much less of the p52 protein
of NF-
B2 than aly/+ mice while
maintaining normal levels of p100 (21). NIK-induced processing in B
cells depends on IKK
(14), and thus IKK
-deficient B cells contain
much less p52 protein (14). Finally, although IKK
-deficient animals
die perinatally, which limits their analysis, it could nevertheless be
shown that these mutant mice are deficient in Peyer's patches
organogenesis (27). Based on these data, we hypothesized that critical
functions of the LT
receptor on stromal cells depend on signaling
via NIK, IKK
, and NF-
B2 and thus may involve processing of p100.
To test for this possibility, we subjected MEFs to an agonistic
antibody directed against the LT
R. We investigated with Western analyses for the expression of the NF-
B2 proteins p100 and p52, as
well as the NF-
B1 proteins p105 and p50, the inhibitor of NF-
B
(I
B)
, I
B
, RelA, RelB, and c-Rel at six time points during an 8-h stimulation with anti-LT
R antibodies (Fig.
1). We also tested for expression of
these proteins at 15 min and 8 h of stimulation with TNF
. The
experiments revealed a marked decrease in p100 and a concomitant
increase in p52, beginning just before 4 h and maximal by 8 h
of stimulation via the LT
R. No such changes in p100 and p52 levels
were seen with TNF
stimulation. TNF
instead caused the nearly
complete degradation of I
B
and the partial degradation of
I
B
by 15 min of stimulation; I
B
levels were partly restored
by 8 h of stimulation. LT
R stimulation induced only a partial
degradation of I
B
, which showed a delayed onset when compared
with that induced by TNF
. The amounts of I
B
began to increase
again after 2 h of stimulation via the LT
R and were above
starting levels by 8 h. Most likely this was due to increased synthesis in response to activated NF-
B, in the absence of continued degradation of this inhibitor (see below). We failed to observe consistent changes in the amounts of the other proteins analyzed in
Fig. 1, with the exceptions of RelB, whose amounts were increased, and
p105, whose amounts were modestly decreased after 8 h of
stimulation with TNF
. In contrast to p52, the amounts of the p50
form of NF-
B1 did not increase after LT
R stimulation. These data
indicated that LT
receptor stimulation in MEFs caused processing of
p100 to generate p52 and a more modest degradation of the I
B
inhibitory protein, implying engagement of two pathways to activate
NF-
B.

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Fig. 1.
LT receptor
engagement induces processing of p100/NF- B2 to
p52 as well as I B
degradation in wild-type MEFs. Wild-type MEFs were
stimulated with TNF or with agonistic anti-LT receptor antibodies
for the times shown. Total extracts were prepared by SDS lysis and
subjected to SDS-PAGE followed by Western analysis with antibodies
against NF- B and I B proteins as indicated.
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LT
R-induced Processing of p100 Depends on Protein Synthesis,
NIK, and IKK
but Not IKK
or IKK
--
Light and the
membrane-bound LT
2
are natural ligands of the LT
receptor. In addition to the agonistic antibody, Light could also be
shown to induce processing of p100 to p52 in MEFs, whereas platelet-derived growth factor did not (Fig.
2). Given the delayed onset of
processing, we asked whether the underlying mechanisms might involve
intermediate steps requiring protein synthesis. Light-induced
processing of p100 was sensitive to the protein synthesis inhibitor
cycloheximide (Fig. 2), and this result was confirmed when cells were
stimulated with the agonistic antibody to the LT
receptor (data not
shown). Thus, signal-induced processing of p100 required the new or
continued synthesis of a protein, which could explain the slow onset of
processing upon stimulation.

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Fig. 2.
LT
receptor-induced processing of p100 depends on
de novo protein synthesis. Wild-type MEFs were
stimulated for 8 h with anti-LT receptors, Light, or
platelet-derived growth factor (PDGF), or they were
preincubated with cycloheximide (CHX) and stimulated for
8 h with Light or left without added stimulus, as indicated. Whole
cell extracts were analyzed for the presence of the p100 and p52
proteins of NF- B2 using SDS-PAGE. ns denotes a
nonspecific protein that is variably detected.
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Next, we investigated the mechanism underlying LT
R-induced
processing by taking advantage of mutant mice impaired or lacking in
various signaling components. We generated MEFs from
aly/aly mice, which carry a mutation in NIK.
Agonistic antibodies to the LT
R failed to induce processing of p100
to p52 in aly/aly MEFs (Fig.
3A). Therefore, NIK was
required for LT
R-mediated processing of p100, consistent with the
ability of NIK to induce processing in transfected cells and the
impaired LT
R-induced NF-
B transcriptional activity in
NIK-mutated and NIK-deficient MEFs (27, 28).

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Fig. 3.
IKK and NIK are
required for p100 processing, but IKK ,
IKK (Nemo) or NF- B1
are not. As shown in A, MEFs deficient in IKK ,
IKK , IKK , or NF- B1 or mutated in NIK
(aly/aly) were stimulated with anti-LT
receptor antibodies for 8 h or not treated as indicated. p100 and
p52 proteins of NF- B2 were detected by Western analysis of whole
cell lysates. Because expression levels of NF- B2 were low in the
IKK -deficient cells, a long exposure of the Western blot for these
mutant cells is also shown in the lower panel. ns
denotes a nonspecific protein that is variably detected. As shown in
B, an inhibitor of IKK reduces resynthesis of p100 but
does not interfere with p100 processing in LT receptor-stimulated
wild-type MEFs. Wild-type MEFs were preincubated for 30 min with
increasing amounts of PS1145 and then stimulated with TNF for 15 min
to detect I B degradation (lower panel), or they were
stimulated for 8 h with anti-LT receptor antibodies to detect
p100 and p52 proteins (upper panel) by Western analysis of
whole cell lysates as indicated. Untreated cells are shown in the
first lane.
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NIK was shown previously to depend on IKK
to induce processing in B
cells, and consistent with this, LT
R signaling also failed to induce
processing in MEFs from IKK
-deficient mice (Fig. 3A; the
8-h-stimulated lane for these mutant cells contained slightly more
protein, but the ratio of p100 to p52 did not change). Interestingly, MEFs from mice lacking IKK
or those lacking IKK
(also known as
Nemo) were permissive for LT
R-induced processing of p100, as were
MEFs deficient in NF-
B1 (Fig. 3A). (IKK
/IKK1-,
IKK
/IKK2-, and Nemo/IKK
-deficient mice are described in Refs.
29-31.) The regulatory subunit IKK
and the two catalytic subunits
IKK
and IKK
together constitute the classical IKK core complex.
Therefore, although LT
R-induced processing did require the IKK
subunit, it was nevertheless independent of the classical, IKK
(Nemo)-containing IKK complex that controls degradation of the small
I
B inhibitors in response to many signals.
By comparison with wild-type MEFs, the amounts of p100 appeared to be
somewhat reduced in NF-
B1- and IKK
-deficient MEFs but were
especially reduced in IKK
-deficient MEFs. Nevertheless, processing
still occurred in response to LT
R stimulation, resulting in a more
substantial depletion of p100 in the mutant versus wild-type cells (a longer exposure of the IKK
-deficient MEFs is shown in Fig.
3A, lower panel). Basal and LT
R-induced
activation of NF-
B via the classical IKK to I
B degradation path
may be required for optimal p100 expression. MEFs deficient in IKK
may have contained especially low levels of p100 because the classical
activation pathway was completely blocked in these mutant cells,
whereas residual activity may have persisted in the IKK
-deficient
mutants due to the presence of IKK
. We also used an IKK
-specific
inhibitor, PS1145, to provide further support for the suggested role of
the classical activation route in maintaining p100 levels while having no role in processing. To control for the activity of this inhibitor, we confirmed a dose-dependent inhibition of TNF
-induced
degradation of I
B
after 15 min of stimulation (Fig.
3B). Increasing amounts of PS1145 also decreased the amounts
of p100 after 8 h of stimulation via the LT
receptor,
presumably due to reduced new synthesis of p100, whereas processing to
p52 was essentially unaffected (Fig. 3B). Therefore, optimal
expression of p100 depended on basal and induced activation of the
classical, IKK-mediated pathway for NF-
B, but processing of p100 did
not and instead only depended on the IKK
subunit.
It remained theoretically possible that the inability to process p100
in IKK
-deficient and NIK-impaired MEFs in response to LT
stimulation was not directly related to loss of IKK
or NIK function.
We therefore tested whether transfection of these mutant and wild-type
MEFs with p100 together with IKK
, IKK
, or NIK could confirm the
conclusions reached with Fig. 2. Overexpression of IKK
and
especially of NIK in wild-type MEFs induced processing of p100 to
generate p52, whereas IKK
did not (Fig.
4A). IKK
and NIK were
similarly able to induce processing in IKK
(Nemo)-deficient (Fig.
4B), IKK
-deficient (Fig. 4D), and NIK-impaired
(aly/aly) (Fig. 4E) MEFs. This
confirmed that the classical Nemo/IKK
pathway was irrelevant for
processing and that the inability of aly/aly MEFs
to allow processing could be overcome simply by supplying wild-type NIK
or IKK
. This latter result also placed IKK
downstream of NIK,
which was confirmed by the fact that overexpression of IKK
in
IKK
-deficient MEFs resulted in processing, whereas overexpression of
wild-type NIK did not (Fig. 4C). Therefore, we concluded
that processing of p100 to p52 as induced by LT
R stimulation
depended on and proceeded via NIK and then IKK
but was independent
of classical IKK
(Nemo) and IKK
-dependent NF-
B
activation; classical IKK activity did, however, help maintain p100
levels.

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Fig. 4.
p100 processing induced by transfected NIK
requires IKK but not IKK
or IKK . Wild-type (WT) MEFs
(A) or MEFs lacking IKK (B), IKK
(C), IKK (D), or MEFs mutated in NIK
(aly/aly) (E) were cotransfected with
a human p100 expression vector together with an empty control vector
( ) or with an expression vector for IKK , NIK, or IKK as shown.
Total lysates were prepared and analyzed for the presence of the human
p100 and p52 NF- B2 p100 proteins.
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LT
R Induces Sequential Activation of RelB and RelA by Distinct
Pathways--
Next, we investigated LT
R-initiated NF-
B
activation in EMSAs designed to determine the composition of activated
NF-
B. Wild-type MEFs contained some basal
B DNA binding activity
composed primarily of p50 homodimers and p50-RelA heterodimers (faster
and slower migrating shifted bands, respectively), as assessed in EMSA
supershift experiments with antibodies to the various NF-
B subunits
(Fig. 5A). Approximately equal
amounts of extracts were loaded, and this was confirmed in separate
EMSAs in which DNA binding activity to the cognate site for the
Octamer-1 transcription factor was assessed (data not shown). LT
R
stimulation for 2 h resulted in increased amounts of DNA binding
activity composed primarily of p50-RelA and, to a lesser degree,
p50-RelB (Fig. 5B; RelA supershifts marked). After 8 h
of stimulation, the binding activity of p50-RelA dimers had decreased,
whereas p50-RelB activity had increased further, and p52-RelB activity
could be detected as well (Fig. 5C; RelB and p52 supershifts
marked). In addition, p50 homodimer binding activity appeared to have
increased somewhat. No significant c-Rel DNA binding activity was noted
in these MEFs, although this antibody was able to detect c-Rel binding
in lymphoid cells (data not shown).

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Fig. 5.
LT receptor
stimulation activates B DNA binding activity
initially composed primarily of RelA-containing and then of
RelB-containing dimers. Wild-type MEFs were stimulated with
anti-LT receptor antibodies (AB) for 0 (A), 2 (B), or 8 h (C). Nuclear extracts were
prepared, preincubated with the indicated antibodies, mixed with a
radiolabeled, NF- B-specific DNA probe, and subjected to a
non-denaturing PAGE (EMSA analysis). The antibody supershifts obtained
with anti-RelA antibodies after 2 h of stimulation and with
anti-RelB antibodies and anti-p52 antibodies after 8 h of
stimulation are marked with short vertical lines.
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The EMSA supershifts with 8-h-stimulated extracts revealed
predominantly p50-containing complexes with an apparently much smaller
contribution of p52-containing complexes. One must consider, however,
that the EMSA assays are not quantitative and do not necessarily
correlate with the degree to which a given NF-
B dimer has been
released from cytoplasmic inhibition. Different dimers bind standard
B DNA elements with varying strength, and p52-containing dimers in
particular have not been carefully tested for preferred binding sites.
Furthermore, the various antibodies used differ in strength of binding
and may in addition be differentially affected in the supershift
assay. It is possible, therefore, that these EMSA assays underestimated
the amounts of p52-containing complexes in particular. This notion was
supported by nuclear-cytoplasmic fractionation experiments, which
revealed considerable migration of p52 and RelB into nuclei starting by
2 h of LT
R stimulation and increasing thereafter, whereas the
amount of RelA in nuclei was highest after 2 h of stimulation,
declining thereafter (Fig. 6A;
RelB migrated as two closely spaced bands with the upper band preferentially translocating to nuclei). In addition, RelB
coimmunoprecipitation experiments confirmed that RelB was associated
with p52 in the nucleus (Fig. 6B; coimmunoprecipitated p52
is marked by an asterisk). Together the data revealed an
early activation of p50-RelA dimers, which decreased after 2 h,
whereas activation of RelB dimers continued to increase past 2 h.

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Fig. 6.
Induced nuclear translocation of RelB and p52
during 8 h of LT receptor-mediated
stimulation of wild-type and NEMO-deficient fibroblasts. Wild-type
(WT) (A and B) or
NEMO/IKK -deficient (C) MEFs were stimulated with
anti-LT R antibodies for the times shown, and nuclear (N)
and cytoplasmic (C) extracts were analyzed for the
presence of RelA, RelB, and p52 by Western analyses in panels
A and C. As shown in B, cytoplasmic and
nuclear extracts were also immunoprecipitated (IP) with
anti-RelB antibodies and analyzed for the presence of
coimmunoprecipiated p52. Co denotes a polyclonal control
antibody to NEMO and thus unrelated to RelB. Coimmunoprecipitated p52
is marked by an asterisk. The last lane
represents a direct p52 Western analysis of the 8-h-stimulated cell
lysate that was run on the same gel as the immunoprecipitated material
to identify p52. The background bands in panel B are due to
the immunoprecipitating antibodies present in the immunoprecipitates.
The nuclear and cytoplasmic extract lanes contained approximately equal
amounts of proteins, but on a per cell basis, the nuclear lanes
represented ~8 times more cells than the cytoplasmic lanes.
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We speculated that the partial and transient degradation of I
Bs seen
early after LT
R exposure (see above) might be responsible for the
early and transient increase in the p50-RelA binding activity, whereas the processing of p100 might be responsible for the late rise
in p52-RelB binding activity and possibly also in p50-RelB binding
activity. To test this theory, we investigated the activation of
NF-
B with EMSA assays in the mutant MEFs. LT
R-mediated
stimulation of IKK
- and IKK
(Nemo)-deficient MEFs for 8 h
resulted in strong activation of p50-RelB and, to an apparently lesser
degree, p52-RelB, similar to what was observed in wild-type MEFs (Fig.
7, A and B,
respectively; RelB supershifts marked). Contrary to results with
wild-type MEFs, no activation of RelA-containing dimers (primarily p50-RelA) was observed (Fig. 7, A and B), not
even after 2 h of stimulation (data not shown; see also Fig.
6C) These results indicated that the classical activation
pathway via IKK
/IKK
(Nemo) was not required for DNA binding
activation of p50-RelB or p52-RelB but was required for activation of
p50-RelA.

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Fig. 7.
LT receptor-induced
activation of RelA complexes is primarily dependent on
IKK and IKK , whereas
activation of RelB is primarily dependent on IKK
and NIK. Nuclear extracts from 8-h LT receptor-stimulated
MEFs deficient in IKK (A), IKK (B), IKK
(C), or mutated in NIK (aly/aly)
(D) were analyzed for NF- B DNA binding activity with
EMSAs with or without ( ) supershifting antibodies (AB), as
indicated and as described in the legend for Fig. 5. Anti-RelA and
anti-RelB supershifts are marked.
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To confirm that LT
R-mediated stimulation of IKK
(Nemo)-deficient
MEFs caused translocation of both p52 and RelB into nuclei, we also
performed nuclear-cytoplasmic fractionation experiments. As shown in
Fig. 6C, RelB and p52 entered nuclei of these mutant cells
and continued to do so during the course of stimulation, similar to
what was seen with wild-type cells (Fig. 6A), whereas RelA
failed to be translocated into nuclei, as expected for these mutant
cells, which also served as a control for the experiments.
We next tested the relevance of the alternative pathway in the
activation of
B binding activity in response to LT
R stimulation. IKK
-deficient and NIK-impaired (aly/aly) MEFs
failed to activate p50-RelB or p52-RelB after 8 h of stimulation
(Fig. 7, C and D, respectively). As in wild-type
MEFs, p50-RelA was activated after 2 h of stimulation in both
mutant MEFs (data not shown) and was still clearly detected after
8 h (Fig. 7, C and D; RelA supershifts marked). Together these results demonstrated that activation of p50-RelA required IKK
and IKK
(Nemo) but not IKK
or NIK. On the other hand, activation of p50-RelB and p52-RelB required IKK
and
NIK but not IKK
or IKK
. We also note that activation of p50-RelA
appeared to be somewhat prolonged in the absence of IKK
or NIK,
whereas activation of p50-RelB and p52-RelB seemed to be slightly
enhanced in the absence of IKK
and IKK
at early times (data not shown).
p100 Processing Activates p52-RelB and p50-RelB Complexes--
The
data described indicated that the alternative pathway of activation via
NIK and IKK
and, by extension, processing of p100 were responsible
for the activation of not only p52-RelB but also for the activation of
p50-RelB. Although p52-RelB dimers could result from processing of
p100-RelB complexes, it was less obvious how p50-RelB dimers might be
activated. To investigate underlying mechanisms further, we analyzed
LT
R-induced activation in NF-
B2-deficient MEFs since these cells
lack the p100 inhibitor to begin with. In these mutant MEFs, p50-RelB
was already basally activated in the absence of any added stimulus, and
this binding activity was increased slightly further with stimulation
via the LT
R (Fig. 8A; RelB
supershifts marked). The basal activity suggested that it was not the
act of processing of p100 per se but the absence of the p100
inhibitor that led to p50-RelB binding activity. RelB is reported to
preferentially associate with p100 (32), and in the absence of p100,
RelB is presumably free to associate with p50. We speculated that
LT
R stimulation of NF-
B2-deficient MEFs may have led to a further
increase above basal levels of p50-RelB DNA binding activity as a
result of new synthesis of both RelB and NF-
B1 induced via the
classical NF-
B activation route. In support of the theory that RelB
complexes were easily activated in the absence of p100, we demonstrated
that TNF
stimulation activated p50-RelB DNA binding activity to
extremely high levels in NF-
B2-deficient MEFs (Fig. 8B,
lower panel). By comparison, TNF
-stimulation of wild-type
MEFs, which contain p100, did not result in such high activation of
RelB complexes (Fig. 8B, upper panel; RelB
supershifts in wild-type and NF-
B2-deficient MEFs are marked). The
RelB activity observed after long term stimulation of wild-type with
TNF
(Fig. 8B, upper panel) was most likely due
to the high amounts of RelB protein induced via the classical NF-
B
activation pathway, some of which may have escaped sequestration by
p100 (Fig. 1). These results supported the notion that the absence or
presence of p100 was the key to whether p50-RelB dimers were readily
formed or not. Together these results suggested that NIK and IKK
were required for activation of p50-RelB because they led to processing
of p100 and thus removal of the preferred and inhibitory binding
partner for RelB.

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Fig. 8.
A, LT receptor-induced NF- B
activation in NF- B2-deficient MEFs. Nuclear extracts from wild-type
MEFs left untreated (0 h) or stimulated for 8 h were analyzed for
NF- B DNA binding activity with EMSAs with or without ( )
supershifting antibodies (AB) as indicated and as described
in the legend for Fig. 5. Anti-RelB supershifts are marked. As shown in
B, TNF strongly activates RelB DNA-binding complexes in
NF- B2-deficient MEFs. Wild-type (WT) and
NF- B2-deficient MEFs were stimulated for 8 h with anti-LT R
antibodies with and without cycloheximide (CHX) or with
TNF as indicated. EMSAs were performed with or without anti-RelA and
anti-RelB supershifting antibodies as indicated and as described in
panel A and in the legend for Fig. 5. Anti-RelB supershifts
after TNF treatment are marked.
|
|
The experiments of Fig. 8B also demonstrated that
LT
R-induced activation of RelB DNA binding activity in wild-type
MEFs was dependent on protein synthesis (upper panel),
consistent with the dependence of p100 processing on protein synthesis.
We observed some RelB binding activation even in the presence of the
protein synthesis inhibitor cycloheximide when NF-
B2-deficient MEFs
were stimulated with the LT
R (Fig. 8B, lower
panel). In the absence of p100 (NF-
B2-deficient MEFs), RelB
proteins are likely to be associated with p105 and p50. It is possible
that basal or induced turnover of the inhibitory p105 might have led to
a further release and thus activation of RelB complexes, as p105 would
not be replaced in this situation. LT
R-induced activation of RelA
complexes was noticeably enhanced in the presence of cycloheximide in
wild-type (Fig. 8B, upper panel) and especially
in NF-
B2-deficient MEFs (lower panel), presumably due to
the complete loss of relevant inhibitors in the absence of resynthesis.
 |
DISCUSSION |
The study presented here shows that LT
R-mediated stimulation of
mouse embryo fibroblasts resulted in the engagement of two separate
signaling pathways, leading to activation of distinct NF-
B
complexes. We established that the initial or classical activation was
mediated by IKK
- and IKK
-dependent degradation of
I
B
and subsequent liberation of primarily p50-RelA complexes, independent of NIK and IKK
. We further demonstrated that the second
or alternative activation was mediated by NIK- and
IKK
-dependent processing of the inhibitory p100 protein
of NF-
B2, independent of IKK
and IKK
/Nemo. This latter pathway
led to activation of primarily p50-RelB and p50-RelA dimers. These
conclusions were derived from analyses of LT
R-stimulated wild-type
and mutant MEFs impaired in NIK or lacking IKK
, IKK
, IKK
, or
NF-
B2. LT
R-induced p50-RelA activation was modest and transient,
consistent with an only partial and transient loss of I
B
induced
by the classical pathway. With time of stimulation, amounts of I
B
increased to above starting levels to again inhibit RelA complexes. In
contrast, DNA binding activity of RelB complexes increased and began to dominate the NF-
B binding activity. Stimulation of the LT
R on fibroblasts, therefore, initiated two separate pathways to sequentially activate different NF-
B complexes. The change in the types of NF-
B complexes activated is likely to result in a corresponding change in the genes targeted with time during the course of stimulation via LT
R. The data presented here identify the LT
R as a
physiologic inducer of the second pathway of activation. The results
furthermore clarify the importance of this pathway since long term
B
binding activity in response to LT
R stimulation was entirely
dependent on p100 processing.
A prior analysis of NIK-deficient MEFs discovered gene induction
defects specific to stimulation via the LT
R but not the TNF
-receptor (28). Since the LT
R-induced
B binding activity analyzed at early times after stimulation was not impaired in the
NIK-deficient MEFs (it is dominated by the classical activation path as
shown here), it was speculated that LT
R-induced changes in
transactivation potential might have been defective in the absence of
NIK. Although it remains possible that NIK is also required to enhance
transactivation in response to LT
R stimulation, our data suggest
that the lack of processing of p100 in NIK-deficient MEFs and thus the
lack of sustained activation of RelB complexes in particular could also
explain the loss of gene induction observed.
The alternative activation path involving NIK, IKK
, and p100
processing activated not only p52-RelB complexes, which could be
generated directly from p100-RelB, but also activated p50-RelB. Activation of this latter complex was a consequence of removal of p100
via processing, the preferred and inhibitory binding partner for RelB,
thus liberating RelB to instead associate with NF-
B1 proteins,
including the constitutively generated p50. The mere absence of p100 in
NF-
B2-deficient MEFs was already sufficient to lead to basal
activation of p50-RelB (which was not observed in any other MEF), and
this dimer was further activated in the NF-
B2-deficient MEFs upon
stimulation via the LT
R, and in particular, upon stimulation with
TNF
. This was likely the result of new induced synthesis of RelB by
the classical activation route, which was particularly noticeable after
extensive stimulation with TNF
(Fig. 1), and it may, in addition,
have resulted from some stimulation-dependent degradation
of p105 (12), the only known remaining inhibitor of RelB in the absence
of p100 (small I
Bs are not thought to be physiologic inhibitors of
RelB, although they could theoretically have contributed to some
inhibition in the absence of p100). Based on these data, we conclude
that the mere processing of p100 indirectly led to activation of
p50-RelB complexes without the need of any additional NIK- and
IKK
-mediated signals in the process.
The observed sensitivity of p100 processing to protein synthesis
inhibitors is intriguing. It suggested that induced or continued synthesis of one or more proteins was required. Such a mechanism is
consistent with the observed long delay in p100 processing, in all
experiments requiring 2 or more h of LT
R stimulation before a clear
increase in p52 was apparent. Continuous synthesis of p100 could be
required, especially if processing is somehow linked to translation.
Also, continuous or induced synthesis of NIK could be required,
especially since cells may express only small amounts of NIK normally.
Mechanisms that increase the amounts of NIK might be sufficient to
induce processing, based on the fact that transfected NIK is highly
active even in the absence of any added signals (Fig. 4). Nevertheless,
induced or continued synthesis of other proteins cannot be ruled out.
MEFs in which the alternative pathway was impaired appeared to show a
more sustained activation of RelA complexes, whereas MEFs in which the
classical pathway was impaired appeared to have activated RelB
complexes more rapidly. Although these quantitative assessments need to
be independently confirmed, they do raise the interesting possibility
that the two pathways may compete in wild-type cells. IKK
might be
limiting and thus may not have been immediately available to the second pathway.
The results project a dynamic interplay of the pathways and complexes
activated with time of stimulation. Loss of any of the components of
the classical activation pathway (especially loss of IKK
) appeared
to reduce the amounts of p100/NF-
B2 present in MEFs. In addition to
NF-
B2, RelB, c-Rel, and NF-
B1 are also known targets of NF-
B.
Activation via the classical route may be needed to maintain NF-
B
components, including NF-
B2 and RelB, to allow maximal effects of
the alternative activation pathway. Different signals may involve the
activation paths to different degrees and to different effects when
viewed over the course of prolonged stimulation, depending on cell type.
Recently, we discovered that ligation of the B cell-activating factor
(BAFF)-receptor by BAFF on B cells induces processing of the NF-
B2
protein p100 to generate p52 (33). Although the requirements and
effects of processing in B cells could not be investigated as
thoroughly as was possible with MEFs here, p100 processing in B cells
could be shown to depend on NIK and protein synthesis, to be
independent of IKK
(Nemo), and to lead to activation of RelB
complexes. In addition, in a recent study published after completion of
our manuscript, Dejardin et al. (34) report findings similar
to ours here with LT
R-stimulated fibroblasts. Although LT
R is
able to activate the classical pathway as well as p100 processing (Ref.
34 and our study), BAFF does not appear to significantly stimulate the
classical activation pathway in B cells, although it very effectively
induces p100 processing. Engagement of this second or alternative
activation path by BAFF in developing splenic B cells was shown to
contribute to survival of these cells. Although the biologic roles of
LT
R-mediated activation are not fully characterized, they include
differentiated functions of stromal cells in lymphoid organs. Based on
overlapping defects present in mice deficient in LT
R signaling, NIK
function, IKK
, and NF-
B2, it is likely that LT
R-mediated
processing of p100 in stromal cells contributes to the communication
between stromal cells and lymphoid cells and helps to organize
secondary lymphoid organs. Downstream targets of p100 processing
in stromal cells include the B lymphocyte chemoattractant (BLC)
(CXCL13) chemokine (34), which is in agreement with previous reports
demonstrating that induced expression of this chemokine is suppressed
in NF-
B2-deficient stromal cells (18) and that mice deficient in
LT
R signaling, NIK function, or NF-
B2 are severely impaired in
formation of B cell follicles (6, 7, 18-25).