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Department of Microbiology and Immunology, G. W. Hooper Foundation, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and -2,
phosphatidylinositol (PI) 3 kinase, the Ras-Raf-mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase
(MEK)-extracellular signal-regulated kinase (ERK) mitogen-activated
protein (MAP) kinase pathway, and phosphorylation of the Vav
proto-oncogene product (1, 2, 3). The resulting signals
quickly reach the nucleus and alter gene expression. The ultimate
effects on the B cell are profound and vary depending on the maturation
state of the cell and on the additional signals the cell receives. Ag
contact with immature B cells typically causes them to enter into an
anergic state or undergo apoptosis, responses that promote
immunological tolerance to self-Ags. In contrast, mature B cells
contacting Ag enter G1 phase of the cell cycle
and up-regulate many proteins involved in adhesion and Ag presentation
to Th cells. Strong BCR stimulation can also induce B cell
proliferation (3). Th cells enhance this proliferation and
induce B cell differentiation into the Ab-secreting plasma cell state
by providing cell-cell contact signals via CD40 and by releasing
cytokines, such as IL-4 and IL-5 (3).
Both genetic and pharmacologic approaches have been used to determine
how individual signaling pathways participate in these biological
responses to Ag. For example, wortmannin, which inhibits PI 3 kinase,
has been shown to block BCR-induced apoptosis in a human immature B
cell line (4). In addition, gene disruption experiments
demonstrate that PI 3 kinase participates in B cell development and
proliferation as well (5, 6). Experiments with
pharmacologic agents that mimic the second messengers resulting from
PLC- activation, and with mutant cell lines defective in this
pathway, have demonstrated its importance for BCR-induced growth arrest
and apoptosis of B cell lines (7, 8, 9, 10) and proliferation of
mature splenic B cells (11, 12). Moreover, mice and cell
lines with targeted mutations in the signaling proteins Vav (13, 14) and HS1 (15, 16) both exhibit decreased B cell
responses to Ag.
By comparison, relatively little is known about the role(s) of the Ras-Raf-MEK-ERK MAP kinase pathway in B cell responses to Ag. When in its active GTP-bound state, Ras activates the serine/threonine protein kinase Raf, which phosphorylates and activates the protein kinases MEK1 and MEK2. The MEK1/2 kinases in turn phosphorylate and activate the classical p44 and p42 MAP kinases, also called ERK 1 and 2, respectively (17). This pathway has been found to be important in many cell types for receptor-induced biological responses, such as proliferation, growth arrest, and differentiation (17, 18, 19, 20, 21, 22). In B cells, Ras-ERK MAP kinase activation correlates with BCR-induced growth arrest and apoptosis in immature B cells, and cell-cycle entry and activation in mature B cells (3, 23, 24). We used two structurally and mechanistically distinct MEK1/2 inhibitors, PD98059 and U0126 (25, 26, 27), to investigate the role that this pathway plays in these BCR-induced events. Both inhibitors suppressed BCR-induced activation of the ERK MAP kinases in all B cells examined. These inhibitors also suppressed up-regulation of the early response gene product Egr-1 in mature splenic B cells and in the WEHI-231 immature B cell line and partially blocked the induction of the CD44 adhesion protein and CD69 activation marker in mature splenic B cells. In addition, both inhibitors blocked BCR-induced proliferation of mature splenic B cells. In contrast, MEK-ERK inhibition failed to suppress BCR-induced growth arrest or apoptosis of WEHI-231 cells and did not inhibit BCR-induced apoptosis in primary immature splenic B cells. Thus, the Ras-ERK MAP kinase pathway is important for mediating some, but not all, specific B cell responses to Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Affinity-purified goat-anti-mouse IgM was obtained from
Jackson ImmunoResearch (West Grove, PA). The anti-IgD mAb H
a/1
was generously provided by Fred Finkelman, University of Cincinnati, as
a mouse ascites fluid. Mouse rIL-4 was from Genzyme (Cambridge, MA).
Hybridomas HO13.4 (anti-Thy1), 53.6.172 (anti-CD8), and GK1.5
(anti-CD4) were obtained from American Type Culture Collection
(Manassas, VA). Anti-ERK2 (C-14), anti-c-Jun N-terminal kinase
(JNK) 1 (C-17), and anti-Egr-1 (C-19) Abs were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Anti-ACTIVE MAP kinase polyclonal
Ab (pTEpY) was purchased from Promega (Madison, WI). PE- and
FITC-conjugated RA3–6B2 (anti-B220), FITC-conjugated GL-1
(anti-CD86), FITC-conjugated anti-CD44, FITC-conjugated
anti-CD69, and anti-CD16/CD32 were purchased from PharMingen
(San Diego, CA). 5-Bromo-2'-deoxyuridine (BrdUrd) was purchased from
Sigma (St. Louis, MO). Anti-BrdUrd-FITC was purchased from Becton
Dickinson (San Jose, CA). Avidin-TriColor was obtained from Caltag
(South San Francisco, CA). [3H]Thymidine was
purchased from NEN (Boston, MA). PD98059 and the GST-Elk-1 fusion
protein were purchased from New England Biolabs (Beverly, MA). U0126
was purchased from Calbiochem (La Jolla, CA). The GST-Jun1–79(1–79) fusion
protein was prepared as described (28). PD98059 and U0126
were dissolved in DMSO and stored at -20°C until used.
Cell culture
Splenic B cells were cultured in RPMI 1640 medium supplemented with 10% FCS (Life Technologies, Rockville, MD), 2 mM pyruvate, 20 mM glutamine, 50 µM 2-ME, and penicillin/streptomycin. WEHI-231 cells were cultured as previously described (2). In all experiments, PD98059, U0126, or DMSO carrier was added to the splenic B cells or WEHI-231 cells 1 h before stimulation, unless otherwise indicated. Cells were stimulated and cultured in medium that always contained PD98059, U0126, or an equivalent volume of DMSO carrier.
Isolation and apoptosis of immature splenic B cells
Three-week-old BALB/c mice were obtained from The Jackson
Laboratory (Bar Harbor, ME). Mice were given 500-rad whole-body
irradiation 13.5 days before sacrifice (29). A single cell
suspension of the spleen cells in either ice-cold RPMI 1640 or DMEM
with 10% FCS was treated with an equal volume of 17 mM Tris-HCl and
1.44% ammonium chloride for 5 min on ice to lyse erythrocytes. The T
cells and IgD+ B cells were lysed by incubating
in a cocktail containing anti-CD4, anti-CD8, anti-Thy1.2,
and Ha/1 anti-IgD ascites on ice for 30 min, followed by
complement lysis at 37°C for 30 min. The isolated immature
splenocytes were IgMhigh,
IgDlow, B220high, and
negative for Gr1, CD3, and Mac-1 as shown by flow cytometry. The
immature splenic B cells at a density of 2–4 x
106/ml were incubated for 30 min to 1 h with
20 µM PD98059 and then stimulated with various concentrations of
anti-IgM in the presence or absence of 20 µM PD98059 for 18
h. The apoptosis of immature splenic cells was quantified by annexin
V/PI staining (Oncogene Research Products, Cambridge, MA) following the
manufacturer’s protocol.
Isolation and stimulation of mature splenic B lymphocytes
Female (C57BL/6 x DBA/2) F1 (BDF1) mice from Simonsen Laboratories (Gilroy, CA) or Charles River Breeding Laboratories (Hollister, CA) were sacrificed at 2–4 mo of age. Small resting B cells were isolated from the spleen as described (30). Cells were collected from the 60%/70% Percoll interphase, washed, and rested in normal medium at 37°C for 30 min to 2 h before treatment with DMSO or inhibitors. The purity of the splenic B cell preparations was typically at least 96% B220+, as verified by flow cytometry.
Splenic B cells (1 x 107 cells/ml) were stimulated with goat anti-mouse IgM and/or IL-4 (50 U/ml) for the indicated times and washed twice with ice-cold PBS containing 1 mM Na3VO4 before lysis.
ERK2 and JNK immunocomplex protein kinase assays
Cells were stimulated for 4 min (ERK2 assays) or for 15 min (JNK assays), and subsequently lysed as previously described (31). Analysis of the kinetics of activation of these MAP kinases indicated that strong inductions were reproducibly seen at these times. Anti-JNK and anti-ERK2 immunoprecipitations were performed as described (28). Immunocomplex protein kinase assays were performed on the immunoprecipitates as described (30), with GST-Elk-1 as the substrate for ERK2 and GST-Jun1–79(1–79) as the substrate for JNK. Reaction products were resolved by SDS-PAGE, transferred to nitrocellulose, and detected by autoradiography. Quantitation of kinase activity was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). To calculate inhibition of kinase activity by PD98059, we used the formula: Percent inhibition = (1 – ((stimpd - unstimpd)/(stim - unstim))) x 100. Blots were reprobed with anti-ERK2 Abs (1:1000) to ensure that all reactions contained equal amounts of ERK2.
Egr-1 and anti-phosphoMAP kinase immunoblotting
For examining Egr-1 up-regulation, cells (5 x 106 cells/lane) were stimulated for 1 h and lysed in SDS-PAGE sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose. The anti-Egr-1 Ab was used at 1:500 for detection. General SDS-PAGE and immunoblotting procedures were as described (2). For anti-phosphoMAP kinase immunoblotting time course experiments, 1 x 107 splenic B cells/ml or 2.5 x 106 WEHI-231 cells in 0.5 ml were stimulated with 10 µg/ml goat anti-mouse IgM for the indicated times, washed, and lysed in SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-ACTIVE MAP kinase Ab (pTEpY) (diluted 1:1000). For anti-IgM dose-response experiments, cells were stimulated with the indicated concentrations of anti-IgM for 4 min before washing, lysis, SDS-PAGE and immunoblotting.
Growth arrest and apoptosis of WEHI-231 cells
For growth arrest, WEHI-231 cells were plated in triplicate at 2 x 104 cells/well in 96-well plates and stimulated with goat-anti-mouse IgM for 28 h. Cells were pulsed with [3H]dT (1 µCi/well) for the last 4 h, harvested onto glass-fiber filters (Wallac, Gaithersburg, MD), and the amount of incorporated [3H]dT was determined using a Betaplate reader (Wallac). For apoptosis, WEHI-231 cells were plated at 1.5 x 105 cells/ml in 12-well plates, stimulated, and cultured for 48 h. Cell survival was measured by ability to exclude 1 µg/ml propidium iodide, as determined by flow cytometry, or by annexin V staining following protocols supplied by the manufacturer.
Proliferation and survival of mature splenic B cells
Proliferation of murine splenic B cells was measured either by incorporation of [3H]thymidine or incorporation of BrdUrd. For [3H]dT incorporation assays, splenic B cells were plated in triplicate in 96-well plates (2.5 x 106 cells/ml), in the presence of anti-IgM ± 50 U/ml IL-4, and cultured for 45 h. Cells were pulsed with [3H]dT for the last 5 h of culture, and incorporation was determined as described above. In vitro BrdUrd-labeling experiments were performed as described (30). Briefly, RBC-depleted splenocytes were plated at 5 x 105 cells/ml, stimulated, and cultured for 48 h. BrdUrd (10 µM) was added during the final 24 h. Cells were fixed, permeabilized with 0.5% Tween 20 in PBS, the DNA denatured, and the cells subsequently were costained with anti-B220 and anti-BrdUrd and analyzed by flow cytometry. The data are presented as the percentage of B220+ cells that are BrdUrd+. For survival experiments, RBC-depleted splenocytes or purified splenic B cells were cultured for 45 h. B220+ cells were then tested by flow cytometry for their ability to exclude 1 µg/ml propidium iodide.
Up-regulation of cell surface molecules
RBC-depleted splenocytes were cultured at 5 x 105 or 1 x 106 cells/ml. Cells were stimulated with goat anti-mouse whole Abs or F(ab')2 fragments for 18 h. Cells were harvested, Fc receptors blocked with anti-CD16/CD32, and subsequently costained with anti-B220 and anti-B7-2 (CD86), anti-CD44, or anti-CD69 and analyzed by flow cytometry. Only live (as determined by propidium iodide exclusion or by forward- and side-scatter profiles) B220+ cells were analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro protein kinase assays were performed to determine whether
PD98059 inhibited the BCR-induced activation of the ERK2 form of MAP
kinase, as expected. As previously reported (23, 32, 33),
stimulation of the WEHI-231 immature B cell line with anti-IgM Abs
substantially increased the activity of ERK2 (Fig. 1
A). This activation was
inhibited in a dose-dependent manner by PD98059 at a 4-min time point
(Fig. 1A). PD98059 also inhibited ERK2 activation over a
24-h time course (see below and data not shown). At low concentrations
of stimulating Ab, ERK2 activation was always greatly inhibited by
5–20 µM PD98059 and in some experiments completely blocked.
Inhibition by 10–20 µM PD98059 was substantial but not absolute at
higher doses of stimulating Ab, consistent with previous results in
other systems (26). Likewise, 5–10 µM PD98059
suppressed ERK2 activation in purified murine splenic B cells
stimulated with anti-IgM (Fig. 1B) and anti-IgM plus
IL-4 (Fig. 1C). IL-4 did not reproducibly increase or
decrease BCR-induced ERK2 activation at a 4-min time point (Fig. 1C) and it did not alter the magnitude and kinetics of
BCR-induced ERK2 activation over a 2-h time course (data not shown).
Moreover, PD98059 was equally as effective at blocking BCR-induced ERK2
activity in the presence or absence of IL-4 (compare Fig. 1, H and I). In contrast, PD98059 in this dose range
did not decrease or increase the BCR-induced activation of JNK, another
MAP kinase protein that is downstream of the MEK family member MKK4
(34), in purified splenic B cells (Fig. 1D) or
in WEHI-231 B cells (data not shown). PD98059 also did not block the
BCR-induced activation of the tyrosine kinase Syk or the appearance of
tyrosine phosphoproteins in the lysates of BCR-stimulated B cells (data
not shown). Therefore, PD98059 seemed to inhibit MEK1/2, and, thus, ERK
specifically in B cells, as it does in other cell types up to
concentrations of at least 50 µM (25, 26, 35, 36).
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E) and in WEHI-231 B cells
(Fig. 1F). Similar to the above protein kinase assays, the
inhibition of ERK phosphorylation provided by PD98059 was significant
but incomplete. (However, note that by the end of the time course, ERK
phosphorylation was inhibited back down to levels approximating
baseline.) In contrast to PD98059, U0126 blocked BCR-induced
phosphorylation of ERK completely or almost completely at all time
points in both cell types. Both inhibitors also blocked ERK
phosphorylation over a wide range of stimulating Ab in purified B cells
(Fig. 1G) and in WEHI-231 cells (data not shown). As with
the time course assays, U0126 inhibited ERK phosphorylation more
completely than did PD98059. PD98059 inhibits the BCR-induced up-regulation of Egr-1, CD44, and CD69
BCR-stimulated B cells up-regulate the expression of many
transcription factors (38, 39). This up-regulation leads
to changes in the expression of proteins important for cell-cycle
regulation, cell-cell adhesion, and activation of T cells. One
BCR-induced transcription factor is Egr-1, which is important for the
BCR-induced expression of the adhesion proteins, CD44 and ICAM-1
(40, 41). In addition, putative Egr-1 binding sites exist
in the promoter of the gene encoding the human CD69 activation marker
(42). BCR-induced transcription of a reporter gene linked
to the egr-1 promoter can be blocked by dominant negative
forms of Ras and Raf (39). Therefore, we wished to
determine whether BCR-induced Egr-1, CD44, and CD69 protein expression
required MEK activity. PD98059 treatment (20 µM) greatly suppressed
anti-IgM-induced up-regulation of Egr-1 in mature splenic B cells
(Fig. 2A) and in WEHI-231 B
lymphoma cells (Fig. 2B). In addition, both PD98059 and
U0126 partially inhibited the BCR-induced up-regulation of CD44 and
CD69 in mature splenic B cells (Fig. 2, C–E). In contrast,
PD98059 and U0126 did not inhibit BCR-induced expression of B7-2 (CD86)
(Fig. 2F), a protein that provides an important
costimulatory signal for Th cells (43).
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BCR stimulation induces WEHI-231 B cells to arrest in the
G1 phase of the cell cycle and then undergo
apoptosis (7, 8, 9, 44, 45). To determine whether the MEK-ERK
pathway is important for these events, we pretreated WEHI-231 cells
with varying amounts of PD98059 or U0126 before stimulation with
anti-IgM Abs. No inhibition of BCR-induced growth arrest was seen,
as assessed by incorporation of [3H]thymidine
(Fig. 3, A and B).
However, 20 µM PD98059 and 10 µM U0126 did reproducibly decrease
basal proliferation of WEHI-231 cells, suggesting that the MEK-ERK
pathway may be required for the proliferation of this cell line.
PD98059 and U0126 also failed to inhibit the BCR-induced apoptosis of
these cells, as determined by propidium iodide exclusion (Fig. 3, C and D), and by annexin V staining (data not
shown). These data suggest that the MEK-ERK pathway is not required for
anti-IgM-mediated growth arrest or apoptosis in WEHI-231 cells.
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A).
Similar results were obtained when apoptosis was judged by failure to
exclude propidium iodide, representing a late step in apoptosis (data
not shown). PD98059 treatment did not decrease the fraction of immature
B cells that were apoptotic upon stimulation with anti-IgM, whereas
it did partially inhibit ERK2 activation (by 50–72% in the experiment
shown in Fig. 4B), as seen in the other B cell
populations tested. It should be noted that PD98059 treatment by
itself often increased the fraction of cells that had initiated
apoptosis. In some experiments, this effect was small, or, as in the
example shown in Fig. 4A, moderate. In other experiments,
the fraction of immature B cells initiating apoptosis was similar in
the cultures treated with PD98059 alone, anti-IgM alone, and the
cultures containing both agents. It may be that stimulation of the
Ras-ERK MAP kinase pathway by some element of the culture conditions
(serum, for example) provides a survival signal, such that inhibition
of this pathway leads to greater apoptosis even in the absence of BCR
stimulation.
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In contrast to its effect on immature B cells, BCR
engagement causes mature B cells to enter the G1
phase of the cell cycle and to proliferate. Addition of T cell-derived
cytokines such as IL-4 enhances this proliferative response
(47, 48, 49). We wished to determine whether the MEK-ERK
pathway is important for BCR-induced proliferation of murine splenic B
cells. Anti-IgM treatment induced B cell proliferation in a
[3H]thymidine incorporation assay, and PD98059
blocked this proliferation in a dose-dependent manner (Fig. 5A). In agreement with earlier
reports (47, 48, 49), the addition of IL-4 greatly enhanced
the proliferation of the anti-IgM-stimulated B cells (Fig. 5B, note different scale on x-axis). PD98059 was
also able to suppress B cell proliferation in the presence of IL-4, but
to a lesser degree than when cells were stimulated with anti-IgM
alone. IL-4 increases [3H]thymidine
incorporation in this assay both by acting as a B cell survival factor
and by increasing the percentage of B cells that enter the cell cycle
(47, 48, 49). We investigated whether a block in MEK-ERK
activity would inhibit one or both of these effects. As shown in Fig. 5C, the percentage of B cells that entered S phase in
response to anti-IgM plus IL-4 was significantly diminished by
treatment with either PD98059 or U0126. U0126 proved to be the more
potent inhibitor, because 10 µM U0126 was sufficient to block
proliferation essentially completely. In contrast, neither inhibitor
substantially altered B cell survival in the presence of IL-4 (Fig. 5D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D and
data not shown) (25, 26, 35, 36). U0126 also specifically
inhibits MEK1/2, without blocking the activation of JNK, p38 MAP
kinase, or several other protein kinases (27, 50, 51). In
vitro immunocomplex kinase assays demonstrated that PD98059 was able to
substantially inhibit BCR-induced ERK2 activity in a dose-dependent
manner (Fig. 1, A–C). Likewise, anti-phosphoMAP kinase
blots determined that U0126 and PD98059 both inhibited BCR-induced
ERK1/2 activation over a wide range of times and doses of stimulation
(Fig. 1, E–G). Treatment with PD98059 also inhibited the
BCR-induced expression of the early response gene product Egr-1,
demonstrating the importance of the MEK-ERK kinase pathway for the
expression of this transcription factor. This result is consistent with
previous studies demonstrating the requirement of promoter serum
response elements (SREs) for egr-1 expression
(52), and the ability of dominant negative mutants of Ras
and Raf to block egr-1 promoter-driven reporter gene
expression in response to BCR cross-linking (39).
Anti-IgM-induced proliferation of splenic B cells was also
substantially inhibited by PD98059 and U0126, in the absence and
presence of IL-4. Thus, MEK1/2 activity and presumably ERK activity
play important roles in BCR-induced proliferation. In contrast,
this pathway is not required for the BCR-induced expression of B7-2
(CD86) in mature splenic B cells, for growth arrest and apoptosis of
the immature WEHI-231 B cell line, or for BCR-induced
apoptosis of immature splenic B cells. In addition to demonstrating
that some BCR-induced effects are not dependent on the MEK-ERK
signaling pathway, these results demonstrated that PD98059 and U0126
did not exert significant toxicity or nonspecific inhibition of B cell
functions. Moreover, because these two structurally and mechanistically
distinct inhibitors gave very similar results, this strongly suggests
that they are indeed working via the MEK-ERK MAP kinase pathway and not
via some other unknown actions.
Our results indicating that activation of the Ras-ERK MAP kinase
pathway is important for proliferation of splenic B cells are
consistent with results obtained in other cell types, such as
fibroblasts and PC12 cells, where this pathway also contributes to
cellular proliferation (17, 18, 19, 20, 21, 22). The mechanism by which
ERK promotes BCR-mediated proliferation is not yet defined, although it
is likely to involve changes in gene expression; BCR stimulation
rapidly induces changes in expression of several important cell cycle
regulators, such as cyclins, cyclin-dependent kinases, and cell-cycle
inhibitors (53, 54, 55). The ERK kinases phosphorylate and
activate several transcription factors, including Elk-1 and SAP-1a,
which help enhance transcription of genes whose promoters contain SREs
(34). Indeed, the egr-1 early response gene
contains several SREs and is rapidly induced upon BCR cross-linking,
and ERK activity was required for Egr-1 expression (Fig. 2).
In addition to the Ras-ERK MAP kinase pathway, HS1, Vav, and the
PLC- pathway are also important for BCR-mediated proliferation
(11, 12, 13, 14, 15). Just as ERK kinases activate Elk-1 and Sap-1a,
each of the above signaling pathways are likely to activate other
pre-existing transcription factors. For example, the PLC- pathway
activates the serine/threonine phosphatase calcineurin, which
dephosphorylates and activates NFAT (56). Together, these
pre-existing but newly activated transcription factors induce
expression of early response genes, many of which, like
egr-1, encode transcription factors themselves. These
secondary transcription factors likely control the expression of many
other genes important for events such as proliferation and interaction
with Th cells (3). Indeed, Egr-1 has been implicated in
the BCR-induced expression of CD44 and ICAM-1 (see below). Different
signaling pathways, then, could regulate the expression of different
cell cycle genes by virtue of the transcription factors they activate.
Some cell-cycle genes may require the activation of transcription
factors controlled by one signaling pathway and others by multiple
pathways.
The induction of proliferation by the BCR is known to be dramatically
enhanced by IL-4. We have found that this enhancement is not paralleled
by an increase in ERK activation (Fig. 1C and data not
shown). Thus, IL-4 appears to promote B cell proliferation by its
action on other signaling pathways, perhaps those involving IRS-1,
IRS-2, or Stat-6 (57, 58, 59, 60, 61). Interestingly, high
concentrations of PD98059 only partially inhibited proliferation in
response to anti-IgM and IL-4, whereas it completely or almost
completely inhibited proliferation to similar or even higher doses of
anti-IgM alone. This difference in proliferative inhibition
occurred despite the fact that ERK activation was suppressed equally
well in B cells stimulated in the absence or presence of IL-4 (Fig. 1, H and I). Thus, it appears that BCR-induced B
cell proliferation has a decreased requirement for MEK/ERK signaling
events in the presence of IL-4. However, this proliferation of B cells
was still dependent on a low level of ERK activation, because it was
completely blocked by the more effective MEK1/2 inhibitor, U0126.
MEK-ERK activity appears to be required for cell-cycle entry (Fig. 5C) rather than for survival (Fig. 5D).
BCR-stimulated mature B cells also up-regulate adhesion molecules and a
variety of other cell surface proteins, some of which are important for
promoting interaction with Th cells. MEK-ERK inhibition failed to block
up-regulation of B7-2 (CD86) (Fig. 2F), a protein that
provides a costimulatory signal to T cells through its interaction with
CD28 (43). In contrast, BCR-induced up-regulation of the
transcription factor Egr-1 was suppressed by PD98059, as mentioned
above. Egr-1 expression has been implicated in the up-regulation of the
adhesion proteins CD44 and ICAM-1 (40, 41). CD44 has been
proposed to facilitate B cell migration to and retention in secondary
lymphoid follicles, where B cells interact with T cells
(40), and ICAM-1 expression helps mediate adhesion between
Ag-presenting B cells and Th cells during T cell activation, and
subsequent stimulation of the B cells (3, 62).
Up-regulation of CD44 expression was partially blocked by PD98059 (Fig. 2C) and U0126 (Fig. 2D), consistent with the
hypothesis that Egr-1 plays a significant role in this induction. These
data provide evidence that in addition to its role in BCR-induced
proliferation, the Ras-ERK MAP kinase pathway is important for the
up-regulation of specific proteins involved in cell-cell interactions
and B cell trafficking. In addition, putative Egr-1 binding sites are
found in the human CD69 promoter (42), and the Ras pathway
is involved in its up-regulation in T cells (63, 64). Our
data demonstrate that CD69 up-regulation is MEK1/2-dependent in murine
B cells (Fig. 2E) and are consistent with a role for Egr-1
in its up-regulation.
These data are also consistent with recently published results that PD98059 inhibits the BCR-induced up-regulation of Egr-1 mRNA (65). However, in contrast to Egr-1, this group found that BCR-induced induction of Egr-2 mRNA was not significantly blocked by PD98059. The persistence of Egr-2 expression in PD98059-treated B cells may explain why CD44 and CD69 up-regulation is only partially inhibited by PD98059 and U0126.
Whereas in most situations the Ras-ERK MAP kinase pathway seems to be
involved in promoting cell growth or differentiation, this pathway has
been implicated in growth arrest in murine fibroblasts
(20, 21, 22), and ERK activity also has been reported to be
important for apoptosis in some instances (24, 66, 67).
However, we observed no inhibition in BCR-induced growth arrest or
apoptosis in WEHI-231 B cells treated with PD98059 or with U0126, which
blocks BCR-induced MEK-ERK activity almost completely (Fig. 3).
Moreover, 20 µM PD98059 also failed to inhibit BCR-induced apoptosis
in immature splenic B cells (Fig. 4A), although this same
concentration of PD98059 dramatically blocked BCR-induced proliferation
and Egr-1 and CD69 up-regulation in mature splenic B cells.
In contrast to our results, Lee and Koretzky (24) found
that overexpression of MKP-1, a phosphatase that can dephosphorylate
and inactivate ERK1/2, did block apoptosis of WEHI-231 cells. This
conclusion was based on experiments examining apoptosis at early times
when <20% of cells had undergone apoptosis, so it is possible that
MKP-1 overexpression delayed but did not prevent apoptosis. However, it
should be noted that we did not observe inhibition of apoptosis by
PD98059 even at relatively early times when apoptosis had occurred in a
minority of cells (data not shown). Alternatively, it is possible that
MKP-1 overexpression exerted its effects by dephosphorylating other MAP
kinases, such as JNK or p38 MAP kinase. MKP-1 has been demonstrated to
dephosphorylate and inhibit the activity of JNK and p38 in many cell
types, including lymphocytes (68, 69, 70, 71), and BCR
cross-linking has been reported to activate JNK and p38 (Fig. 1D) (23, 33, 72, 73, 74, 75). Moreover, Graves and
colleagues (74) have presented data indicating that JNK
and p38 (73, 74) MAP kinases are involved in BCR-induced
apoptosis in human B cell lines, although contrasting data have been
presented that p38 is not required for apoptosis in WEHI-231 cells
(75). If MKP-1 overexpression inactivates JNK and/or p38
in B cells, as it does in other cell types, this could explain the
block in BCR-induced apoptosis reported by Lee and Koretzky
(24). Indeed, MKP-1 blocks UV-induced apoptosis in U937
human leukemia cells, apparently by inhibiting JNK activity
(70). In contrast to MKP-1 overexpression, PD98059 and
U0126 appear to inhibit ERK activity specifically without inhibiting
JNK or p38 (Fig. 1) (25, 26, 35, 36, 50, 51). Moreover,
neither inhibitor blocked BCR-induced apoptosis in our experiments,
even when BCR-induced ERK activity was blocked almost completely (Figs. 3 and 4). Thus, whereas JNK and/or p38 activity may be required for
BCR-induced apoptosis of immature B cells, we conclude that ERK
activity likely is not required.
The data presented here demonstrate that the Ras-ERK MAP kinase pathway plays an important role in a subset of B cell responses to Ag. BCR-induced proliferation and up-regulation of specific proteins important for B cell function are dependent on activation of this pathway. In contrast, anti-IgM-induced growth arrest and apoptosis do not appear to require MEK-ERK activity. BCR cross-linking activates many signaling pathways in both immature and mature B cells; however, it appears that particular biological responses to Ag require only some of these events.
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Acknowledgments |
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a/1 mAb. We also thank V.
Chan, A. Finn, C. Fitzer-Attas, M.
L. MacKichan, T. Tian, and C. Burke for critical
review of the manuscript. |
Footnotes |
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2 Current address: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112.
3 Current address: University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
4 Address correspondence and reprint requests to Dr. Anthony L. DeFranco, G. W. Hooper Foundation, Box 0552, University of California, San Francisco, San Francisco, CA 94143-0552.
5 Abbreviations used in this paper: BCR, B cell Ag receptor; PLC, phospholipase C; PI, phosphatidylinositol; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; BrdUrd, 5-bromo-2'-deoxyuridine; JNK, c-Jun N-terminal kinase; SRE, serum response element.
Received for publication November 29, 1999. Accepted for publication January 8, 2001.
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