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Departments of
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Pharmacology,
Biochemistry, and
Microbiology, University of Washington, Seattle, WA 98195
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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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The CD95/Fas receptor, a member of the TNF receptor family, also makes an important contribution to determining the lymphocyte life span (9, 10). Cross-linking Fas with agonistic Abs or Fas ligand results in rapid apoptosis of many cell types (reviewed in 11 . Fas-induced apoptosis plays an important role in activation-induced cell death and elimination of autoreactive B cells (reviewed in 2 . The intracellular domain of CD95/Fas contains a death domain that recruits other death domain-containing proteins, including the adapter protein FADD/MORT-1. FADD/MORT-1, in turn, recruits a member of the caspase family of cysteine proteases termed caspase 8 (FLICE/MACH) (12, 13, 14). Caspase 8 is then thought to undergo autoproteolytic activation, initiating a cascade of proteolytic events involving downstream caspases such as caspase 1 (ICE) and caspase 3 (CPP32/Yama) (15, 16). This caspase cascade, by cleaving critical targets, plays a central role in the induction of apoptosis by a wide variety of stimuli (reviewed in 17 . However, the mechanisms regulating the initiation of caspase activity by receptors outside the TNF receptor family and the identities of critical caspase substrates remain to be determined.
As with other cell fate decisions, it is likely that protein phosphorylation/dephosphorylation mechanisms play an important role in the initiation and progression of apoptosis. Recently, two families of mitogen-activated protein kinase (MAPK) have been implicated in apoptotic signaling (reviewed in 18 . These pathways, the stress-activated protein kinase (SAPK) or c-Jun amino-terminal kinase pathway and the p38 MAPK pathways, are strongly activated by stressful stimuli such as proinflammatory cytokines, UV light, and osmotic shock (19, 20, 21, 22). SAPK and p38 MAPK are also activated during apoptosis induced by nerve growth factor withdrawal of PC12 cells, UV irradiation, Fas cross-linking in human T lymphoma cells or neuroblastoma cells, and BCR ligation in human B lymphoma cells (23, 24, 25, 26, 27, 28). The ability of cysteine protease inhibitors such as ZVAD-fmk to block activation of SAPK and p38 MAPK by many apoptotic stimuli suggests that caspases may play an important upstream role in activating these pathways (25, 26).
Whether the SAPK and p38 MAPK pathways play an active role in apoptosis is controversial. Several studies have apparently dissociated SAPK and p38 MAPK activity from the induction of apoptosis (29, 30, 31). However, others have provided strong evidence to support an apoptotic role for the SAPK and p38 MAPK pathways (23, 32, 33, 34). For example, overexpression of MKK6b, an upstream activator of p38 MAPK, has been shown to induce apoptosis in Jurkat T cells (34). Furthermore, MKK6b activity was found to be required for Fas-induced apoptosis (34). Thus, depending on the stimuli and cell type, activation of the SAPK/p38 MAPK pathways may be sufficient to induce cell death. However, the precise cellular roles of these cascades in the cellular response to apoptotic stimuli are not clear. In this study we present data suggesting that p38 MAPK plays a differential role in BCR- and Fas-induced apoptosis of human B lymphoma cells and discuss a model to rationalize these findings.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Goat F(ab')2 anti-µ and anti-
sera
were obtained from Jackson ImmunoResearch (West Grove, PA) and Southern
Biotechnology (Birmingham, AL). The anti-Fas Ab IPO-4 (35) was a
gift from Dr. Svetlana Sidorenko (Kavetsky Institute, Kiev, Ukraine).
SB203580 and PD98059 were purchased from Calbiochem (San Diego, CA).
Rabbit polyclonal antiserum raised against MAPKAP kinase-2 was a gift
from Dr. Philip Cohen (University of Dundee, Dundee, U.K.). Rabbit
polyclonal antiserum raised against SAPK (C17) was obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal Ab against p38
MAPK was provided by Dr. Jeremy Saklatvala (Kennedy Institute of
Rheumatology, London, U.K.). ZVAD-fmk was purchased from Kamiya
Biomedical (Tukwila, WA). The CPP32 substrate peptide was obtained from
Peptides International (Louisville, KY). The B104 human B lymphoma
line, provided by Dr. Mitsufumi Mayumi (Kyoto University Hospital,
Kyoto, Japan), was grown in culture as previously described (36). The
BJAB human B lymphoma line, provided by Dr. Vishva Dixit (Genentech,
South San Francisco, CA), was maintained as previously described
(37).
Immunoprecipitation and kinase assays
After incubation with the indicated stimuli, 5 to 10 x
106 cells were lysed for 15 min on ice in 1 ml of
lysis buffer (20 mM HEPES, pH 7.4; 2 mM EGTA; 50 mM
ß-glycerophosphate; 1% Triton X-100; 10% glycerol; 1 mM DTT; 1 mM
PMSF; 25 µg/ml leupeptin; 25 µg/ml aprotinin; 2 mM
Na2VO4; and 10 mM NaF). Cell debris was removed
by centrifugation at 10,000 x g for 10 min at 4°C.
SAPK, p38 MAPK, or MAPKAPK-2 was then immunoprecipitated by addition of
2 µg of antiserum for 3 h followed by 50 µl of protein-A
Sepharose (1/1) slurry for the final hour. The beads were pelleted by
centrifugation and then washed three times each in lysis buffer, wash
buffer (500 mM LiCl; 100 mM Tris-Cl, pH 7.6; 0.1% Triton X-100; and 1
mM DTT), and assay buffer (20 mM
3-[N-morpholino]-2-hydroxypropanesulfonic acid (MOPS) pH 7.2; 2 mM
EGTA; 10 mM MgCl2; 0.1% Triton X-100; and 1 mM DTT). The
beads were left as a 1/1 suspension in assay buffer, and 20 µl of the
appropriate substrate was added. For SAPK and p38 MAPK assays, 0.3
mg/ml of either GST-Jun (for SAPK) or GST-ATF2 (for p38 MAPK) was
added. Kinase reactions were initiated by the addition of 15 µl of
32P-labeled Mg-ATP solution (50 mM MgCl2, 500
µM ATP, and 10 µCi of [-32P]ATP) and were
conducted at 30°C for 20 min. Reactions were stopped by the addition
of 25 µl of 6x Laemmli sample buffer and boiling for 3 min. Samples
were separated on a 10% SDS-PAGE gel and, after drying, were subjected
to autoradiography. Quantitation by densitometry was performed with an
imaging densitometer (Bio-Rad, Hercules, CA). For MAPKAPK-2 assays, 0.3
mg/ml substrate peptide (KKLNRTLSVA) was added. Kinase reactions were
initiated by the addition of 15 µl of 32P-labeled MgATP
solution (50 mM MgCl2, 500 µM ATP, and 10 µCi of
[-32P]ATP) and were conducted at 30°C for 20 min.
Reactions were stopped, and 32P incorporation was
quantified by spotting 50 µl of the assay mixture onto p81 paper,
washing in 0.1% phosphoric acid, and counting as previously
described (38).
Cell death and flow cytometric assays
Dead cells were quantified with the use of annexin V flow cytometry as previously described (39). The annexin V assay exploits the fact that an early event during apoptosis of many cells is a loss of membrane lipid asymmetry, resulting in the exposure of phosphatidylserine in the outer leaflet of the plasma membrane. Briefly, cells were incubated with FITC-conjugated annexin V and counterstained with propidium iodide to exclude necrotic cells (Clontech, Palo Alto, CA). The cells were subsequently analyzed using a Becton Dickinson FACStar Plus flow cytometer (San Jose, CA). Other apoptosis assays employed included trypan blue dye exclusion and Hoechst 33342 7-amino-actinomycin D flow cytometry (27). These techniques yielded results similar to those obtained with annexin V (data not shown). Where indicated, cells were pretreated for 3 h with 10 µM ZVAD-fmk.
Caspase assays
Following stimulation, cells were washed once in PBS and resuspended at 2 x 108/ml in hypotonic lysis buffer (50 mM NaCl; 40 mM ß-glycerophosphate; 10 mM HEPES, pH 7.0; 5 mM EGTA; and 2 mM MgCl2). The lysate was then subjected to four freeze-thaw cycles before centrifugation at 10,000 x g for 10 min each (100 mM HEPES, pH 7.5; 10% sucrose; 0.1% 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonic acid; 10 mM DTT; and 0.1 mg/ml OVA). Protein concentrations were determined, and 25 µg of cell extract was incubated for 1 h at 37°C with either 50 µM Ac-DEVD-AMC or YVAD-AMC. Protease activity was determined by monitoring the release of 7-amino-4-trifluoromethyl coumarin at an excitation wavelength of 400 nm and an emission wavelength of 510 nm with the use of a CytoFluor II 96-well plate spectrofluorometer (PerSeptive Biosystems, Framingham, MA). Relative caspase activity was determined by dividing the activity observed at each time point by the values detected at time zero.
Data presentation
All experiments shown are representative of between three and five repetitions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). This increased
annexin V binding was blocked by pretreatment with ZVAD-fmk, a
cell-permeable peptide inhibitor of caspases (Fig. 1A). Treatment of BJAB cells with anti-Fas Abs
also induced a ZVAD-fmk-sensitive increase in annexin V binding (Fig. 1B). Thus, both BCR- and Fas-induced apoptosis, as
measured by loss of membrane assymetry, are dependent upon caspase
activity.
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, A and
B). One possible explanation for the relative delay
in apoptosis in B104 cells is that initiation of the cell death process
requires synthesis of new genes and proteins. Consistent with this
hypothesis, BCR-induced apoptosis was almost completely blocked by
preincubation with the RNA synthesis inhibitor actinomycin D (Fig. 2C). In contrast, actinomycin D did not inhibit
anti-Fas-induced apoptosis (Fig. 2D). Similar
results were obtained with the protein synthesis inhibitor
cycloheximide (data not shown). These data show that BCR-induced
apoptosis, in contrast to Fas-induced apoptosis, is dependent on
transcriptional and translational events.
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A). To gain further
insight into the roles of these kinase pathways in BCR- and Fas-induced
apoptosis, we compared the effects of these agonists on SAPK and p38
MAPK activity in B104 and BJAB cells. Both anti-IgM and
anti-Fas induced a slow and sustained increase in SAPK and p38 MAPK
activity that peaked about 8 h after treatment (Fig. 3, A and B). However, the kinetics of SAPK and p38
MAPK activation in response to BCR ligation were delayed relative to
those observed upon Fas cross-linking. In this respect, the increase in
SAPK and p38 MAPK activities stimulated by these treatments parallels
the kinetics observed for increased annexin V binding. Activation of
SAPK and p38 MAPK are among the earliest measurable events associated
with apoptosis in these cells.
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, A and
B). These results indicates that the sustained
activation of both SAPK and p38 MAPK by ligation of either BCR or
CD95/Fas is dependent on the proteolytic activity of caspases.
We next sought to determine the role of p38 MAPK in BCR- and
Fas-induced apoptosis using a p38 MAPK inhibitor. The pyridinyl
imidazole p38 MAPK inhibitor, SB203580, has been shown to specifically
block p38 MAPK activity by a variety of stimuli in many different cells
(41, 42). SB203580 appears to be specific for the 
and ß isoforms
of p38 MAPK (42, 43, 44, 45). The mechanism of SB203580 action involves
reversible binding to p38 MAPK itself. Thus, to confirm inhibition of
p38 MAPK in vivo, it is necessary to measure an event downstream of p38
MAPK. One such event is the activation of MAPKAPK-2 (38). Figure 4A shows that anti-IgM
treatment of B104 cells resulted in a late and sustained activation of
MAPKAPK-2 activity. The kinetics of MAPKAPK-2 activation closely
resemble those observed for p38 MAPK in these cells, consistent with
its status as a downstream target for p38 MAPK. Preincubation of B104
cells with 10 µM SB203580 almost completely abolished the MAPKAPK-2
response to BCR ligation, indicating that p38 MAPK activity was
profoundly inhibited. Under these conditions, BCR-induced apoptosis was
also significantly inhibited (Fig. 4B). For example,
SB203580 reduced the percentage of apoptotic cells observed after
12 h of anti-IgM treatment from approximately 75 to 20%.
Pretreatment of BJAB cells with SB203580 also completely blocked
anti-Fas-induced activation of MAPKAPK-2 (Fig. 4C). However, in contrast to the effects on
BCR-induced apoptosis, SB203580 did not inhibit Fas-induced apoptosis
(Fig. 4D). The dose response for SB203580 inhibition
of anti-IgM-induced MAPKAPK-2 activity closely matched that
observed for inhibition of apoptosis, with a half-maximal concentration
of approximately 2 µM. This provides further support for the
hypothesis that p38 MAPK plays an important role in BCR-induced
apoptosis of B104 cells (Fig. 5).
Consistent with our previous results suggesting that the classical MAPK
pathway does not play a determinant role in anti-IgM-induced
apoptosis of B104 cells (27), the PD98059 inhibitor of MAP kinase
kinase did not inhibit apoptosis in response to anti-IgM (data not
shown).
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A). Whereas increased
DEVD-specific protease activity was detectable within 1 h after
Fas ligation, anti-IgM-induced caspase activity was not detected
until 3 to 4 h after BCR cross-linking. The kinetics of
BCR-induced caspase activity paralleled the kinetics of apoptosis (Fig. 2) and SAPK/p38 MAPK activation (Fig. 3) and were delayed relative to
Fas-induced caspase activation (Fig. 6A). Little
activity was detected against a YVAD peptide in response to either
anti-IgM or anti-Fas (data not shown). This result is
consistent with a low level of activity of caspase 1 and related
caspases.
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B). Similar results were
obtained with cycloheximide (data not shown), suggesting that
activation of DEVD-specific caspases in response to BCR cross-linking
is dependent on the induction of gene expression and protein synthesis.
Because BCR-induced apoptosis also requires p38 MAPK activity, it is
possible that p38 MAPK may function upstream of caspase activation. To
test this hypothesis, the ability of anti-IgM to induce
DEVD-specific caspase activity was examined in B104 cells pretreated
with SB203580. SB203580 pretreatment significantly blocked BCR-induced
caspase activity (Fig. 6B). Neither actinomycin D nor
SB203580 inhibited caspase activation induced by Fas
cross-linking in BJAB cells (data not shown). Collectively, these
findings establish a role for p38 MAPK in BCR-induced apoptosis both
upstream and downstream of caspase activity. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise caspase targets responsible for activation of the SAPK and
p38 MAPK pathways are not known. However, it is reasonable to speculate
that there might be upstream components of these protein kinase
cascades that are targets for caspase-mediated proteolytic cleavage and
activation. In this respect, known caspase substrates include D4-GDI, a
GDP dissociation inhibitor for Rho family GTPases, DNA-dependent
protein kinase, and the 
, 
, and 
isoforms of protein kinase C
(46, 47, 48, 49, 50). More recently, several protein kinases that are thought to
function in mammalian MAPK pathways have been identified as caspase
substrates. For example, p21-activated kinase 2 and MEKK1, have been
shown to be activated by caspase-mediated proteolysis (51, 52).
Additional studies will be required to determine the contributions of
these proteins and other caspase targets to caspase-dependent SAPK and
p38 MAPK activation during BCR- and Fas-induced apoptosis.
Our kinetic comparisons suggest that BCR-induced caspase activation is
delayed relative to Fas-induced caspase activation. This delay may
reflect a requirement for macromolecular synthesis to occur before
apoptosis. The ability of actinomycin D or cycloheximide to inhibit
BCR-induced caspase activation and apoptosis supports this hypothesis.
Actinomycin D and cycloheximide promote apoptosis in many cells. For
example, actinomycin D sensitizes some cells to TNF--mediated
apoptosis by blocking the ability of the TNF receptor to induce an
NF
B-mediated cell survival pathway (29, 53). However, actinomycin D
has also been found to inhibit apoptosis induced by nerve growth factor
withdrawal (54, 55), suggesting that the effects of actinomycin D may
depend on cell type and stimulus. The ability of actinomycin D to block
BCR-induced apoptosis could be mediated by either blocking the
expression of factors that promote cell survival or inhibiting the
induction of proapoptotic components. Candidates for such targets
include transcription factors, components of the cellular protein
synthesis machinery, and proteins that function in cellular apoptotic
pathways. The sensitivity of immature B cells to apoptosis has recently
been shown to correlate with increased expression levels of caspase
3/CPP32 (56, 57). Thus, up-regulation of the expression level of
caspases themselves is one potential explanation for inhibition of
BCR-induced apoptosis by actinomycin D.
SB203580, a pyridinyl imidazole inhibitor of p38 MAPK, has allowed the
role of p38 MAPK in the regulation of cellular processes such as
cytokine production, glucose transport, and transcriptional regulation
to be determined (41, 58, 59, 60, 61). SB203580 appears to be highly specific
for p38 MAPK, inhibiting the and ß isoforms of p38 MAPK, but not
the more distantly related p38/SAPK3 or SAPK4 (42, 43, 44, 45). SB203580
significantly inhibited BCR-induced apoptosis in B104 cells. Consistent
with previous reports of the effects of SB203580, the concentration
required for 50% inhibition of apoptosis was 
2 µM (41, 42). Since
results obtained with SB203580 do not discriminate between events
mediated directly by p38 MAPK and those mediated by downstream targets
of p38 MAPK, it is possible that MAPKAPK-2 or related kinases perform
critical functions during BCR-induced apoptosis. SB203580 has
previously been shown to inhibit apoptosis of neural cells induced by
either trophic factor withdrawal or glutamate treatment and sodium
salicylate-induced apoptosis of fibroblasts (59, 60, 61). The ability of
SB203580 to inhibit BCR-induced apoptosis in B104 cells, but not
anti-Fas-induced apoptosis in BJAB cells, suggests that the p38
MAPK pathway plays a differential role in BCR- and Fas-induced
signaling pathways (Ref. 26 and our results). In this respect, we
cannot rule out the possibility that the differential effects of
SB203580 are a result of cell type differences. It appears likely that
the involvement of p38 MAPK in apoptosis may be dependent on both the
cell type and the stimulus used. Our results, demonstrating an
important role for p38 MAPK in BCR-induced apoptosis, raise a question
regarding the role of SAPK. Since SAPK is insensitive to inhibition by
SB203580 in vitro and in vivo (42), SAPK activation appears to be
insufficient to induce apoptosis in these cells.
The identities of the substrates for p38 MAPK that function during apoptosis are unknown. However, in light of the differential sensitivity of BCR- and Fas-induced apoptosis to actinomycin D and cycloheximide, p38 MAPK substrates may function in pathways that regulate transcription and/or translation. Previous studies employing SB203580 have implicated p38 MAPK in the regulation of both transcriptional and translational events. For example, p38 MAPK has been shown to phosphorylate the transcription factors Elk-1 and ATF-2 that participate in regulation of the serum response element and activator protein-1 (62, 63, 64). The mechanism by which SB203580 blocks production of inflammatory cytokines appears to be at the level of translation rather than at that of transcription (41). The effects of SB203580 on BCR-induced apoptosis could reflect a role for p38 MAPK at either the transcriptional or the translational level. An alternative hypothesis is that components of the cellular apoptotic machinery are direct targets for kinases in the p38 MAPK pathway. Such targets could include caspases themselves, caspase regulators, or components of cell survival pathways such as members of the Bcl-2 family.
One interpretation of the effects of ZVAD-fmk and SB203580 is that p38
MAPK may function both upstream and downstream of caspases during
BCR-induced apoptosis. A hypothesis to account for these data is that
p38 MAPK may function as part of a positive feedback loop that serves
to amplify the apoptotic response to BCR ligation (see Fig. 7). A similar model has been proposed to
explain the role of MKK6b in Fas-induced apoptosis of Jurkat cells.
Huang et al. (34) showed that expression of an activated mutant of
MKK6b induced apoptosis and that a dominant negative MKK6b mutant
inhibited Fas-induced apoptosis. However, SB203580 did not inhibit
apoptosis induced by MKK6b. To account for these results, the authors
suggested the existence of a positive feedback loop that comprises MKK6
but not p38 MAPK. Our data concerning BCR-mediated apoptosis differ
from these findings in one important respect. The ability of ZVAD-fmk
to block p38 MAPK activation and of SB203580 to inhibit the induction
of caspase activity clearly implicate p38 MAPK as both a target and a
positive effector of the BCR-induced apoptotic response. One
explanation for these apparently paradoxical results is that MKK6 may
function in both Fas- and BCR-mediated apoptotic pathways. However, in
the case of Fas-induced apoptosis, MKK6 targets other than
SB203580-inhibitable p38 MAPK may function to amplify the response. In
contrast, BCR-induced apoptosis may depend on the activity of an MKK6
target, such as the or ß isoforms of p38 MAPK, that is sensitive
to SB203580 (Fig. 7). Studies are underway to identify the signaling
components that mediate caspase-dependent activation of SAPK and p38
MAPK and the critical p38 MAPK targets that function during BCR-induced
apoptosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jonathan Graves, Department of Immunology, University of Washington, HSB Box 357370, Seattle, WA 98185.
3 Abbreviations used in this paper: BCR, B cell receptor; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; MKK, MAPK kinase; MAPKAPK-2; MAPK-activated protein kinase-2; GST, glutathione S-transferase; ATF-2, activated transcription factor-2; YVAD-AMC, acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin.
Received for publication January 7, 1998. Accepted for publication March 3, 1998.
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 R. P. Singh, P. Dhawan, C. Golden, G. S. Kapoor, and K. D. Mehta One-way Cross-talk between p38MAPK and p42/44MAPK. INHIBITION OF p38MAPK INDUCES LOW DENSITY LIPOPROTEIN RECEPTOR EXPRESSION THROUGH ACTIVATION OF THE p42/44MAPK CASCADE J. Biol. Chem., July 9, 1999; 274(28): 19593 - 19600. [Abstract] [Full Text] [PDF]
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A. Jiang, A. Craxton, T. Kurosaki, and E. A. Clark Different Protein Tyrosine Kinases Are Required for B Cell Antigen Receptor-mediated Activation of Extracellular Signal-Regulated kinase, c-Jun NH2-terminal Kinase 1, and p38 Mitogen-activated Protein Kinase J. Exp. Med., October 5, 1998; 188(7): 1297 - 1306. [Abstract] [Full Text] [PDF]
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A. Craxton, G. Shu, J. D. Graves, J. Saklatvala, E. G. Krebs, and E. A. Clark p38 MAPK Is Required for CD40-Induced Gene Expression and Proliferation in B Lymphocytes J. Immunol., October 1, 1998; 161(7): 3225 - 3236. [Abstract] [Full Text] [PDF]
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M.-Y. Cao, F. Shinjo, S. Heinrichs, J.-W. Soh, J. Jongstra-Bilen, and J. Jongstra Inhibition of Anti-IgM-induced Translocation of Protein Kinase C beta I Inhibits ERK2 Activation and Increases Apoptosis J. Biol. Chem., June 29, 2001; 276(27): 24506 - 24510. [Abstract] [Full Text] [PDF]
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R. Marches, E. S. Vitetta, and J. W. Uhr A role for intracellular pH in membrane IgM-mediated cell death of human B lymphomas PNAS, March 13, 2001; 98(6): 3434 - 3439. [Abstract] [Full Text] [PDF]
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) or were pretreated for
3 h with 20 µM ZVAD-fmk (
) before stimulation for the
indicated time with anti-IgM (1 µg/ml). B, BJAB cells
were untreated (•) or were pretreated for 3 h with 20 µM
ZVAD-fmk (
) before stimulation for the indicated time with
anti-Fas (1 µg/ml). C, B104 cells were untreated (
)
before stimulation for the indicated time with anti-IgM (1
µg/ml). B, BJAB cells were untreated (•) or were
pretreated for 3 h with 12.5 µM actinomycin D (
) before
stimulation for the indicated time with anti-Fas (1 µg/ml).
