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*
The Walter and Eliza Hall Institute, Melbourne, Australia; and
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland
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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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Conflicting results have been published regarding the role of JNK signaling in the control of apoptotic cell death (3, 4, 5). Overexpression of certain components of the JNK signaling pathway were shown to induce apoptosis in Jurkat T lymphoma cells or fibroblasts, and dominant negative (DN) mutants of these proteins were reported to block UV irradiation or cisplatin-induced killing of these cells (6, 7, 8). However, the requirement of JNK signaling in drug-induced cell death does not seem to be a general one (9).
Ligation of the death receptors Fas (also called APO-1 or CD95), TNF-R1, DR3, DR4, DR5, or DR6 leads to activation of the JNK pathway in many cell types (1, 5, 10, 11, 12, 13). In the case of Fas, many mechanisms have been reported to lead to JNK activation. One mechanism involves caspase cleavage-mediated activation of MEKK1, a JNK kinase kinase, that can activate the JNK upstream regulators SEK1/MKK4 and MKK7 (14). Elevation of ceramide levels in response to Fas cross-linking was also reported to activate JNK, but this has been challenged (3, 15, 16, 17). Fas was reported to engage the JNK pathway via death domain associated protein (DAXX)-mediated activation of ASK1/MKK5 (18, 19). Overexpressed DAXX was shown to interact with the death domain (DD) in Fas and to potentiate anti-Fas Ab-induced apoptosis and JNK activation in 293 and HeLa cells. The C-terminal fragment of DAXX (aa 628–739) was reported to be sufficient for Fas binding and to act as a dominant interfering mutant, inhibiting Fas-induced apoptosis and JNK activation (18). The DD in Fas has also been reported to undergo homotypic interaction with the DD of receptor interacting protein (RIP) kinase (20). RIP, in turn, binds the adapter molecule RIP-associated ICH-1/CED-3-homologous protein with a death domain (RAIDD) to recruit and activate caspase-2 zymogens via homotypic interaction of their caspase recruitment domain (CARD) (21). RIP can also bind to the TNF-R1 downstream effector TNF-receptor associated factor 2 (TRAF-2) (22). TRAF-2 deficient cells are unable to induce JNK in response to TNF, but are normally sensitive to anti-Fas Abs and hypersensitive to TNF, indicating that JNK activation is dispensable for death receptor-transduced apoptosis (23, 24).
The question of whether JNK signaling is a consequence of activating the death program or is an independent event is still unclear. In Jurkat T lymphoma cells, application of the broad spectrum caspase inhibitor zVADfmk inhibited Fas-induced apoptosis and JNK activation at low concentrations of anti-Fas Ab, but failed to block JNK activation when higher concentrations of this Ab were used (25). However, other investigators demonstrated that zVADfmk effectively inhibited JNK activation in the same cell type at even higher concentrations of anti-Fas Ab (26), suggesting that caspase activity is a prerequisite for Fas-mediated JNK activation. In contrast, in neuroblastoma cells and 293 human embryonic kidney fibroblasts, Fas-induced JNK activation was unaffected by zVADfmk treatment (5, 18, 19).
Mice lacking distinct components of the JNK signaling cascade have been analyzed to investigate its role in cell death control. Hippocampal neurons from JNK3-/- animals were protected from kainic acid-induced cell death and seizures, and immature CD4+8+ thymocytes from JNK2-/- animals were reported to have increased resistance to TCR/CD3 activation-induced cell death (AICD) (27, 28). Mice lacking JNK1 had elevated numbers of Th 1 cells, which were also abnormally resistant to AICD (29). Thymocytes of both strains of mutant mice were normally sensitive to Fas-induced apoptosis. AICD requires Fas and Fas ligand (FasL) (30). It therefore appears that JNK1 and JNK2 are not needed for Fas signaling, but play a role in FasL expression. The latter has been confirmed by several studies of the promoter of the FasL gene (31, 32). In contrast to these results, it has been reported that in animals lacking the JNK upstream regulator, MKK4/SEK1, thymocytes showed enhanced sensitivity to Fas-mediated cell death (33), but this has been challenged (34, 35).
To date, all these studies took advantage of cross-linking Abs to Fas. This may have produced misleading results, since it has been documented that anti-Fas Abs do not perfectly mimic the physiological ligand of Fas (36). We investigated the impact of soluble, cross-linked, or membrane-bound FasL on JNK activation and cell death in the lymphoid cell lines, CEM and SKW6. Furthermore, we analyzed the dependence of FasL-mediated JNK activation on caspases and the intracellular protein DAXX by analyzing cell lines stably expressing dominant inhibitors of these proteins. We also investigated the role of the signal transducers DAXX, RIP, and RAIDD in FasL-induced apoptosis in these lymphoid cells.
Our results demonstrate that cross-linked or membrane-bound, but not soluble FasL induces JNK activity and apoptosis, suggesting that aggregation of many Fas receptors is necessary to induce these events. While Fas-induced apoptosis required caspase activity, FasL-mediated JNK activation was only partially dependent on caspases and was completely independent of DAXX. Furthermore, Fas-mediated apoptosis in lymphoid cells does not require DAXX, RIP, or RAIDD, which have been proposed to play a role in Fas-induced cell death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CEM and Jurkat human T acute leukemia-derived lines and the human B lymphoblastoid SKW-6 as well as the mouse B lymphoma cell line CH1 were used in this investigation. Neuro2A neuroblastoma cells and a derivative clone transfected with a murine FasL expression construct have been described (36). CEM and Jurkat cells were cultured in RPMI 1640 medium, and Neuro2A, SKW6, and CH1 cells were cultured in the high glucose version of DMEM, both supplemented with 10% (v/v) heat-inactivated FCS (TRACE Bioscience Castle Hill, NSW, Australia), 250 µM L-asparagine (Sigma, St. Louis, MO), 50 µM 2-ME (Sigma), and 100 µg/ml penicillin/streptomycin (Life Technologies, Gaithersburg, MD) at 37°C in a humidified atmosphere containing 10% CO2.
Expression constructs and cell transfection
Expression vectors for dominant interfering mutants of DAXX, RIP, or RAIDD were generated by PCR amplification of aa 628–739 of mouse DAXX, aa 120–199 of human RAIDD, and aa 5–553 of mouse RIP. The products were subcloned into pEF-based expression vectors incorporating N-terminal EE- (37), FLAG- (Sigma), or I-SPY-epitope tags (38). PCR was performed using proof-reading Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA), and the sequences of all constructs were verified by automated sequencing (ABI Perkin-Elmer, Foster City, CA). Cells were transfected by electroporation, and drugs for selection of transfectants (3–10 µg/ml puromycin, 0.5–2 mg/ml neomycin or hygromycin) were added after 36 h of culture. Drug-resistant lines were cloned with the single-cell deposition unit of the FACStarPlus cell sorter (Becton Dickinson, Mountain View, CA). The expression construct for FLAG-tagged CrmA and FADD-DN have been described (39, 40).
Determination of Jun kinase activation and immunoblotting
Cells (1–2 x 106/ml) were resuspended in 50 µl of lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, and 1 mM EGTA, pH 7.5, supplemented with 1 mM Na3VO4; 1 mM NaF; 25 µg/ml aprotinin, leupeptin, and pepstatin; Pefabloc; and 1% Triton X-100). Samples were cleared by centrifugation (16,000 x g, 5 min, 4°C). Proteins were size fractionated by SDS-PAGE (14%) under reducing conditions in Tris/glycine-buffered gels (NOVEX, San Diego, CA) and transferred onto polyvinyl-difluoridon membranes (Millipore, Bedford, MA) by electroblotting. Membranes were blocked for >12 h in PBS containing 0.05% Tween-20 and 5% skim milk at 4°C. The anti-phospho-Jun kinase mAb G-9 (New England Biolabs, Beverley, MA) was diluted 1/2000 in PBS containing 0.05% Tween-20 and 5% skim milk for immunolabeling. Peroxidase-conjugated rabbit anti-mouse Ig Abs (Silenus, Paris, France) served as secondary reagent (1/1000), and the ECL chemiluminescence system (Amersham, Arlington Heights, IL) was used for detection. Membranes were reprobed with polyclonal rabbit antiserum sc-571 specific for JNK1 and JNK2 (Santa Cruz Biotechnology, Santa Cruz, CA) to demonstrate equal protein loading of the gel. Mouse anti-human FADD mAb (PharMingen, San Diego, CA), mouse anti-human caspase-8 mAb (PharMingen), and rabbit anti-human caspase-3 antiserum (PharMingen) were diluted 1/1000 in PBS containing 0.05% Tween-20 and 5% skim milk. Anti-human 70-kDa heat shock protein mAb was a gift from R. Anderson (Peter MacCallum Cancer Research Institute, Melbourne, Australia) and was used at 0.1 µg/ml in PBS containing 0.05% Tween-20 and 5% skim milk to demonstrate equal protein loading of the gel.
Immunofluorescence staining and flow cytometric analysis
Detection of FLAG-CrmA, B-tag RAIDD-DN, EE-RIP-DN, FLAG-FADD-DN, or EE-DAXX-DN proteins in cells was performed by cytoplasmic immunofluorescence staining (39). Cells (0.2 x 106) were washed in PBS, resuspended, and fixed for 10 min at room temperature in 1 ml 1% paraformaldehyde in PBS. After washing with PBS, cells were resuspended in FACS buffer (balanced salt solution, 2% FCS, 10 mM sodium azide, and 0.3% saponin) containing anti-FLAG M2 (Sigma), anti-I-SPY (Silenus), or anti-EE mAbs (Babco, Richmond, CA; 1/1000). After 30-min incubation on ice, cells were washed in FACS buffer containing 0.03% saponin (Sigma) and incubated with FITC-labeled goat anti-mouse Ig Ab (1/100 in FACS buffer containing 0.3% saponin) for 30 min on ice. After a final wash, the cells were resuspended in 200 µl of FACS buffer, and 5000 cells/sample were analyzed on a FACScan (Becton Dickinson). Parental cells stained with the relevant epitope tag-specific Abs and the secondary FITC-coupled Ab served as negative controls.
Cell death analysis
The percentage of viable cells in culture was determined by staining cells with 2 µg/ml propidium iodide or annexin V-FITC (Roche, Indianapolis, IN), used at the manufacturer’s recommended concentrations, and analyzing the samples on a FACScan. As cell death-inducing stimuli for in vitro studies we used membrane-anchored mouse FasL (coculture with Neuro2A FasL cells), 100 ng/ml FLAG-tagged recombinant human FasL (41), 100 ng/ml JO2 anti-mouse Fas mAb (PharMingen), or 100 ng/ml anti-human Fas mAb APO-1 (42) and 50 µg/ml of the topoisomerase inhibitor VP-16 (Sigma). Recombinant FasL was aggregated by addition of 0.5 µg/ml monoclonal anti-FLAG M2 Ab. Abs to Fas were cross-linked by 100 ng/ml protein A (Amersham Pharmacia, Piscataway, NJ).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Application of the cross-linking anti-human Fas Ab CH11 was
shown to induce activation of JNK in several cell lines, but the effect
of the physiological ligand, FasL, was never tested. We treated human T
lymphoma-derived CEM cells and human B lymphoblastoid SKW6 cells with
graded concentrations of recombinant FasL (1, 10, and 100 ng/ml) and
determined JNK activity after 90 min using a phospho-JNK-specific Ab.
Soluble FasL did not induce JNK activity, while application of the
cytotoxic drug VP-16 did so in both cell lines (Fig. 1
a). However, when an
anti-FLAG M2 mAb was used to cross-link recombinant FasL, activation of
JNK was readily observed in both CEM and SKW6 cells (Fig. 1b). Similarly, the anti-Fas Ab APO-1 induced JNK
activity most efficiently when aggregated by addition of protein A
(Fig. 1b). As previously reported (36), cell
death in CEM cells was only observed when FasL or anti-APO-1 mAb
were cross-linked, but not by application of FasL or APO-1 mAb alone
(Fig. 1c). In SKW6 cells, induction of apoptosis required
aggregated FasL, but could also be mediated by APO-1 mAb alone (Fig. 1c). Coculture with Neuro2A cells, which were engineered to
express membrane-bound FasL, but not with Neuro 2A cells transfected
with a control vector, rapidly induced JNK activity and apoptosis in
CEM cells (Fig. 1, d and e). These results
demonstrate that, as for cell death, aggregation of many Fas receptors
is required to induce JNK activation in lymphoid cells.
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To investigate whether FasL-mediated JNK activation requires
caspase activity we used CEM cells and SKW6 cells stably expressing
CrmA, an inhibitor of caspase-8, or pretreated parental cells with the
pan-caspase inhibitor zVADfmk (100 µM) for 30 min before application
of FasL. Expression of CrmA was confirmed by cytoplasmic
immunofluorescence staining of the cell lines and immunoblotting of
cell lysates using FLAG epitope tag-specific Abs (Fig. 2a).
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b). However, longer exposure of filters
revealed that JNK activation became apparent in CrmA-expressing cells
treated with cross-linked FasL or APO-1 mAb, although to a much lower
extent compared with that in parental cells (Fig. 2, b and
c). While zVADfmk reduced FasL-induced JNK activation in
SKW6 cells efficiently, it repeatedly failed to do so in CEM cells,
even when lower doses of cross-linked FasL were applied (25 and 50
ng/ml) and when the dosage of the caspase inhibitor was increased up to
300 µM (Fig. 2d and data not shown). Increased expression
levels of the Fas-signaling components FADD, pro-caspase-8, or
pro-caspase-3 or decreased zVADfmk uptake did not account for the
reduced ability of zVADfmk to inhibit JNK activation in CEM cells.
Comparison of FADD, pro-caspase-8, and pro-caspase-3 revealed that all
three proteins are expressed at comparable levels in CEM and SKW6 cells
(Fig. 2e). Moreover, the amount of zVADfmk permeating the
cell membrane was sufficient to prevent FasL-mediated processing of
pro-caspase-3 in both cell types, but failed to block FasL-mediated JNK
activity in CEM cells (Fig. 2, d and f). In CEM
and SKW6 cells VP-16-mediated JNK activation was also unaffected by
zVADfmk even at concentrations of up to 300 µM, and expression of
CrmA also had no impact (Fig. 2g and data not shown).
Consistent with previous reports (39, 42), CrmA expression
drastically reduced FasL-induced, but not VP-16-induced, apoptosis,
while zVADfmk inhibited both FasL- as well as VP-16-induced apoptosis
in CEM (Fig. 3a) or SKW6 (Fig. 3b) cells. These observations indicate that FasL-induced JNK
activation can occur in a caspase-dependent and caspase-independent
manner, whereas VP-16 treatment can induce JNK in the absence of
caspase activity.
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Our current understanding of Fas signaling is based almost exclusively on experiments with Abs to Fas. Since these reagents do not reliably mimic the physiological ligand, the proposed mechanisms of Fas signaling identified by using anti-Fas Abs may need re-evaluation (36).
The roles of DAXX, RIP, RAIDD, and FADD in FasL-induced apoptosis were
investigated in CEM, SKW6, Jurkat, and CH1 cells stably expressing
dominant-interfering mutants of these signal transducers.
Dominant-negative DAXX or RIP contained the regions necessary for
binding to the DD in Fas, but lacked the regions required for
downstream apoptosis signaling (18, 20). Dominant-negative
RAIDD contained the DD with which it binds to RIP, but lacked the CARD
region needed for interaction with pro-caspase-2 (21). The
expression construct for FLAG-tagged dominant-negative FADD comprising
aa 80–208 of human FADD has been described (36). Stably
transfected clones expressing readily detectable levels of these
dominant-interfering mutants (Fig. 4a and data not shown) were
exposed to a range of apoptotic stimuli. Dominant-negative DAXX, RIP,
or RAIDD afforded no protection against membrane-bound FasL, aggregated
soluble FasL, cross-linked Abs to Fas, or staurosporine in the cell
types tested (Fig. 4, b–d, and data not shown). Coculture
with Neuro2A-FasL cells or treatment with as little as 10 ng/ml
aggregated anti-Fas Abs killed most of the transfected lymphoma
lines within 24 h. In contrast, FADD-DN efficiently prevented cell
death induced by cross-linking of Fas (Fig. 4, b–d).
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e). These results indicate that DAXX,
RIP, and RAIDD are dispensable for Fas-transduced apoptosis in lymphoid
cells and that DAXX is not required for FasL-induced JNK
activation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). It has previously been shown that FasL can only kill cells
efficiently when cross-linked or when it is presented in its
membrane-bound form (41, 44). This may indicate that
FasL-induced JNK signaling only occurs when the cell death program is
engaged. Alternatively, cell killing and JNK activation could be
independent, but both Fas signaling cascades can only be activated by
extensive receptor aggregation. The Fas apoptosis signaling pathway
requires recruitment of the adapter protein FADD and autocatalytic
activation of caspase-8 (45). Caspase-8, the most upstream
caspase in death receptor signaling, can be inhibited by the cowpox
virus protein CrmA, and like most other known caspases it is sensitive
to the tripeptide inhibitor zVADfmk (45, 46). Consistent
with previous reports (36, 39), both inhibitors
significantly reduced FasL-induced apoptosis in CEM and SKW6 cells
(Fig. 3). In contrast, and as expected, only zVADfmk inhibited
VP-16-induced apoptosis because this cell death pathway requires
CrmA-insensitive caspases such as caspase-9 (47, 48). CrmA
reduced FasL-induced activation of JNK signaling in CEM and SKW6 cells,
but inhibition was incomplete (Fig. 2 and data not shown). Although
zVADfmk reduced FasL-induced JNK activation in SKW6 cells, it did so
poorly in FasL-treated CEM cells even when lower doses of FasL and
higher zVADfmk concentrations were used (Fig. 2). This difference was
not caused by either higher expression levels of FADD,
pro-caspase-8, or pro-caspase-3 or reduced drug uptake in CEM cells.
All three Fas-signaling components were expressed at similar
amounts, and zVADfmk blocked caspase-3 processing in CEM cells as
efficiently as in SKW6 cells (Fig. 2, e and f).
In addition, caspase inhibition by zVADfmk had no impact on
VP-16-mediated JNK activation (Fig. 2 and data not shown). These
observations indicate that FasL can engage caspase-dependent as well as
caspase-independent pathways leading to JNK activation, and it is
possible that different cell types preferentially activate one or the
other pathway in response to FasL. In this context it is interesting to
note that caspase-8 was shown to be able to activate JNK independent of
its enzymatic activity via interaction with TRAFs and subsequent
activation of MEKK1. However, this study is only based on
overexpression analysis and lacks proof that this interaction can also
occur when all components are present at physiological levels
(10). Nevertheless, it is possible that Fas-mediated JNK
activation is not only a stress response of the insulted cell, but,
under certain circumstances, may be an active signaling event. This is
also supported by the fact that JNK signaling seems to occur
exclusively upstream of and/or independent of caspases in VP-16-treated
lymphocytes (Fig. 2).
Fas ligation-induced JNK activation was reported to occur upstream of
caspase activation via the intracellular protein DAXX. DAXX originally
was identified in a yeast two-hybrid screen and was shown to bind to
Fas and signal to the JNK cascade via the apoptosis signal-regulating
kinase, ASK1 (18, 19). However, in our analysis of several
lymphoid cell lines, expression of a dominant-interfering mutant of
DAXX had no impact on FasL-induced JNK activation and apoptosis (Fig. 4). This demonstrates that DAXX plays no physiological role in
FasL-mediated JNK signaling and apoptosis, at least in lymphoid cells.
Although expression of DAXX-DN was confirmed by FACS analysis and
immunoblotting, it cannot be excluded that higher levels of expression
might have had an effect. It is, however, interesting to note that DAXX
was shown to reside exclusively in the nucleus and to interact with
CENP-C, a centromere binding protein, and the transcription factor Pax3
(49, 50). Most convincingly, mice lacking DAXX die in
utero due to abnormally increased apoptosis in critical organs, and
cells from these embryos are normally sensitive to treatment with
anti-Fas Abs (51). Collectively, these results suggest
that DAXX plays no essential role in controlling Fas-mediated
apoptosis. Considering the high rates of apoptosis in
DAXX-/- embryonic
tissues, an anti-apoptotic role for DAXX in other apoptosis
pathways appears likely.
Our experiments using dominant-interfering mutants of RIP and RAIDD
demonstrate that these proteins are also dispensable for FasL-induced
apoptosis, at least in lymphoid cells (Fig. 4). Consistent with our
results, thymocytes from RIP-deficient mice are normally sensitive to
treatment with anti-Fas Abs. Instead, RIP is essential for
transmitting NF-
B-activating signals from TNF-R1 (52).
The biological function of RAIDD is currently unknown. The presence of
a CARD region and the ability to interact with pro-caspase-2 suggest
that RAIDD acts in a presently unknown apoptosis signaling pathway
(21)
Since at least some FasL-induced JNK signaling occurs independent of
caspase activity, it appears that it is not simply a consequence of
apoptosis, but may have another function. It even appears possible that
it might confer a protective signal. In the case of TNF-R1 signaling,
the death-inducing property of the receptor was genetically separated
from its ability to activate JNK and NF-B (11). Cells
lacking the TNF-R1 downstream effector TRAF2 were hypersensitive to
TNF-induced apoptosis and unable to activate the JNK pathway,
demonstrating that JNK activity is dispensable for death
receptor-mediated apoptosis (23). Studies with lymphocytes
lacking the adapter protein FADD demonstrated that this adapter is not
only required for death receptor-induced apoptosis, but is also
essential for the transmission of mitogen-induced proliferative signals
(40, 53). Perhaps, if all death receptor pathways to
apoptosis are blocked, for instance by molecules such as cellular
FLICE-inhibitory protein (cFIP) (54), JNK together with
NF-B could contribute to cell proliferation or cell
differentiation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andreas Strasser, The Walter and Eliza Hall Institute, Royal Melbourne Hospital, Victoria 3050, Australia.
3 Abbreviations used in this paper: MAPK, mitogen-activated kinase; FasL, Fas (APO-1/CD95) ligand; JNK, c-Jun N-terminal kinase; MEKK1, JNK kinase kinase; TNF-R1, TNF receptor 1; ASK, apoptotic signal-regulating kinase; DN, dominant negative; DD, death domain; CARD, caspase recruitment domain; AICD, activation-induced cell death; wt, wild type; CrmA, cytokine response modifier A; TRAF-2, TNF-receptor associated factor 2; DAXX, death domain associated protein; RIP, receptor interacting protein; RAIDD, RIP-associated ICH-1/CED-3-homologous protein with a death domain.
Received for publication January 19, 2000. Accepted for publication May 18, 2000.
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References |
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B activation prevents cell death. Cell 87:565.[Medline]
B and JNK activation and apoptosis through distinct pathways. J. Biol. Chem. 274:30603.B activation and regulates lymphocyte proliferation and survival. Immunity 7:703.[Medline]
B activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7:715.[Medline]
and calcineurin is sufficient for Fas ligand expression and induction of a CrmA-sensitive apoptosis pathway in Jurkat T cells. Eur. J. Immunol. 29:3549.[Medline]
B but not Fas/APO-1-initiated apoptosis. EMBO J. 15:6189.[Medline]
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