| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Apoptosis is characterized by morphological changes in the cell, including shrinkage, chromatin condensation, membrane blebbing, and formation of apoptotic bodies (reviewed in Ref. 2). Central to the controlled disassembly of the cell is the activation of caspases, a family of cysteinyl aspartate-specific proteases (reviewed in Ref. 3). Caspases are constitutively expressed and are present in cells as inactive precursors (procaspases or zymogens) that consist of three domains: a prodomain and a large and small subunit. Based on the length of their prodomain, caspases are subdivided into initiator (i.e., caspase-8 and -9) and effector caspases (i.e., caspase-3, -6, and -7). To obtain full proteolytic activity, a precursor has to be cleaved between the domains. The large and the small fragment then combine to form an active enzyme. Triggering of the caspase cascade takes place in multicomponent complexes either at the plasma membrane (death-inducing signaling complexes at death receptors, e.g., CD95) or in the cytoplasm (apoptosomes), where the initiator caspase-8 and caspase-9 are activated, respectively (reviewed in Refs. 2 and 3). The active initiator caspases subsequently cleave and thereby activate the downstream effector caspases. Consecutive proteolytic degradation of a broad spectrum of cellular targets including poly(ADP-ribose) polymerase (PARP) or inhibitor of caspase-activated DNase ultimately leads to cell death.
Apoptosomes are high-m.w. complexes containing at least apoptotic protease-activating factor 1 (Apaf-1), cytochrome c (Cyt c), dATP, and caspase-9 (Ref. 4 and reviewed in Ref. 3). Apaf-1, procaspase-9, and dATP are located in the cytoplasm, whereas Cyt c is normally sequestered in the mitochondria. When released, Cyt c in concert with dATP triggers a conformational change in Apaf-1, leading to its oligomerization. Procaspase-9 is simultaneously recruited into this nascent complex and is activated by cleavage. This pathway will be referred to as the mitochondrial/caspase-9 pathway. Release of Cyt c from mitochondria follows a wide variety of stress signals, including UV, oxidative stress, anticancer drugs, and growth factor withdrawal (reviewed in Ref. 5). Oligomerization of the proapoptotic "multidomain" Bcl-2 family members Bak or Bax in the outer mitochondrial membrane seems to be obligatory to initiate mitochondrial dysfunction by most if not all of these stimuli (6, 7, 8, 9). In contrast, antiapoptotic Bcl-2 family members such as Bcl-2, Bcl-xL, and A1 appear to stabilize mitochondria by inhibiting the formation of such oligomers (10, 11).
Engaging the BCR induces apoptosis in a variety of B cells, including primary human activated memory cells and various B cell lines. Mitochondria seem to link the BCR signals to the downstream apoptosis-inducing machinery, i.e., the caspases (12, 13, 14). In keeping with this, primary mature B cells lacking A1 are highly sensitive to BCR-mediated apoptosis (15).
For WEHI 231, a murine B lymphoma serving as a model for BCR-induced apoptosis at the immature stage of B cell development, we and others have shown that ectopic expression of antiapoptotic Bcl-2 proteins protects against deleterious effects of BCR cross-linking, thereby arguing for a mitochondrial gateway to apoptosis in tolerance induction (14, 16, 17, 18, 19, 20, 21). Again, the physiological importance is highlighted in transgenic mice overexpressing Bcl-xL in the context of a transgenic self-reactive BCR. B cells in these animals efficiently escaped developmental arrest and deletion (22). Recently, however, Ruiz-Vela et al. (20) presented data suggesting that the activation of the effector caspase-7 is independent of Cyt c release and processing of caspase-9.
In this report, we used retrovirally transduced WEHI 231 cells to assess the importance of the mitochondrial/caspase-9 pathway for BCR-induced apoptosis. We show that A1 inhibits processing of caspase-9 and caspase-7. In addition, a mutant of caspase-9 with an inactivated enzymatic center, dominant negative caspase-9 (dnC9), strongly interferes with processing of caspase-7, degradation of PARP, and oligosomal fragmentation of DNA. Both A1 and dnC9 alleviate apoptosis induced by cross-linking the BCR. These results suggest that mitochondria and caspase-9 are critically involved in BCR-triggered caspase activation and apoptosis in immature B cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
WEHI 231 cells (murine B cell lymphoma line) and 293T cells (human embryonic kidney cell line 293 expressing SV40 T Ag) were obtained from American Type Culture Collection (Manassas, VA). The B cell line was maintained in RPMI 1640 medium supplemented with 4.8 mM L-glutamine, 1 mM sodium pyruvate, 1x nonessential amino acids (Life Technologies, Rockville, MD), 5% (v/v) FCS, streptomycin (0.2 mg/ml), penicillin (400 U/ml), and 2-ME (5 x 10-5 M). The 293T cells were propagated in DMEM supplemented with 10% (v/v) FCS and streptomycin/penicillin as mentioned above.
Reagents
Anti-IgM (AffiniPure F(ab')2 goat anti-mouse IgM) was obtained from ICN Pharmaceuticals (Costa Mesa, CA) and used at 7.5 µg/ml. Biotinylated anti-IgM (AffiniPure F(ab')2 goat anti-mouse IgM; working concentration, 2 µg/ml) used for survival experiments was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Etoposide was purchased from Calbiochem (La Jolla, CA).
Plasmids
The bicistronic retroviral vector pEYZ/MCS containing a chimeric
selection marker consisting of the enhanced yellow fluorescent (Y) and
the zeocin resistant (Z) proteins was previously described
(19). pEGN/MCS and pEGZ/MCS are analogous to pEYZ/MCS but
contain the green fluorescent marker (G) fused to either the neomycin
(N)- or the zeocin (Z)-resistant protein. Oligonucleotides encoding a
flag-peptide were inserted into the MCS of pEGN/MCS (
pEGN/MCS-F) and
pEYZ/MCS (pEYZ/MCS-F) to routinely flag-tag encoded proteins at the
C terminus. Caspase-7 and caspase-9 cDNAs were obtained by conventional
RT-PCR technique from RNA isolated from WEHI 231 cells. The mutant
forms of caspase-7 and caspase-9 were generated by PCR techniques. Both
mutants encode a protein with a cysteine to serine mutation at their
active center. All cDNAs were inserted into the EcoRI and
BamHI of pEGN/MCS-Flag or pEYZ/MCS-Flag using
EcoRI/BamHI restriction sites for caspase-7 and
EcoRI/BglII restriction sites for caspase-9
cDNAs. pEYZ/FmA1 was described previously (19). The
pEGZ/hBcl2 expression vector encoding human Bcl2 was generated by
releasing a full-length cDNA of hBcl2 from pBluescript KS-Bcl2 (a gift
from J. Troppmair, Institut für Medizinische Strahlenkunde und
Zellforschung, University of Würzburg, Würzburg,
Germany) by EcoRI digestion and inserting it into the
EcoRI restriction site of pEGZ/MCS.
Transfection/infection assays and cell sorting
Recombinant retroviral particles were generated by using the pHIT packaging system essentially as described by Soneoka et al. (23). Transfection of 293T cells and infection of WEHI 231 cells have been described previously (19). Transduced cells were highly enriched (>80%) by treatment with the adequate antibiotics (250 µg/ml zeocin or 1 mg/ml G418) and/or by FACS (FACSVantage; BD Biosciences, Mountain View, CA).
Determination of live cells
The percentage of live cells was determined as the number of events falling into the region of the live gate in a forward light scatter/side light scatter analysis on a FACS. To determine a stringent live gate, cell cultures were supplemented with 4 µg/ml propidium iodide and were incubated for 15 min at 37°C. Cells excluding propidium iodide were considered as live cells (19).
Western blot analysis
Whole-cell lysates were prepared by lysing 3 x 105 cells in 25 µl of 6x Laemmli buffer. Mitochondria containing cellular fractions and cytoplasmic fractions were prepared as described by Heibein et al. (24). Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA), and analyzed by Western blotting. Polyclonal anti-caspase-7 (catalog no. 9492) and anti-caspase-9 (catalog no. 9504) Abs were obtained from New England Biolabs (Beverly, MA). The monoclonal mouse anti-PARP (Ab-2) Ab (catalog no. AM30) was purchased from Calbiochem. The monoclonal mouse anti-Cyt c Ab (catalog no. 65981A) was obtained from BD PharMingen (San Diego, CA).
Analysis of DNA fragmentation
Extraction of cellular DNA for the DNA fragmentation assay was conducted by lysing 2 x 105 cells as described by Doi et al. (14). For analysis, DNA equivalent to 5 x 104 cells was electrophoresed in a 2% agarose gel.
Analysis of caspase-3-like activity
Caspase-3-like (caspase-3, -6, and -7) activity in lysates of various WEHI 231 populations was determined by means of a colorimetric substrate (catalog no. 235400; Calbiochem) following the manufacturer’s instructions.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cross-linking of the Ag receptors on WEHI 231 cells induces growth
arrest and apoptosis. We monitored cell death after BCR cross-linking
as an increase in the number of propidium iodide-positive cells (Fig. 1
A). In WEHI 231 cells,
induction of apoptosis particularly correlates with processing, i.e.,
activation of caspase-7 (18). To confirm this observation
by Bras et al. (18) for the WEHI 231 cell line used in our
laboratory, we analyzed cleavage of endogenous procaspase-7 (p35) after
BCR cross-linking. One day after stimulation, we observed significant
amounts of the large fragment of caspase-7 (p20), which is indicative
of active caspase-7 (Fig. 1B). Degradation of intact PARP
(116 kDa) to the 85-kDa apoptotic fragment further supports the notion
that caspases are indeed activated in WEHI 231 cells after BCR ligation
(Fig. 1B, lower panel). In addition, we observed
DNA laddering characteristic of apoptotic cells (Fig. 1C)
and indicative for caspase-3 activation (see Discussion).
Also, we directly measured a rise in caspase-3-like activity in cell
extracts of Ag receptor-stimulated WEHI 231 cells (Fig. 4D
and data not shown).
|
|
D). Increased expression of procaspase-7 did not influence
proliferation of the cells or their response to apoptosis-inducing
agents (Abs to the BCR or etoposide; data not shown). As observed with
endogenous caspase-7, we found strong cleavage of ectopically expressed
caspase-7 and degradation of PARP 
24 h after BCR ligation (Fig. 1E). Absence or presence of a flag tag at the C terminus of
caspase-7 did not influence processing (data not shown). In most of our
experiments, significant amounts of processed caspase-7 (p20) were
detected, starting at 20–24 h poststimulation (Fig. 1F and
data not shown).
Experiments by Ruiz-Vela et al. (20) suggest that cleavage
of caspase-7 is initiated by an autoproteolytic process. If so,
mutation of the active center of procaspase-7 should inhibit or at
least slow down the cleavage of the proform of caspase-7. Therefore, a
mutant of caspase-7 was constructed, in which the cysteine of the
active center was changed to a serine, which renders the protein
enzymatically inactive. When we compared the processing of ectopically
expressed wild-type procaspase-7 with the mutant, no strong difference
was found after BCR ligation (Fig. 2).
Taking into account that overexpression of enzymatically inactive
caspase-7 was 15- to 30-fold compared with the endogenous enzyme, we
conclude that strong auto and/or transprocessing of caspase-7 is rather
unlikely.
|
Cleavage of effector caspases such as caspase-7 is normally
dependent on the preceding activation of an initiator caspase. Because
we and others have described the protective role of Bcl-2 family
members, which are known to stabilize mitochondria, on Ag
receptor-stimulated WEHI 231 cells (14, 16, 17, 18, 19, 20, 21),
activation of the mitochondrial-dependent initiator caspase-9 was
examined. To date, only very low BCR-induced caspase-9 activity, but no
processing, has been observed for this initiator caspase (18, 20). This does not necessarily argue against a role for
caspase-9 in this pathway, because caspase-9 can be activated without
prior proteolytic processing (25). Accordingly, we
reevaluated this issue by analyzing WEHI 231 cell populations
retrovirally transduced with procaspase-9. As shown in Fig. 3, triggering this population of WEHI 231
cells via the BCR induces low but unambiguous cleavage of caspase-9
from its proform (p47) to the large fragment of the active caspase
(p35). This shows for the first time that caspase-9 is not only
activated but is also processed after BCR ligation in WEHI 231 and
opens the possibility that caspase-9 might initiate BCR-induced
activation of downstream caspases.
|
To investigate whether caspase-9 processing is only secondary to
an ongoing apoptotic process or is actually required for caspase-7
processing, we generated populations of WEHI 231 cells overexpressing
dnC9 (CS exchange in the active center, also refered to as
kinase-dead caspase-9) in cells previously transduced with
caspase-7. Compared with endogenous caspase-9, transduced dnC9
was 25- to 50-fold overexpressed in different populations (data not
shown). As a control, only the relevant selection marker was
coexpressed together with caspase-7. Double-positive cell populations
were enriched on the basis of green (coexpressed with caspase-7) and
yellow (coexpressed with dnC9) fluorescent marker proteins by FACS. An
example of the purity of the sorted cells is shown in Fig. 4A. When the BCR was ligated
on cells expressing the enzymatically inactive mutant of caspase-9,
processing of caspase-7 was indeed strongly reduced (Fig. 4B, upper panel).
To functionally test for caspase activation in these cells, we again
looked for degradation of PARP and the appearance of DNA laddering
after BCR ligation. The dnC9 clearly protected WEHI 231 cells against
both destructive processes, i.e., it inhibited PARP cleavage (Fig. 4B, lower panel) and fragmentation of the
chromosomal DNA (Fig. 4C). Furthermore, we compared cell
extracts of dnC9 transduced and control cells for effector caspase
activity. We used a colorimetric substrate, which is cleaved by
caspase-3-like (caspase-3, -6, and -7) activity. Consistent with the
finding that dnC9 blocks BCR-mediated PARP and DNA degradation, we
found that the presence of overexpressed dnC9 reduced BCR-inducible
caspase-3-like activity to almost undetectable levels over the whole
period of observation (Fig. 4D and data not shown). Thus,
our results suggest that activation of caspase-9 is central and
obligatory for the activation of the whole cascade of downstream
effector caspases.
Antiapoptotic members of the Bcl-2 family prevent BCR-inducible caspase activation
We previously showed that CD40 signaling, which alleviates
BCR-mediated apoptosis in WEHI 231 cells (26), boosts the
expression of the antiapoptotic Bcl-2 family member A1
(19). Furthermore, ectopic expression of A1 protects these
cells against the deleterious consequence of BCR-induced signals
(19). To address whether damage of mitochondria is
upstream of caspase-9 activation, we first tested whether A1 can
stabilize mitochondrial function in WEHI 231. As expected, A1 was able
to reduce the production of reactive oxygen species after engaging the
BCR on WEHI 231 cells (data not shown). This result is in keeping with
the observation that A1 localizes to mitochondria and stabilizes their
function in epithelial cells (27). Thus, if disruption of
mitochondrial function or integrity is upstream of BCR-mediated
caspase-9 processing in WEHI 231, sufficient levels of A1 should
interfere with the cleavage of procaspase-9. To test this, WEHI 231
cells expressing procaspase-9 in the absence or presence of A1 were
analyzed. Indeed, WEHI 231 cells overexpressing A1 no longer exhibited
caspase-9 processing after BCR ligation, whereas the respective control
populations expressing caspase-9 only in combination with the relevant
selection marker activated this initiator caspase (Fig. 5A). As a further control, we
used etoposide known to activate caspases via the mitochondrial
pathway. Again, A1 strongly inhibited caspase-9 activation (Fig. 5A), thus demonstrating the stabilizing potential of A1 on
mitochondria. In addition, A1 also strongly interfered with caspase-7
activation after BCR ligation, further supporting a central role of
caspase-9 (Fig. 5B). Bcl-2 itself (normally not highly
expressed in WEHI 231), when ectopically expressed had the same effect
as A1 and protected against BCR-stimulated caspase-7 processing (Fig. 5C). Thus, these data indicate that both the initiator
caspase-9 and the effector caspases are downstream of mitochondria in
BCR signal-induced apoptosis.
|
Caspase-9 activation depends on the formation of apoptosomes
consisting of Apaf-1, Cyt c, dATP, and caspase-9
(4). Despite cleavage of caspase-9 in our experiments, we
have not been able to detect significant release of Cyt c
from the mitochondrial intermembrane space to the cytosol after BCR
ligation (Fig. 6A). This was
also true for experiments in which we already observed basal amounts of
Cyt c in the cytoplasm of untreated cells (data not shown).
When we compared the cytosolic and mitochondrial fractions for
caspase-9, we found a possible explanation for this apparent
discrepancy. Whereas only a minor amount of procaspase-9 is found in
the mitochondria-containing fraction of unstimulated cells compared
with the cytosol, it is exactly this fraction in which the large
fragment of caspase-9 (p35) is strongly enriched after BCR stimulation
(Fig. 6, B and C, upper panel,
C9 + eyz). In contrast, almost no processed form of caspase-9
(p47) is found in the cytoplasm. As expected, antiapoptotic A1 strongly
interfered with the formation and the accumulation of cleaved caspase-9
in the mitochondrial fraction (Fig. 6C, lower
panel, C9 + A1). Even though the current level of analysis does
not allow unequivocal allocation of caspase-9, the association with the
mitochondria-enriched fraction is consistent with the notion that minor
amounts of an apoptosome(-like) structure are already deposited on the
surface of mitochondria in unstimulated cells where it can quickly
sense very low amounts of released Cyt c.
|
Our molecular analyses show that dnC9 has the same potential as A1
in inhibiting caspase-7 cleavage and PARP degradation. Because we have
previously shown that A1 interferes with BCR-activated cell death, we
next explored whether dnC9-transduced WEHI 231 cells are also more
resistant to this type of cell death. We approached this question by
determining the survival rate after BCR ligation in A1- and
dnC9-transduced populations relative to a control population. Fig. 7 shows that dnC9 supports, over the
period of 2 days, survival of Ag receptor-stimulated WEHI 231 cells to
the same extend as A1. Again, the data indicate the importance of the
mitochondrial/caspase-9 pathway for BCR-released apoptotic signals.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Caspase-7 has been suggested to be activated by an autocatalytic
process (20). Our experiments addressed this issue at two
levels: 1) the initial activation of wild-type caspase-7 in the
presence of dnC9 (Fig. 4B), and 2) the "amplification"
step with a kinase-dead caspase-7 (Fig. 2). Our observation that a dnC9
totally blocks caspase-7 processing after BCR ligation indicates an
absolute requirement for active caspase-9 in the initial phase of
caspase-7 cleavage. Furthermore, the observation that kinase-dead
caspase-7 is processed to a similar extent as wild-type caspase-7 seems
to argue against strong autocatalytic activation or transprocessing of
this effector kinase. Of course, overexpression of the mutant did not
eliminate the expression of endogenous caspase-7, which would provide
caspase-7 activity after BCR engagement. Even though exogeneous mutant
caspase-7 was 15- to 30-fold more highly expressed than the endogenous
enzyme, our experiments cannot rule out that processed endogenous
caspase-7 contributes to the processing of the inactive mutant.
Besides caspase-7, activation of caspase-3 after BCR ligation seems very likely. Whereas PARP can apparently be cleaved by both caspase-3 and -7 (28, 29), chromosomal DNA degradation observed in our experiments rather specifically points to the activation of caspase-3 because various cell types deficient for this effector caspase (including primary thymocytes, T cells, murine embryonic fibroblasts, and hepatocytes) exhibit strong impairment or even absence of DNA degradation (30, 31). Consistent with our conclusion, caspase-3 activation after BCR ligation has been observed in B cells, including primary human memory B cell blasts and various B cell lines (12, 13, 32). However, in agreement with the finding of Bras et al. (18), we were unable to directly demonstrate processing of procaspase-3 after BCR ligation by Western blotting, even though the caspase-3-specific Ab used in the experiments did detect the large fragment of caspase-3 in extracts of etoposide-treated WEHI 231 (data not shown). It is possible that levels of caspase-3 cleavage below the level of detection are still sufficient for DNA degradation.
Cleavage of effector caspases such as caspase-3 and -7 is normally dependent on the preceding activation of initiator caspases in a death-inducing signaling complex at the plasma membrane (caspase-8) or in an apoptosome in the cytoplasm (caspase-9) (2). Experiments by Yoshida et al. (33) clearly argue against the involvement of caspase-8 in BCR-mediated apoptosis because overexpression of either CrmA (the cowpox virus caspase inhibitor protein known to preferentially block caspase-8) or a dominant negative form of the adapter Fas-associated death domain protein did not block BCR-mediated apoptosis in WEHI 231, whereas it inhibited Fas-dependent cell death. Even though Scott et al. (34) described a Fas-dependent pathway in which caspase-8 activation could not be observed, the inability of dominant negative Fas-associated death domain protein to interfere with BCR-dependent apoptosis makes it rather unlikely that Fas is involved in the initiation of the caspase cascade.
In contrast, caspase activation via the mitochondria should be involved because the protective role of Bcl-2 family members like A1 and Bcl-xL is well established for the immature B cell line WEHI 231 (14, 16, 17, 18, 19, 20, 21). Consistent with this, we show for the first time processing of caspase-9 after BCR ligation. Furthermore, the absence of effector caspase activation in the presence of dnC9 demonstrates an obligatory role of this effector caspase for initiation of the whole caspase cascade. This is in line with the finding that mitochondria are central to BCR-triggered apoptosis in various B cells (12, 13, 14). Interestingly, there is one report on a mature and an immature human B cell line in which the authors place caspase-9 activation downstream of caspase-2 and -3 (32). This result is in seeming contrast to our findings. However, in the human B cell lines analyzed by Chen et al. (32), activation of caspase-2 and -3 occurred rather early, i.e., within 2–4 h. Thus, one explanation to reconcile the data lies in the possibility that the BCR first generates a low-level activation of caspase-2 and -3 via an unknown mechanism. This first wave of caspases would then trigger the strong effector caspase response via caspase-9. We are currently testing this possibility.
Even though dnC9 seems to totally block certain indicators of apoptosis (cleavage of caspase-7, caspase-3-like activity, PARP and DNA degradation), the kinase-dead version of caspase-9 did not completely inhibit cell death. This could indicate: 1) because caspase-9 is downstream of the mitochondria, signals from the BCR can still damage the mitochondria, leading to the release of apoptotic effector molecules like apoptosis-inducing factor, which would kill the cells in a caspase-independent way (35), and 2) blockage of effector caspases is not complete and the undetectable activity of these enzymes is sufficient to eventually kill the cells.
It is intriguing that the large fragment of caspase-9 indicative of an active enzyme is preferentially associated with the mitochondrial preparation in WEHI 231 cells. However, because the mitochondria preparations used in our study contain other cellular compartments including nuclei, it is currently not possible to pinpoint the exact cellular structure to which the active caspase-9 fraction localizes. Several sites are possible.
First, caspase-9 might be found in the nucleus as observed for human
epithelial cells (36). This localization pattern would be
consistent with a need for active caspase-3 in the nucleus to degrade
the inhibitor of caspase-activated DNase to allow DNA cleavage by
caspase-activated DNase (37, 38, 39). However, in the
precedent of epithelial cells, nuclear caspase-9 activation is
downstream of a caspase-3-like activity (36). Because in
WEHI 231 cells BCR-induced caspase-9 cleavage primarily depends on
mitochondria, as indicated by the inhibition through A1 (Figs. 5A and 6C, lower panel), we do not
favor this possibility.
Second, as described, for instance, for primary rat neurons and cardiomyocytes, caspase-9 could also be sequestered together with Cyt c within the mitochondrial intermembrane space (40, 41, 42). In these cells, >80% of all cellular caspase-9 localizes within mitochondria (42). In WEHI 231, however, only a small fraction of all caspase-9 would be locked up in the mitochondria to be released upon stimulation into the cytosol, where the majority of the protein is located anyway. We do not consider this a likely possibility.
Third, the minor fraction of caspase-9 might be localized not within mitochondria but onto the cytoplasmic surface of mitochondria. This localization could potentially sensitize the cells for mitochondria-dependent caspase activation because even very low amounts of released Cyt c would readily "find" caspase-9. According to this scenario, Cyt c would be released from the mitochondria after BCR ligation and would be bound immediately. The resulting activation of this complex would then lead to activation of caspase-9. Subsequently, effector caspases-3 and -7 would be recruited, activated by cleavage, and then released, whereas active caspase-9 would remain in the complex. The model predicts that Apaf-1 or a homolog like the recently described proapoptotic caspase adaptor protein (43) will also be found in this compartment. Alternatively, caspase-9 could be activated, not in an apoptosome(-like) complex, but via a yet unknown mechanism. Current experiments are addressing these issues.
Although damage to mitochondria have been emerging as central to BCR-induced apoptosis, it remains unclear how mitochondria are damaged and if there is one or more players in the game. The "multidomain" proapoptotic Bcl-2 proteins (i.e., Bak and Bax) should be most important because at least one of these proteins seems to be necessary for the induction of apoptosis by many stimuli (10, 44). Besides Bcl-2 proteins, lipids like ceramide or arachidonic acid are also known to impinge on mitochondria after BCR ligation (45, 46, 47). In keeping with this, an unusually low level of ceramide production correlated with resistance to BCR-induced apoptosis in a WEHI 231 variant (48). At the current stage, however, it is not clear whether physiological levels of these compounds are sufficient to induce apoptosis on their own or whether they only set the stage for Bak or Bax, for example, by lowering the threshold for Cyt c release. Clearly, additional experiments are needed to clarify the very important questions of how mitochondria are damaged and how apoptosis is induced after BCR ligation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Ingolf Berberich, Institut für Virologie und Immunbiologie, Versbacher Strasse 7, 97078 Würzburg, Germany. E-mail address: berberich.i{at}mail.uni-wuerzburg.de
3 Abbreviations used in this paper: BCR, B cell Ag receptor; PARP, poly(ADP-ribose) polymerase; Apaf-1, apoptotic protease-activating factor 1; Cyt c, cytochrome c; dnC9, dominant negative caspase-9.
Received for publication October 30, 2001. Accepted for publication February 11, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
m) and cytochrome c release are caspase independent. J. Immunol. 163:4683.This article has been cited by other articles:
|
|
 |
|
  M. Poggianella, M. Bestagno, and O. R. Burrone The Extracellular Membrane-Proximal Domain of Human Membrane IgE Controls Apoptotic Signaling of the B Cell Receptor in the Mature B Cell Line A20 J. Immunol., September 15, 2006; 177(6): 3597 - 3605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Craxton, K. E. Draves, A. Gruppi, and E. A. Clark BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway J. Exp. Med., November 21, 2005; 202(10): 1363 - 1374. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Mielenz, C. Vettermann, M. Hampel, C. Lang, A. Avramidou, M. Karas, and H.-M. Jack Lipid Rafts Associate with Intracellular B Cell Receptors and Exhibit a B Cell Stage-Specific Protein Composition J. Immunol., March 15, 2005; 174(6): 3508 - 3517. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R. Herrin, A. L. Groeger, and L. B. Justement The Adaptor Protein HSH2 Attenuates Apoptosis in Response to Ligation of the B Cell Antigen Receptor Complex on the B Lymphoma Cell Line, WEHI-231 J. Biol. Chem., February 4, 2005; 280(5): 3507 - 3515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. He, Y. Tohyama, K.-i. Yamamoto, M. Kobayashi, Y. Shi, T. Takano, C. Noda, K. Tohyama, and H. Yamamura Lysosome is a primary organelle in B cell receptor-mediated apoptosis: an indispensable role of Syk in lysosomal function Genes Cells, January 1, 2005; 10(1): 23 - 35. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Harnett CD40: A Growing Cytoplasmic Tale Sci. Signal., June 15, 2004; 2004(237): pe25 - pe25. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Katz, C. Lord, C. A. Ford, S. B. Gauld, N. A. Carter, and M. M. Harnett Bcl-xL antagonism of BCR-coupled mitochondrial phospholipase A2 signaling correlates with protection from apoptosis in WEHI-231 B cells Blood, January 1, 2004; 103(1): 168 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Muscarella and S. E. Bloom Cross-linking of Surface IgM in the Burkitt's Lymphoma Cell Line ST486 Provides Protection against Arsenite- and Stress-induced Apoptosis That Is Mediated by ERK and Phosphoinositide 3-Kinase Signaling Pathways J. Biol. Chem., January 31, 2003; 278(6): 4358 - 4367. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
