By
From * The Walter and Eliza Hall Institute of Medical Research, Post Office The Royal Melbourne
Hospital, Victoria 3050, Australia; the Department of Immunology, Saga Medical School,
Nabeshima, Saga 849, Japan; and the § University of California, Berkeley, California 94720
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Abstract |
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Rel and nuclear factor (NF)-B1, two members of the Rel/NF-
B transcription factor family,
are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-
B1 genes, we show that these transcription factors differentially regulate cell cycle progression and
apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive
nfkb1
/
mice, the level of apoptosis in cultures of quiescent nfkb1
/
, but not c-rel
/
, B
cells is higher. The failure of c-rel
/
or nfkb1
/
B cells to proliferate in response to particular
mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2
transgene prevents apoptosis in resting and activated c-rel
/
and nfkb1
/
B cells, but does
not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not
simply a consequence of apoptosis and that Rel/NF-
B proteins regulate cell survival and cell cycle
control through independent mechanisms. In contrast to certain B lymphoma cell lines in which
mitogen-induced cell death can result from Rel/NF-
B-dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel
/
B cells, indicating that target
gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells,
NF-
B1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli
requires different Rel/NF-
B factors to control cell cycle progression and prevent apoptosis.
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Introduction |
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The outcome of lymphocyte responses to antigenic or
mitogenic activation reflects a balance between the
relative rates of cell division, death, and differentiation (1).
Although signal transduction pathways that control cell
survival and proliferation are the subject of intense investigation, in mature B cells little is known about the role of
transcription factors in regulating these processes. Among
the many transcription factors implicated in controlling gene
expression in B lymphocytes (2), the Rel (3) and nuclear
factor (NF)-B1 (4, 5) subunits of the Rel/NF-
B family
have recently been shown to be essential for mitogen-induced
proliferation. Rel/NF-
B transcription factors are homo-
and heterodimeric proteins comprising subunits encoded by a small multigene family related to the c-rel protooncogene (6). These proteins regulate transcription by binding
to decameric sequences (
B motifs) located in the promoters and enhancers of many viral and cellular genes, particularly those encoding proteins involved in immune, acute
phase, and inflammatory responses (6). The five known
mammalian Rel/NF-
B proteins, NF-
B1 (p50, p105), NF-
B2 (p52, p100), RelA (p65), RelB, and Rel share a
highly conserved 300-amino acid NH2-terminal Rel homology domain that encompasses sequences required for
DNA binding, protein dimerization, and nuclear localization (6). The CH2-termini of these proteins are divergent,
with those of Rel, RelA, and RelB containing transcriptional transactivation domains (6, 9). Before stimulation, in
most cells, a large proportion of Rel/NF-
B is retained in
the cytoplasm in an inactive form through association with
a family of inhibitor (I
B) proteins (10, 11). A wide range
of stimuli promote the nuclear translocation of Rel/NF-
B
complexes by a mechanism involving the phosphorylation
of conserved NH2-terminal serine residues in I
B
and
I
B
(12, 13), which targets these I
B proteins for ubiquitin-dependent proteosome-mediated degradation (12).
To determine the physiological roles of the various Rel/
NF-B proteins, mice with inactivated c-rel (3), nfkb1 (4), relb (16, 17), or rela (18) genes have been generated by gene
targeting. rela
/
mice die at day 15 of embryogenesis, apparently as a result of fetal hepatocyte apoptosis (18). In
contrast, Rel (3), RelB (16, 17), and NF-
B1 (4) are not
essential for embryogenesis, but are important in the function of hemopoietic cells. Although differentiation of stem
cells into all hemopoietic lineages appears normal in c-rel
/
and nfkb1
/
mice, Rel and NF-
B1 are involved in controlling genes induced during immune responses. Mature B
cells from both mutant strains are defective in their response to mitogens and antigens (3, 4), whereas T cells and
macrophages from c-rel
/
mice exhibit defects in the production of cytokines and immune regulatory molecules, including IL-2, IL-3, IL-6, GM-CSF, G-CSF, TNF-
, and
iNOS (19, 20). RelB appears to serve a dual role. Naive
RelB
/
mice spontaneously develop hemopoietic lesions
characterized by multifocal inflammatory infiltrates, myeloid hyperplasia, and splenomegaly (16, 17). Although this
phenotype suggests that RelB is important in regulating
genes in hemopoeitic cells that are associated with constitutive or housekeeping functions, RelB
/
mice challenged
with pathogens reveal that RelB is also involved in various
specific and nonspecific immune responses (17).
Rel/NF-B proteins have also been implicated in the
control of apoptosis. For example, loss of RelA renders
embryonic fibroblasts highly sensitive to TNF-
-induced
apoptosis (21) and expression of a trans-dominant I
B
mutant in various cell types markedly increases the death of
these cells when treated with TNF-
, ionizing radiation,
or daudorubicin (22, 23). Although enforced expression of
Rel can overcome TNF-
-induced apoptosis of HeLa cells
expressing the trans-dominant I
B
mutant (24), Rel has
also been implicated as an inducer of cell death. Overexpression of Rel induces apoptosis in chicken bone marrow
cells (25) and high levels of c-rel messenger RNA (mRNA)1
were found in cells undergoing programmed cell death in
the developing chick embryo (25).
Although Rel and NF-B1 have been shown to be important in mitogen-induced B cell proliferation (3, 4), it is
not known whether these proteins are needed for cell cycle
progression, inhibition of apoptosis, or both. Here we examine in detail defects in proliferation and cell survival of B
cells from c-rel
/
and nfkb1
/
mice. The results show
that different mitogenic signals require Rel and/or NF-
B1 to promote G1 progression during B cell division and
that specific NF-
B-like factors are necessary for the survival of quiescent and mitogen activated B cells.
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Materials and Methods |
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Mice.
The generation of the c-relPurification of B Lymphocytes.
Small, resting B lymphocytes were purified from spleens of 4-6-wk-old wild-type c-relB Lymphocyte Activation in Tissue Culture.
B lymphocytes were cultured in the high glucose version of DMEM supplemented with 13 µM folic acid, 250 µM L-asparagine, 50 µM 2-mercaptoethanol, and 10% fetal bovine serum at an initial concentration of 3 × 105/ml. Cells were stimulated in vitro with LPS (Difco, Detroit, MI) at a concentration of 20 µg/ml, affinity purified goat anti-mouse IgM (FabCell Turnover Studies.
Cell turnover was determined by labeling proliferating cells in vivo with the thymidine analogue 5-bromo-2Proliferation and RNA Synthesis Assays.
B cells were cultured as described above in 100 µl at a starting density of 3 × 105 cells/ml in 96-well microtiter plates. Cellular proliferation and RNA synthesis were measured at various times by adding 0.5 µCi of [3H]thymidine or 0.25 µCi of [3H]uridine, respectively, for 6 h, after which cells were harvested onto glass fiber filters. Incorporated radioactivity was quantitated by scintillation counting.Immunofluorescence Staining and Flow Cytometry.
The expression of class II MHC, B7.2, and CD25 was analyzed on resting and anti-IgM- or anti-CD40-treated splenic B cells were stimulated for 24 h. Cells were surface stained with biotinylated monoclonal antibodies M5/114 (anti-class II MHC), GL-1 (anti-B7.2), PC16 (anti-IL-2 receptorCell Cycle Analysis.
Cell division in mitogen stimulated lymphocyte cultures was determined by assessing cellular DNA content using PI staining (29). Cells were first fixed (>8 h at 4°C) in 70% ethanol, treated with 0.5 µg/ml DNase-free RNase A (Boehringer Mannheim, Indianapolis, IN) for 20 min at room temperature, and finally stained with 69 µM PI in 0.1 M sodium citrate (pH 7.4) for 30 min at 4°C. Flow cytometric analysis (10,000 cells/sample) was performed on a FACScan® at a low flow rate (100-300 cells/s). Cell cycle distribution was determined with the Cellfit program (Becton Dickinson) or manual gating.Analysis of DNA and Protein Content.
Simultaneous analysis of cellular DNA and protein content was performed as described previously (30). In brief, 5 × 105 resting or mitogen-activated B cells were first fixed in ice-cold 70% ethanol (>8 h), washed once in phosphate-buffered saline, and resuspended at a cell density of ~106 cells/ml in PBS containing 0.5 µg/ml Hoechst 33342 (HO; Calbiochem Corp., La Jolla, CA), 1 µg/ml pyronin Y (Polysciences, Warrington, PA), and 0.1 µg/ml FITC (isomer 1; BBL Microbiology Systems, Cockeyville, MD). Cells were stained for 30 min and then analyzed by flow cytometry using a modified dual lasar FACS® II. Dead cells were excluded from analyses by gating on forward and side light scatter parameters. A total of 10,000 events were collected and analyzed using an in house computer program.Northern Blot Analysis.
10 µg samples of total RNA isolated from resting or anti-IgM antibody-activated B cells were fractionated on 1% formaldehyde agarose gels, transferred onto Hybond C Extra (Amersham Corp., Arlington Heights, IL) and sequentially hybridized as previously described (31) with a 32P- radiolabeled mouse 1.4-kb XhoI c-myc cDNA (32) and a rat 1.1kb PstI glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA (33). Both probes were labeled by random primer extension to specific activities ranging between 5 × 108 and 109 cpm/µg. ![]() |
Results |
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To assess whether
Rel and NF-B1 have a role in regulating the survival of
primary B cells, we first compared the rate of B cell turnover in normal, c-rel
/
and nfkb1
/
mice. Splenic B
cells isolated from mice administered BrdU in the drinking
water continuously for up to 7 d were analyzed for BrdU
incorporation using flow cytometry (Fig. 1 A). Although
no significant difference was observed in the rate of BrdU
incorporated by surface (s)IgM+sIgD
or sIgM+sIgD+ splenic
B cells after 2 d, by days 4 and 7 of this kinetic analysis, nfkb1
/
B cells had incorporated between two- and fourfold more BrdU than either normal or c-rel
/
B cells. To
determine if the increased turnover of nfkb1
/
B cells in
vivo could be due to enhanced cell death, the survival of
resting splenic B cells from mice of each strain were examined in tissue culture (Fig. 1 B). As expected, normal B
cells did not proliferate in the absence of mitogens (results
not shown), and a significant proportion had died by apoptosis over the course of 72 h. c-rel
/
B lymphocytes behaved indistinguishably from control cells, whereas the frequency of apoptotic cells was higher in cultures of quiescent nfkb1
/
B cells. Collectively these findings suggest that
the survival of resting B cells is dependent on NF-
B1, but
not Rel.
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To determine if the
role of Rel and NF-B1 in B cell proliferation involves the
regulation of cell division and/or apoptosis, we performed
cell cycle and cell death analysis on mitogen-activated splenic B cells from c-rel
/
, nfkb1
/
, and control mice.
Purified B lymphocytes were stimulated with LPS or cross-linking antibodies specific for IgM or the B cell-specific membrane protein, RP (27) in the presence or absence of
cytokines. Cell proliferation and apoptosis were assessed
daily over a 72-h time course by cell counting, [3H]thymidine incorporation, and flow cytometric analysis of cellular DNA content. The proliferation, apoptosis, and cell division data for all mitogenic stimulations is graphically summarized in Fig. 2. The first 24 h of a normal mitogenic response is characterized by apoptosis and a low level of cell
division. The extent of cell death is dependent on the stimulus, varying between 15 (LPS) and 44% (anti-IgM antibody) (Fig. 2 B). Between 24 and 72 h, a reduction in the
frequency of dead cells coincides with an increase in viable cells due to division (Fig. 2, A and C; [3H]thymidine incorporation and the frequency of cells residing in S, and G2/M,
respectively). Consistent with previous findings, c-rel
/
(3)
and nfkb1
/
(4, 5) B cells exhibit overlapping, but distinct,
defects in mitogen responsiveness. c-rel
/
B cells proliferate poorly in response to all single mitogens, whereas
nfkb1
/
B cells react normally to anti-IgM antibody, but
show diminished (10-fold) responsiveness to anti-RP antibody and respond poorly to LPS (3-5% of normal [3H]thymidine incorporation). A combination of the cytokines IL-2, IL-4, and IL-5, which alone do not induce B cell proliferation, but instead maximize the response of normal B cells
to mitogens (34), only rescued some c-rel
/
and nfkb1
/
B cell mitogenic responses such as the anti-RP antibody
stimulation of Rel
/
cells.
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Impaired c-rel/
and nfkb1
/
B cell proliferation coincides with higher than normal levels of apoptosis (Fig. 2
B) and reduced cell division (Fig. 2 C), the extent of which
both vary in a mitogen-specific manner. For example, after
72 h, 93 and 60% of anti-IgM- and anti-RP-stimulated c-rel
/
cells, respectively, have undergone apoptosis compared to 20 and 18% of normal B cells. Of the remaining
viable B cells, <0.5% of anti-IgM or anti-RP antibody-stimulated c-rel
/
cells are in S or G2/M (compared with
30 and 37% of normal cells). LPS stimulation leads to the
death of 34 and 44% of c-rel
/
and nfkb1
/
B cells, respectively, after 72 h (compared with 11% of normal cells),
with 10- and 6-fold fewer of the viable c-rel
/
(3.5%) and
nfkb1
/
(5.5%) cells, respectively, residing in S or G2/M
compared to normal B cells (35%). Cytokines enhance proliferation of c-rel
/
and nfkb1
/
B cells by increasing cell
division and reducing apoptosis (see Fig. 2 C).
As previous studies have demonstrated that Rel is a crucial regulator of several growth factor and cytokine genes (3, 19, 20),
we determined if the impaired cell division and increased apoptosis of mitogen-stimulated c-rel/
B cells resulted
from an inability to produce an autocrine growth factor or
the expression of a growth inhibitory activity. This was assessed by comparing apoptosis and cell cycle progression in
anti-IgM or anti-RP antibody-stimulated cocultures of
Ly5.1 c-rel
/
and Ly5.2 c-rel+/+ B cells. The results in
Fig. 3 show that c-rel+/+ and c-rel
/
cells proliferate or
die by apoptosis at the same rate, regardless of whether the
cells are grown in mixed or separate cultures. Reduced cell
division and increased apoptosis of LPS-stimulated nfkb1
/
B cells was also shown not to be influenced by the presence
of normal cells (Grumont, R.J., results not shown). These
results establish that the cell cycle block and increased cell
death in mitogen stimulated c-rel
/
B cell cultures is due
to a cell autonomous defect that does not involve autocrine
growth factors.
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Mitogenic activation of B lymphocytes is
associated with early changes in several biochemical and
cellular parameters, including increased cell size, elevated
RNA synthesis, and modified expression of cell surface
proteins (35). We investigated which of the early activation
events are affected by the absence of Rel. Normal and
c-rel/
B cells stimulated for 24 h with mitogens and
cytokines were stained with antibodies specific for various
B cell surface markers and then examined by flow cytometry. A representative sample of data from cells stimulated
with anti-IgM or anti-CD40 antibodies plus cytokines (IL-2,
IL-4, and IL-5) is shown in Fig. 4. Forward scatter profiles
indicate that although c-rel
/
B cells enlarge during the
initial 24 h of activation, it is to a lesser extent than normal
B cells. The changes in cell surface marker expression are
complex. B7.2 is upregulated normally on both anti-IgM
and anti-CD40 antibody-stimulated c-rel
/
cells, but
CD25 expression is only weakly elevated on anti-IgM antibody-treated c-rel
/
cells. This difference appears to be
mitogen-specific, as no significant difference in CD25 induction is seen on anti-CD40 antibody-stimulated normal
or c-rel
/
cells. MHC class II expression is increased on
c-rel
/
B cells by mitogen treatment, but to a lesser extent
than on normal B cells. The expression of all other cell surface
markers that were examined on c-rel
/
lymphocytes was
normal (Strasser, A., results not shown) as were those expressed
on mitogen-stimulated nfkb1
/
B cells (Gerondakis, S.,
unpublished results). These results indicate that c-rel
/
B
cells can undergo some early events associated with mitogen-induced activation, but remain blocked at a stage characterized by a cell volume that is intermediate between the
resting and the cycling state.
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The low frequency of
anti-IgM or -RP antibody-treated c-rel/
and LPS-stimulated nfkb1
/
B cells in S and G2/M indicated that these
cells appear to be blocked at an earlier point in the cell proliferative cycle. Although the partial enlargement of mitogen-treated c-rel
/
B cells was consistent with progression
to an early phase in G1 (36), the G1 state can be subdivided into early and late phases; the late phase being characterized by an increased rate of RNA and protein synthesis
(35). To define the point in the cell cycle at which c-rel
/
and nfkb1
/
B cells arrest, RNA synthesis was monitored
in mitogen-treated B cells by [3H]uridine incorporation.
Fig. 5 A shows that RNA synthesis in anti-IgM antibody,
anti-RP antibody, and LPS-stimulated c-rel
/
and LPS-stimulated nfkb1
/
cells is between 10- and 30-fold lower
than in normal cells. Moreover, cytokine enhanced division
of anti-RP antibody-treated c-rel
/
B cells coincides with
a >20-fold increase in RNA synthesis compared with anti-RP antibody-stimulated c-rel
/
B cells. The same trend is
seen in mitogen-stimulated c-rel
/
B cells stained for protein and DNA content (Fig. 5 B). c-rel
/
cells treated
with anti-RP antibody alone fail to progress to G1, whereas
a significant proportion of anti-RP antibody plus cytokine-stimulated c-rel
/
B cells can progress via G1 into S phase
and G2/M (Fig. 5 B). These results demonstrate that
c-rel
/
and nfkb1
/
B cells are blocked at a point early
in G1 that precedes increased RNA and protein synthesis
and that cytokines can partly overcome this cell cycle block
allowing c-rel
/
cells to progress through S plus G2/M.
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To further understand
the mechanism(s) by which Rel and NF-B1 prevent apoptosis in B lymphocytes, we tested whether the expression of
a bcl-2 transgene could inhibit the apoptosis of c-rel
/
and
nfkb1
/
B cells. Survival curves for nonstimulated normal,
c-rel
/
, and nfkb1
/
B cells expressing bcl-2 (Fig. 6 A)
show that in addition to inhibiting the death of normal
quiescent B cells, a finding consistent with previous reports
(39), bcl-2 prevented the enhanced apoptosis of resting
nfkb1
/
B cells. Analysis of apoptosis and cell division in
mitogen-stimulated c-rel+/+bcl-2+ and c-rel
/
bcl-2+ B cell
cultures reveals that bcl-2 also prevented the death of anti-IgM- and anti-RP-treated c-rel
/
B cells (Fig. 6 C), but
failed to promote cell division (Fig. 6, B and D). Collectively, these findings show that the death of B cells resulting
from an absence of Rel or NF-
B1 belong to that class of
apoptosis that can be inhibited by bcl-2.
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Recent studies have shown that
anti-IgM antibody-induced apoptosis in W231 B lymphoma cells appears to result from an inhibition of Rel/
NF-B-mediated c-myc transcription (40, 41). To determine if the loss of Rel or NF-
B1 alters c-myc expression
in primary B cells, the level of c-myc mRNA in normal,
c-rel
/
, and nfkb1
/
B cells before and after stimulation
with anti-IgM antibodies was examined by Northern blot
analysis (Fig. 7). Consistent with previous findings (42), c-myc
mRNA levels are upregulated in normal B cells within 2 h
of stimulation (lane 5) and remain elevated after 4 h (lane 9).
In anti-IgM antibody stimulated W231 cells, an initial increase in induced c-myc expression (1 h) is followed by a
downregulation of c-myc mRNA to near prestimulation
levels by 3 h (40). In contrast, c-myc mRNA levels were
upregulated normally in anti-IgM antibody-activated c-rel
/
(lanes 6 and 10) and nfkb1
/
(lanes 7 and 11) B cells. Furthermore, expression of a bcl-2 transgene, which reduces
anti-IgM antibody-induced apoptosis in c-rel
/
B cells,
did not alter the pattern of c-myc expression (lanes 8 and
12). These results establish that neither Rel nor NF-
B1 alone is essential for anti-IgM-induced c-myc expression in
primary B lymphocytes and that the cell cycle block and
enhanced apoptosis in mitogen-activated c-rel
/
B cells is
not a consequence of reduced c-myc expression.
|
Despite
the inability of c-rel/
B cells to proliferate in vitro in response to a range of discrete mitogens, humoral responses are reduced but not abrogated in c-rel
/
mice (3; data not
shown). Since B cells are normally activated by multiple
signals during an immune response (43, 44), we examined
the proliferative response of c-rel
/
B cells to a range of
mitogen and cytokine combinations. Table 1 summarizes
these results as the amount of [3H]thymidine incorporated
72 h after stimulation. In contrast to single stimuli, certain
combinations of mitogens or mitogens plus cytokines could
partially overcome the c-rel
/
B cell proliferative defect.
For example, anti-IgM antibody plus LPS or anti-RP antibody plus LPS synergistically enhanced c-rel
/
B cell proliferation, whereas anti-RP plus anti-IgM antibodies failed
to augment the response above the level achieved by either of the single mitogens. IL-4 increases anti-RP antibody-induced c-rel
/
B cell proliferation ~70-fold, yet this
cytokine increases the response to LPS only marginally
(two- to threefold). Consistent with previous findings (3),
the proliferation of c-rel+/
B cells in response to the panel
of single and multiple stimuli is intermediate between that
of normal and c-rel
/
B cells (Table 1). Based on these results, B cell mitogens can be categorized into two groups:
those which do or do not cooperate to promote cellular
proliferation in the absence of Rel.
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Discussion |
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We have demonstrated that Rel and NF-B1, subunits
of the Rel/NF-
B family of transcription factors, differentially regulate cell cycle progression and apoptosis in B lymphocytes. NF-
B1 prevents apoptosis in quiescent cells,
whereas both Rel and NF-
B1 are important for the survival of mitogen-activated B cells. Differences in the proliferative defects exhibited by B cells lacking Rel or NF-
B1
also indicate that the requirement of one or both of these
transcription factors for progression through the G1 phase of the cell cycle is dependent on the particular mitogenic
signal.
Although both Rel
and NF-B1 are required for normal B cell proliferation
(3) and Rel/NF-
B1 is the predominant NF-
B-like heterodimer in splenic B lymphocytes (45), the induction of B cell mitogenesis by specific stimuli appears to require different NF-
B-like complexes. This is most clearly
illustrated for anti-IgM antibody-stimulated c-rel
/
and
nfkb1
/
B cells. Anti-IgM antibody-activated nfkb1
/
B
cells proliferate normally despite Rel/RelA heterodimers
and Rel homodimers being the only NF-
B-like factors
upregulated in the nucleus (4; Grumont, R.J., results not
shown). In contrast, c-rel
/
B cells stimulated with this
mitogen fail to proliferate, even though nuclear NF-
B1/
RelA heterodimers and NF-
B1 homodimers are upregulated with normal kinetics (3). Taking into consideration the recent finding that proliferation of anti-IgM antibody-stimulated rela
/
B cells is also impaired (48), this indicates that B cell mitogenesis triggered by engaging the antigen receptor only requires Rel/RelA heterodimers and
Rel homodimers. LPS-dependent B cell proliferation, however requires Rel, RelA, and NF-
B1 (3, 4, 48).
Certain mitogen combinations such as anti-IgM antibody and LPS, anti-RP antibody and LPS, or anti-RP antibody plus cytokines synergistically enhance B cell proliferation by increasing cell division and reducing apoptosis,
whereas others such as anti-IgM and anti-RP antibodies fail
to increase c-rel/
B cell mitogenesis above that promoted by the individual stimuli. These findings are consistent with models in which dual stimuli partly overcome the
proliferative defect as a result of each mitogen activating
distinct intracellular signals required for survival and/or cell
cycle progression. Conversely, it would appear that nonsynergizing mitogens such as anti-IgM plus anti-RP antibodies engage the same or functionally equivalent signaling
pathways. If Rel controls a single pathway crucial for the
division and survival of B cells, then perhaps multiple mitogens can in part bypass the need for Rel by engaging the
pathway downstream of Rel. The molecular basis of mitogen synergy remains to be determined. This could involve
activation of other NF-
B-like complexes, or Rel/NF-
B-independent signal transduction pathways. The second possibility is consistent with the finding that mitogen-activated protein (MAP) and stress-activated protein (SAP) kinases are activated normally in c-rel
/
B (results not
shown).
c-rel/
and nfkb1
/
B cells undergo abortive
mitogenic responses characterized by initial cell enlargement but failure to undergo RNA and protein synthesis.
This indicates that cell cycle arrest occurs at a stage between early and late G1. Consistent with Rel and NF-
B1
controlling cell cycle progression is the recent finding that
the cyclin-dependent kinase Cdk2 can regulate the transcriptional activation of RelA through mutual interaction
with the coactivator p300 (49). Our data also shows that
the involvement of particular Rel/NF-
B subunits in cell
cycle regulation may be cell-type specific. Although NF-
B1 is important for B cell division and NF-
B (NF-
B1/ RelA) is induced in fibroblasts during the G0 to G1 transition (50), mitogen-stimulated primary fibroblasts that lack
NF-
B1 divide normally (Gerondakis, S., unpublished results).
The synergistic action of mitogen combinations in promoting c-rel/
B cell proliferation (e.g., anti-RP antibodies plus cytokines) demonstrates certain signals permit near
normal numbers of c-rel
/
B cells to progress through G1
and complete the cell cycle. Although this indicates that
neither Rel nor NF-
B1 appear to be essential at other
stages of the cell cycle, it remains a possibility that a requirement for these transcription factors at a post-G1 stage of the cell cycle could also be overcome by certain mitogenic signals. Despite dual stimuli increasing the frequency
of dividing c-rel
/
cells, an inability to completely bypass
the Rel-dependent phase in G1 is consistent with only a
subset of splenic B cells responding to the combined stimulus. If true, this would indicate heterogeneity in the control
of cell division amongst primary splenic B cells.
Our results demonstrate for the
first time that NF-B1, like Rel and RelA, is important in
regulating cell survival. In normal B lymphocytes, NF-
B1
is required for the survival of quiescent cells, whereas both
Rel and NF-
B1 prevent apoptosis after mitogenic stimulation. Data from a variety of experimental systems indicate that Rel/NF-
B-like transcription factors can play both
anti- (21-24, 40, 41, this paper) and proapoptotic (25, 51,
52) roles. Such a dual function for particular Rel/NF-
B-
like factors may depend on the cell type and the nature of
the apoptotic signal. For example, the importance of cell
type is emphasized with NF-
B1 protecting resting and
LPS-treated B cells from apoptosis (this paper) while being
dispensible for survival of TNF-
-treated embryonic fibroblasts (21). Quantitative differences in the survival of c-rel
/
and nfkb1
/
B cells treated with different mitogens also emphasizes the complex relationship between
Rel/NF-
B-dependent mitogenesis and apoptosis.
It remains to be determined how Rel and NF-B1 promote the survival of B lymphocytes and which gene(s) crucial to cell survival are regulated by these transcription factors in quiescent and mitogen-stimulated cells. Since Rel
and NF-
B1 can function both as a transcriptional activators or repressors (4, 19, 20), cell survival mediated by these
proteins could result either from the induction of a survival
gene or repression of a cell death gene. Candidate gene(s)
include those specifically dedicated to controlling cell survival, or in the case of mitogen-stimulated c-rel
/
and
nfkb1
/
B cells, these could be cell cycle genes if enhanced levels of death are an indirect consequence of the
impaired expression of genes required for G1 progression.
Support for the former model comes from the finding that
even though mitogen-induced rela
/
B cell proliferation
is impaired, apoptosis is normal (48), suggesting that the
defects in mitogen-treated c-rel
/
and nfkb1
/
B cells
could be the result of Rel and NF-
B1 regulating two distinct groups of genes, one crucial for cell cycle progression and the other apoptosis. Moreover, it appears likely that
Rel/NF-
B factors regulate the expression of more than
one gene important for lymphocyte survival. Although
c-myc is a crucial regulator of cell division and apoptosis
(53) and anti-IgM-treated W231 B lymphoma cells undergo apoptosis due an inhibition of Rel/NF-
B-dependent c-myc transcription (40, 41), the expression of c-myc
was normal in quiescent or anti-IgM-treated c-rel
/
and
nfkb1
/
B cells. This difference may indicate that the regulation of c-myc transcription differs in primary and immortalized B cells. NF-
B-induced cellular inhibitor of
apoptosis protein 2 (c-IAP2) expression was also shown to
be important in preventing the TNF-
-mediated death of
Jurkat T cells (54), yet the absence of Rel or NF-
B1 does
not alter c-IAP2 expression in B cells (results not shown). Ultimately, the identification of Rel/NF-
B-regulated
genes in B cells that are important for cell cycle regulation
and cell survival should offer important insight into the
mechanisms by which different Rel/NF-
B dimers regulate
these processes in response to particular mitogenic signals.
![]() |
Footnotes |
---|
Received for publication 8 October 1997 and in revised form 8 December 1997.
The authors thank Dr. David Huang for technical advice, Dr. Frank Battye and colleagues for assistance with cell sorting, and Ms. Julie Merryfull for animal husbandry. ![]() |
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