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*
Department of Immunology,
Allergy Research Center,
Department of Biochemistry, and
Division of Pathology, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, Tokyo, Japan; and
¶ Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated monocytes and induces cell death in certain tumor
cell lines. In this study, we characterized the TWEAK-induced cell
death in several tumor cell lines that exhibited distinct features.
Although the TWEAK-induced cell death in Kym-1 cells was indirectly
mediated by TNF-
and was inhibited by cycloheximide, the
TWEAK-induced cell death in HSC3 cells or IFN--treated HT-29
cells was not inhibited by anti-TNF- mAb or cycloheximide,
suggesting a direct triggering of cell death via TWEAK receptor in the
latter cell lines. The TWEAK-induced apoptosis in HSC3 cells and
IFN--treated HT-29 cells was associated with caspase-8 and caspase-3
activation. Although a pan-caspase inhibitor,
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, inhibited the
TWEAK-induced cell death in HSC3 cells, it rather sensitized HT-29
cells to TWEAK-induced cell death by necrosis. This necrosis was
abrogated by lysosomal proteinase inhibitors, particularly a cathepsin
B inhibitor,
[L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline
methyl ester. During the process of TWEAK-induced necrosis, cathepsin B
was released from lysosome to cytosol. Although DR3 has been reported
to be a receptor for TWEAK, all TWEAK-sensitive tumor cell lines used
in this study did not express DR3 at either protein or mRNA level, but
did bind CD8-TWEAK specifically. These results indicated that TWEAK
could induce multiple pathways of cell death, including both
caspase-dependent apoptosis and cathepsin B-dependent necrosis, in a
cell type-specific manner via TWEAK receptor(s) distinct from
DR3. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Fas ligand
(FasL)3/CD95
ligand/APO-1 ligand, and TNF-related apoptosis-inducing ligand
(TRAIL)/APO-2 ligand, induce cell death in a variety of tumor cells and
nontransformed cells and are critically involved in tumor suppression,
homeostasis of immune system, and pathogenesis of various diseases
(1, 2, 3). TWEAK has recently been identified as a new
death-inducing ligand belonging to the TNF family (4). It
has also been reported that TWEAK induces proliferation of endothelial
cells and angiogenesis (5). Although TWEAK mRNA has been
found in various tissues and cells (4, 6), its expression
at the protein level and its physiological role remain largely unknown.
We have recently demonstrated that TWEAK is expressed on human
peripheral blood (PB) monocytes upon IFN- stimulation and is
involved in IFN--stimulated monocyte cytotoxicity against
TWEAK-sensitive tumor cells (7). However, the
TWEAK-induced death signaling pathway has not been well
characterized.
So far, the mechanisms of TNF--, FasL-, or TRAIL-induced cell death
have been well characterized. These death-inducing ligands exert their
cytotoxic effects through TNF-R1, Fas, TRAIL-R1/death receptor (DR)4,
and TRAIL-R2/DR5, respectively. Trimeric ligand-induced oligomerization
of the cytoplasmic death domain (DD) of these receptors leads to
recruitment of caspase-8 via Fas-associated DD or a Fas-associated
DD-like adaptor molecule, which activates the caspase cascade,
resulting in apoptosis (8, 9, 10). In some cell types,
TNF- and FasL could induce apoptosis not only via the activation of
caspases, but also via the activation of noncaspase proteinases such as
cathepsins and calpains (11, 12, 13, 14). Furthermore, TNF- and
FasL could also induce necrosis, which appears to be mediated by
production of reactive oxygen intermediates and/or loss of
mitochondrial transmembrane potential
(
m; Refs.
15, 16, 17, 18).
It has been reported that TWEAK induced cell death through
DR3/TRAMP/LARD/APO-3/WSL1 (6). DR3 is a type I membrane
protein belonging to the TNF-R family, and it contains the DD
homologous to TNF-R1, Fas, TRAIL-R1, and TRAIL-R2
(19, 20, 21). However, there are conflicting reports as to the
TWEAK-DR3 interaction. For example, Schneider et al. (22)
have reported that TWEAK could induce cell death in Kym-1 cells lacking
DR3 mRNA expression. In addition, Kaptein et al. (23) have
reported that TWEAK could not bind to DR3 in an in vitro binding assay.
Thus, it remains unclear whether DR3 is really involved in
TWEAK-induced cell death. Furthermore, Schneider et al.
(22) have demonstrated that the TWEAK-induced cell death
in Kym-1 cells was indirectly mediated by the interaction of endogenous
TNF- and TNF-R1. Thus, it also remains unclear whether TWEAK
receptor (TWEAK-R) could directly induce cell death, like TNF-R1
and Fas.
In the present study, we characterized the mechanisms of TWEAK-induced
cell death in several tumor cell lines. We found that TWEAK could
directly induce apoptosis via caspase activation in HSC3 cells
and IFN--treated HT-29 cells, and that TWEAK could also induce
necrosis via a lysosomal cathepsin B pathway when caspases were blocked
by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) in
IFN--treated HT-29 cells. In addition, DR3 expression was not
detectable in all TWEAK-sensitive tumor cell lines examined in this
study. These results indicated that TWEAK could directly induce both
apoptosis and necrosis via death-inducing receptor(s) distinct
from DR3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human colon adenocarcinoma HT-29 cells and human T cell lymphoma Jurkat cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 containing 10% FCS, 100 µg/ml streptomycin and penicillin, and 2 mM glutamine (culture medium). Human oral squamous cell carcinoma HSC3 cells and human gastric adenocarcinoma KATO-III cells were obtained from Japan Cancer Research Bank (Osaka, Japan) and were maintained in the culture medium. Human rhabdomyosarcoma Kym-1 cells were kindly provided by Dr. H. Endo (Jichi Medical School, Tochigi, Japan) and were cultured in DMEM containing 10% FCS, 100 µg/ml streptomycin and penicillin, and 2 mM glutamine.
Reagents
Human IFN- and anti-human TNF- mAb (mAb1) were
purchased from BD PharMingen (San Diego, CA). z-VAD-fmk and
[L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline
methyl ester (CA074 Me) were purchased from Peptide Institute
(Osaka, Japan). Boc-Asp-fluoromethylketone was purchased from
Calbiochem (San Diego, CA). Anti-human Fas mAb (CH-11) and recombinant
soluble human FasL were purchased from MBL (Nagoya, Japan) and Alexis
(San Diego, CA), respectively. Butylated hydroxyanisole (BHA),
L-monomethyl-L-arginine
monoacetate (L-NMMA),
N-(1-iminoethyl)-L-omithine
(L-NIO), and Bafilomycin A1 were purchased from
Wako Pure Chemicals (Osaka, Japan). D609 was purchased from Calbiochem.
Desipramine was purchased from Sigma-Aldrich (St. Louis, MO). CD8-human
TWEAK fusion protein was prepared as described previously
(7). An anti-human DR3 mAb (JD3, mouse IgG1/
) was
generated by immunizing a BALB/c mouse with human (h)DR3-Ig fusion
protein and screening for binding to hDR3-transfected BHK (hDR3/BHK)
and L5178Y (hDR3/L5178Y) cells (described elsewhere).
Cell viability assay
Cells (5 x 103 or 1 x
104 per well) were cultured with or without the
indicated dose of CD8-TWEAK, anti-Fas mAb, or TNF- in the
presence or absence of IFN- (20 ng/ml) for the indicated period in a
flat-bottom 96-well microtiter plate. In some experiments, cells were
pretreated with IFN- (20 ng/ml) for 12 h and/or z-VAD-fmk (50
µM) for 1 h before the CD8-TWEAK treatment. In some experiments,
to inhibit new protein synthesis, cells were pretreated with
cycloheximide (CHX; 2 µg/ml) for 1 h before the CD8-TWEAK
treatment. This dose of CHX was not toxic for the cells, but it could
inhibit new protein synthesis by at least 86% in the cells used in
this study, as estimated by the uptake of
35S-methionine into proteins. Cell viability was
then determined by measuring the metabolic activity using
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,
4-disulfophenyl)[2H]tetrazolium monosodium
salt (WST; Wako Pure Chemicals) or the standard crystal violet assay as
described previously (7).
Fluorogenic substrate assay for caspase activity
The activity of caspases was measured as described by Vercammen et al. (16) with minor modifications. After various treatments, cells (1 x 106) were resuspended in the lysis buffer (0.5% Nonidet P-40, 250 mM NaCl, 50 mM Tris-HCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 100 µM PMSF). The lysates were centrifuged at 15,000 x g for 15 min, and the supernatants were collected. The extracts (40 µg of total protein) were incubated in 100 µl of the cell-free system buffer (10 mM HEPES, pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM PMSF, and 1 mM DTT) with 100 µM of the fluorogenic peptide substrates acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide (Ac-DEVD-MCA), acetyl-Ile-Glu-Thr-Asp-4-methyl-coumaryl-7-amide (Ac-IETD-MCA), acetyl-Leu-Glu-His-Asp-4-methyl-coumaryl-7-amide (Ac-LEHD-MCA), or acetyl-Tyr-Val-Ala-Asp-4-methyl-coumaryl-7-amide (Ac-YVAD-MCA; Peptide Institute) to measure caspase-3-, -8-, -9-, or -1-like activity, respectively. The release of fluorescent aminomethylcoumarin was measured for 1 h at 5-min intervals on a Fluoroskan Ascent (Labsystems, Helsinki, Finland). Data are expressed as the increase in fluorescence as a function of time.
Western blot analysis for caspase activation
HSC3 cells and IFN- (20 ng/ml)-treated HT-29 cells (1 x
106) were incubated with or without CD8-TWEAK
(100 ng/ml) for 6 or 24 h, respectively. Then the cells were lysed
in 200 µl of the lysis buffer. The extracts (30 µg of total
protein) were subjected to 15% SDS-PAGE for caspase-3 and p18
processed form of caspase-8, or 12% SDS-PAGE for pro-form and p41, p43
processed forms of caspase-8. After blotting onto polyvinylidene
difluoride membranes, the caspase fragments were detected with rabbit
anti-human caspase-3 Ab (kindly provided by Drs. R. Takahashi and
J. C. Reed, Burnham Institute, La Jolla, CA), mouse anti-human
caspase-8 mAb (5F7, MBL), or mouse anti--tubulin mAb (Oncogene
Science, Cambridge, MA) and then developed by ECL Plus (Amersham
Pharmacia Biotech, Piscataway, NJ).
Measurement of DNA hypoploidy by flow cytometry
DNA hypoploidy was measured as described by Nicoletti et al. (24) with minor modifications. In brief, cells (1 x 106) were stained in 900 µl of 50 µg/ml propidium iodide in 0.1% Triton X-100, 4 mM sodium citrate (pH 7.2), and 450 µg/ml RNase for 10 min at 4°C, and then 100 µl of 1.5 M NaCl was added. Samples were incubated in the dark at 4°C for 30 min and then analyzed on a FACSCalibur (BD Biosciences, San Jose, CA). Data were processed by using the CellQuest program (BD Biosciences).
Electron microscopy
After various treatments, cells (3 x 106) were fixed with 2% glutaraldehyde in PBS for 2 h and then with 2% OsO4 for 2 h before embedding in Epon 812. Thin sections were prepared using a MT-5000 ultramicrotome (DuPont Pharmaceuticals, Wilmington, DE), stained with uranyl acetate followed by lead citrate, and then observed on a JEM1230 electron transmission microscope (JEOL, Tokyo, Japan).
Subcellular fractionation and Western blot analysis for cathepsin B
HSC3 cells and IFN- (20 ng/ml)-treated HT-29 cells (2 x
107) were incubated with z-VAD-fmk (50 µM) and
CD8-TWEAK (100 ng/ml) for the indicated periods. Cytosolic fractions
(S-100) were prepared as described by Yang et al. (25)
with minor modifications. Cell pellets were resuspended in 400 µl of
0.25 M sucrose containing 5 mM sodium phosphate buffer (pH 7.2). The
cells were homogenized by passing them through a 23-gauge needle 60
times, followed by two rounds of centrifugation at 750 x
g for 10 min at 4°C to remove the nuclei and unbroken
cells. The supernatants (enucleared whole-cell lysates) were then
centrifuged at 10,000 x g for 30 min at 4°C to
remove lysosomes and mitochondria. The resulting supernatants were
further centrifuged at 100,000 x g for 1 h at
4°C. The final supernatants (cytosolic fractions) were collected and
then concentrated by 5% TCA, including 0.02% deoxycholic acid
precipitation. The cytosolic fractions (30 µg) and the whole-cell
lysates (30 µg) were subjected to 12% SDS-PAGE, blotted onto
polyvinylidene difluoride membranes, and probed with rabbit
anti-cathepsin B Ab (26) or mouse anti--tublin
mAb (Oncogene Science), followed by detection with ECL Plus (Amersham
Pharmacia Biotech).
Flow cytometric analysis for DR3 and TWEAK-R expression
Cells (1 x 106) were incubated with 0.5 µg of CD8-TWEAK or biotinylated anti-human DR3 mAb for 1 h at 4°C, followed by PE-labeled anti-human CD8 mAb (BD PharMingen) or PE-labeled avidin (BD PharMingen), respectively. After washing with PBS, the cells were analyzed on a FACSCalibur, and data were analyzed by using the CellQuest program.
Northern blot analysis for DR3 mRNA
Northern blot analysis was performed as described previously
(27). In brief, tumor cells were cultured in the presence
or absence of IFN- (20 µg/ml) for 12 h. Total RNA was
extracted from the cells by using RBA STAT-60 (Tel-Test, Friendswood,
TX) according to the manufacturer’s instruction. Ten micrograms each
of denatured RNA was electrophoresed in a 1.5% agarose gel containing
6.6% formaldehyde and then blotted onto a nylon membrane. The membrane
was hybridized with an [-32P]dCTP-labeled
1100-bp BamHI/XbaI fragment containing human
TRAMP/DR3 cDNA (kindly provided by Dr. J. Tschopp, Lausanne University,
Epalinges, Switzerland) at 65°C in ExpressHyb hybridization
solution (Clontech Laboratories, Palo Alto, CA), and then washed twice
in 2x SSC/0.1% SDS at 65°C for 15 min. The autoradiogram was
analyzed on a BAS2500 (Fuji Film, Tokyo, Japan).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others have shown that TWEAK induced cell death in certain
tumor cell lines in the presence or absence of IFN- (4, 7). Recently, Schneider et al. (22) have reported
that the TWEAK-induced cell death in Kym-1 cells was indirectly
mediated by the interaction of endogenous TNF- and TNF-R1. Thus, we
first tested whether TWEAK-induced cell death was generally mediated by
endogenous TNF- in several TWEAK-sensitive tumor cell lines. As
previously reported (4, 7, 22), Kym-1 and HSC3 cells were
sensitive to TWEAK in the absence of IFN-, and HT-29 and KATO-III
cells were sensitive to TWEAK in the presence of IFN- (Fig. 1
A). The TWEAK-induced cell
death in all these cell lines was specifically abrogated by a
neutralizing anti-human TWEAK mAb CARL-1 (Ref. 7 and
data not shown). Consistent with the observation by Schneider et al.
(22), the CD8-TWEAK-induced cell death in Kym-1 cells was
completely inhibited by neutralizing anti-TNF- mAb. In contrast,
anti-TNF- mAb did not inhibit the TWEAK-induced cell death in
HSC3, HT-29, or KATO-III cells (Fig. 1A). To further examine
whether endogenous FasL or TRAIL might be involved in the TWEAK-induced
cell death in HSC3, HT-29, and KATO-III cells, we tested the effect of
neutralizing anti-FasL (NOK-1; Ref. 28) and
anti-TRAIL (RIK-2; Ref. 29) mAbs on the TWEAK-induced
cell death in these cells. However, the mixture of anti-TNF-,
anti-FasL, and anti-TRAIL mAbs did not significantly inhibit
the TWEAK-induced cell death in HSC3, HT-29, and KATO-III cells (data
not shown). These results indicated that the TWEAK-induced cell death
was not generally mediated indirectly by endogenous TNF-, FasL, or
TRAIL.
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, FasL, and TRAIL, we next
examined the effect of a protein synthesis inhibitor CHX on
TWEAK-induced cell death. As shown in Fig. 1
B, the CHX
treatment abrogated the TWEAK-induced cell death in Kym-1 cells, but
not in HSC3, HT-29, or KATO-III cells. The TWEAK-induced cell death in
HSC3 cells was rather enhanced by the CHX treatment. These results
suggested that TWEAK could induce cell death directly via its receptor
on HSC3, HT-29, and KATO-III cells independently of de novo synthesis
of an endogenous death factor. Activation of caspases by TWEAK
The death-inducing ligands such as TNF-, FasL, and TRAIL can
induce apoptosis via activation of caspases (1, 2, 8, 9, 10).
However, it remains unknown whether TWEAK could activate caspases.
Thus, we next investigated whether caspases were activated in the
course of TWEAK-induced cell death in HSC3 cells and IFN--treated
HT-29 cells. We also examined anti-Fas- or TNF--induced cell
death in these cells for comparison. HSC3 cells were sensitive to TWEAK
and anti-Fas mAb, but not TNF- (Fig. 2A). HT-29 cells were sensitive to TWEAK,
anti-Fas mAb, and TNF- only when pretreated with IFN- (Fig. 2A). We then examined caspase activities in the cell lysates
by using fluorogeneic peptide substrates, Ac-DEVD-MCA, Ac-IETD-MCA,
Ac-LEHD-MCA, and Ac-YVAD-MCA for caspase-3, -8, -9, and -1,
respectively. In HSC3 cells, anti-Fas mAb rapidly activated
caspase-3, -8, and -9, which peaked at 3 h (Fig. 2B).
TWEAK also activated these caspases with somewhat delayed kinetics,
which peaked at 6 h. In HT-29 cells, anti-Fas mAb rapidly
activated caspase-3, -8, and -9, which peaked at 3 h (Fig. 2B). In contrast, TWEAK and TNF- activated caspase-3 and
-8 with greatly delayed and sustained kinetics, which gradually
increased from 6 to 36 h. TWEAK, but not TNF-, activated
caspase-9 significantly. These differences in kinetics of caspase
activation were mostly correlated with rapid kinetics of TWEAK- or
anti-Fas-induced cell death in HSC3 cells, which reached a plateau
within 12 h, and slower kinetics of TWEAK- and TNF--induced
cell death in HT-29 cells, which gradually increased over 36 h
(Fig. 2A).
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C, TWEAK stimulation could
induce processing of 36-kDa procaspase-3 into p20 and p17 in HSC3
cells, and into p24 and p20 in HT-29 cells. A substantial part of
procaspase-8 was processed into p18 in TWEAK-stimulated HSC3 cells, and
into p43, p41, and p18 in TWEAK-stimulated HT-29 cells (Fig. 2C). Taken together, these results indicated that TWEAK
could activate caspase-8 and caspase-3 in HSC3 cells and
IFN--treated HT-29 cells. Effect of z-VAD-fmk on TWEAK-induced cell death and apoptosis
To assess whether the TWEAK-induced cell death was dependent on
the caspase activation, we next examined the effect of a pan-caspase
inhibitor, z-VAD-fmk, on the TWEAK-, anti-Fas-, or TNF--induced
cell death in HSC3 cells and IFN--treated HT-29 cells. In addition,
we also examined whether TWEAK could induce DNA hypoploidy as an
indication of apoptosis in a caspase-dependent manner. As shown in Fig. 3, A and B, both
cell death and DNA hypoploidy were blocked by z-VAD-fmk in TWEAK- or
anti-Fas-stimulated HSC3 cells, suggesting that the TWEAK-induced
HSC3 cell death was primarily due to caspase-dependent apoptosis with
nuclear disintegration like that caused by Fas-mediated signaling. In
HT-29 cells, z-VAD-fmk also blocked both cell death and DNA hypoploidy
when stimulated by anti-Fas mAb or TNF- (Fig. 3, A
and B). Recently, it has been reported that z-VAD-fmk
abrogated apoptosis, but not cell death, in T cell lines induced by a
high dose of cross-linked recombinant soluble FasL (sFasL) or TNF-
(18, 33). However, in HT-29 cells, z-VAD-fmk abrogated
both cell death and apoptosis even when stimulated with a high dose of
sFasL (1000 ng/ml), anti-Fas mAb (5000 ng/ml), or TNF- (1000
ng/ml; data not shown). In contrast, z-VAD-fmk rather markedly enhanced
TWEAK-induced cell death (Fig. 3A), despite efficiently
blocking DNA hypoploidy (Fig. 3B). Similar results were
obtained with another pan-caspase inhibitor, Boc-Asp-fluoromethylketone
(data not shown). These results indicated that caspase inhibition could
abrogate sFasL-, anti-Fas mAb-, or TNF--induced cell death, but
rather enhanced TWEAK-induced cell death in HT-29 cells.
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, the
TWEAK-induced HSC3 cell death was associated with typical apoptotic
morphological changes such as chromatin condensation, cell shrinking,
and apoptotic bodies, like anti-Fas mAb-induced apoptosis. These
changes were completely abrogated by z-VAD-fmk. In HT-29 cells, TWEAK,
anti-Fas mAb, and TNF- also induced apoptotic morphological
changes. These changes induced by anti-Fas mAb and TNF- were
completely abrogated by z-VAD-fmk. Notably, the TWEAK-stimulated HT-29
cells in the presence of z-VAD-fmk exhibited a necrotic morphology,
which was characterized by loss of plasma membrane integrity without
apparent damage to nuclei. These results indicated that TWEAK could
primarily induce apoptosis in HSC3 and HT-29 cells, but it could also
induce necrosis in HT-29 cells when caspases were inactivated.
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In contrast to apoptosis, signaling pathways leading to necrosis
have not been well characterized. Recently, it has been reported that
mitochondrial stress-induced reactive oxygen intermediates were
responsible for TNF-- or anti-Fas mAb-induced necrosis, which
was abrogated by antioxidants such as BHA (15, 16, 17). Thus,
we tested whether BHA could inhibit the TWEAK-induced necrosis in
z-VAD-fmk-treated HT-29 cells. Although BHA completely inhibited the
TNF--induced necrosis in mouse fibrosarcoma L929 cells (data not
shown) as previously reported (16), it did not inhibit the
TWEAK-induced necrosis in HT-29 cells, which was characterized by cell
death without DNA hypoploidy (Fig. 5A). Moreover, we could not
observe apparent morphological changes of mitochondria by electron
microscopy or release of cytochrome c from mitochondria to
cytosol by Western blot analysis in the necrotic HT-29 cells (data not
shown). These results suggested that mitochondria stress might not be
critically responsible for the TWEAK-induced necrosis. It has also been
reported that oxidative stress, such as NO, and sphingolipids, such as
ceramide and sphingosine, could provoke necrosis (34, 35).
Thus, we next examined the effect of various compounds, including
inducible NO synthase inhibitors, L-NMMA
and L-NIO, and acidic sphingomyelinase
inhibitors, desipramine and D609, on the TWEAK-induced necrosis. As
shown in Fig. 5A, the TWEAK-induced necrosis was not
significantly blocked by these inhibitors. It has also been reported
that lysosomal stresses, such as autophagy or release of lysosomal
enzymes, were involved in necrosis (36, 37).
Furthermore, it has recently been reported that a lysosomal cysteine
proteinase, cathepsin B, was involved in TNF--induced cell death
(11, 12). Thus, we next examined the effect of a specific
inhibitor of cathepsin B, CA074 Me (38), on the
TWEAK-induced necrosis. As shown in Fig. 5A, CA074 Me almost
completely abrogated the TWEAK-induced necrosis in HT-29 cells. It has
been reported that pretreatment with a vacuolar
H+-ATPase inhibitor, Bafilomycin A1, raised
lysosomal pH, resulting in degradation of lysosomal proteinases,
including cathepsin B (39). We observed that the 12-h
pretreatment with Bafilomycin A1 also completely abrogated the
TWEAK-induced necrosis in HT-29 cells (Fig. 5A). These
results suggested that cathepsin B played a critical role in the
TWEAK-induced necrosis in HT-29 cells.
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B, we observed the only two-chain form of
the mature cathepsin B in the whole cell lysate of HT-29 cells by
Western blot analysis, and the TWEAK stimulation did not increase the
amount of cathepsin B. Subcellular fractionation showed that a
substantial amount of the two-chain form of the mature cathepsin B was
released to cytosol in the TWEAK-stimulated HT-29 cells (Fig. 5B). In HSC3 cells that did not undergo TWEAK-induced
necrosis, we observed both single-chain form and two-chain form of
cathepsin B in the whole-cell lysate. However, these active forms of
cathepsin B could not be released to cytosol by TWEAK stimulation
(Fig. 5B). These results suggested that the TWEAK-induced
necrosis was mediated by cathepsin B released from lysosomes. Expression of TWEAK-R and DR3 in TWEAK-sensitive tumor cell lines
It has been reported that TWEAK bound to and induced cell death
via DR3 (6). However, there are conflicting results as to
the TWEAK-DR3 interaction (23). To determine the
involvement of DR3 in the TWEAK-induced cell death observed in the
present study, we finally estimated the expression of DR3 on Kym-1,
HSC3, HT-29, and KATO-III cells by cell surface staining with an
anti-human DR3 mAb (JD3) that we recently established. This mAb was
generated against hDR3-Ig fusion protein and specifically reacted with
hDR3-transfected L5178Y (hDR3/L5178Y) cells (Fig. 6A) and hDR3/BHK cells (not
shown). It also reacted with anti-CD3-stimulated PB T cells, but
not with unstimulated PB T cells (Fig. 6A). As shown in Fig. 6B, this anti-DR3 mAb did not react with Kym-1, HSC3,
HT-29, or KATO-III cells even after IFN- pretreatment. It has been
reported that DR3 has various splicing variant forms (21).
Because we could not rule out the possibility that the anti-DR3 mAb
we used could not recognize a certain splicing variant form of DR3, we
further examined DR3 mRNA expression in these cell lines by Northern
blot analysis. As shown in Fig. 6C, the expression of DR3
mRNA was not be detectable in these tumor cell lines. We next examined
the expression of TWEAK-R on these cell lines by binding of CD8-TWEAK
detected by PE-labeled anti-human CD8 mAb. As shown in Fig. 6B, all the TWEAK-sensitive tumor cell lines, including
Kym-1, HSC3, HT-29, and KATO-III, but not TWEAK-resistant Jurkat cells,
bound CD8-TWEAK irrespective of IFN- pretreatment. The binding of
CD8-TWEAK to these cell lines was specific because it was abrogated by
preincubation with a neutralizing anti-TWEAK mAb, CARL-1 (data not
shown). These results suggested that the TWEAK-induced cell death in
these cell lines was mediated by TWEAK-R other than DR3. It should be
also noted that CD8-TWEAK did not bind to hDR3/L5178Y cells or
anti-CD3-stimulated PB T cells expressing DR3 on cell surface,
suggesting that DR3 might not be a receptor for TWEAK.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TWEAK could directly induce
apoptosis in HSC3 cells and IFN--treated HT-29 cells by activating
caspase-8 and caspase-3, like anti-Fas mAb and TNF-. The
pan-caspase inhibitor z-VAD-fmk abrogated both the TWEAK-induced
apoptosis and cell death in HSC3 cells. In HT-29 cells, z-VAD-fmk
abrogated the TWEAK-induced apoptosis characterized by DNA hypoploidy
and chromatin condensation, but was sensitized to death by necrosis via
a cathepsin B pathway. In addition, no detectable levels of DR3, which
has been reported to be a receptor for TWEAK, were observed in all the
TWEAK-sensitive tumor cell lines tested in this study. These results
are the first indication that TWEAK could directly induce not only the
caspase-dependent apoptosis but also the cathepsin B-dependent necrosis
in a cell type-specific manner via an as-yet-undefined receptor(s)
distinct from DR3.
A recent study by others (22) has demonstrated that the
TWEAK-induced cell death was indirectly mediated by the interaction of
endogenous TNF- and TNF-R1 in Kym-1 cells lacking DR3. It has also
been reported that TNF-R2-, CD30-, or CD40-mediated cell death was
indirect and dependent on de novo synthesis of endogenous
death-inducing ligands such as TNF-, FasL, and TRAIL in some tumor
cell lines (41, 42). These results suggested that the
putative TWEAK-R other than DR3 might be a non-DD-containing receptor
like CD30 and CD40. However, in this study, we observed that the
TWEAK-induced cell death in HSC3 cells and IFN--treated HT-29 cells
could not be inhibited by neutralizing anti-TNF- mAb,
anti-FasL mAb, anti-TRAIL mAb, or CHX. Moreover, TWEAK induced
caspase-8 and caspase-3 activation with similar kinetics to
anti-Fas mAb or TNF- in these cells. These results strongly
suggested that TWEAK could directly induce cell death via a
DD-containing receptor other than DR3.
Recently, we have reported that TWEAK was expressed on
IFN--stimulated monocytes and that it contributed to their
cytotoxicity against TWEAK-sensitive tumor cells (7). As
shown in Fig. 1A, TWEAK exerted cytotoxic activity against
some tumor cell lines only in the presence of IFN-. A previous study
in the murine system indicated a critical role for monocytes in tumor
rejection in vivo that was evoked by endogenous IFN-
(43). In this respect, endogenous IFN- might exert a
synergistic antitumor effect by up-regulating the expression of TWEAK
on tumor-infiltrating monocytes and by sensitizing tumor cells to
TWEAK-induced cell death in vivo. However, the mechanism by which
IFN- sensitizes the tumor cells to TWEAK is still unclear. IFN-
has been reported to modulate cell death by inducing several
apoptosis-related genes, including TNF-R1, Fas, and caspase-1
(44, 45). However, we could not observe either the
up-regulation of TWEAK-R (Fig. 6B) or the TWEAK-induced
caspase-1 activation (Fig. 2B) in IFN--treated HT-29
cells. It has been reported that inhibition of NF-B activation
sensitized some tumor cells to TNF--, TRAIL-, or FasL-induced cell
death (46). Thus, it is possible that IFN- sensitized
HT-29 cells to TWEAK-induced cell death by inhibiting TWEAK-induced
NF-B activation. We observed that TWEAK could activate NF-B in
HT-29 cells, but IFN- did not affect the NF-B activity (data not
shown). In HT-29 cells, IFN- was essential not only for
TWEAK-induced apoptosis, but also for necrosis (data not shown),
suggesting that IFN- modulates a common signaling component in
apoptosis and necrosis. Further studies are now under way to determine
the molecular mechanism for the IFN- action in TWEAK-induced
apoptosis and necrosis.
In this study, we found that the inhibition of caspases by z-VAD-fmk
sensitized IFN--treated HT-29 cells to TWEAK-induced necrosis. A
similar sensitization to the TWEAK-induced necrosis by z-VAD-fmk was
also observed in IFN--treated colon cancer cell lines, SW620 and
Colo205 (our unpublished data). The signaling pathways leading to
necrosis are not well understood. We revealed that cathepsin B was
responsible for the TWEAK-induced necrosis in HT-29 cells. It is well
known that cathepsin B is a lysosomal cysteine proteinase that
contributes to proteolysis of proteins taken up by phagocytosis.
Recently, some papers have shown that cathepsin B is involved in
TNF--induced cell death (11, 12). For example,
Guicciardi et al. (11) have reported that cathepsin B
contributed to TNF--induced hepatocyte apoptosis and that cathepsin
B-deficient mice were resistant to TNF--mediated hepatitis. More
recently, Foghsgaard et al. (12) have reported that
cathepsin B acted as an essential downstream mediator of TNF-induced
and caspases-mediated apoptosis in several tumor cell lines. In this
study, we demonstrated that cathepsin B was responsible for
TWEAK-induced necrosis independently of caspase activation. Moreover,
we demonstrated that cathepsin B was released to cytosol by TWEAK
stimulation. However, target molecules for the released cathepsin B
leading to necrosis remains unknown. Guicciardi et al.
(11) have reported that cathepsin B could increase the
release of cytochrome c from mitochondria via unknown
cytosolic factor in a cell-free system. However, we could not observe
the cytochrome c release from mitochondria to cytosol during
the TWEAK-induced necrosis in HT-29 cells (data not shown). Further
biochemical analysis will be needed to identify the target molecule(s)
cleaved by cathepsin B and leading to necrosis. Recently, Holler et al.
(33) have reported that FasL-, TRAIL-, or TNF-- induced
necrosis was mediated by receptor-interacting protein (RIP). We also
observed that the TWEAK-induced necrosis could be abrogated by 1
µg/ml geldanamycin (our unpublished data), which has been reported to
degrade RIP by disruption of heat-shock protein 90 interacting with RIP
(33, 47). However, further studies are needed to determine
the involvement of RIP in TWEAK-induced necrosis in HT-29 cells.
A previous study has shown that TWEAK could induce cell death via DR3
(6). It has been reported that DR3 expression was observed
preferentially in lymphocytes (19, 20) and is rapidly
up-regulated in human PB lymphocytes upon stimulation with PMA plus
IL-2 (20). In this study, we demonstrated that cell
surface expression of DR3 was induced on anti-CD3 mAb-stimulated PB
T cells (Fig. 6A). These results suggested that DR3 might
regulate activated T cells. Consistent with this notion, a recent study
using DR3-deficient mice has revealed that DR3 was involved in
anti-CD3-induced thymocyte apoptosis and negative selection during
thymocyte development (48). Thus, TWEAK might regulate
activated T cells via DR3. However, we could not observe CD8-TWEAK
binding to anti-CD3-stimulated PB T cells (Fig. 6A). In
addition, CD8-TWEAK did not induce either cell death or proliferation
in anti-CD3 mAb-stimulated PB T cells (our unpublished data).
Moreover, we could not observe CD8-TWEAK binding to DR3 transfectants
(Fig. 6A). Similar results have been obtained by Kaptein et
al. (23). These results suggest that TWEAK is not a
physiological ligand for DR3. Conversely, we could not detect the
expression of DR3 in all the TWEAK-sensitive tumor cell lines used in
this study at the protein or mRNA levels, while they did bind CD8-TWEAK
(Fig. 6). This suggests that TWEAK could induce cell death via a
TWEAK-R distinct from DR3.
The putative TWEAK-R remains to be identified. Our present results
indicate that the TWEAK-R expressed on HSC3 cells acts like Fas to
rapidly activate caspase-8 and caspase-3 (Fig. 2B), leading
to cell death by apoptosis (Figs. 3 and 4). In contrast, the TWEAK-R
expressed on HT-29 cells acts like TNF-R1 to slowly activate caspase-8
and caspase-3 (Fig. 2B), leading to cell death primarily by
apoptosis (Figs. 3 and 4). However, the z-VAD-fmk treatment did not
induce necrosis in TNF--stimulated HT-29 cells, indicating a unique
feature of TWEAK-R, leading to the cathepsin B-mediated necrosis. It is
also worth noting that the TWEAK-R expressed on Kym-1 cells did not
induce cell death directly because the TWEAK-induced cell death in
Kym-1 cells was completely abrogated by anti-TNF- mAb (Fig. 1A). It remains to be determined whether these distinct
features of TWEAK-induced cell death are mediated by distinct TWEAK-Rs
or whether a single TWEAK-R transmits differential signals in
particular cellular contexts. Further studies are now under way to
address these possibilities.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hideo Yagita, Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address: hyagita{at}med.juntendo.ac.jp
3 Abbreviations used in this paper: FasL, Fas ligand; TRAIL, TNF-related apoptosis-inducing ligand; DR, death receptor; h, human; TWEAK-R, TWEAK receptor; PB, peripheral blood; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; Ac-DEVD-MCA, acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide; Ac-IETD-MCA, acetyl-Ile-Glu-Thr-Asp-4-methyl-coumaryl-7-amide; Ac-LEHD-MCA, acetyl-Leu-Glu-His-Asp-4-methyl-coumaryl-7-amide; Ac-YVAD-MCA, acetyl-Tyr-Val-Ala-Asp-4-methyl-coumaryl-7-amide; CHX, cycloheximide; BHA, butylated hydroxyanisole; CA074 Me, [L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester; L-NMMA, L-monomethyl-L-arginine monoacetate; L-NIO, N-(1-iminoethyl)-L-omithine; WST, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)[2H]tetrazolium monosodium salt; sFasL, soluble FasL; RIP, receptor-interacting protein; DD, death domain.
Received for publication August 15, 2001. Accepted for publication November 5, 2001.
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