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Departments of
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Microbiology and
Pharmacology, University of Washington, Seattle, WA 98195; and
The Kennedy Institute of Rheumatology, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation and gene expression. Cross-linking CD40 rapidly
stimulates both p38 MAPK and its downstream effector, MAPKAPK-2.
Inhibition of p38 MAPK activity in vivo with the specific
cell-permeable inhibitor, SB203580, under conditions that completely
prevented MAPKAPK-2 activation, strongly perturbed CD40-induced
tonsillar B cell proliferation while potentiating the B cell receptor
(BCR)-driven proliferative response. SB203580 also significantly
reduced expression of a reporter gene driven by a minimal promoter
containing four NF-B elements, indicating a requirement for the p38
MAPK pathway in CD40-induced NF-B activation. However, CD40-mediated
NF-B binding was not affected by SB203580, suggesting that NF-B
may not be a direct target for the CD40-induced p38 MAPK pathway. In
addition, SB203580 selectively reduced CD40-induced CD54/ICAM-1
expression, whereas CD40-dependent expression of CD40 and CD95/Fas and
four newly defined CD40-responsive genes cIAP2, TRAF1, TRAF4/CART and
DR3 were unaffected. Our observations show that the p38 MAPK pathway is
required for CD40-induced proliferation and that CD40 induces gene
expression via both p38 MAPK-dependent and -independent
pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the earliest detectable signaling events following CD40 engagement are activation of the Src protein tyrosine kinase (PTK) member Lyn, phosphoinositide 3-kinase (PI 3-kinase) stimulation and Ras activation (23, 24, 25). In addition, CD40 engagement activates two subfamilies of the stress-activated protein kinases, the c-jun amino-terminal kinase (JNK/SAPK; Refs. 26–29) and p38 MAPK (28, 30), while inducing little or no activation of the more distantly related extracellular signal-regulated mitogen-activated protein kinases (ERK; Refs. 26–28). However, the role(s) of either JNK or p38 MAPK in the biologic functions of CD40 remain uncharacterized.
Considerable attention has been focused toward investigating the role of p38 MAPK in the regulation of gene expression at both transcriptional and translational levels. Thus, CHOP (GADD 153), a stress-activated member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors (31), and MEF2C, a member of the myocyte enhancer factor 2 family of transcription factors (32), are phosphorylated by p38 MAPK. In addition, MAPKAPK-2, a downstream target for p38 MAPK, phosphorylates CREB in response to FGF and arsenite (33). At the transcriptional level, the p38 MAPK pathway is required for c-jun and c-fos mRNA expression in response to UV light and anisomycin (34) and IL-1ß-induced cyclooxygenase-2 mRNA synthesis (35).
Thus, since engagement of the CD40 receptor leads to activation of a
variety of transcription factors, including NF-B, AP-1, NFAT, and
STATs 3 and 6 (36, 37, 38, 39, 40, 41), we investigated the role of the p38 MAPK
pathway in CD40-induced transcriptional activation using the specific,
cell-permeable p38 MAPK inhibitor, SB203580 (42, 43). Furthermore, we
tested the role of the p38 MAPK pathway in CD40-induced gene expression
at the transcriptional level.
Our results demonstrate that CD40 activates the p38 MAPK pathway in
both human tonsillar B cells and multiple B cell lines. We show that
the p38 MAPK pathway is required, at least in part for CD40-induced
NF-B activation and that the induction of CD40-responsive genes
occurs via both p38 MAPK-dependent and -independent pathways. Moreover,
we describe a role for the p38 MAPK pathway in CD40-driven
proliferation of mature tonsillar B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mAb used in these studies were: G28-5 (IgG1) to human CD40
(44), 1C10 to murine CD40 (45), G19-4 (IgG1) to CD3 (46), G28-7 (IgG1)
to CD22 (47), G28-1 (IgG1) to CD37 (48), G28-8 (IgG1) to Bgp95, which
is defined by our single agonistic Ab and does not yet have a CD
designation (49), and LB-2 (IgG2b) to CD54 (50). We elected
to use the G28-5 mAb to CD40 in these studies since we have previously
shown that G28-5 anti-CD40 induces equivalent activation of both
NF-B and SAPK when compared with CD40L (27, 37). F(ab')2
fragments of goat anti-mouse IgM were purchased from Jackson
ImmunoResearch Laboratories (West Grove, PA). Rabbit polyclonal
anti-p38 MAPK antiserum prepared against the C-terminal peptide
ISFVPPLDQEEMES was generated as described (51). Rabbit polyclonal
anti-JNK1 (C-17) antiserum, rabbit polyclonal anti-p50, p65 and
c-rel antiserum, and rabbit polyclonal anti-Stat1 (E-23)
antiserum were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Sheep polyclonal anti-MAPKAPK-2 antiserum was purchased from
Upstate Biotechnology (Lake Placid, NY). Affinity-purified rabbit
polyclonal anti-c-jun Ab was from New England Biolabs
(Beverly, MA). In vitro transcription and RNase protection assay kits
were obtained from PharMingen (San Diego, CA). GST-c-jun
(5-89) and GST-ATF2 were expressed in Escherichia coli, and
the fusion proteins were purified as described (27). SB203580 was
purchased from Calbiochem (San Diego, CA). EMSA oligonucleotide probes
synthesized by Life Technologies (Gaithersburg, MD), were annealed and
end-labeled with [
-32P]ATP using T4 polynucleotide
kinase (Promega, Madison, WI), followed by spin-column purification
(Chromaspin TE-10, Clontech, Palo Alto, CA). Atlas human cDNA
expression arrays were purchased from Clontech.
Cell lines and human B Cells
The murine B lymphoma cell line WEHI-231 and the human Burkitt’s lymphoma line Daudi were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin, 1 mM pyruvate, and nonessential amino acids. In addition, 50 µM 2-ME was added to all murine cell lines. Dense and buoyant human tonsillar B-enriched cells were isolated as described (52).
Immune complex assay for p38 MAPK
B cells (5–10 x 106 per sample) were
resuspended in complete RPMI 1640 medium to a density of 1 x
106/ml and stimulated with either anti-CD40 (1 µg/ml)
or goat anti-mouse IgM (10 µg/ml) for the indicated times.
Incubations were terminated by rapid dilution with approximately 40 to
50 ml ice-cold PBS and centrifuged at 500 x g for 5
min at 4°C. The supernatants were aspirated, and cells were lysed by
resuspension and brief vortexing (5 s) with 0.1 ml per 1 x
106 cells of RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl,
10 mM Na4P2O7, 25 mM sodium
ß-glycerophosphate, 1 mM EDTA, 1% (w/v) Nonidet P-40, 0.5% (w/v)
sodium deoxycholate, 0.1% (w/v) SDS, 1 mM PMSF, 1 mM
Na3VO4, 10 µM E-64, 1 µg/ml pepstatin, 10
µg/ml aprotinin, and 10 µg/ml leupeptin). Following incubation on
ice for 15 min, the lysed cells were centrifuged at 16,000 x
g for 10 min at 4°C. The lysates were added to 20 µl
packed protein A-Sepharose beads, and 5 µl rabbit polyclonal
anti-p38 MAPK antiserum was added. Following constant mixing by
inversion for 3 h at 4°C, immune complexes were pelleted at
4°C by centrifugation at 16,000 x g for 5 min. The
beads were washed twice each with i) 1 ml of RIPA, 0.1 mM
Na3VO4 and ii) 1 ml of p38 MAPK assay buffer
(25 mM HEPES, pH 7.4, 25 mM sodium ß-glycerophosphate, 25 mM
MgCl2, 2 mM DTT, 0.1 mM Na3VO4).
p38 MAPK activity was quantified by immune complex assay in a final
volume of 90 µl p38 MAPK assay buffer at 25°C for 20 min using 3
µg GST-ATF2 and 20 µM ATP containing 10 µCi
[-32P]ATP. Incubations were terminated with an equal
volume of 2x SDS sample buffer. After boiling for 5 min and brief
centrifugation, samples were resolved by 10% SDS-PAGE. The gels were
stained for 30 min with 50% methanol, 10% acetic acid, 0.005%
Coomassie brilliant blue G-250, destained for 30 min with 40%
methanol, 10% acetic acid, 3% glycerol, and dried before
autoradiography at -70°C for 3 to 6 h.
Immune complex assay for JNK1 (SAPK)
JNK1 activity was quantified by immune complex assay using GST-c-jun (5–89) as a substrate, as described previously (27).
p38 MAPK in gel kinase assay
p38 MAPK immunoprecipitates prepared as described above were
mixed with 15 µl 4x SDS-PAGE sample buffer and applied without
boiling onto a 1.5-mm thick 10% SDS-PAGE minigel copolymerized with
0.2 mg/ml MBP. Following resolution, the gel was washed twice for 30
min each with 100 ml buffer A (50 mM HEPES, pH 7.6, 5 mM 2-ME)
containing 20% (v/v) isopropanol to remove SDS. Isopropanol was
subsequently removed by washing twice for 30 min each with 100 ml
buffer A, and proteins were denatured by two consecutive washes for 15
min each with buffer A plus 6 M urea. The gel was washed sequentially
for 15 min each at 4°C with i) buffer A plus 3 M urea, ii) buffer A
plus 1.5 M urea, and iii) buffer A plus 0.75 M urea to renature
proteins; urea was subsequently removed with three washes for 15 min
each at 4°C and a final wash overnight at 4°C with buffer A
containing 0.05% (w/v) Tween 20. Following two washes for 30 min each
at 30°C with kinase buffer (20 mM HEPES, pH 7.6, 20 mM
MgCl2, 2 mM DTT), the gel was incubated for 30 min at
30°C with 10 ml 20 µM ATP plus 100 µCi
[-32P]ATP. The gel was finally washed 6 to 10 times
for 30 min each with 100 ml of 5% (w/v) TCA and 1% (w/v)
Na4P2O7 to remove unincorporated
[-32P]ATP; it was then stained with Coomassie
brilliant blue, destained, dried, and exposed to film.
Immune complex assay for MAPKAPK-2
Cell lysates (100 µl per 1 x 106 cells) were
prepared from 5 to 10 x 106 Daudi, WEHI-231, or human
tonsillar B cells stimulated with either 1 µg/ml anti-CD40 or 10
µg/ml anti-IgM as described for JNK1 (27). Lysates were mixed
with 2 to 4 µg anti-MAPKAPK-2 serum and incubated at 4°C with
constant inversion for 3 h at 4°C with the addition of 10 µl
packed protein G-Sepharose for the final 90 min. Immunoprecipitates
were washed twice each with 1 ml JNK lysis buffer containing 1 mM DTT
and 0.25 mM Na3VO4, 1 ml JNK assay buffer plus
1 mM DTT and 0.25 mM Na3VO4 and 1 ml MAPKAPK-2
assay buffer (20 mM MOPS, pH 7.2, 25 mM ß-glycerophosphate, 25 mM
MgCl2, 2 mM EGTA, 1 mM DTT, 0.25 mM
Na3VO4). Following brief incubation of the
immune complexes with 125 µM MAPKAPtide (KKLNRTLSVA) for 5 min at
30°C, in vitro kinase reactions (total volume 80 µl) were initiated
by the addition of 0.1 mM ATP containing 10 µCi
[-32P]ATP. After 30 min at 30°C, an aliquot of the
reaction mixture (60 µl) was spotted onto phosphocellulose paper
(2.5 x 2.5 cm; Whatman P81) and washed 4 to 5 times for 10 min
each with 75 mM phosphoric acid (approximately 20 ml/filter) to remove
unincorporated [-32P]ATP followed by a single wash
with acetone. Filters were dried, and 32P incorporation
into MAPKAPtide was quantified by liquid scintillation counting.
Western blot analysis of c-jun phosphorylation
B cells (5 x 106 per sample) were resuspended in complete RPMI 1640 medium to a density of 2 x 106/ml and pretreated with either solvent vehicle (DMSO) or SB203580 (0–20 µM) for 30 min at 37°C before stimulation with either anti-CD40 (1 µg/ml) or a combination of PMA (50 ng/ml) and ionomycin (250 ng/ml) for 15 min. Samples were immediately cooled on ice and centrifuged at 500 x g for 5 min at 4°C. Supernatants were aspirated, and 200 µl JNK lysis buffer containing 1x SDS sample buffer was added. Following transfer to microfuge tubes, samples were lysed by vortexing for 5 min at 4°C, heated at 95 to 100°C for 5 min, and briefly cooled on ice. Following centrifugation at 14,000 x g for 5 min, 50 µl aliquots were resolved by 10% SDS-PAGE. Resolved proteins were electrotransferred overnight to nitrocellulose membranes in 25 mM Tris, pH 8.5, 0.2 M glycine, 20% methanol at 25 V. Blots were blocked for at least 10 h with 1x TBST plus 5% nonfat dry milk, and c-jun phosphorylation was analyzed essentially according to the manufacturer’s instructions using a polyclonal anti-c-jun Ab, except that the primary Ab dilution was 1:750.
Electrophoretic mobility shift assays (EMSA)
Nuclear extracts were prepared from Daudi cells as reported previously (37) and divided into 5- to 10-µl aliquots before storage at -70°C. Once thawed, individual aliquots were never refrozen and reused. For EMSA, binding reactions contained 10 mM HEPES, pH 8.0, 20 mM NaCl, 4 mM MgCl2, 0.1 mM EDTA, 17.5% (v/v) glycerol, 0.5 µg poly(dI-dC), and 1 mM DTT. Upon brief thawing on ice, equal quantities of nuclear protein were immediately added to the binding buffer. After 10 min incubation at 25°C, 50,000 cpm 32P-labeled oligonucleotide probe was added, and the binding reactions (final volume 10 µl) were continued for an additional 30 min at 25°C. Following the addition of 1 µl of 0.5x TBE, 10% (v/v) Ficoll, aliquots (0–2.5 µl) were loaded onto preelectrophoresed (90 min) 4% native polyacrylamide gels (1 mm thick) prepared in 0.5x TBE buffer and resolved for approximately 2 to 3 h with 120 V constant voltage at 4°C. Gels were fixed in 10% (v/v) acetic acid for 15 min, dried, and exposed to film at -70°C. Supershift analyses were performed by preincubating 1 µg of either anti-p50, anti-p65, anti-c-rel, or a nonspecific rabbit control Ab (anti-Stat1) with the nuclear extract for 30 min at 25°C before addition of the radiolabeled oligonucleotide probe.
Transient transfection and reporter gene expression
Transient transfection of the Daudi B lymphoblastoid line was performed using DEAE-dextran as described previously (37), except that, following the addition of 9 vol RPMI/10% FCS, incubations were continued for 1 to 2 h at 37°C. After culture for 36 to 48 h, cells were pretreated with 0 to 20 µM SB203580 (final DMSO concentration 0.1% (v/v)) for 1 h before stimulation for 6 h at 37°C in a humidified incubator with either 0 to 1 µg/ml anti-CD40 or isotype-matched control Ab (anti-CD3, anti-CD22, or anti-CD37). Cell lysates were prepared according to the manufacturer’s instructions (Promega), and luciferase activity was quantified using a luminometer (Berthold Analytical Instruments, Nashua, NH). The data are expressed as the -fold increase in luciferase activity relative to enzyme activity from unstimulated cells using equivalent quantities of protein.
Analysis of CD40-induced gene expression using Atlas human cDNA expression arrays
Daudi cells (50–150 x 106; 1 x
106 cells/ml) were stimulated for either 0 or 2 h with
anti-CD40 (1 µg/ml) following a 30-min pretreatment with SB203580
(20 µM) or DMSO solvent vehicle (final concentration 0.1% (v/v)).
Total RNA was isolated using a guanidine isothiocyanate-based method
(Isoquick kit, Orca Biosciences, Bothell, WA) according to the
manufacturer’s instructions except that residual genomic DNA was
digested with 10 U RNase-free DNase and the mixture was subsequently
re-extracted twice as described in the instruction manual.
Poly(A)+ RNA was isolated from total RNA using a single
round of oligo(dT) latex bead chromatography according to the
manufacturer’s instructions (Oligotex Kit; Qiagen, Chatsworth, CA).
[
-32 P]dATP-labeled cDNA was subsequently prepared
from approximately 1 µg poly(A)+ RNA, purified to remove
unincorporated [-32 P]dATP using chromaspin 200
DEPC-H2O columns, and equivalent quantities (5 x
106 cpm) of [32P]-labeled cDNA were
hybridized for 16 to 20 h at 68°C with the human cDNA expression
array in a hybridization oven according to the manufacturer’s
instructions (Clontech). Following a series of washes at 68°C as
recommended by the manufacturer, the expression arrays were exposed to
BioMax MS film for 0 to 3 days at -70°C.
Northern blot analysis of CD54/ICAM-1 and CD40 expression
Total cellular RNA was prepared with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. RNA (5 µg) was resolved by denaturing electrophoresis in 0.8% formaldehyde agarose gels, transferred to a nylon membrane by capillary blotting, and cross-linked by UV irradiation. Hybridization was performed at 42°C in 50 mM sodium phosphate, pH 6.5, 50% formamide, 1x Denhardt’s solution, 5x SSC, and 250 µg/ml denatured salmon sperm DNA. The blots were washed to a final stringency of 0.1 x SSC, 0.2% (w/v) SDS at 50°C, and exposed to autoradiographic film at -70°C. cDNA fragments of human CD54/ICAM-1 and CD40 were 32P-labeled using random primers according to standard procedures.
Multi probe RNase protection assays
RNA was isolated from Daudi cells (5–10 x 10 6 cells) using a guanidine isothiocyanate-based method (Isoquick kit, Orca Biosciences), followed by a 30-min incubation at 37°C with 5 to 10 U RNase-free DNase (Promega) to digest residual genomic DNA. Total RNA was subsequently prepared using an RNA clean-up protocol supplied with the RNeasy kit (Qiagen). Total RNA was quantified by spectrophotometry at 260 nm, and 2 µg was dried by brief vacuum centrifugation. RNase protection assays were performed according to the manufacturer’s protocol, using either [32 P]-labeled hAPO-3 or hAPO-5 template sets, and protected fragments were resolved by denaturing PAGE in 0.5x TBE. After drying for 60 min at 80°C, gels were exposed to film at -70°C for varying times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since the engagement of TNF receptors such as TNFR2 and CD95/Fas stimulates JNK and p38 MAPK activities (53) and CD40 engagement itself strongly activates JNK (26, 27, 28, 29), we investigated whether CD40 ligation also stimulates p38 MAPK.
Daudi B cells were stimulated with anti-CD40 mAb for various times
and proteins were immunoprecipitated with antiserum prepared against
the carboxyl terminus of p38 MAPK (51). In vitro kinase assays were
subsequently performed on the immune complexes using a GST-ATF2 fusion
protein, a p38 MAPK substrate in vitro (53). Figure 1
A illustrates that CD40
cross-linking strongly activated p38 MAPK or other coimmunoprecipitated
kinase(s), whereas other binding or nonbinding isotype-matched control
Abs (anti-CD22, anti-CD37, and anti-CD3) either failed to
stimulate or only weakly stimulated kinase activity. To evaluate the
specificity of the anti-p38 MAPK antiserum, anti-p38
MAPK-immune complexes were resolved by SDS-PAGE, and an in gel kinase
assay was performed in which MBP, an alternative p38 MAPK substrate in
vitro (53), was copolymerized within the polyacrylamide gel. A single
CD40-stimulated, 40-kDa immunoprecipitated MBP kinase was observed
(Fig. 1B), which is entirely consistent with p38 MAPK, which
electrophoreses as a 40-kDa kinase on SDS-PAGE (42, 54); maximal
activation was observed 15 min following CD40 engagement and persisted
for at least 60 min. Both PMA and a combination of both PMA and
ionomycin also stimulated the 40-kDa MBP kinase (Fig. 1B),
in accord with earlier studies (53). The 40-kDa MBP kinase was
unequivocally resolved from both ERK1 (p44) and ERK2 (p42), two MAPKs
of similar molecular masses, neither of which was stimulated by CD40
cross-linking and both of which were observed only in cell
lysates and postimmunoprecipitate fractions (data not shown). A minor
MBP kinase activity of 56 kDa, which was not activated upon CD40
ligation but was apparently stimulated by both PMA and PMA/ionomycin,
was also coimmunoprecipitated with p38 MAPK immune complexes (Fig. 1B); its identity remains to be elucidated. Sequential
immunoprecipitation of other mammalian MAPKs (ERK1, ERK2, and JNK1),
followed by in vitro kinase assays of anti-p38 MAPK immune
complexes, resulted in no significant reduction of GST-ATF2
phosphorylation compared with assays performed with anti-p38 MAPK
immunoprecipitates alone (data not shown), suggesting that the
immunoprecipitated kinase was distinct from either ERK or JNK. In
addition, anti-p38 MAPK immune complexes did not phosphorylate
GST-c-jun, an excellent substrate for JNK (data not shown).
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C). IL-4, which is a potent regulator of human B cell
growth and differentiation (56), did not significantly affect total
anti-CD40-mediated p38 MAPK activation, although IL-4 treatment may
induce earlier p38 MAPK stimulation in response to CD40 cross-linking
(Fig. 1C). Anti-CD40 and anti-IgM both stimulate p38 MAPK in WEHI-231 B cells; lack of correlation with either an apoptotic or rescue signal
The murine immature B lymphoma cell line WEHI-231 represents a
convenient model cell system for studying the functional interaction of
BCR and CD40 signaling pathways, since cross-linking CD40 prevents
BCR-induced growth arrest in the G1 phase of the cell cycle
and subsequent apoptosis via signaling pathway(s) involving
Bcl-XL induction (57, 58). Cross-linking CD40
with the 1C10 anti-CD40 mAb rapidly elevated p38 MAPK activity
within 5 min, with a maximal fourfold activation observed after 15 min,
followed by a steady decline in activity (Fig. 2). A similar transient activation of
JNK1 upon CD40 ligation of WEHI-231 cells was also observed (data not
shown; Refs. 28, 29). BCR engagement also transiently stimulated p38
MAPK, albeit less strongly than with anti-CD40, with a maximal
twofold activation after 10 min (Fig. 2). Simultaneous engagement of
the BCR and CD40 appeared to weakly potentiate CD40-induced p38 MAPK
activation at all times following receptor cross-linking but failed to
stimulate p38 MAPK in a sustained manner (Fig. 2). Our data suggest
that rapid and transient p38 MAPK activation, at least in the WEHI-231
cell line, does not correlate with either the ability of BCR ligation
to induce programmed cell death or the role of CD40 to rescue cells
from BCR-induced apoptosis. These observations are consistent with the
inability of the p38 MAPK inhibitor, SB203580, to block
anti-IgM-induced apoptosis in WEHI-231 cells (30).
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To independently confirm that both CD40 and BCR cross-linking
stimulate the p38 MAPK pathway, we investigated whether anti-CD40
and anti-IgM activated MAPKAPK-2 activity in Daudi, WEHI-231, and
human tonsillar B cells. Both MAPKAPK-2 and its recently identified
homologue, MAPKAPK-3, are phosphorylated and activated by p38 MAPK in
vivo (43, 59). CD40 cross-linking rapidly and transiently stimulated
MAPKAPK-2 in Daudi cells, with a maximal three- to fourfold activation
following 10 to 20 min stimulation (Fig. 3A). Neither of the
isotype-matched Abs to CD3 or CD22 significantly elevated MAPKAPK-2
activity, showing that the response to CD40 was stimulus specific (Fig. 3B). Similarly, both CD40 and BCR engagement
stimulated MAPKAPK-2 in WEHI-231 cells. Anti-CD40
induced a two- to threefold maximal activation whereas anti-IgM
induced a smaller 1.5- to 2-fold increase in MAPKAPK-2 activity (Fig. 3D), consistent with the ability of each stimulus to
activate p38 MAPK (Fig. 2). The kinetics of both BCR- and CD40-induced
MAPKAPK-2 activation were also similar to those for p38 MAPK, with peak
stimulation after 10 to 20 min (Fig. 3, A and D).
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, C and E); 1 µM SB203580
completely abolished MAPKAPK-2 stimulation in response to either BCR or
CD40 ligation (Fig. 3, C and E). In contrast to
both B cell lines examined, dense human tonsillar B cells expressed
relatively higher levels of constitutive MAPKAPK-2 activity (Fig. 3F). Nevertheless, anti-CD40 induced a 1.5-fold
stimulation of MAPKAPK-2 activity in dense human tonsillar B
lymphocytes (Fig. 3F). Pretreatment with SB203580
dose-dependently inhibited MAPKAPK-2 activity to levels that were
significantly less than those in unstimulated cells, showing that
CD40-induced MAPKAPK-2 activity is strongly p38 MAPK
dependent (Fig. 3F).
Since CD40 cross-linking also strongly stimulates JNK (26, 27, 28, 29), a
highly related MAPK family member, it was important to exclude the
possibility that the p38 MAPK inhibitor SB203580 blocked CD40-induced
JNK activation. Hence, since SB203580 is a reversible p38 MAPK
inhibitor and is thus washed away from kinases during cell lysis and
immunoprecipitation, we tested whether SB203580 abrogated
anti-CD40-induced c-jun phosphorylation, a downstream
target for JNK in vivo. Both anti-CD40 and PMA/ionomycin induced a
similar transient JNK activation, with maximal stimulation after 15 min
as detected by both in vitro immune complex kinase assays with
GST-c-jun as an exogenous JNK substrate (data not shown) and
c-jun Western blot analysis using anti-c-jun
antiserum (Fig. 3G). Moreover, SB203580 at concentrations
(0–20 µM) that totally blocked anti-CD40-mediated
MAPKAPK-2 activation, did not significantly attenuate either
anti-CD40 or PMA/ionomycin-induced c-jun phosphorylation
(Fig. 3G).
In addition, although CD40 engagement does not stimulate either ERK1 or ERK2 in Daudi cells (Ref. 27; data not shown), we examined whether SB203580 inhibited the ERK pathway in B cells by testing the effect of SB203580 upon anti-IgM-induced rsk2 activation, which is a MEK-ERK-dependent pathway (data not shown), using an in vitro immune complex kinase assay with ribosomal S6 peptide as a specific substrate. While anti-IgM stimulated rsk2 activity three- to fourfold, enzyme activity was not inhibited by 0 to 20 µM SB203580 (data not shown). Thus, our results indicate that SB203580 does not inhibit either JNK or ERK signaling pathways and are consistent with earlier studies that show that SB203580 is a highly specific p38 MAPK inhibitor (42, 43).
The p38 MAPK pathway positively regulates CD40-dependent proliferation but negatively controls BCR-dependent B cell proliferation in tonsillar B cells
Since the p38 MAPK pathway is required for both IL-2- and
IL-7-driven T cell proliferation and a primary response of
CD40-activated B cells is the stimulation of B cell growth (56, 60), we
examined the effect of SB203580 on anti-CD40-induced proliferation
of isolated buoyant and dense tonsillar B cells. Preincubation with
SB203580, at concentrations that completely perturbed CD40-mediated
MAPKAPK-2 activation (Fig. 3F), dose-dependently inhibited
anti-CD40-induced proliferation in both buoyant and dense tonsillar
B cells with similar IC50 values of approximately 1 µM
(Fig. 4, A and B).
In contrast, SB203580 strongly potentiated anti-IgM-mediated
tonsillar B cell proliferation in either buoyant or dense B cell
subfractions (Fig. 4, A and B). Equivalent doses
of SB203580 either did not or moderately increased
anti-Bgp95-driven proliferation in buoyant or dense tonsillar B
cells under identical experimental conditions (Fig. 4, A and
B), providing further support for the specificity and lack
of cytotoxicity of the compound.
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B activation is regulated by the p38 MAPK
pathway via a mechanism that does not involve NF-B binding
CD40 cross-linking in B lymphocytes induces rapid and persistent
NF-B activation (37). Since the p38 MAPK pathway is required for
TNF--induced NF-B activation (61) and CD40 is also a member of
the same TNFR superfamily, we tested the effect of SB203580 on
CD40-induced reporter gene expression of a construct containing four
copies of a NF-B binding site following transient transfection into
Daudi B lymphocytes. CD40 cross-linking increased NF-B-dependent
reporter gene expression between 8- and 12-fold following stimulation
for 6 h, similar to previous studies (Fig. 5A; 37 . SB203580
pretreatment consistently reduced anti-CD40-induced NF-B
reporter gene expression by approximately 50% at all doses of
anti-CD40 tested (Fig. 5A), suggesting a partial
requirement for the p38 MAPK pathway in CD40-mediated NF-B
transcriptional activation. In contrast, SB203580 had no effect on
PMA-induced NF-B-dependent reporter gene expression (Fig. 5B), indicating that the requirement for the p38 MAPK
pathway was stimulus specific. We also evaluated the potential
contribution to NF-B activation of another MAPK signaling pathway,
the MEK-ERK pathway using the specific MEK1 inhibitor PD 98059, which
prevents the activation of MEK1 by Raf (62). Under conditions where PD
98059 (100 µM) almost completely abrogated anti-IgM-induced ERK2
activation in Daudi B cells (data not shown), 0 to 100 µM PD 98059
either had no significant effect or slightly activated NF-B reporter
gene expression (data not shown).
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B requires multiple biochemical events
including inducible phosphorylation and subsequent degradation of
IB, nuclear translocation and binding of NF-B to
sequence-specific DNA elements, and inducible phosphorylation of
NF-B subunits such as p65/RelA (63). Thus, we tested the effect of
SB203580 on CD40-induced NF-B DNA binding in Daudi B cells by EMSA,
using a NF-B-specific [32P]-labeled oligonucleotide.
Anti-CD40 induced detectable increases in binding of at least three
specific protein-DNA complexes within 15 min following stimulation
(Fig. 5C). EMSA supershift analysis with specific antiserum
for different NF-B/Rel family members suggested that the upper (I)
and middle complexes (II) consisted of both p65 and c-rel
whereas the lower complex (III) comprises a mixture of both p50 and
c-rel (Fig. 5C); the possibility that these
complexes may contain other NF-B/Rel members such as RelB or p52
cannot be excluded. Pretreatment of the Daudi cells with 0 to 10 µM
SB203580 for 30 min failed to prevent CD40-induced NF-B DNA binding,
demonstrating that the p38 MAPK pathway does not appear to regulate
either the release of IB from NF-B, the nuclear translocation of
NF-B, or its subsequent DNA binding (Fig. 5D). A requirement for the p38 MAPK pathway in CD40-induced CD54/ICAM-1 expression but not in CD40, CD95/Fas, DR3, TRAF1, TRAF4/CART, or cIAP2 expression
NF-B plays an important role in the cytokine-inducible
regulation of gene expression at the transcriptional level of various
cell adhesion molecules, including CD54/ICAM-1 (64), CD62E/E-selectin
(65, 66), and CD106/VCAM-1 (67). Thus, in preliminary studies the
effect of SB203580 pretreatment upon CD40-induced expression of
multiple genes including CD54/ICAM-1 was examined in Daudi B cells
using an Atlas human cDNA expression array.
CD40 cross-linking strongly increased steady-state levels of both
CD54/ICAM-1 and itself (CD40) at the transcriptional level within
2 h (Fig. 6A). Moderate
up-regulation of the C-C chemokines MIP-1 and MIP-1ß and the
chemokine and chemoattractant receptor CCR2 were also apparently
evident following anti-CD40 treatment, while expression of the TNFR
superfamily member, CD27, was moderately reduced (Fig. 6A).
A similar CD40-induced down-regulation of CD27 mRNA levels was
previously reported in chronic lymphocytic leukemia (CLL) B cells (68).
Moreover, SB203580 pretreatment of the Daudi cells significantly
reduced anti-CD40-mediated CD54/ICAM-1 expression while exhibiting
no effect upon CD40-induced up-regulation of its own mRNA (Fig. 6B, circles 4 and 2, respectively). To quantitatively
analyze the effect of SB203580 upon anti-CD40-induced CD54/ICAM-1
and CD40 RNA levels, Northern blot analysis was performed. Similar to
results with the cDNA expression array, SB203580 again partially
inhibited CD40-induced CD54/ICAM-1 expression by more than 50%, while
anti-CD40-mediated up-regulation of its own transcript was
unaffected (Fig. 6C).
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A). In addition,
anti-CD40 induced selective up-regulation of both TRAF1 and
TRAF4/CART, whereas the steady-state mRNA levels of both TRAF2 and -3
remained unaffected (Fig. 7A). Since the p38 MAPK pathway
may be required for the differential up-regulation of IAP and TRAF
family members, we tested the effect of SB203580 upon CD40-mediated
elevations in cIAP2, TRAF1, and TRAF4/CART transcript levels.
Pretreatment with SB203580, under conditions that blocked
CD40-dependent MAPKAPK-2 activation, had no significant effect upon the
up-regulation of either cIAP2, TRAF1, or TRAF4/CART expression in
response to anti-CD40 stimulation (Fig. 7A).
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B). CD40 cross-linking also induced a weak, but
reproducible, increase in steady-state levels of DR3, another TNFR with
a death domain, while the mRNA levels of other TNFR members and
associated proteins tested were not significantly affected (Fig. 7B). However, pretreatment of the Daudi cells with SB203580
did not significantly reduce either anti-CD40-induced CD95/Fas or
DR3 expression (Fig. 7B). Collectively, these results
indicate the the p38 MAPK pathway is required, at least in part, for
CD40-mediated CD54/ICAM-1 expression but not CD40-induced cIAP2, TRAF1,
TRAF4/CART, CD95/Fas, CD40 or DR3 expression. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C, 3F). Thus, our results both agree with
and expand upon recent reports demonstrating that CD40 cross-linking
transiently stimulates the p38 MAPK pathway in WEHI-231 lymphoma and
mouse splenic B cells (28, 30). In the immature WEHI-231 cell line,
CD40 and BCR engagement have opposing effects upon cell fate (57).
Thus, the ability of both anti-CD40 and anti-IgM to activate
p38 MAPK and one of its targets, MAPKAPK-2, indicates that activation
of the p38 MAPK pathway does not correlate with either an apoptotic or
survival signal (Figs. 2 and 3D). This result is further
supported by the inability of SB203580 to suppress BCR-mediated cell
death in WEHI-231 cells (30). In apparent contrast,
anti-IgM-induced apoptosis in human B104 lymphoma cells correlated
with BCR-induced delayed and sustained activation of p38 MAPK (72).
Moreover, anti-IgM-induced cell death was strongly inhibited by the
p38 MAPK inhibitor, SB203580 (73). It seems plausible that the kinetics
of BCR-induced p38 MAPK activation may correlate with the differential
requirement for p38 MAPK in BCR-mediated apoptosis, or, alternatively,
the requirement for p38 MAPK activation in anti-IgM-induced cell
death is cell type specific. It is possible that a block in p38
MAPK-dependent cell death may contribute to the increase in BCR-induced
proliferation observed in the presence of SB203580 with human tonsillar
B cells (Fig. 4).
The recent identification of the pyridinyl imidazole, SB203580, as a
highly specific and cell-permeable inhibitor of p38 MAPK has enabled us
to study the role of the p38 MAPK pathway in CD40-responsive B cells. A
role for the p38 MAPK pathway has previously been identified using
SB203580 in diverse cellular processes such as LPS- and TNF--induced
cytokine production (42, 61), UV- and anisomycin-induced
c-jun and c-fos expression (34), IL-2- and
IL-7-mediated T cell proliferation (60), glutamate- (74) and
BCR-induced apoptosis (73), and FGF-, arsenite- and UVC-mediated
CREB/ATF-1 phosphorylation (33, 75). SB203580 appears to be specific
for p38 MAPK, since it selectively inhibits both p38 MAPK and -ß,
but not the isoform, and exhibits no significant effect upon
multiple other related kinases, including multiple members of the ERK
and JNK families and their upstream activators (43, 76, 77, 78).
Our observation that SB203580 specifically blocked CD40-induced
proliferation, under conditions where CD40-dependent MAPKAPK-2
activation was suppressed, while potentiating anti-IgM-driven
proliferation of tonsillar B lymphocytes (Fig. 3F, 4),
suggests that the p38 MAPK pathway may either positively or negatively
regulate proliferative responses in a stimulus-dependent manner.
Moreover, the requirement for p38 MAPK activity in CD40-mediated
proliferation supports the idea of a role for p38 MAPK in nonstress
responses. A requirement for p38 MAPK activity in IL-2- and
IL-7-induced T cell proliferation, another mitogenic response, has also
been recently shown (60).
One important role of CD40 is its ability to regulate gene expression
at the transcriptional level. CD40 engagement up-regulates steady-state
levels of various transcripts including the anti-apoptotic Bcl-2
homologue Bcl-XL (58), TNF and TNFR family members such as
LT and CD70 (68, 79), as well as adhesion molecules such as
CD54/ICAM-1 (80). However, the signaling pathways and identities of key
signaling intermediates which mediate CD40-induced gene expression are
poorly characterized.
Under experimental conditions where SB203580 completely prevented
anti-CD40-mediated activation of the immediate p38 MAPK target,
MAPKAPK-2 (Fig. 3C), the p38 MAPK inhibitor significantly
reduced CD40-induced CD54/ICAM-1 expression, suggesting a role for the
p38 MAPK pathway in CD40-regulated gene expression at the
transcriptional level (Fig. 6). The possibility that SB203580 inhibits
CD40-induced increases in steady-state CD54/ICAM-1 mRNA levels
nonspecifically is unlikely since SB203580 failed to prevent the
up-regulation of multiple other genes (CD40, CD95/Fas, DR3, cIAP2,
TRAF1, TRAF4/CART) in response to CD40 engagement (Figs. 6 and 7).
Furthermore, the p38 MAPK inhibitor did not inhibit either
anti-CD40-induced NF-B binding or anti-IgM-mediated
CREBSer133 phosphorylation in the same cells (Fig. 5D; data not shown). The possibility that SB203580 inhibits
another CD40-responsive protein kinase is unlikely, since the inhibitor
is without effect on either JNK or ERK or multiple other related
protein kinases including the homologues, p38 MAPK (SAPK3) and
SAPK4, which share 60% identity with p38 MAPK and -ß (Fig. 3G; Refs. 43, 76, 81). However, the possibility of
other targets cannot be completely excluded. In summary, these results
suggest that CD40 induces gene expression via both p38 MAPK-dependent
and -independent pathways in B lymphocytes.
Our results indicate that CD40-mediated NF-B
trans-activation is also a target for the p38 MAPK pathway
(Fig. 5A). However, CD40-induced NF-B binding was not
modulated by SB203580, suggesting that the p38 MAPK pathway selectively
regulates the trans-activation potential of NF-B (Fig. 5D). A similar requirement for the p38 MAPK pathway in
NF-B activation via an unknown mechanism independent of its effect
on DNA binding activity has also been reported in response to TNF-
(61, 82). While the p38 MAPK inhibitor did not prevent TNF--induced
phosphorylation of either p65 and p50 subunits or its precursor p105
subunit (61), it remains possible that the p38 MAPK pathway may
regulate CD40-dependent NF-B activation via phosphorylation of the
trans-activation domains of either p50, p65, or p105
subunits. Alternatively, the p38 MAPK pathway may regulate NF-B
activation in response to CD40 through phosphorylation of another
transcription factor that is required for NF-B
trans-activation. Indeed, the p38 MAPK pathway is required
for phosphorylation of other transcription factors such as ATF-1 and
CREB (33, 75), CHOP (31), and myocyte enhancer factor MEF2C (32)
in response to a variety of stimuli.
Our studies have also identified a number of novel CD40-responsive genes that may perform roles in the regulation of cell fate, including two TNF receptors, CD40 and DR3, the TNFR-associated factors TRAF1 and TRAF4/CART, and cIAP2, a member of the inhibitor of apoptosis protein family. CD40 is generally considered to play a positive role in B cell activation, proliferation and apoptotic rescue. Thus, the ability of CD40 to up-regulate DR3 expression appears paradoxical since DR3 both contains a cytoplasmic "death domain" and mediates cell death (3). However, these observations are analogous to the ability of CD40 ligation to up-regulate CD95/Fas expression and thereby enhance the sensitivity of B cells to CD95/Fas-mediated apoptosis (70, 71). Thus, CD40-induced DR3 expression may promote increased sensitivity to DR3 ligand-induced cell death, a possibility we are currently investigating.
CD40 engagement differentially increased steady-state mRNA levels of
cIAP2, while not significantly modifying the levels of other family
members (XIAP, NAIP, cIAP1; Fig. 7A). Since TNF- also
selectively up-regulates cIAP2 expression in T lymphocytes (83), our
findings suggest that NF-B-inducing TNF receptors may share the
capacity to up-regulate cIAP2 in response to ligand binding. Moreover,
our results suggest that one putative mechanism whereby CD40 may
mediate B cell rescue, in addition to the induction of
Bcl-XL expression (58), is via up-regulation of cIAP2. The
possibility that IAPs also physically associate with the
ligand-inducible CD40 receptor complex via interactions with TRAFs,
similar to the TNFR complex, merits further study (69, 84). Thus, CD40
ligation may both make cells more susceptible to apoptosis in response
to either CD95/Fas or DR3 receptor ligation, and also enhance their
capacity to be rescued from apoptosis via increased expression of
cIAP2.
Rather surprisingly, CD40 engagement specifically increased the
steady-state mRNA levels of TRAF1 and TRAF4/CART, whereas the levels of
TRAF2 and TRAF3, which associate with the CD40 cytoplasmic tail
(85, 86, 87), were unaffected (Fig. 7A). Although TRAF1
overexpression inhibits Ag-induced cell death of CD8+ T
lymphocytes, the molecular details of its mechanism of action remain to
be elucidated (88). Perhaps the up-regulation of TRAF1 in response to
CD40 cross-linking also serves as an anti-apoptotic signal in B
cells to either promote the recruitment of anti-apoptotic proteins,
such as IAPs, or alternatively prevent the association of proapoptotic
molecules with TRAF-TNFR complexes. The role of TRAF4/CART in CD40
function remains to be explored. In contrast to TRAF2 and TRAF3, which
are ubiquitously expressed, TRAF4/CART transcripts are apparently
highly restricted to some primary breast carcinomas and metastatic
axillary lymph nodes (89).
In summary, we have shown that the stress-activated protein kinase, p38
MAPK, is rapidly and transiently stimulated following CD40 engagement
in B lymphocytes. Furthermore, we have demonstrated a role for the p38
MAPK pathway in CD40-induced tonsillar B cell proliferation and NF-B
activation using the specific and cell-permeable p38 MAPK inhibitor,
SB203580. In addition, we demonstrated that CD40 regulates gene
expression via both p38 MAPK-dependent and -independent pathways.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew Craxton, Box 357330, Department of Microbiology, University of Washington, Seattle, WA 98195. E-mail address:
3 Abbreviations used in this paper: DR, death receptor; CD40L, CD40 ligand; BCR, B cell Ag receptor; MAPK, mitogen-activated protein kinase; JNK, c-jun amino-terminal kinase; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated protein kinase; FGF, fibroblast growth factor; MAPKAPK-2, MAPK-activated protein kinase-2; CREB, cAMP response element-binding protein; AP-1, activating protein-1; GST, glutathione S-transferase; ATF, activated transcription factor; EMSA, electrophoretic mobility shift assay; TRAF, TNFR-associated factor;MBP, myelin basic protein; IAP, inhibitor of apoptosis protein; MIP, macrophage inflammatory protein; TRAIL, TNF-related apoptosis-inducing ligand; DC, dendritic cell.
Received for publication January 2, 1998. Accepted for publication May 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Curr. Biol. 6:1669.[Medline]
2. J. Exp. Med. 179:673.
