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Department of Immunology and Bacteriology, University of Glasgow, Glasgow, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 h) Erk-MAP kinase signal
which activates a phospholipase A2 pathway that we have
previously shown to mediate collapse of mitochondrial membrane
potential, resulting in depletion of cellular ATP and cathepsin B
execution of apoptosis. Rescue of BCR-driven apoptosis by CD40
signaling desensitizes such early extracellularly regulated kinase
(Erk) signaling and hence uncouples the BCR from the apoptotic
mitochondrial phospholipase A2 pathway. A second role for
Erk-MAP kinase in promoting the growth and proliferation of WEHI-231
immature B cells is evidenced by data showing that proliferating and
CD40-stimulated WEHI-231 B cells exhibit a sustained cycling pattern
(8–48 h) of Erk activation that correlates with cell growth and
proliferation. This growth-promoting role for Erk signaling is
supported by three key pieces of evidence: 1) signaling via the BCR,
under conditions that induce growth arrest, completely abrogates
sustained Erk activation; 2) CD40-mediated rescue from growth arrest
correlates with restoration of cycling Erk activation; and 3) sustained
inhibition of Erk prevents CD40-mediated rescue of BCR-driven
growth arrest of WEHI-231 immature B cells. Erk-MAP kinase can
therefore induce diverse biological responses in WEHI-231 cells
depending on the context and kinetics of
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine B cell lymphoma cell line WEHI-231 is widely used as a model
for immature B lymphocyte clonal deletion not least because it has a
cell surface phenotype of an immature B lymphocyte
(sIgM+, sIgD+/low,
FcRlow, Faslow, and MHC
class IIlow). Moreover, WEHI-231 B cells undergo
growth arrest and apoptosis after BCR ligation (2, 3, 4) and
can be rescued from BCR-mediated apoptosis by costimulation via CD40.
The signaling mechanisms underlying such apoptosis and rescue remain to
be precisely defined, but we have recently shown that the BCR couples
to up-regulation of cytosolic phospholipase A2
(cPLA2) expression, induction of mitochondrial
phospholipase A2 activity, arachidonic
acid-mediated collapse of 
m, and depletion
of cellular ATP under conditions of apoptotic, but not proliferative,
signaling via the BCR (5). Importantly, disruption of
m, ATP depletion, and apoptosis can be
prevented by rescue signals via CD40 (5). In addition, it
is clear that CD40-mediated induction of Bcl-xL
plays a key role in protecting WEHI-231 cells from BCR-driven apoptosis
(6, 7, 8, 9). However, the key upstream regulators of these
signaling events have not been defined given that many of the early
signaling events following ligation of the BCR that result in either
proliferation or apoptosis of B cells are similar. For example,
although Erk-MAP kinase is generally considered to be a mitogenic
signal, and the stress-activated kinases, Jun N-terminal kinase (Jnk)
and p38 MAP kinase, have often been implicated in apoptotic signaling,
all of these MAP kinases have been reported to be activated following
BCR ligation in immature and mature B cells (10, 11, 12, 13).
Similarly, all of these MAP kinases have also been shown to be
activated in B lymphocytes following CD40 ligation (11, 12, 14). Indeed, whereas BCR-mediated Erk signaling has been
proposed to be associated with apoptotic signaling in WEHI-231 B cells
(15), CD40 has been reported to preferentially activate
Jnk and p38 MAP kinase cascades (11). However, recent
papers by Berberich et al. (14) and Sutherland et al.
(11) have suggested that BCR and CD40 ligation may lead to
different patterns of the type or kinetics of MAP kinase family
activation depending on the maturation state of the cell suggesting
that the overall balance of MAP kinase activation could determine B
lymphocyte fate.
We now report that Erk-MAP kinase plays differential roles in
BCR-induced apoptosis and CD40-mediated rescue of WEHI-231 immature B
cells. For example, we find that the BCR couples to an early (2 h)
Erk-MAP kinase signal which activates the mitochondrial phospholipase
A2 pathway which results in apoptosis in WEHI-231
immature B cells. In contrast, CD40 signaling only marginally activates
Erk at these early time points, and indeed costimulation with
anti-Ig and anti-CD40 desensitizes this early Erk signaling and
uncouples the apoptotic mitochondrial phospholipase
A2 pathway. However, normal proliferating and
CD40-stimulated WEHI-231 B cells exhibit a sustained cycling pattern of
Erk activation over 48 h that correlates with cell cycle
progression, cell growth, and proliferation. This proposal of an
additional role for Erk in WEHI-231 B cell growth is supported by the
findings that apoptotic signaling via the BCR completely abrogates such
sustained Erk activation and that CD40-mediated rescue from apoptosis
and growth arrest correlates with restoration of this late cycling
pattern of Erk activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine B cell lymphoma, WEHI-231 was cultured in RPMI 1640 containing 5% FCS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) (RPMI complete) at 37°C in 5% CO2. RPMI complete for WEHI-231 B cells was additionally supplemented with 2-ME (50 µM). All cell culture reagents were obtained from Life Technologies (Paisley, U.K.).
PD98059 and propidium iodide (PI) were obtained from Calbiochem (La Jolla, CA). U0126 was obtained from Promega (Southampton, U.K.). SB230580 was obtained from Alexis Biochemicals. [6-3H]Thymidine (5 Ci/mmol) was obtained from Amersham International (Aylesbury, U.K.). All other reagents were obtained from Sigma (Poole, U.K.).
Phospho-specific p44/p42 MAP kinase (Erk-MAP kinase/pErk), p44/42 MAP kinase (Erk), phospho-specific SAPK/Jnk (pJnk), SAPK/Jnk (Jnk), phospho-specific stress-activated protein kinase kinase (pSEK), SEK, phospho-specific c-Jun (pJun), c-Jun (Jun), phospho-specific p38 MAP kinase (pp38), p38 MAP kinase (p38), and anti-rabbit Ig-HRP Abs were obtained from New England Biolabs (Hitchin, U.K.). Purified monoclonal anti-IgM Abs (anti-mouse µ-chain) and anti-CD40 Abs were produced from the B7.6 and FGK45 hybridomas, respectively, as described previously (16).
Measurement of DNA synthesis
For measurement of DNA synthesis, WEHI-231 cells (104 cells/well) were cultured in triplicate in round-bottom microtiter plates in RPMI 1640 supplemented with glutamine (2 mM), sodium pyruvate (1 mM), 1% nonessential amino acids, 2-ME (50 µM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 5% FCS, in the presence of the appropriate agonist in a total volume of 200 µl. Cells were cultured at 37°C in a 5% (v/v) CO2 atmosphere at 95% humidity for 48 h. [3H]Thymidine (0.5 µCi/well) was added 4 h before harvesting of the cells with an automated cell harvester (Molecular Devices, Sunnyvale, CA). Incorporated label was estimated by liquid scintillation counting and is represented as dpm ± SEM.
Cell stimulation and whole cell lysate preparation
WEHI-231 cells (107 cells) were stimulated
as required in RPMI 1640 supplemented with glutamine (2 mM), sodium
pyruvate (1 mM), 1% nonessential amino acids, 2-ME (50 µM),
penicillin (100 U/ml), streptomycin (100 mg/ml), and 5% FCS. In all
experiments, except in the indicated panels of Fig. 4
, stimulations
described as medium contained 5% FCS. In Fig. 4, cells denoted as
serum free were washed twice with RPMI 1640 and then cultured overnight
at 37°C in serum-free medium (RPMI 1640 supplemented with 0.5 mg/ml
BSA, 50 µM 2-ME, 1 mM pyruvate, and 2 mM glutamine) before being
stimulated (17). Reactions were terminated by the addition
of 2x ice-cold modified radioimmunoprecipitation assay lysis
buffer (50 mM Tris (pH 7.4), 150 mM sodium chloride, 2% (v/v) Nonidet
P-40, 0.25% (w/v) sodium deoxycholate, 1 mM EGTA) plus 10 mM sodium
orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, chymostatin (10
µg/ml), leupeptin (10 µg/ml), antipain (10 µg/ml), and pepstatin
A (10 µg/ml)), and lysates were solubilized for 30 min on ice before
centrifugation at 12,000 rpm for 15 min. The resulting supernatants
(whole cell lysate) were stored at -20°C before being used for
immunoprecipitation or Western blot analysis.
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Equal protein loadings of lysates (determined by BSA protein
assay (Pierce, Rockford, IL)) were resolved on 10% SDS-PAGE, followed
by transfer onto a polyvinylidene difluoride filter membrane
(Millipore, Bedford, MA). Membranes were blocked in PBS, 0.1% Tween
20, 10% nonfat milk for 1 h at 25°C or overnight at 4°C.
Membranes were incubated with rabbit anti-pErk or anti-Erk
(both 1/2000) or with anti-p38 MAPK, JNK, SEK, and c-Jun (1/2000)
Abs for 2 h at 25°C or overnight at 4°C followed by 1 h
incubation at 25°C with a donkey anti-rabbit IgG-HRP Ab (1/5000
dilution in PBS, 0.1% Tween 20). Protein bands were visualized by
incubation with the ECL system (Amersham International). Relative band
densities were determined by the use of the Gel-Pro analysis program.
The anti-phospho-Erk Abs used recognized p42 and p44 MAP kinase
(Erk2 and Erk1, respectively) only when catalytically activated by
phosphorylation at the TEY motif corresponding to T202/Y204 on human
Erk1. Likewise, the anti-Erk Abs recognized both Erk1 and Erk2 as
indicated in Fig. 2. However, in WEHI-231 B cells, phospho-Erk1 and
Erk1 activity was generally barely detectable; thus, for consistency,
all other figures are annotated simply as phospho-Erk or Erk.
Similarly, although the anti-Jnk Abs can recognize both p46 and p54
Jnk, only phospho-p54Jnk was detectable; hence, these blots are labeled
as Jnk or pJnk.
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Whole cell lysates were prepared from WEHI-231 cells (1 x
107 cells) stimulated with medium, anti-Ig
(10 µg/ml), anti-CD40 (10 µg/ml), or anti-Ig and
anti-CD40 (both 10 µg/ml) for the indicated time.
MEK1/2-containing immune complexes were prepared from lysates (100
µg) using an anti-MEK1/2 Ab (New England Biolabs) and protein
G-Sepharose beads. The MEK1/2 immune complexes were assayed for MEK
activity using the MEK1 assay kit (TCS Biologicals, Boltoph Claydon,
U.K.), and 0.5 U human activated MEK1 was used as the positive control
sample. Briefly, the immune complex samples, or human activated MEK1,
were incubated with assay buffer (20 mM MOPS (pH 7.2), 25 mM
-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM
DTT), Mg2+-ATP mixture (75 mM magnesium chloride
and 500 µM ATP in assay buffer ADB) and 1 µg of inactive
GST-p42 MAP kinase at 30°C for 30 min. Samples were then analyzed by
SDS-PAGE using 10% SDS minigels. Western blotting was performed using
anti-phospho-Erk1/2 (New England Biolabs) to detect phosphorylated
GST-p42 MAP kinase (62 kDa). The amount of phosphorylated GST-p42 MAP
kinase is proportional to the activity of MEK1/2 in the sample. Blots
were stripped and reprobed with anti-MEK1/2 (New England Biolabs)
to check the loading of the gel.
Cell cycle arrest and analysis by laser scanning cytometry (LSC)
Cells were pretreated for 24 or 40 h with aphidicolin (5
µg/ml) or olomoucine (50 µM). The cells were then washed twice in
medium before being analyzed by FACS or LSC as indicated in Fig. 10.
Alternatively, the cells were then stimulated (resuspended at 2 x
106/ml for Western blotting or at 2 x
105/ml for LSC) with anti-Ig (10 µg/ml),
anti-Ig (10 µg/ml) plus anti-CD40 (10 µg/ml), or medium
alone for up to 48 h as indicated before processing for Western
blot (see above) or LSC analysis. For LSC, the cells were attached to
microscope slides by cytocentrifugation at 600 rpm for 4 min in a
Shandon Cytospin centrifuge (Shandon, Pittsburgh, PA), then fixed in
4% formaldehyde in PBS for 10 min at room temperature, washed with
PBS, and permeabilized with 2% FCS, 2 mM EDTA (pH 8.0), 0.01% w/v
saponin for 5 min at room temperature. The slides were then washed
three times with PBS and incubated with a blocking solution containing
10% goat serum, 1% BSA, and 0.02% sodium azide in PBS for 10 min.
Subsequently, a 50-µl aliquot of a 1% BSA-PBS solution containing a
1/250 dilution of anti-phospho p44/42 MAP kinase Ab (Cell Signaling
Technology, Beverly, MA) was placed on top of the site with the
attached cells on the microscope slide and incubated for 30 min. The
sites were washed three times with 1% BSA-PBS and incubated with a
50-µl aliquot of a 1% BSA-PBS solution containing a 1/100 dilution
of anti-rabbit FITC-conjugated secondary Ab in 5 µg/ml PI
containing RNase A (200 µg/ml) for 30 min. Cells were washed a
further three times in 1% BSA-PBS and then allowed to dry in the dark
before being mounted in Vectashield (Vector Laboratories, Burlingame,
CA) without 4',6'-diamidino-2-phenylindole and left in the dark at
4°C until analysis on the LSC using CompuCyte Software (CompuCyte,
Cambridge, MA).
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Cells (5 x 105) were harvested at required time intervals. Cells were washed twice, resuspended in 100 µl PI stain (0.1% w/v sodium (tri)citrate, 0.1% v/v Triton X-100, 50 µg/ml PI) and incubated at 4°C for 10 min and then at room temperature for at least 30 min. Cells were analyzed on a FACSCalibur (BD Biosciences, Mountain View, CA) using CellQuest software (BD Biosciences).
cPLA2 assays
cPLA2 activity was assessed by measurement of [3H]arachidonic acid release as described previously (18). This activity was blocked by the inhibitor arachidonyl trifluoromethyl ketone (selective for iPLA2 and cPLA2) excluding a role for sPLA2. Briefly, cells (106/ml) were prelabeled overnight with RPMI, 10% FCS medium containing 1 µCi/ml [3H]arachidonate. Before each experiment, cells were washed, resuspended in fresh isotope-free medium, and cultured for a further hour at 37°C. The cells were then washed three times in HBSS, pH 7.4, containing 2% (w/v) BSA and 10 mM glucose, resuspended in this buffer at 107 cells/ml, and equilibrated for 30 min at 37°C. Cells (106/assay) were then stimulated with the appropriate agent for the indicated time at 37°C. Reactions were terminated by the addition of 1 ml ice-cold methanol and 15 µl glacial acetic acid followed by a further 0.5 ml methanol and 0.75 ml chloroform, and the cells were extracted for 30 min on ice. Phases were split by the addition of chloroform and water, and the chloroform phase was then dried under vacuum. For measurement of [3H]arachidonate levels, 30 µl samples (prepared in chloroform-methanol (2:1)) were spotted onto Silica Gel 60 thin layer plates and developed in hexane-diethyl ether-formic acid (80:20:2, by volume). After exposure to iodine vapor, arachidonate was identified by comparison with standards, the plate was sprayed lightly with water, and the corresponding silica gel was scraped from the plate and assayed for radioactivity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B cell lymphoma WEHI-231 is a widely used model system for
investigating the signaling mechanisms underlying clonal selection of
normal IgM+IgD- immature B
cells. We and others have reported that ligation of sIgM on the
WEHI-231 immature B cell line induces growth arrest (suppression of DNA
synthesis and cell cycle arrest) which is maximal at concentrations of
anti-Ig between 0.1 and 1 µg/ml. Moreover, anti-Ig at
concentrations 
1 µg/ml induces apoptosis as indicated by disruption
of mitochondrial potential, annexin V staining, DNA laddering, and
subdiploid DNA content analysis (3, 5, 19). In contrast,
treatment with anti-CD40 alone either weakly promotes or has little
effect on DNA synthesis in WEHI-231 cells (5). Moreover,
anti-CD40, at concentrations 1.0 µg/ml, is effective in
completely rescuing sIg-stimulated WEHI-231 cells from growth arrest
(Fig. 1A and results not
shown) and apoptosis (Fig. 1, B–D). This rescue from
apoptosis is reflected by abrogation of BCR-mediated disruption of
mitochondrial potential, annexin V staining, and DNA fragmentation
(5).
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A). Analysis of the kinetics
of such Erk activation showed that anti-Ig (10 µg/ml) treatment
resulted in the phosphorylation of Erk (pErk) as early as 1 min, with
maximal Erk phosphorylation at 30 min (Fig. 2B). In
contrast, anti-CD40 (10 µg/ml) was shown to induce little or no
Erk phosphorylation over similar time ranges (Figs. 2C and
3A), findings consistent with previous studies (11, 12). Moreover, costimulation of WEHI-231 B cells with
anti-Ig (10 µg/ml) and anti-CD40 (10 µg/ml) resulted in a
more transient and weaker Erk-MAP kinase signal than that observed
under conditions of sIg-mediated growth arrest and apoptosis (Figs. 2D and 3A).
In contrast, neither anti-Ig nor anti-CD40, either alone or in
combination, stimulated p38 MAP kinase activity above basal levels over
a 30-min time period (Fig. 3B). Likewise, little or no
change in Jnk activity in terms of dually phosphorylated Jnk expression
could be detected (Fig. 3C). Moreover, little or no
activation (phosphorylation) of the upstream regulator of Jnk, SEK1, or
its downstream effector, c-Jun, could be detected in response to either
anti-Ig or anti-CD40 signals (Fig. 3C). However,
anti-Ig was found to stimulate a weak, delayed transient activation
of p54 Jnk, but not p38 MAP kinase, which was apparent between 1 and
2 h poststimulation before returning to basal levels (Fig. 4, A and B).
Although at first sight these results may seem rather contradictory to
previous studies reporting strong activation of Jnk and p38 MAP kinase
via CD40 or the BCR in WEHI-231 B cells, these earlier studies were
performed under serum-free conditions which may have primed BCR
activation of the stress-activated kinases (11, 12).
Indeed, we have analyzed Jnk and p38 activity under serum-free and
serum-supplemented conditions, and these data show that the BCR is
coupled to Jnk activity (maximal at 60 min) in serum-free but not
serum-supplemented medium. Similarly, these data suggest that the BCR
is also weakly coupled to p38 activity under serum-free conditions
(Fig. 4).
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Taken together, the above results could suggest that the BCR coupling to early (within 1 h) Erk-MAP kinase signaling transduces apoptosis of WEHI-231 cells and that costimulation with CD40 may rescue such B cells from growth arrest and apoptosis, at least in part, by uncoupling the BCR from this Erk-MAP kinase-dependent apoptotic pathway. We therefore investigated whether pharmacological inhibitors of MEK (PD98059 and U0126), the upstream regulator of Erk-MAP kinases, would prevent BCR-mediated apoptosis of WEHI-231 cells.
To verify the ability of PD98059 and U0126 to inhibit the activity of
Erk-MAP kinase in WEHI-231 cells, we tested whether these reagents
blocked anti-Ig-stimulated dual phosphorylation of Erk and hence
activation in WEHI-231 cells stimulated via the BCR. As shown in Fig. 5, A and B, PD98059
and U0126 were effective in the inhibition of Erk phosphorylation in
both a time (pretreatment before BCR ligation) and dose-dependent
manner. Optimal inhibition was achieved when the cells were pretreated
with either inhibitor for between 90 min and 3 h, and under these
conditions BCR-stimulated Erk-MAP kinase activity was completely
suppressed (Fig. 5, A and B, and results not
shown). Neither of these inhibitors, however, appeared to be effective
in longer term cultures of WEHI-231 cells given that PD98059 and U0126
proved only partially capable of blocking Erk-MAP kinase activity over
periods of 6–24 h (Fig. 5C), indicating that sustained
inhibition of MEK over such time periods required multiple treatment of
the cells with these reagents.
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and
results not shown). In contrast, the p38 MAP kinase inhibitor
SB203580 had no effect on BCR-mediated apoptosis (data not shown),
results consistent with our finding that anti-Ig did not
significantly stimulate p38 MAP kinase under these conditions (Fig. 3B).
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) that the MEK inhibitor, PD98O59, also
inhibits BCR coupling to cPLA2, suggesting that
this early Erk-MAP kinase signal couples the BCR to
cPLA2-dependent apoptosis.
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Although the MEK inhibitors PD98059 and U0126 blocked BCR-mediated
apoptosis, these reagents did not relieve anti-Ig-induced arrest in
the G0-G1 phase of the cell
cycle (Fig. 6). These results suggested that Erk signaling, distinct
from the early transient Erk response we have proposed to be associated
with BCR-driven apoptosis, might play a role in the sustained
proliferative response observed in untreated or CD40-rescued WEHI-231 B
cells.
To test this, we investigated whether WEHI-231 B cells exhibited
differential Erk signals during 48 h under conditions of normal
growth, BCR-driven growth arrest, or CD40-mediated rescue (Fig. 8). We found that normal proliferating or
anti-CD40-treated WEHI-231 cells exhibited strong dual
phosphorylation of Erk, but not Jnk or p38, between 8 and 48 h,
whereas protein levels of Erk, Jnk, and p38 expression remained
constant (Fig. 8A and results not shown). This strong dual
phosphorylation did not reflect a sustained elevated level of Erk
activation but rather exhibited a cycling pattern of dual
phosphorylation of Erk with peaks between 8–16 h and 32–48 h (Fig. 8D and results not shown) suggesting that cycling Erk
activation was required for sustained progression of WEHI-231 cells
throughout the cell cycle. In contrast, whereas apoptotic
concentrations of anti-Ig induced a strong transient activation of
Erk consistent with the apoptotic Erk signal described above, all Erk
activity 2 h poststimulation was ablated (Fig. 8B).
Moreover, costimulation with anti-CD40 not only reduced the early
transient Erk response but also restored (onset 8–24 h) and enhanced
the late cycling pattern observed in unstimulated cells (Fig. 8C). That
Erk activation within 8–24 h is critical for CD40-mediated rescue from
BCR-driven growth arrest may be inferred from our finding that WEHI-231
B cells can still be rescued from anti-Ig-mediated growth arrest
when anti-CD40 is added 8 h, but not 24 h,
poststimulation via the BCR (Fig. 8E).
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To investigate whether cycling Erk activity contributed to the
survival and/or growth of WEHI-231 B cells, we tested the effect of
sustained abrogation of MEK1/2, and hence Erk, activity, on WEHI-231
DNA synthesis and apoptosis. We found that sustained treatment of
WEHI-231 B cells with U0126, PD98059 (results not shown), or U0126 plus
PD98059 (Fig. 9A) not only
appeared to enhance anti-Ig-mediated growth arrest but also almost
completely blocked the DNA synthesis observed in unstimulated,
anti-CD40-treated, or anti-Ig plus anti-CD40-treated cells
(Fig. 9A and results not shown). To ensure that these
results reflected the contribution of the late (8 h) cycling Erk
pool, we investigated the effects of a single dose of PD95089 (Fig. 9C) or U0126 (Fig. 9D). These latter results
confirmed our earlier cell cycle analysis (Fig. 6), which showed that
blocking early Erk signals had only marginal effects on normal or
CD40-rescued growth of WEHI-231 B cells. Taken together, these results
suggest a role for late (8 h) Erk signaling both in the basal
proliferation of WEHI-231 cells and in the rescue of WEHI-231 cells
from anti-Ig-mediated growth arrest by CD40.
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B). This analysis revealed that
sustained blockage of MEK1/2 activity and growth of unstimulated
WEHI-231 B cells led to apoptosis. The possibility that this apoptosis
was due to inhibitor toxicity was ruled out by the finding that
although sustained blockage of MEK1/2 activity in WEHI-231 cells
treated with anti-CD40 or anti-Ig plus anti-CD40 induced
profound growth arrest (Fig. 9A), it did not induce
apoptosis in either case. These results therefore suggest that although
signaling via CD40 induces signals which can compensate for ablation of
Erk activity in terms of survival, additional signals including Erk are
required to rescue anti-Ig-mediated growth arrest. A role for cyclical Erk-MAP kinase activation in cell cycle progression of WEHI-231 B cells?
The MEK inhibitor data supported the proposal that cycling Erk
activity contributed to the survival and/or growth of WEHI-231 B cells,
presumably by promoting cell cycle transition. To investigate how
cycling Erk activation correlates with cell cycle progression, we
analyzed Erk signaling after arrest and release (removal of cell cycle
blockers by washing) of cells from either the G1
or S phases of the cell cycle using the cell cycle blockers olomoucine
(21, 22) and aphidicolin (23, 24),
respectively (Fig. 10A).
Although enriched populations of G1 and S phase
cells were obtained, it was not possible to obtain fully synchronized
populations as increasing either the concentrations of these cell cycle
blockers or the length of preincubation with these reagents simply led
to the induction of apoptosis, particularly with aphidicolin.
Nevertheless, as with the asynchronous cells, subsequent treatment with
anti-Ig alone induced inhibition of Erk activity within 16–48 h
following removal of cell cycle blockers and subsequent release from
either G1 or S phase arrest (Fig. 10B). In contrast, costimulation with anti-CD40 induces
strong Erk activation during this time period (Fig. 10B).
These results therefore supported our proposal that anti-Ig induced
growth arrest of WEHI-231 cells by suppressing late Erk activity
associated with cell cycle progression and that anti-CD40 rescued
such cells by promoting Erk activity to drive mitosis. To address more
directly the role of Erk activity in driving cell cycle progression, we
next analyzed intracellular staining of phospho-Erk in conjunction with
cell cycle analysis (using PI) by LSC. These latter data showed that
the majority of cycling cells (i.e., those transiting
G1, S phase, or G2-M)
showed phospho-Erk staining (Fig. 10C and Table I). However, <50% of newly divided
cells demonstrated phospho-Erk staining (Fig. 10C and Table I). Moreover, treatment with anti-Ig or olomoucine to induce growth
arrest in G0-G1 reduced the
percentage of such cells expressing phospho-Erk to 30–35% (Table I
and results not shown). These results, which indicate that BCR- or
olomoucine-mediated growth arrest in G1
correlates with inhibition of Erk activity, suggest that suppression of
late (>2–4 h) Erk activation is also likely to be necessary for
preventing transition through G1. Release of the
olomoucine block followed by culture in medium alone showed synchronous
transition of cells through G2-M within 48 h
with >85% of such mitotic cells exhibiting Erk activation.
Interestingly, stimulation of these cells with anti-Ig plus
anti-CD40 resulted in asynchronous transition of the cells through
cycle (all stages exhibiting >50% cells expressing phospho-Erk)
within 48 h presumably due to the ability of anti-CD40 to
promote growth and division (Table I).
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To address how BCR signaling abrogates cycling Erk-MAP kinase
activation to achieve cell cycle arrest, we investigated the effect of
anti-Ig on the activity of MEK, the upstream regulator of Erk. We
found that although the cycling Erk activity was inhibited by BCR
signaling, MEK activity (Fig. 11) was
comparable in untreated and BCR-stimulated cells between 12 and 48
h as indicated both by MEK phosphorylation and by in vitro MEK kinase
assays. These data therefore suggested that BCR signaling did not
uncouple the upstream regulators of Erk-MAP kinase but rather induced
negative feedback mechanisms that could override ongoing activation of
Erk. Consistent with this, our data suggest that BCR signaling inhibits
cycling Erk activity in WEHI-231 B cells by up-regulating expression of
the MAP kinase phosphatase, Pac-1, and by promoting its association
with Erk-MAP kinase (Fig. 11). Anti-CD40 treatment does not appear to
down-regulate Pac-1 expression per se but prevents Pac-1 association
with Erk (Fig. 11 and results not shown). Use of this mechanism to
regulate B cell proliferation is consistent with our previous findings
that immunomodulatory products secreted by filarial nematodes can
elicit B cell unresponsiveness by priming uncoupling of BCR-Erk-MAP
kinase signaling by the MAP kinase phosphatase, Pac-1
(25).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 h) Erk-MAP
kinase signal (Figs. 2, 3, 6, and 8) which activates a phospholipase
A2 pathway (Fig. 7) that mediates arachidonic
acid-mediated collapse of m and depletion
of cellular ATP, resulting in cathepsin B execution of apoptosis
(5). Rescue of BCR-driven apoptosis by CD40 signaling
desensitizes this early Erk signaling (Figs. 2 and 3) and uncouples the
apoptotic mitochondrial phospholipase A2 pathway
(Fig. 7). Consistent with this early Erk-MAP kinase signal playing a
key role in the commitment of WEHI-231 cells to apoptosis is the
finding that attenuation of the early Erk signal prevents BCR-mediated
coupling to phospholipase A2 and resultant
apoptosis (Figs. 6 and 7).
We have also identified a second role for Erk-MAP kinase in promoting
the growth and proliferation of WEHI-231 immature B cells.
Proliferating and CD40-stimulated WEHI-231B cells exhibit a sustained
cycling pattern (8–48 h) of Erk activation (presumably in response to
serum- or autocrine factors) which correlates with cell cycle
progression, cell growth, and proliferation (Fig. 8). This distinct
role for Erk signaling in the regulation of WEHI-231 B cell growth is
supported by three key pieces of evidence: 1) apoptotic signaling via
the BCR completely abrogates sustained Erk activation (Fig. 8); 2)
CD40-mediated rescue from apoptosis and growth arrest correlates with
restoration of this late cycling pattern of Erk activation (Fig. 8);
and 3) sustained inhibition of the Erk activator MEK prevents
CD40-mediated rescue of BCR-driven growth arrest of WEHI-231 immature B
cells (Fig. 9). That such cycling Erk activation directly contributes
to cell cycle progression is demonstrated by the finding that both BCR-
and olomoucine-driven growth arrest in the
G0-G1 phase of the cell
cycle correlates with suppression of Erk activity (Fig. 10 and Table I). Such growth arrest therefore presumably reflects suppression
of Erk-MAP kinase-inducible components of the cell cycle machinery
(such as cyclin D) required for G1-S phase
transition. Alternatively, abrogation of Erk signaling may relieve
down-regulation of p27kip1-mediated inhibition
of cell cycle progression (26, 27, 28, 29). Our studies therefore
demonstrate that Erk-MAP kinase can induce distinct biological
responses in WEHI-231 B cells, depending on the kinetics of activation
and the context of the downstream signaling machinery.
Interestingly, the onset (8–24 h) of the sustained Erk-MAP kinase
signal restored by CD40-signaling correlates with the final time window
in which anti-Ig-treated WEHI-231 B cells can be effectively
rescued from growth arrest by anti-CD40 treatment (Fig. 8). This
finite time frame for CD40-mediated rescue is reminiscent of the
recently described progression of anti-Ig stimulated immature B
lymphocytes through a series of "temporal windows" resulting,
finally, in the induction of apoptosis (10). Such an
orderly progression through initiation of an apoptotic program,
followed by a delay in which the fate of the cell can be redirected is
believed to allow external signals, such as those delivered through
CD40, to determine the fate of the cell. Additionally, this temporal
window is believed to allow self-reactive immature B lymphocytes time
to initiate receptor editing so that they can alter their receptor
specificity and re-enter the B lymphocyte pool before further
maturation. However, failure to successfully rearrange their BCR or
receive external help will ultimately result in deletion of these cells
from the B lymphocyte pool (10). The ability of WEHI-231
cells to develop through similar temporal windows in which short term
BCR-Erk-MAP kinase signaling similarly initiates a negative signal
that, in the absence of an additional signal such as anti-CD40,
ultimately leads to cell death suggests that maintenance of cycling Erk
signaling could be a key element in the survival of immature cells
during this selection process.
CD40 stimulation of WEHI-231 cells has been shown to up-regulate the
antiapoptotic protein Bcl-xL (30)
which has been widely proposed to play a major role in the rescue of
WEHI-231 cells from BCR-mediated apoptosis (4, 6, 7, 9).
Although Erk signaling has previously been shown to promote
up-regulation of Bcl-xL in other systems
(31), it is likely that in WEHI-231 B cells CD40
up-regulates Bcl-xL and rescue from apoptosis by
Erk-independent mechanisms given that we have found that CD40 can
rescue WEHI-231 cells from BCR-mediated apoptosis even in the presence
of sustained abrogation of MEK activity (Fig. 9). In contrast,
sustained abrogation of MEK activity prevents CD40-mediated rescue of
BCR-driven growth arrest of WEHI-231 B cells (Fig. 9). Taken together,
these findings could provide a molecular mechanism to explain earlier
reports that whereas overexpression of Bcl-xL
could rescue WEHI-231 B cells from BCR-driven apoptosis but not growth
arrest, CD40 signaling could abrogate such apoptosis and promote growth
and proliferation (7, 9). Indeed, Erk-independent
up-regulation of Bcl-xL by anti-CD40 could
explain why unstimulated, but not CD40-stimulated WEHI-231 B cells
undergo apoptosis in the sustained presence of MEK inhibitors. However,
it is possible that such Erk-independent rescue from apoptosis may
reflect the involvement of other antiapoptotic molecules that have been
implicated in CD40-mediated rescue from BCR-induced apoptosis. In
particular, a recent study has highlighted the role of another member
of the Bcl-2 family, the antiapoptotic protein A1 (32).
For example, overexpression of this protein was shown to render
WEHI-231 cells resistant to BCR-mediated apoptosis, and CD40
stimulation was also shown to increase A1 RNA expression
(32). However, the role of endogenous A1 in WEHI-231 cells
remains to be explored, and the molecular mechanisms leading to its
expression and activation to be elucidated.
In addition to our finding of differential roles for Erk-MAP kinase in mediating both apoptotic and proliferative responses in WEHI-231 cells, dual roles for Erk-MAP kinase in the induction of negative and positive selection have also recently been reported for thymocytes (33). Similarly, although Erk activation has been widely established to be crucial for T cell activation, recent studies have shown Erk to play a critical role in cytokine unresponsiveness (anergy) and activation-induced cell death (34, 35, 36, 37). How Erk differentially signals to regulate these distinct physiological responses is not known, but in the T cell systems, sustained hyperactivation of Erk appears to be associated with anergy and apoptosis. In contrast, in our WEHI-231 system, rescue and growth signals appear to be associated with sustained, albeit cycling, Erk activity. An additional factor may be the ability of Erk to act in both cytosolic and nuclear compartments and hence target differential downstream effector elements. Indeed, similarly to previously published findings (38, 39), we have preliminary results to suggest that whereas CD40 stimulation enhances nuclear Erk-MAP kinase activity, BCR stimulation results predominantly in cytoplasmic Erk-MAP kinase activity in WEHI-231 B cells. That CD40 signaling promotes nuclear Erk-MAP kinase activity may fit with current models suggesting that CD40 rescue signals promote transcription factor activation and new protein synthesis (40). In contrast, a cytoplasmic location for BCR-mediated Erk-MAP kinase activity is necessary for activation of cPLA2 and initiation of the mitochondrial apoptotic pathway in WEHI-231 B cells. Although early BCR-coupled Erk activity in mature B cells also seems to be predominantly located in the cytosol, these signals are not apoptotic, presumably because cPLA2 is not expressed or activated in the cells (18). Thus, Erk-MAP kinase signaling may induce diverse biological responses in B cells depending on the kinetics and amplitude of activation, the subcellular localization of the pool of Erk used, and the maturation stage-dependent context of the downstream signaling machinery recruited.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206.
3 Address correspondence and reprint requests to Dr. Margaret M. Harnett, Department of Immunology, University of Glasgow, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, U.K. E-mail address: M.Harnett{at}bio.gla.ac.uk
4 Abbreviations used in this paper: BCR, B cell Ag receptor; cPLA2, cytosolic phospholipase A2; Erk, extracellularly regulated kinase; Jnk, Jun N-terminal kinase; MAP kinase, mitogen-activated protein kinase; MEK, ERK-MAP kinase kinase; PI, propidium iodide; sIg, surface Ig; LSC, laser scanning cytometry; SEK, stress-activated protein kinase kinase.
Received for publication April 2, 2001. Accepted for publication February 19, 2002.
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