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B in B Cells in the Absence of IB Degradation1
Department of Microbiology, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel family of transcription factors are
involved in many aspects of B lymphocyte development and function.
NF-B is constitutively active in these cells, in contrast with most
other cell types. In the inactive form, NF-B/Rel proteins are
sequestered in the cytoplasm by members of the IB family of NF-B
inhibitors. When activated, NF-B is translocated to the nucleus, a
process that involves the phosphorylation and proteasomal degradation
of IB proteins. Thus, NF-B activation is accompanied by the rapid
turnover of IB proteins. We show that while this "classical"
mode of NF-B activation is a uniform feature of IgM+ B
cell lines, all IgG+ B cells analyzed contain nuclear
NF-B yet have stable IB
, IBß, and IB
.
Furthermore, Iß levels are at least 10 times lower in
IgG+ B cells than in IgM+ B cells, an
additional indication that the regulation of constitutive NF-B
activity in these two types of B cells is fundamentally different.
These data imply the existence of a novel mechanism of NF-B
activation in IgG+ B cells that operates independently of
IB degradation. They further suggest that different isoforms of the
B cell receptor may have distinct roles in regulating NF-B
activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel
transcription factors are involved in many processes that are important
for B cell development, differentiation, and function
(1, 2, 3, 4). NF-B was originally described as a constitutive
nuclear factor in B cells and an inducible factor in pre-B cells that
is required for the expression of the Ig light chain
()3 gene (2, 5, 6). In addition to the regulation of expression, NF-B
also plays a role in the rearrangement of the Ig locus
(7), suggesting a central role of NF-B in the initial
stages of B cell receptor expression. NF-B is important in the
rescue of B cells from apoptosis and in the maintenance of cell
cycling, events important in the survival, selection, and maturation of
B cells (8, 9, 10). NF-B has also been implicated in the
initiation of Ig isotype switching (11, 12), suggesting a
role in adaptive humoral immune responses.
NF-B is now known to be ubiquitously expressed and to play a major
role in controlling the expression of proteins involved in immune,
inflammatory, and acute phase responses (4, 13). NF-B
activity is primarily regulated through nuclear translocation (5, 6, 13, 14, 15, 16, 17). In its inactive form, NF-B is sequestered in the
cytoplasm, bound by members of the IB family of inhibitor proteins,
which include IB, IBß, IB
, and the recently identified
IB (16, 18, 19, 20). Activation occurs when NF-B is
released from IB and translocated to the nucleus. Nuclear
translocation of NF-B can be induced by a variety of stimuli,
including TNF-, IL-1, LPS, and phorbol esters such as PMA (1, 3). These stimuli trigger the phosphorylation of IB, which is
followed by the ubiquitination of IB and its subsequent degradation
through the proteasome (17, 21, 22). This sequence of
events has been called the classical pathway of NF-B activation, and
most signals that activate NF-B converge into this pathway
(17, 23). The IB proteins identified to date that are
the targets of this pathway include IB, IBß, and IB.
Each of these IB proteins contains two highly conserved serine
residues in their N-terminal domain that serve as substrates for IB
kinase and are necessary for NF-B activation (20, 21, 22).
A key feature of the classical pathway is that IB proteins are
unstable and exhibit shortened half-lives during NF-B
activation.
In most cells, NF-B activation is transient because NF-B induces
the transcription of IB, among other target genes (13, 15). Consequently, NF-B activation normally results in a
negative feedback loop, in which newly synthesized IB leads to
the resequestration of NF-B in the cytoplasm (15).
However, in the B lineage, NF-B activation becomes constitutive at
the transition from the pre-B to the B cell stage (5, 24).
Although much of the NF-B remains sequestered in the cytoplasm of
primary B cells and B cell lines, these cells contain constitutive
levels of nuclear NF-B that can be increased upon stimulation
(2, 5, 6, 25, 26, 27, 28, 29). Using the immature B cell line,
WEHI231, as a model system, several groups have shown that both
IB and IBß undergo rapid degradation (30, 31, 32, 33).
The degradation of these IB proteins has been shown to be required
for the presence of constitutive nuclear NF-B, suggesting that
NF-B activation in WEHI231 cells occurs via the classical pathway
(33). Whether this cell line serves as a general model for
the constitutive activation of NF-B in B cells is not known.
In the present study we evaluated the stability of IB, IBß,
and IB in a number of B cell lines to determine whether the rapid
turnover of IB is a general feature of constitutive NF-B
activation in the B lineage. Although several B cell lines, including
WEHI231, exhibited enhanced degradation of IB and IBß, other
B cell lines showed no evidence of rapid IB turnover despite the
presence of nuclear NF-B. The differences in IB turnover
correlated with the isotype of the surface Ig receptor. Although all B
cells exhibiting unstable IB and IBß were
IgM+, the B cells with stable IB expressed
surface IgG. These data suggest the presence of an alternative
mechanism of constitutive NF-B activation in
IgG+ B cells that is independent of the
degradation of IB and IBß. Interestingly, while IB was
stable in all B cell lines, IgG+ B cells
expressed much lower levels of IB than the
IgM+ B cells, suggesting that reduced levels of
IB may play a role in NF-B activation in
IgG+ B cells. We also suggest that the B cell
receptor plays a role in regulating the different mechanisms of
constitutive NF-B activation in B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A panel of murine B cell lymphoma lines representing different stages of development and differentiation was used in this study; many of these have been previously described (34, 35, 36). The IgM+ B cell lines WEHI231 and CH31 are representative of immature B cells that are susceptible to tolerance induction (37). CH27 (38) and CH12-LBK (39) are both representative of mature IgM+ B cell lines. A20 and M12 (40) (provided by L. Glimcher, Harvard School of Public Health, Boston, MA) are IgG+ B cell lymphoma cells that are representative of mature B cells. LK (41) (provided by A. Marshak-Rothstein (Boston University, Boston, MA)) is a B cell hybridoma derived by fusion of splenic B cells from B10.BR mice with the B cell lymphoma, L10.A. We found that the LK cell line expresses surface IgG but not IgM. CH12.LX2.1A7 (42) (provided by Dr. L. Arnold, University of North Carolina, Chapel Hill, NC) is a subclone of the CH12 B cell lymphoma that has undergone spontaneous class switching and expresses surface IgG but lacks IgM. All Ig isotypes were confirmed by Western blot and/or FACS analysis. The pre-B cell lines 70Z/3 and Bine4.8 (the latter provided by H.-M. Jäck, University of Erlangen-Nurnberg, Erlangen-Nurnberg, Germany) and the T cell hybridoma BW5147 (43) (provided by D. Woodland, St. Jude Children’s Research Hospital, Memphis, TN) served as controls. All cell lines were maintained in DMEM containing 7% FBS (Life Technologies, Gaithersburg, MD) and 50 µM 2-ME (Sigma, St. Louis, MO) and supplemented as previously described (44). In some experiments cells were treated with cycloheximide (CHX; 50 µg/ml, Sigma), LPS (50 µg/ml; Escherichia coli 055:B5, Difco, Detroit, MI), or PMA (50 nM; Sigma) for various periods of time as described in Results.
Immunoblotting
Cells were washed in ice-cold BSS and lysed at 1 x
105 cells/µl in ice-cold whole cell lysis
buffer (150 mM NaCl, 10 mM Tris (pH 7.50, and 1% Nonidet P-40)
containing 1 mM DTT, 1 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml
leupeptin, 0.5 µg/ml pepstatin, 50 mM NaF, and 1 mM
Na3VO4. Cell lysates were
incubated for 30 min on ice and centrifuged at 13,000 x
g at 4°C for 15 min, and supernatants were snap-frozen in
liquid nitrogen. For the lysates used to determine IB expression
levels, protein concentrations were determined by Bradford assay
(Bio-Rad, Hercules, CA). Lysates from equivalent cell numbers or
equivalent amounts of protein were fractionated on 10% SDS-PAGE. Gels
were transferred to nitrocellulose membranes (Micron Separations,
Westboro, MA), and membranes were stained with Ponceau S (Sigma) to
ensure equivalent loading and transfer. Membranes were probed with the
appropriate Abs and developed with the ECL system (Amersham, Arlington
Heights, IL). Autoradiographs were quantified using a Molecular
Dynamics densitometer with ImageQuant software (Molecular Dynamics,
Sunnyvale, CA). All primary Abs were developed in rabbit and were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). They included
Abs against IB (sc-371), IBß (sc-945), IB (sc-7155),
RelA (sc-109), RelB (sc-226), c-Rel (sc-71), and NF-B1 (sc-114). In
some experiments the anti-IB Ab detected an additional
protein that migrated slightly more slowly than IB. This protein
was stable after PMA or LPS activation (see Fig. 5
). It is unclear
whether this protein represents a stable phosphoisoform of IB or
is an unrelated cross-reacting protein.
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Cells were washed in BSS, resuspended at 4–5 x
106 cells/ml in complete DMEM lacking cysteine
and methionine, and incubated at 37°C for 1 h. Cells were
labeled for 1 h (or indicated times) by addition of 60–100
µCi/ml of [35S]cysteine and
[35S]methionine (New England Nuclear, Boston,
MA). Cells were washed in BSS and chased with complete DMEM for the
times indicated in the respective experiment. Cells were washed twice
in BSS, and whole cell lysates were prepared as described above.
Lysates were precleared using normal rabbit serum and protein
A-Sepharose (Sigma). IB protein was immunoprecipitated with Ab
sc-371 and protein A-Sepharose. Precipitated material from the
preclearing step and from the IB immunoprecipitations was
separated by 10% SDS-PAGE, and gels were analyzed by autoradiography
or phosphorimaging. The m.w. standards were
14C-labeled High Range standards (Life
Technologies). We found that the Ab to IBß (sc-945) did not
immunoprecipitate IBß (not shown).
Preparation of nuclear and cytoplasmic extracts
Nuclear extracts were prepared according to standard procedures
(45). Cells (1–4 x 108) were
washed once with BSS and once with hypotonic buffer (10 mM HEPES (pH
7.9), 1.5 mM MgCl2, 10 mM KCl, 1 mM PMSF, and 1
mM DTT), resuspended in hypotonic buffer, and incubated on ice for 10
min. Cells were homogenized using a Dounce homogenizer (Kontes,
Vineland, NJ) until >90% lysis was detected as determined by trypan
blue exclusion. Nuclei were collected by centrifugation for 5 min at
3000 x g. Supernatants were saved for the preparation
of cytoplasmic extracts. Nuclei were washed once in hypotonic buffer
and resuspended in low salt buffer (20 mM HEPES (pH 7.9), 0.02 mM KCl,
1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1 mM
PMSF, and 1 mM DTT). An equal volume of high salt buffer (same
composition as low salt buffer, but containing 800 mM KCl) was added
dropwise with occasional vortex mixing. The nuclei were incubated for
30 min at 4°C and centrifuged for 30 min at 13,000 x
g, and supernatants were snap-frozen in liquid nitrogen. All
nuclear extracts were free from cytoplasmic contamination as determined
by Western blot with an Ab against the cytoplasmic protein NF-B
p105, with the exception of the WEHI231 extracts, which contained about
3–5% of cytoplasmic contamination.
Cytoplasmic extracts from above were supplemented with cytoplasmic extract buffer to a final concentration of 40 mM HEPES (pH 7.9) and 150 mM KCl, incubated for 30 min on ice, and centrifuged for 30 min at 13,000 x g, and supernatants were snap-frozen. The protein concentration of all extracts was determined by Bradford assay and further controlled by separating equal aliquots of the samples via SDS-PAGE, transfer to a nitrocellulose membrane, and staining of the membrane with Ponceau S.
DNA-protein binding studies
A photoreactive, radiolabeled B-specific oligonucleotide
probe (B-pd) was prepared by primer extension labeling with
[32P]dCTP (New England Nuclear) and
bromodeoxyuridine (Sigma) as previously described (46).
For gel-shift assays, 20 µg of nuclear extracts were added to a
reaction mix containing final concentrations of 20 mM HEPES (pH 7.9),
50 mM KCl, 1 mM EDTA, 5% glycerol, 5 mM DTT, 250 µg/ml BSA, and the
nonspecific competitors pd(N)6 (250 µg/ml;
Amersham) and poly(dI-dC) (250 µg/ml, Amersham) in a total volume of
20 µl. Radiolabeled probe (5 x 104 cpm)
was added, and the reactions were incubated at 20°C for 20 min.
DNA-protein complexes were analyzed by gel retardation on native 5%
acrylamide gels as previously described (47) and were
visualized by phosphorimaging.
For UV cross-linking analyses, 50 µg of nuclear extracts were
incubated for 20 min in a total volume of 50 µl in the same reaction
mix as that described above, except containing 5 x
105 cpm of the B-pd probe, and were irradiated
for 20 min with a UV light source (300 nm; Fotodyne, Hartland, WI). The
reaction was diluted in lysis buffer, and equal aliquots were subjected
to immunoprecipitation with Abs against either RelA or c-Rel.
Precipitated material was analyzed by SDS-PAGE and autoradiography
together with an aliquot of each cross-linking reaction. RelB was not
detectable by this analysis due to the failure of RelB to be
efficiently cross-linked or to the inability of the anti-RelB Ab to
precipitate RelB-DNA adducts.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B degradation
We first sought to determine whether the paradigm established
using WEHI231 cells was generally applicable and whether other B cell
lines showed accelerated degradation of IB proteins. We therefore
analyzed the stability of IB proteins in a panel of murine B cell
lines using two different approaches. In the first, the stability of
IB proteins was determined by treatment of cells with the protein
synthesis inhibitor CHX. Degradation of the respective IB proteins
was monitored by Western blot analysis of protein extracts prepared at
different times following CHX treatment.
As expected, IB and IBß were rapidly degraded in WEHI231
cells (Fig. 1). The half-lives of these
proteins were calculated to be 50 and 140 min, respectively. Similar
results were obtained using another IgM+ B cell
line, CH27. In striking contrast, IB and IBß were
significantly more stable in two IgG+ B cell
lines, A20 and M12, and one IgG+ B cell
hybridoma, LK, with half-lives of >4 h (Fig. 1). These half-lives were
similar to or even longer than those found in a pre-B cell line
(Bine4.8) and a T cell line (BW5147; Fig. 1). Pre-B cells and T cells
generally do not contain significant levels of constitutive nuclear
NF-B, and their IB proteins are stable in the absence of stimuli
(5, 32, 48, 49, 50, 51, 52) (see also Fig. 3). In contrast to IB
and IBß, IB was stable in all cell lines analyzed, including
WEHI231. This suggests that IB degradation in WEHI231 cells is
controlled independently of IB and IBß.
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B was also investigated using pulse-chase
analysis. Representative results are shown in Fig. 2. In the IgM+ cell
lines WEHI231, CH31, and CH12-LBK, IB was unstable, with a
half-life of 40–55 min. In contrast and in complete agreement with the
results obtained using the CHX block, IB was very stable in A20
and M12 cells, exhibiting a half-life of 4 h or longer. We
conclude that IB and IBß are not rapidly degraded in all B
cell lines. Interestingly, the only apparent difference between the B
cells with short-lived and those with stable IB and IBß is
their surface Ig isotype. All IgM+ cells
exhibited rapid degradation of IB and IBß, while all
IgG+ cells contained stable IB proteins.
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B is efficiently translocated to the nucleus in B cells with
stable IB
B cells normally express constitutive levels of nuclear NF-B
and express all known mammalian members of the NF-B/Rel
transcription factor family, including NF-B1, NF-B2, RelA, RelB,
and c-Rel (2, 25, 26, 27, 28, 31). These proteins exist as
homodimers or heterodimers; the latter are predominantly formed between
either NF-B1 or NF-B2 and RelA, RelB, or c-Rel (2, 13). RelA and c-Rel serve as receptors for IB, IBß,
and IB, and consequently, complexes containing these Rel proteins
are normally retained in the cytoplasm in the absence of stimuli
(16, 18, 19, 20, 53). Because the vast majority of the known
pathways of NF-B activation converge in the degradation of IB
or IBß, we were especially interested in determining whether the B
cell lines with stable IB also contained nuclear RelA and c-Rel. We
also included RelB in this analysis, since it is expressed at high
levels in B cells and has a functional trans-activation
domain as do RelA and c-Rel. However, it should be noted that not all
NF-B complexes containing RelB can be inhibited by IB
(54).
We first analyzed the B cell lines for the presence of nuclear DNA
binding NF-B by gel-shift assay. This assay can distinguish between
different NF-B complexes, with complexes comprised of NF-B1 or
NF-B2 homodimers migrating faster than NF-B/Rel heterodimers
(47). As controls, we used the pre-B cell lines 70Z/3 and
Bine4.8, which, as expected, contained little nuclear NF-B (Fig. 3
A).
All the B cell lines contained nuclear NF-B, including the B cell
lines that exhibited stable IB (Fig. 3A). The complexes
detected were consistent with the presence of Rel-containing
heterodimers, suggesting that these NFB complexes are efficiently
translocated to the nucleus in B cells with stable IB.
To directly demonstrate the presence of RelA, RelB, and c-Rel in the
nucleus of B cells with stable IB, two types of experiments were
performed. In the first, nuclear extracts from
IgM+ and IgG+ B cell lines
were cross-linked to a B probe by UV irradiation, and RelA- and
c-Rel-containing DNA-protein adducts were identified by
immunoprecipitation and SDS-PAGE (Fig. 3, B and
C). Importantly, in all cell lines nuclear DNA binding RelA
and c-Rel could be detected by this method, albeit at different
concentrations. The IgM+ cell lines WEHI231,
CH27, and CH12-LBK had consistently higher levels of c-Rel than the
IgG+ B cell lines A20 and M12. In contrast, Rel A
levels did not correlate with Ig phenotype or IB half-life. WEHI231
and M12 cells had the highest RelA levels, followed by CH27 and CH12
cells, while RelA was barely detectable in A20 cells. A direct
comparison between the cell line with the highest (WEHI231) and that
with the lowest (A20) content of nuclear RelA and c-Rel is shown in an
independent UV cross-link experiment, in which the respective bands are
separated at a higher resolution (Fig. 3C). This experiment
clearly shows the presence of both RelA and cRel in the nuclei of A20
cells. RelB could not be detected by this method, but its presence in
nuclear extracts could be confirmed by Western blot analysis (Fig. 3D).
To obtain an independent measure of the relative efficiency of NF-B
translocation in the two groups of B cell lines, we estimated the
nuclear fraction of total RelB and c-Rel by Western blot of nuclear and
cytoplasmic extracts (Fig. 3D). Nuclear RelA levels were too
low to be detected by this method. From the four cell lines used in
these experiments, two were IgM+ and had unstable
IB and IBß proteins (WEHI231 and CH12), and two were
IgG+ and exhibited stable IB (A20 and M12).
All these B cells contained nuclear RelB and c-Rel, although some
differences in the proportions of these NF-B constituents were
observed. The cell lines with unstable IB contained a higher
proportion of c-Rel in the nucleus (
20% of the total) than cell
lines with stable IB (6–7%). This is consistent with the
results of the UV cross-linking experiments (Fig. 3B), which
suggested the presence of higher levels of nuclear c-Rel in WEHI231
than in A20 cells. Both of the IgG+ cell lines
contained nuclear RelB in somewhat higher proportions (30–70% of the
total) than cells with unstable IB (<25%). Taken together, these
data confirm that RelA, RelB, and c-Rel, the three NF-B members that
are transcriptional activators, are constitutively present in the
nucleus of IgG+ B cells. They also demonstrate
that nuclear translocation of RelA and c-Rel, which are the targets for
cytoplasmic sequestration by IB, IBß, and IB, can
occur without the accelerated degradation of these IB proteins.
B cells with stable IB have a slower rate of IB
synthesis
For a cell to maintain homeostatic levels of a protein, the
protein must be synthesized at the same rate as it is being degraded.
We therefore reasoned that the differences in IB turnover in the
various B cell lines would be accompanied by different IB
synthesis rates. The relative rates of IB biosynthesis in WEHI231
and A20 cells were therefore determined by following the incorporation
of radiolabeled amino acids into IB, which was quantified by
immunoprecipitation and phosphorimaging. As predicted, WEHI231 cells
synthesized IB at a much faster rate than A20 cells (Fig. 4). Two types of control experiments
showed that this was not true for all proteins. First, we fractionated
labeled proteins by SDS-PAGE and determined the incorporation of
radioactivity into an abundant random protein of 55 kDa. As shown in
Fig. 4, this protein was synthesized more rapidly in A20 cells than in
WEHI231 cells. In addition, we determined the total incorporation of
radiolabel into proteins in these two cell lines using a beta counter
and again found that A20 cells incorporated label faster than WEHI231
cells (data not shown). We conclude that the reduced rate of IB
synthesis in A20 cells is not due to a lower general translation
efficiency, but, rather, is specific for IB and reflects the
requirement for a lower rate of synthesis of IB due to its
stability in these cells.
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B degradation in B cells with stable
IB
We next wanted to determine whether IB turnover could be
stimulated in the B cell lines, A20 and M12, that otherwise contained
stable IB. We used LPS and PMA, since these agents are known to
induce IB degradation and NF-B activation in pre-B cells and T
cells (5, 51, 52). A20 and M12 cells were treated with CHX
in the presence or the absence of LPS or PMA, and IB levels were
assessed by Western blot analysis. As shown above (Fig. 1), IB
levels remained unchanged for >4 h in the absence of stimuli. In
contrast, IB was almost completely degraded after 4 h in the
LPS-treated cells (Fig. 5). Consistent
with previous reports (51, 52), PMA induced an even more
rapid turnover of IB than LPS, resulting in complete degradation
of the total IB pool of M12 cells in <1 h (Fig. 5). The
stability of IB in A20 cells was unaffected by PMA treatment,
possibly because these cells are refractory to its effects (data not
shown). These results demonstrate that the ability to signal IB
degradation is not defective in A20 and M12 cells and suggest that the
classical pathway of NF-B translocation can be activated.
IgG+ and IgM+ B cells express comparable
levels of IB and IBß, but strikingly different levels of
IB
We next compared the steady state levels of IB, IBß,
and IB in the various B cell lines. We were particularly
interested in determining whether the different half-lives of IB
and IBß detected in IgM+ and
IgG+ B cell lines would result in different
expression levels of these proteins. As shown in Fig. 6, all cell lines tested expressed high
steady state levels of IB and IBß. Although the expression
levels varied somewhat between the different cell lines, they did not
correlate with the respective half-lives. For example, one
IgG+ cell line, A20, expressed the highest levels
of IB, but the other IgG+ cell line, M12,
expressed equal or lower levels of IB than
IgM+ cells.
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B were
significantly different in IgM+ and
IgG+ B cells (Fig. 6). In
IgM+ cells, IB was expressed at high
levels, similar to those of IB and IBß. In contrast,
IB was expressed at extremely low levels in all
IgG+ cells, including LK (see Fig. 7B), even though it was as
stable as IB and IBß in these cells. These results indicate
that not only the stability (see Fig. 1) but also the steady state
levels of these three IB proteins can be differentially
regulated.
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B
stability and IB levels
To better establish the correlation among Ig isotype, IB
stability, and IB expression levels, we analyzed a spontaneous
class-switched variant of the IgM+ CH12 B cell
lymphoma, CH12.LX2.1A7, which expresses surface IgG and not IgM
(42). In these cells we found the stability of IB to
be significantly increased in the IgG+ variant
compared with that in IgM+ CH12 cells (Fig. 7A). In addition, the levels of IB were considerably
lower in the IgG+ variant than in the
IgM+ cell line and were similar to the levels
found in other IgG+ B cells (Fig. 7B).
Thus, the differences in IB stability and IB expression
levels detected between several IgM+ and
IgG+ B cell lines are also observed in two CH12
subclones expressing these different Ig isotypes. These results
strongly suggest that the Ig isotype plays a role, either direct or
indirect, in the differential regulation of IB proteins in
IgM+ and IgG+ B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B signaling pathway is the release of
NF-B from its inhibitor, IB, and its subsequent translocation to
the nucleus (13, 16, 17). The release of IB is
accompanied by its degradation, a process that is the hallmark of
NF-B activation. This pathway has been well documented during the
transient activation of NF-B, but it has also been proposed to
function in the constitutive activation of NF-B in B cells, based on
studies of the immature B cell line, WEHI231. In these cells, IB
and IBß are rapidly degraded (30, 31, 32, 33). Although the
precise mechanism of NF-B translocation in these cells remains
unknown, several studies find that the degradation of IB,
IBß, or both is required for nuclear translocation of NF-B
(33, 55, 56). However, despite the central role that
NF-B plays in regulating many aspects of B cell differentiation and
function, detailed studies of other B cell lines have not been
conducted, and the mechanism for constitutive NF-B activation in B
cells remains largely unknown. Here we provide evidence that different
B cell lines use distinct strategies for the nuclear translocation of
NF-B, and that this difference may be associated with the isotype of
the B cell receptor.
Like WEHI231, we found that IB and IBß were rapidly degraded
in several other B cell lines, including CH31, CH12-LBK, and CH27, all
of which are IgM+. In striking contrast, IB
and IBß were very stable in the three B cell lines tested, A20,
M12, and LK (Fig. 1), all of which express surface IgG. In these cells,
IB and IBß were at least as stable as in pre-B and T cell
lines, which do not express significant quantities of nuclear NF-B
(32, 51, 52) (see also Fig. 1). At the very least, these results
demonstrate that the rapid degradation of IB proteins is not a
general feature of all B cell lines. They also suggest that the
stability of IB and IBß may be a general feature of
IgG+ B cells, or at least of B cells that are not
IgM+.
Despite the stability of IB, IBß, and IB in the
IgG+ B cell lines, they contained constitutive
nuclear NF-B, including complexes containing RelA and c-Rel (Fig. 3)
that are normally susceptible to cytoplasmic retention by IB
(16, 18, 19, 20, 53). The presence of these complexes is in
striking contrast to other cells, such as pre-B cells and T cells, that
have stable IB proteins but do not contain activated NF-B
(32, 51, 52). To our knowledge, this is the first
demonstration that constitutive translocation of NF-B complexes
containing RelA and c-Rel can occur without accelerated degradation of
IB, IBß, or IB. We therefore conclude that these
IgG+ B cell lines use a novel mechanism of
nuclear NF-B translocation that is independent of the degradation of
these IB proteins.
The presence of nuclear RelB in IgG+ B cells is
perhaps less surprising than the presence of RelA and c-Rel, since the
nuclear translocation of RelB-containing complexes is regulated very
differently. NF-B p52/RelB heterodimers are not inhibited by IB,
while NF-B p50/RelB heterodimers are only poorly retained by
IB and are not bound by IBß (54). Thus, the
effect that the novel mechanism of NF-B activation in
IgG+ B cells has on modulating levels of nuclear
RelB, if any, cannot be determined at this time.
Although IB and IBß were degraded at different rates in
IgM+ and IgG+ B cell lines,
they were expressed at comparable levels in these two groups of cells.
In contrast, IB was very stable in both types of B cell lines,
suggesting that the turnover of IB is regulated independently of
that of IB and IBß, at least in IgM+ B
cells. Despite the similarity in stability of IB in the different
cell lines, it was expressed at high levels only in the
IgM+ B cells. All IgG+ B
cells expressed very low levels of IB, estimated to be at most
10% of the levels in IgM+ cells. These data
indicate that IB expression is regulated independently of
IB and IBß and is also regulated differently in the
IgM+ and IgG+ B cells. We
therefore propose that the newly identified member of the IB family,
IB, may play a distinct role in regulating NF-B activity.
The nature of the novel mechanism of NF-B translocation in the
IgG+ B cell lines from this study remains to be
determined. Several possibilities exist to account for the presence of
Rel-containing complexes in the nucleus of these cells. For example,
NF-B may dissociate from one or more IB proteins without their
degradation. Such a possibility has been suggested in studies of T cell
activation after pervanadate treatment (57). It is also
possible that a portion of the cytoplasmic NF-B is associated with
an as yet unidentified IB protein, one that is being degraded and
thus releases NF-B for nuclear translocation. This would imply that
the degradation of this putative IB protein occurs independently of
IB, IBß, and IB. A third possibility is that the
efficient retention of NF-B requires a minimal level of total IB
proteins in the cytoplasm. Thus, a significant reduction in the level
of any IB protein would allow the translocation of NF-B complexes
to the nucleus after the assembly and processing of their precursors.
IB has been shown to be similar to IB and IBß in its
specificity, in that it binds complexes containing RelA or c-Rel
(19, 20). Thus, it is possible that the levels of IB
and IBß are insufficient to compensate for the low levels of
IB in IgG+ B cells. On the other hand,
IB has been reported to preferentially bind homodimers of RelA
and c-Rel, rather than heterodimers formed with NF-B1 (19, 20). Thus, even the presence of high levels of IB and
IBß might be unable to sequester these complexes if levels of
IB are too low. In either case, NF-B translocation may be
permitted in IgG+ cells without prior engagement
of IB. At this point, none of these proposed mechanisms can be ruled
out, and further studies are required to distinguish between
them.
What might the consequences be of using two different mechanisms of
NF-B translocation? It is possible that they provide a tool to
differentially regulate the composition of nuclear NF-B and thus the
pattern of NF-B dependent target genes. While our analysis has not
revealed any dramatic differences in nuclear NF-B composition
between the two groups of B cells, it remains possible that their
different mechanisms of NF-B activation may lead to subtle changes
that are not immediately obvious from the current results. The various
NF-B complexes differ in their preference for B sites from
different promoters and also in their trans-activation
potentials (reviewed in Ref. 58). Thus, even small changes
in NF-B composition might alter the expression pattern of
NF-B-dependent genes. Such changes might explain why the
IgG+ B cells had slower rates of IB
synthesis and lower steady state levels of IB. The expression of
both these IB proteins is regulated at least in part by NF-B
(19, 20, 59, 60, 61).
What leads to the different mechanisms of NF-B activation? It is
striking that the only apparent difference between cells with unstable
IB and cells with stable IB is reflected in their surface Ig
receptors. All IgM+ cells had unstable IB
and IBß regardless of their stage of differentiation. In these
studies we used representatives of both immature (WEHI231, CH31) and
mature (CH27, CH12-LBK) IgM+ B cells, which we
found had similar rates of IB degradation. In sharp contrast, all
IgG+ cells analyzed contained stable IB and
IBß. Consequently, it is unlikely that the differences observed
between IgM+ and IgG+ B
cells simply reflect their states of differentiation, but rather
correlate with the surface Ig phenotype. This idea is further supported
by the finding that IB stability and IB levels are
different in a subclone that has undergone spontaneous class switching
from IgM to IgG compared with the parental cell line.
These findings strongly suggest that the Ig receptor itself may play a
direct or an indirect role in regulating IB and IBß
stability and IB expression levels. For example, surface IgM, but
not IgG, may have the ability to constitutively stimulate IB
turnover, possibly by activating IB kinase (reviewed in Ref.
21). Such IgM-mediated IB turnover might occur in the
absence of ligand binding. Indeed, Rajewsky and colleagues
(62) have recently shown that surface IgM expression is
required for the survival of B cells in the periphery. Their studies
imply that surface IgM delivers a constitutive signal that cannot be
provided by other mechanisms in the absence of B cell stimulation.
Given the important role that NF-B plays in cell survival and
protection from apoptosis (63), we predict that this
survival signal leads to IB degradation, and thus NF-B
activation, providing a testable hypothesis for the role of IgM in B
cell survival in the periphery.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefan Doerre, Department of Microbiology, R509, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. E-mail address:
3 Abbreviations used in this paper: , Ig light chain; CHX, cycloheximide.
Received for publication November 13, 1998. Accepted for publication April 13, 1999.
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References |
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