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B During Primary B Cell Differentiation1
*
MSZ, Institut für Medizinische Strahlenkunde und Zellforschung, and
Pathologisches Institut, Universität Würzburg, Wurzburg, Germany;
Institute for Immunology, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B (NF-B) in
the process of primary B cell differentiation in vitro. In this system,
NF-B is strongly induced when B cells develop from the pre-B cell to
the immature B cell stage. Unlike the typical NF-B activation in
response to exogenous stimuli, induction proceeds with a slow time
course. NF-B induction is only observed in B cells that undergo
differentiation, not in Rag2-deficient cells. Nuclear DNA binding
complexes predominantly comprise p50/RelA heterodimers and, to a lesser
extent, c-Rel-containing dimers. The increase in NF-B binding
activity is accompanied by a slow and steady decrease in IBß
protein levels. Interestingly, absolute RelA protein levels remain
unaffected, whereas RelB and c-Rel synthesis is induced. The reason for
preferential nuclear translocation of RelA complexes appears to be
selective inhibition by the IBß protein. IBß can efficiently
inhibit p50/RelA complexes, but has a much reduced ability to interfere
with p50/c-Rel DNA binding both in vitro and in vivo. Interestingly,
p50/RelB complexes are not at all targeted by IBß, and
coimmunoprecipitation experiments show no evidence for an association
of IBß and RelB in vivo. Consistent with these observations,
IBß cotransfection can inhibit p50/RelA-mediated
trans-activation, but barely affects p50/RelB mediated
trans-activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B3
was originally discovered as a DNA binding activity that was
constitutively present in mature B cells and plasma cells, but was
present in a latent inducible form in pre-B cells and many other cell
types (1, 2). Molecular cloning of genes encoding NF-B proteins
revealed the presence of a family of related proteins (the NF-B/Rel
family of transcription factors) that can bind to the B motif as
homo- and/or heterodimers (for recent reviews, see Refs. 3 and 4). To
date, five family members have been identified and characterized in
some detail: p50 (p105/NFKB1), p52 (p100/NFKB2), RelA, RelB, and c-Rel
(5). The activity of these proteins is largely determined by specific
inhibitor proteins, which are responsible for cytoplasmic retention and
inhibition of DNA binding. Biochemical analyses and molecular cloning
revealed two types of IBs, designated IB
and IBß (6, 7, 8).
IB
, a third related inhibitor protein was cloned very recently
(9). In addition, the C-terminal domains of the NF-B1 and NF-B2
precursor proteins, which are proteolytically removed to generate the
mature p50 and p52 proteins, respectively, have structural homologies
with the IB proteins and also function as inhibitors (10, 11). In
some B cell lines, the C-terminal domain of NF-B1 is indeed
expressed as a separate IB entity and was termed IB
(12). A
further protein, Bcl3, can function both as specific inhibitor of p50
and p52 homodimer DNA binding as well as a transcriptional coactivator
with these homodimers, presumably due to the formation of metastable
ternary complexes present in the nuclei (13, 14, 15).
Mature B cell-specific and ubiquitous inducible NF-B functions could
recently be assigned to distinct members of this NF-B/Rel family.
Whereas RelA-containing complexes are typically seen upon induction of
many different cell types by a variety of stimuli, the
lymphoid-specific constitutive complexes predominantly contain RelB
heterodimers (16, 17). Interestingly, c-Rel was shown to be
constitutively present in several cell lines representing mature B
cells or plasma cells, at a low level in primary spleen extracts, and
also as an inducible species in B and T lymphoid cells (17, 18, 19, 20, 21, 22).
The molecular switch that accompanies pre-B to B cell maturation, which
is associated with the occurrence of constitutively NF-B binding
proteins, has yet not been characterized. Several hypotheses have been
put forward to explain constitutive vs inducible DNA binding
activities. In addition to the preferential expression of RelB (and
also c-Rel) in cells of the lymphoid lineage, reduced sensitivity of
RelB to IB inhibition, which is most likely due to a
lymphoid-specific modification of the RelB protein, has been suggested
(23, 24). A further mechanism that could contribute to the constitutive
presence of nuclear NF-B binding proteins in mature B and plasma
cells could be the increased turnover of IB protein in mature B
cells compared with pre-B cells (21, 25). An alternative explanation
for the low levels of nuclear NF-B proteins in pre-B cells was
suggested recently. Essentially all murine pre-B cell lines that had
been characterized for the presence of NF-B had been established by
transformation with the Abelson retrovirus. Using a ts mutant of the
Abelson virus, it was suggested that the abl-oncogene might
directly or indirectly interfere with NF-B activation (26). In
support of this conclusion, they detected significant amounts of
constitutive B binding activity in primary lymphocyte cultures
(Whitlock-Witte cultures). However, a disadvantage of this culture
system is the fact that in addition to supporting the growth of pre-B
cells, these cultures simultaneously allow differentiation of B cells
and, therefore, represent a mixture of B cells representing different
maturation stages (27, 28).
We have employed a more defined B cell culture and differentiation
system that was developed recently (29). In the presence of IL-7 and
stromal cells, pre-B cells can be continuously propagated. These cells
typically have undergone DH-JH rearrangements
on both alleles (30). After removal of IL-7 these cells continue heavy
chain and light chain rearrangements and differentiate to surface
Ig-positive, immature B cells. We used this system and showed that,
like their transformed counterparts, primary pre-B cells have very low
levels of nuclear NF-B, demonstrating that these earlier findings
were not only due to abl oncogene transformation. In
addition, we used the IL-7 withdrawal/differentiation scheme to show
that upon differentiation nuclear NF-B levels increase with an
unusually slow time course. Analysis of the steady state levels of
IB proteins during differentiation suggests that slow degradation of
IBß contributes to the observed NF-B increase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The different pre-B cell lines were established and propagated in Iscove’s modified Dulbecco’s medium-based serum-free medium containing 100 to 200 U/ml IL-7 on gamma-irradiated ST-2 stromal cells (29). For initiation of the differentiation program, pre-B cells were washed three times in medium without IL-7 and then cultured as described above on stromal cells in medium lacking IL-7 for the time indicated. NIH-3T3 and COS cells were grown in DMEM supplemented with 10% FCS.
COS cells were transfected by electroporation (31) with 0.5 µg of p50
expression vector and 3 µg of RelA, RelB, or c-Rel expression
vectors. NIH-3T3 cells were transfected by calcium phosphate
coprecipitation using a total of 20 µg of DNA. Transfections
contained 4 µg of luciferase reporter plasmid, 6 µg of NF-B
expression vectors (pRc/CMV based) as described in the figure legends,
and 1, 3, or 9 µg of pRc/CMV-IB expression vector (always brought
to 9 µg with the empty expression vector). One microgram of a
CMV-lacZ reporter was included in all transfections to
normalize for differences in transfection efficiencies. Transfections
were harvested after 24 h, and luciferase and ß-galactosidase
activities were determined (32).
The murine IBß cDNA was cloned by reverse transcription-PCR from a
murine B cell line (S194) using primers deduced from the published
IBß sequence (7). The cDNA was inserted into pRc/CMV (Invitrogen,
San Diego, CA) and used for transfection experiments as well as in
vitro transcription/translation.
Cell separations
For selection of B220-positive splenic B cells, hemolytic Gey’s solution was first used to remove RBC from a splenic cell suspension. After Gey lysis, splenic lymphocytes were centrifuged through a FCS cushion and washed with PBS, 5 mM EDTA, and 0.5% BSA. For sorting of B220-positive B cells a magnetic separation kit was used according to the manufacturer’s instructions (Miltenyi Biotec). Surface IgM-positive cells were selected from differentiated pre-B cell cultures after 3 days of IL-7 withdrawal. The cells were first passed over a Ficoll gradient to enrich for living cells and then were positively selected using rat anti-mouse IgM Abs coupled to magnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Selected cell populations were analyzed by FACS using FITC-labeled rat anti-mouse IgM and phycoerythrin-labeled rat anti-B220 (PharMingen, San Diego, CA).
Cell extracts, conditions for EMSA, Western immunoblots, and immunoprecipitations
For preparation of nuclear and cytoplasmic extracts (33), pre-B cells were lysed in sucrose buffer I (100 µl/107 cells, 0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.5 mM PMSF, and 0.5% Nonidet P-40). Nuclei were pelleted by centrifugation (500 x g, 5 min, 4°C), and 0.22 vol of 5x cytoplasmic extraction buffer (0.15 M HEPES (pH 7.9), 0.7 M KCl, and 0.015 M MgCl2) was added to the supernatant (=cytoplasm), microcentrifuged at 12,000 x g for 15 min at 4°C, and transfered to a fresh tube. Pelleted nuclei were washed in sucrose buffer I lacking Nonidet P-40, microcentrifuged (500 x g, 5 min, 4°C), and resuspended in low salt buffer (20 µl/107 cells, 20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2, 0.02 M KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF). To extract nuclei, sequentially high salt buffer (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2, 0.8 M KCl, 0.2 mM EDTA, 1% Nonidet P-40, 0.5 mM DTT, and 0.5 mM PMSF) was added, incubated for 20 min on ice, diluted 1/2.5 with diluent (25 mM HEPES (pH 7.6), 25% glycerol, 0.1 mM EDTA, and 0.5 mM PMSF), and microcentrifuged (12,000 x g, 15 min, 4°C). Supernatant (=nuclear extract) was transfered to fresh tubes.
Generation of whole cell extracts and conditions for EMSAs have been
previously described (16, 24). For supershift experiments, Abs were
preincubated with 5 µg of protein extract for 10 min at room
temperature. Probe was added together with poly(dI-dC), and after an
additional incubation for 10 min at room temperature, samples were
loaded onto prerun gels. For all whole cell or nuclear extracts that
were analyzed for their B binding activities, parallel EMSA
reactions with an octamer probe were performed to control for integrity
and concentration of the extracts. For in vitro inhibition of
NF-B/Rel complexes, wheat-germ-translated IB and IBß
proteins were produced (Promega, Madison, WI). In vitro translated
proteins were diluted 1/3, 1/9, and 1/27 in buffer containing 25 mM
HEPES (pH 7.7), 100 mM KCl, 1 mM EDTA (pH 8.0), and 20% glycerol,
supplemented with 1 mM DTT and 1 mM PMSF. One microliter of each
dilution was preincubated with 1 µg of protein extract for 10 min at
room temperature. After addition of probe and nonspecific competitor,
reactions were incubated for 10 min at room temperature and then loaded
onto prerun gels.
Western blot analyses were conducted using 40 µg of protein extract. Proteins were blotted onto polyvinyldifluoride membrane, and the membrane was blocked with 7.5% dry milk including 0.2% Tween-20. Subsequent washes were performed in PBS/0.2% Tween-20. Proteins detected by the primary Ab were visualized by the use of an enhanced chemiluminescence assay (Amersham (Arlington Heights, IL) or Boehringer Mannheim (Mannheim, Germany)). Abs used for supershifts, Western immunoblots, and immunoprecipitations were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), except for the p50-specific Ab, which was a gift from Dr. Rodrigo Bravo, Bristol Myers Squibb (Princeton, NJ).
For coimmunoprecipitations, 2 x 107 pre-B cells were washed once with PBS and lysed at 4°C for 30 min under rotation with 1 ml of lysis buffer (50 mM NaF, 250 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.5 mM EDTA, 0.001% sodium azide, 0.1% Triton X-100, 20% glycerol, 50 mM Tris (pH 8.0), 1 mM PMSF, and 10 µg/ml leupeptin). After microcentrifugation (12,000 x g, 15 min, 4°C) supernatants were transfered to fresh tubes and incubated with 5 µg of Abs (Santa Cruz) in the presence or the absence of the corresponding peptides (4 µg) for 2 h on ice. Equilibrated protein A-agarose beads (50 µl; Pierce Chemical Co., Rockford, IL) were added and incubated for an additional 1 h. The beads were collected and washed three times with lysis buffer before analysis on 12% SDS-PAGE.
RNA analyses
Preparation of total cellular RNA and conditions for Northern
blot hybridizations were previously described (34). The probes used
were derived from the murine Ig light chain (35) and murine
BOB.1/OBF.1 (36, 37).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
We analyzed primary pre-B cell lines, which grow in the presence
of IL-7 on bone marrow-derived stromal cells, for the presence of
active NF-B. Cells that can be expanded by this method represent
very early, committed stem cells of the B lineage (29). Virtually all
cells in these cultures express PB76, an early B-lineage marker. Both
the and the 
light chain genes are in germ-line configuration,
but all the cells show DH-JH
rearrangements on either one or both alleles. When nuclear extracts
from these cells were compared with either an Abelson virus-transformed
pre-B cell line (PD31) or a pre-B cell line transformed by a retrovirus
bearing an activated Ha-ras oncogene (HAFTL), the same low
levels of constitutive nuclear NF-B complexes were observed (Fig. 1
A). Three main
complexes were distinguishable. The amounts of detectable complexes
were drastically lower than those in typical mature B cell lines or in
B220-sorted splenic B cells (Fig. 1B and data not shown).
The observed complexes were specific for the B motif, as they could
be efficiently competed for by an excess of wild-type, but not mutant,
B binding sites (data not shown).
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B complexes in splenic B cells have
previously been identified to be predominantly p50/RelB heterodimers
(16, 17, 24). We therefore analyzed the identity of the weak
constitutive complexes in pre-B cells using specific Abs. Whereas the
RelA-specific Ab removed the upper complex, a slightly faster migrating
complex reacted with the RelB-specific Ab. The lowest complex was
completely abolished by anti-p50 Abs, which also diminished the
other two complexes (Fig. 1
C). Although no clear
supershift was observed with the c-Rel-specific Abs, they do remove a
complex migrating between the RelA- and RelB-specific complexes. Given
that a complex of similar mobility remains after supershifting with the
p50-specific Ab, this suggests that c-Rel might be predominantly
complexed with RelA as RelA/c-Rel heterodimer in these pre-B cells.
Identical results were obtained when the constitutively active
complexes from Abelson virus-transformed PD31 cells were characterized
(data not shown). In summary, these results demonstrate that both
primary and transformed pre-B cells contain low levels of constitutive
NF-B binding complexes, consisting of the various Rel family
members.
Differentiation-associated nuclear translocation of NF-B
We next wanted to determine whether nuclear NF-B levels would
be affected upon differentiation of the pre-B cells. Upon removal of
IL-7, cells stop proliferation, and some of the cells differentiate to
surface IgM-positive, immature B cells within 72 h (29). Strong
induction of NF-B binding activity was observed upon IL-7 withdrawal
(Fig. 2A). In contrast
to conventional NF-B induction by a multitude of exogenous stimuli,
which is typically a very rapid event taking only minutes, NF-B
induction following IL-7 withdrawal was slow. No significant induction
was detectable at early time points, but induction could be seen at
18 h of IL-7 withdrawal and continued to increase until 48 h
(Fig. 2A).
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B is
seen in the sIgM+ population, these cells were enriched by
magnetic bead separation. This procedure resulted in a population
containing about 70–80% sIgM+ cells. The identical
increase in NF-B binding activity was detected in this enriched cell
population, suggesting that the increase in NF-B activity
accompanies the differentiation step (Fig. 2B).
Specific Abs were used to analyze which NF-B/Rel family members were
induced under these conditions. The majority of the inducible B
binding activity reacted with a RelA-specific Ab, suggesting that
p50/RelA represented the main inducible complex. In addition, some
c-Rel-containing complexes, most likely RelA/c-Rel heterodimers, were
detected in the induced DNA binding complexes. In contrast, the amount
of RelB-containing complexes remained low and unaltered during this
differentiation process (Fig. 2C).
An important issue to resolve was whether the observed increase in
NF-B activity was associated with the differentiation program or
merely some sort of stress response due to growth factor deprivation.
We first analyzed NF-B induction in a pre-B cell clone expressing
the bcl-2-transgene. These cells show a similar
differentiation as normal cells upon IL-7 withdrawal, but they are
protected from apoptosis (38). A similar level of NF-B induction as
that with normal cells was obtained, albeit with a slightly retarded
time curve (Fig. 3A).
The reasons for this delay are presently unclear. We then analyzed
pre-B cell lines derived from rag-2-deficient (Rag2T) mice.
These cells have been demonstrated to show some signs of
differentiation upon IL-7 withdrawal, but due to the lack of DNA
rearrangements, these cells cannot differentiate to sIgM+
immature B cells (39). Upon IL-7 withdrawal, only a very weak induction
of active NF-B at 24 h was observed (Fig. 3A). The same result was obtained when Rag2T pre-B
cells expressing the bcl-2 transgene were used. As expected,
differentiation by IL-7 withdrawal resulted in induction of -RNA
levels in wild-type and bcl-2-transgenic cells, whereas no
increase was seen for the rag-2-deficient cells (Fig. 3B). These results suggest that strong activation of
NF-B specifically occurs in pre-B cells that have the ability to
differentiate to the sIgM+ stage.
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B in the rag-2-/-
cells could be due to the fact that these cells lack expression of
NF-B/Rel proteins. We therefore analyzed extracts from the various
pre-B cell lines by Western immunoblotting with Abs specific for
different NF-B/Rel family members. All cell lines contained
comparable levels of RelA, RelB, and c-Rel proteins (Fig. 3C). Similarly, IB levels were also
indistinguishable in the various cell lines, suggesting that the
rag-2 deficiency did not directly imbalance the level of
NF-B/IB protein expression. Furthermore, the NF-B/Rel proteins
in these cell lines were all functional and inducible. Hydroxyl
radicals have been shown to be involved in most pathways leading to
NF-B induction. Therefore, NF-B induction can be achieved in most
cell types by treating them with H2O2 (40) This
experiment revealed that both rag-2-positive and negative
cell lines all contained comparable levels of inducible NF-B
proteins (Fig. 3D).
IBß degradation correlates with NF-B induction
The kinetics of NF-B induction in this B cell differentiation
system are clearly different from those of most other NF-B
inductions. In most cases, stimulation of cells results in a very
rapid, typically transient induction of p50/RelA (3, 4). In contrast,
the induction that we detected in B cells showed very slow kinetics,
and the induced complexes did not disappear within the time period
analyzed. To determine whether this might be due to a consistent
overproduction of RelA or permanent down-regulation of IB, we
analyzed NF-B/Rel protein levels by Western immunoblots.
Interestingly, neither the RelA nor the IB protein levels showed
any alterations in any of the cell lines tested (Fig. 4A). In contrast, both
RelB and c-Rel protein levels clearly increased in the wild-type and
bcl-2 transgenic cells, but not in either of the two
rag-2-deficient cell lines. Interestingly, induction of RelB
accumulation is again delayed in the bcl-2 transgenic pre-B
cells similar to the appearance of nuclear p50/RelA.
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B/Rel proteins in undifferentiated pre-B cells, immature B cells
generated by IL-7 withdrawal, and B220 sorted primary B cells from
spleens. Whereas comparable amounts of RelA were observed at all
stages, immature and mature B cells contained significantly increased
levels of both RelB and c-Rel (Fig. 4B, left panel).
In fact the amounts of c-Rel and RelB protein present in the cells
differentiated by IL-7 removal for 72 h were almost identical with
the amounts detected in the mature splenic B cells. We also analyzed
the amounts of the different Rel proteins in the
sIgM+-selected fraction and observed an identical increase,
suggesting that these induced expression levels accompany pre-B to
immature B cell differentiation (Fig. 4B, right
panel).
The results from the EMSA experiments had demonstrated that p50/RelA-
and, to a lesser extent, c-Rel-containing complexes appear in the
nucleus as a consequence of B cell differentiation, whereas no
significant increase in p50/RelB can be seen (Fig. 2). Given the strong
induction of RelB protein expression observed, these findings suggest
that the RelB protein accumulates preferentially in the cytoplasm. We
therefore analyzed nuclear and cytoplasmic fractions of pre-B cells and
cells differentiated for 3 days by IL-7 withdrawal for the presence of
the different Rel proteins. Evidently, there is an increase in the
amounts of nuclear p50, RelA, and also c-Rel. In contrast, the newly
synthesized RelB protein as well as most of c-Rel are primarily
retained in the cytoplasm (Fig. 4C). Interestingly,
although the induction of nuclear DNA-binding NF-B activity
increases significantly during this differentiation process, only a
small amount of the cytoplasmic NF-B pool is mobilized. A similar
result was obtained previously when the transformed pre-B cell line
70Z/3 was differentiated to the immature B cell stage by LPS treatment.
This also resulted in a strong induction of nuclear NF-B DNA binding
activity of RelA and c-Rel complexes without showing a reduction of the
cytoplasmic RelA pool (41). The amounts of cytoplasmic p105/NFKB1, the
precursor for p50, did not change during the 3-day differentiation
period (data not shown).
The apparent lack of IB degradation during B cell differentiation
suggested that this NF-B induction might proceed via an alternate
pathway compared with typical NF-B activation. We therefore
investigated whether IBß might be involved. Pre-B cells, immature
B cells, and B220-positive splenic B cells were analyzed for their
IB and IBß expression levels by Western immunoblot. Whereas
IB levels were virtually identical at all stages, steady state
levels of IBß were clearly reduced after 3 days of differentiation
in the absence of IL-7 (Fig. 5A, left
panel). This reduction was also seen in the
sIgM+-selected fraction (Fig. 5A, right
panel). The slow induction of nuclear p50/RelA observed in
this differentiation system suggested that IBß degradation should
also proceed gradually. We therefore investigated the kinetics of
IBß degradation by analyzing protein levels at different time
points after IL-7 withdrawal. IBß protein levels were reduced
about twofold after 24 h and continued to decrease to about 15 to
25% within 72 h (Fig. 5B). The time course of
IBß loss was again delayed in the bcl-2 transgenic
pre-B cells (data not shown), supporting the conclusion that IBß
degradation is involved in the observed induction of NF-B during B
cell differentiation.
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Bß is a selective inhibitor of RelA-containing
complexes
Why does degradation of IBß lead to a rather specific
accumulation of nuclear RelA, when at the same time the absolute level
of RelB significantly increases in the cytoplasm? A potential
explanation could be that IBß is a more selective inhibitor than
IB and primarily targets RelA- and c-Rel-containing complexes.
Previous interaction studies had shown that IBß is associated with
both RelA and c-Rel in B cell lines (7). To address whether IBß is
associated with all Rel proteins in the primary pre-B cells, we
performed coimmunoprecipitations with subsequent immunoblotting of the
immune complexes. IB- and IBß-containing complexes were
immunoprecipitated and then analyzed by immunoblots with RelA, RelB, or
c-Rel-specific Abs. This analysis revealed that RelA and c-Rel are
associated with both IB and IBß inhibitors. In contrast,
RelB was only found associated with IB, not IBß (Fig. 6A).
The specificity of the immunoprecipitation reaction was controlled by
inclusion of an excess of antigenic peptide used to generate the
IB- and IBß-specific Abs. This result suggests that IBß
does not function as an inhibitor of RelB complexes in pre-B cells.
Therefore, increased cytoplasmic RelB complexes must be associated with
a different type of inhibitor protein.
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Bß is as such
incapable of interacting with RelB or whether the pre B cell
environment is responsible for this observed differential interaction.
We therefore performed in vitro inhibition experiments. The different
p50/Rel heterodimer complexes were generated by transfection of the
respective expression vectors in COS cells and challenged with in vitro
translated IB and IBß proteins. In these experiments,
IB was an efficient inhibitor of complexes containing all three
types of Rel proteins (Fig. 6B). In contrast,
IBß specifically inhibited p50/RelA complexes, but no inhibition
of RelB or c-Rel complexes was detectable. The failure to inhibit
p50/RelB was consistent with the previous result showing a lack of
association between IBß and RelB. The observation that p50/c-Rel
complexes likewise were not inhibited by IBß was surprising,
however. Association of IBß with p50/RelA without inhibiting DNA
binding was previously demonstrated for the nonphosphorylated form of
IBß (42). However, in the experiments described here, the
identical IBß protein efficiently inhibits p50/RelA but does not
target p50/c-Rel.
To exclude the possibility that this might be due to incomplete
modification of the IBß protein in the in vitro translation
experiment, we performed transient cotransfections. A B-dependent
reporter construct was cotransfected with expression vectors for p50
and the different Rel family members in the absence or the presence of
increasing amounts of IB or IBß expression vectors. These
results convincingly demonstrate that IB is an
efficient inhibitor of the transcriptional activity of all
three Rel protein complexes (Fig. 6C). In contrast
and consistent with the in vitro inhibition experiments, IBß shows
a strong selectivity for inhibiting RelA-mediated transcription, but is
rather inefficient at inhibiting the transcriptional activity of
p50/RelB or p50/c-Rel. Whereas p50/RelA-mediated transcription is
reduced about 100-fold, p50/RelB- and p50/c-Rel-dependent transcription
are only reduced by 2.5- and 6-fold, respectively. A potential
explanation for the observed interaction of IBß with c-Rel in vivo
yet the failure to efficiently inhibit c-Rel both in vitro as well as
upon cotransfection in vivo could be that the observed association of
IBß with c-Rel in extracts involves RelA/c-Rel heterodimers. It
has been demonstrated by a variety of groups that RelA and c-Rel
efficiently heterodimerize in vivo and are responsible for
transcriptional activation of a subset of NF-B target genes (43, 44). We therefore analyzed the ability of IBß to inhibit
transcription driven by cotransfection of RelA and c-Rel expression
vectors. Evidently, these heterodimers are subject to IBß
inhibition (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. In
both cases these complexes represent predominantly p50/RelA and
p50/RelB heterodimers. Upon removal of the growth-supporting factor
IL-7, these pre-B cells differentiate to surface IgM-positive, immature
B cells (29). In the process of this differentiation, a slow increase
in NF-B was observed. This induction is mainly due to activation of
p50/RelA and, to a lesser extent, c-Rel-containing heterodimers,
whereas p50/RelB complexes remained virtually unaffected. The
observation that p50/RelA heterodimers represent the predominant
species upon primary B cell maturation is consistent with the earlier
report of Singh and colleagues (26). They had analyzed NF-B proteins
in a different primary pre-B cell culture system (Whitlock-Witte
cultures). This culture system is more heterogeneous than the one
employed here, as in the Whitlock-Witte system pre-B cells continuously
differentiate, and a mixed population of pre-B and mature B cells is
always present. The strong constitutive signal observed in that system
therefore most likely was derived from the more mature B cells present
in the population.
It had been suggested that the half-life of the IB protein is
decreased in mature B and plasma cells compared with that in pre-B
cells and that this alteration in the IB turnover rate might be
important for the increased levels of constitutively active NF-B
(21, 25). Although we have not addressed this question in detail in our
system, our results are consistent with this interpretation. IB
is one of the target genes of NF-B, and when steady state levels of
mRNA levels for IB were analyzed, a clear increase was observed
(data not shown). In contrast, the amounts of stable IB protein
remained virtually unaffected during the differentiation. This finding
suggests an increase in the turnover rate of the IB protein. We
could, however, identify a second mechanism involved in the induction
of NF-B during B cell development. IBß levels significantly
decrease in the process of differentiation and the time course of
IBß loss mirrors the induction of DNA-binding NF-B complexes.
We note, however, that at early time points (18–24 h), IBß levels
are only reduced 2-fold, whereas DNA-binding NF-B is induced about
10- to 15-fold. Our experiments do not distinguish between an active
degradation of IBß and a reduced synthesis of IBß over the
period of B cell differentiation. However, the net result is the
obvious reduction in steady state levels of IBß protein. The
conclusion that specific IBß loss contributes to the observed
activation of predominantly p50/RelA is additionally supported by the
inhibition of the specificity of IBß for RelA-containing
complexes. Moreover, when fibroblasts from RelA-deficient mice were
analyzed for IB levels, a significant down-modulation of IBß,
but not IB, levels was observed, again in line with a specific
association between RelA and IBß (45).
A different role for IBß in regulating the induction of NF-B in
B cells was suggested recently. Upon long-lasting induction of 70Z/3
pre-B cells with LPS, it was found that reappearing IBß is
predominantly unphosphorylated (42). This unphosphorylated IBß can
associate with p50/RelA, but does not interfere with nuclear
translocation or with DNA binding. In fact, the authors suggested a
protective function for the NF-B/IBß heterotrimers, in that
they can escape inhibition by IB. A role of unphosphorylated
IBß in the normal pre-B to B cell differentiation process remains
to be investigated.
Our observation that IBß is a much more selective inhibitor of
NF-B proteins than IB has several important physiologic
consequences. In combination with the apparently distinct degradation
pathways for IB and IBß, which were obvious in our studies
as well as in the previous report (7), a fine-tuned regulation of
specific NF-B activities can be achieved. To date, not all the
details of the IBß degradation pathway have been elucidated. For
IB it is known that the various inducing signals result in the
phosphorylation of IB on two serine residues in the N-terminus,
Ser32 and Ser36 (46, 47). This
phosphorylation apparently targets IB-P for the ubiquitin
conjugation enzymes, and polyubiquitinated IB is then rapidly
degraded by the proteasome (48, 49, 50). IBß also contains conserved
phosphorylation sites in the N-terminus, which have been shown to be
important for inducible phosphorylation and degradation (51). Recent
data suggest that IB and IBß are targeted by the same
IB-kinase (IKK/CHUK); the efficiency of phosphorylation of
IBß in vitro was reduced compared with that of IB, however
(52). Clearly, there must be specific intracellular mediators that
distinguish between IB and IBß and therefore lead to
specific degradation of either one or the other. The signaling cascade
that ultimately triggers IBß degradation is largely unknown. It is
unlikely that IL-7-mediated signals are directly involved in this
scenario. Signaling via IL-7 and IL-7R would have to provide a negative
signal to the cells preventing NF-B activation. However, as removal
of IL-7 from rag-2-deficient pre-B cells did not result in a
comparable NF-B induction, arguing for a more complex regulation. It
is more likely that in the process of differentiation the cells
activate an endogenous program that leads to IBß destabilization
and therefore to preferential p50/RelA induction.
A critical role for NF-B induction during B cell differentiation was
recently demonstrated in an independent set of experiments. When
different transformed pre-B cell lines were stably transfected with a
dominant negative version of IB (lacking the N-terminus needed
for inducible degradation), rearrangement and transcription of the
Ig locus were blocked (53). Given the broad inhibition spectrum of
IB, induction of all NF-B complexes was inhibited in these
cells. Therefore, no information could be derived from these
experiments as to which NF-B/Rel family members are induced and
which IB proteins are degraded under physiologic conditions.
We were surprised to find that IBß efficiently associates with
c-Rel complexes, yet when tested for direct inhibition of DNA binding
and transcriptional activity of p50/c-Rel heterodimers, it is
apparently quite inefficient. A reduced inhibition level for p50/c-Rel
heterodimers compared with p50/RelA upon COS cell cotransfection was
noted in the original paper describing the cloning of IBß (7). A
potential explanation for the discrepancy between association and
inhibition could be the following hypothesis. The observed association
between IBß and c-Rel in vivo could be specific for RelA/c-Rel
heterodimers, c-Rel homodimers, or other heterodimers distinct from
p50/c-Rel. In support of this hypothesis we showed that transcriptional
activation mediated by RelA/c-Rel heterodimers could be efficiently
inhibited by IBß. Furthermore, when we performed coprecipitation
experiments from pre-B cells with RelA-specific Abs, we always observed
a large amount of associated c-Rel and vice versa (data not shown).
This suggests that pre-B cells contain significant quantities of these
RelA/c-Rel heterodimer complexes, which could explain the observed
IBß association. Additionally, it was shown recently that
phosphorylated IBß can efficiently inhibit DNA binding of c-Rel
homodimers, both in vitro and in vivo (54). Again, these authors noted
obvious differences between inhibition of RelA (RelA homodimers) vs
c-Rel homodimers by IBß, in line with the observations described
here. Consistent with this result, we found that IBß was more
efficient in reducing c-Rel-driven transcription compared with
RelB-dependent activation (Fig. 6C). This could be due
to the fact that the activity that we scored there was derived from
both p50/c-Rel heterodimers as well as c-Rel homodimers. The latter of
the complexes should be inhibited by IBß (54). A specific
association between RelA and IBß in pre-B cells was recently also
suggested by Whiteside and colleagues, who noted that >90% of the
RelA protein was complexed to IBß in 70Z/3 cells (9).
Our finding that RelB protein levels increase during B cell
differentiation accompanying the increased levels of active NF-B
together with additional evidence suggest that relb, like
c-rel, is also a target gene of NF-B. Firstly,
relb was originally cloned as an immediate early gene
induced by serum in starved fibroblasts (55). Secondly, we have
recently characterized the defect in a pan-NF-B-deficient murine
plasma cell line (56, 57). We could show that S107 cells, which
completely lack nuclear B binding proteins, contain cytosolic RelA
and c-Rel proteins that cannot be induced by any of the known
treatments inducing NF-B. The primary defect in these cells
therefore seems to be located in the pathway leading to NF-B
induction. Interestingly, RelB is not at all expressed in these cells,
consistent with an essential role of NF-B for RelB expression.
Finally, the induction kinetic of RelB expression during pre-B cell
differentiation closely follows the induction of NF-B (p50/RelA) in
our system, again suggesting that NF-B is a positive regulator of
RelB expression.
The primary B cell differentiation system does not recapitulate the
complete B cell maturation. The cells generated in this system
typically do not express IgD on the surface and were shown to have a
much reduced ability to respond to mitogenic stimuli (30). In line with
this observation, there is a clear difference between mature primary B
cells and the cells generated in this system with respect to
constitutively active NF-B. Whereas both cell types express large
quantities of the RelB and c-Rel proteins, the predominant DNA binding
species is p50/RelB in the case of mature B cells and p50/RelA in the
immature B cells generated by in vitro differentiation (this study and
Refs. 16 and 17). It is unclear how the RelB complexes are retained in
the cytoplasm of the immature B cells. RelB could lack the specific
modification that allows it to escape IB-inhibition as suggested
previously (24). Alternatively, another IB-protein, such as the
recently identified IB, might be involved in retaining
RelB.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas Wirth, MSZ, Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg Versbacher Str. 5, 97078 Wurzburg, Germany. E-mail address:
3 Abbreviations used in this paper: NF-B, nuclear factor-B; EMSA, electrophoretic mobility shift assay; sIg, surface Ig.
Received for publication April 29, 1997. Accepted for publication November 5, 1997.
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References |
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