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Departments of
* Immunology,
Biochemistry, and
Respiratory Medicine,
Division of Pathology, Central Laboratory of Medical Sciences, and
¶ Allergy Research Center, Juntendo University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-stimulated monocytes and induces multiple pathways of cell
death, including caspase-dependent apoptosis, cathepsin B-dependent
necrosis, and endogenous TNF-
-mediated cell death, in a cell
type-specific manner. However, the TWEAK receptor(s) that mediates
these multiple death pathways remains to be identified. Recently,
fibroblast growth factor-inducible 14 (Fn14) has been identified to be
a TWEAK receptor, which was responsible for TWEAK-induced proliferation
of endothelial cells and angiogenesis. Because Fn14 lacks the
cytoplasmic death domain, it remains unclear whether Fn14 can also
mediate the TWEAK-induced cell death. In this study, we demonstrated
that TWEAK could induce apoptotic cell death in Fn14 transfectants. A
pan-caspase inhibitor,
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, rather sensitized the
Fn14 transfectants to TWEAK-induced cell death by necrosis via reactive
oxygen intermediates and cathepsin B-dependent pathway. By using newly
generated agonistic anti-Fn14 mAbs, we also observed that Fn14 is
constitutively expressed on the cell surface of all TWEAK-sensitive
tumor cell lines, and can transmit the multiple death signals.
Moreover, an anti-Fn14 mAb that blocks TWEAK-Fn14 interaction could
totally abrogate TWEAK binding and TWEAK-induced cell death in all
TWEAK-sensitive tumor cell lines. These results revealed that the
multiple pathways of TWEAK-induced cell death are solely mediated by
Fn14. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-stimulated human monocytes and is involved in
their cytotoxicity (6). We and others have also
demonstrated that TWEAK could induce multiple pathways of cell death in
different cellular contexts (4, 7). In Kym-1 cells,
TWEAK-induced cell death was indirectly mediated by endogenously
produced TNF-, like that mediated by other TNFR family members
lacking the death domain
(DD)3 such as TNFR2,
CD30, and CD40 (8, 9). In HSC3 cells and IFN--treated
HT-29 cells, TWEAK could directly induce apoptosis via caspase
activation (7), like that mediated by DD-containing TNFR
family members such as TNFR1, Fas, TNF-related apoptosis-inducing
ligand (TRAIL)-R1/DR4, and TRAIL-R2/DR5 (1, 2). In HSC3
cells, a pan-caspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) abrogated
both the TWEAK-induced apoptosis and cell death (7). In
HT-29 cells, z-VAD-fmk abrogated the TWEAK-induced apoptosis, but
rather sensitized to death by necrosis via a lysosomal cathepsin B
pathway (7). These TWEAK-sensitive tumor cell lines did
not express DR3/TRAMP/LARD/APO-3/WSL1 (4, 7), which is a
DD-containing TNFR family member (10, 11) previously
reported to be a receptor for TWEAK (12). These results
suggested the existence of death-inducing TWEAK receptor(s) distinct
from DR3, which remains to be identified. It also remains to be
determined whether these distinct modes of TWEAK-induced cell death are
mediated by distinct TWEAK receptors or whether a single TWEAK receptor
transmits differential signals in particular cellular contexts. Recently, Wiley et al. (13) have identified a TWEAK receptor from HUVEC cDNA library by expression cloning using soluble rTWEAK. The TWEAK receptor turned out to be a fibroblast growth factor-inducible 14-kDa protein, Fn14, which was originally identified by a differential display approach to search for growth factor-inducible molecules in murine NIH3T3 fibroblasts (14, 15). Fn14 is a distantly related TNFR family member, which contains only one cysteine-rich domain in the extracellular region and a TNFR-associated factor (TRAF) binding domain, but not the DD, in the cytoplasmic region (13). Wiley et al. (13) have reported that cross-linking of Fn14 could induce proliferation of HUVEC, and that soluble Fn14 inhibited epidermal growth factor-induced endothelial cell migration in vitro and fibroblast growth factor-2-induced angiogenesis in vivo. These results suggested that Fn14 is responsible for the TWEAK-induced endothelial cell migration, proliferation, and angiogenesis. However, it remains unclear whether Fn14 is also responsible for the TWEAK-induced cell death in tumor cell lines we observed. In this study, we demonstrated that TWEAK could induce cell death in Fn14 transfectants and that the multiple pathways of TWEAK-induced cell death in several tumor cell lines were solely mediated by Fn14.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Human colon adenocarcinoma HT-29, human T lymphoma Jurkat, human hepatoma SK-Hep-1, and mouse T lymphoma L5178Y were obtained from American Type Culture Collection (ATCC, Manassas, VA) and 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 and mouse mastocytoma P815 were obtained from Japan Cancer Research Bank (Osaka, Japan) and maintained in the culture medium. Human rhabdomyosarcoma Kym-1 was kindly provided by H. Endo (Jichi Medical School, Tochigi, Japan) and cultured in DMEM containing 10% FCS, 100 µg/ml streptomycin and penicillin, and 2 mM glutamine. HUVEC was obtained from Clonetics (San Diego, CA) and cultured in endothelial cell growth medium-2.
Reagents
Human IFN- and anti-human TNF- mAb (mAb1) were
purchased from BD PharMingen (San Diego, CA). z-VAD-fmk and a cathepsin
B-specific inhibitor,
[L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline
methyl ester (CA074 Me), were purchased from Peptide Institute (Osaka,
Japan). An antioxidant, butylated hydroxyanisole (BHA), and a
cell-permeable fluorogenic probe for reactive oxygen intermediates
(ROI), dihydrorhodamine (DHR) 123, were purchased from Wako Pure
Chemicals (Osaka, Japan) and Molecular Probes (Eugene, OR),
respectively. Boc-Asp-fluoromethylketone (Boc-D-fmk) was purchased from
Calbiochem (La Jolla, CA). Soluble human CD8-human TWEAK fusion protein
(CD8-TWEAK) was prepared as described previously (6).
Preparation of human Fn14 transfectants
Human Fn14 (hFn14) cDNA was prepared by RT-PCR amplification of total RNA from HUVEC with an oligonucleotide primer corresponding to the first six codons as the 5' primer and that corresponding to the last six codons as the 3' primer, according to the published sequence (14). XhoI and NotI sites were introduced into the 5' and 3' primers, respectively. After XhoI and NotI digestion, the PCR product of 390 bp was subcloned into XhoI- and NotI-digested pEF/myc/cyto (Invitrogen, San Diego, CA). After confirmation of nucleotide sequence, hFn14/pEF/myc/cyto vector was transfected into L5178Y and P815 by electroporation (350 V, 800 µF) with a Gene Pulser (Bio-Rad Laboratories, Hercules, CA). After selection with 1 mg/ml G418 and cloning by limiting dilution, stable transfectants, designated as hFn14/L5178Y and hFn14/P815, were selected by cell surface staining with CD8-TWEAK.
Cell viability assay
Cells (5 x 103/well) were cultured
with or without the indicated doses of CD8-TWEAK or anti-Fn14 mAbs
for the indicated period in a flat-bottom 96-well microtiter plate. For
HT-29, IFN- (20 ng/ml) was included. In some experiments, cells were
pretreated with z-VAD-fmk (10 or 50 µM), BHA (100 µM), and/or CA074
Me (10 or 50 µM) for 1 h before the CD8-TWEAK treatment. The
cell viability was then determined by measuring the metabolic activity
using
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)[2H]tetrazolium
salt (WST; Wako Pure Chemicals), as described previously
(6). The viability was calculated as follows:
(A450/655-treated cells -
A450/655
medium)/(A450/655-untreated cells -
A450/655 medium) x 100.
Fluorogenic substrate assay for caspase activity
Activity of caspases was measured, as described previously (7). After CD8-TWEAK treatment, 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 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.
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 an MT-5000 ultramicrotome (DuPont, Wilmington, DE), stained with uranyl acetate, followed by lead citrate, and then observed on a JEM1230 electron transmission microscope (JEOL).
Measurement of ROI by flow cytometry
Cells (2 x 105/well) were pretreated with BHA (100 µM), CA074 Me (10 µM), or medium alone for 1 h, and then cultured with DHR123 (1 µM) and CD8-TWEAK (100 ng/ml) in the presence or absence of z-VAD-fmk (10 µM) for the indicated period in a 24-well plate. The level of R123, a fluorescent product of DHR oxidation, was measured on a FACSCalibur (BD Biosciences, San Jose, CA), and the data were analyzed by using the CellQuest program (BD Biosciences). Data were expressed as the ratio of fluorescence intensity in the treated cells to that in untreated cells.
Subcellular fractionation and Western blot analysis for cathepsin B
After various treatments, cytosolic fraction (S-100) was prepared as described previously (7). The cytosolic fraction (20 µg) and the whole cell lysate (20 µg) were subjected to 12% SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with rabbit anti-cathepsin B Ab (16) or mouse anti-actin mAb (Biomedical Technologies, Stoughton, MA), followed by detection with ECL Plus (Amersham Pharmacia Biotech, Piscataway, NJ).
Western blot analysis for I
B degradation
The IB degradation was estimated by Western blotting, as
described previously (17). hFn14/L5178Y cells were treated
with CD8-TWEAK (250 ng/ml) in the absence or presence of z-VAD-fmk (10
µM) for the indicated periods. Then the whole cell lysate (10 µg)
was subjected to 12% SDS-PAGE, blotted onto polyvinylidene difluoride
membrane, and probed with rabbit anti-IB Ab (sc-371; Santa
Cruz Biotechnology, Santa Cruz, CA), followed by detection with ECL
Plus (Amersham).
Generation of anti-hFn14 mAbs
Six-week-old female BALB/c mice (Clear Japan, Tokyo,
Japan) were immunized by i.p. injection of hFn14/P815
(107 cells) four times at 10-day intervals. Three
days after the final immunization, the splenocytes were fused with P3U1
mouse myeloma cells, as described previously (18). After
hypoxanthine/aminopterin/thymidine selection, the mAbs that exhibited
cytotoxic activity against hFn14/L5178Y in the presence of z-VAD-fmk
(10 µM) were screened. Four mAbs (ITEM-1, ITEM-2, ITEM-3, and
ITEM-4) were identified by their cytotoxic effects and cloned by
limiting dilution. ITEM-1 (mouse IgG1/), ITEM-2 (mouse IgA/),
ITEM-3 (mouse IgG2b/), and ITEM-4 (mouse IgG2b/) were purified
from culture supernatants by caprylic acid and ammonium sulfate
precipitation method (19).
Flow cytometric analysis for Fn14 expression
Cells (1 x 106) were incubated with 0.5 µg of CD8-TWEAK or biotinylated mAbs 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 the data were analyzed by using the CellQuest program.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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To characterize the functional properties of Fn14, we first
generated stable transfectants expressing full-length hFn14 cDNA. We
used L5178Y and P815 for the transfection, because these cells did not
bind CD8-TWEAK as estimated by flow cytometry (Fig. 1
A). The resulting
hFn14/L5178Y and hFn14/P815 cells exhibited a high CD8-TWEAK binding
(Fig. 1A), which was totally abrogated by a neutralizing
anti-human TWEAK mAb CARL-1 (6) (data not shown).
These results confirmed that Fn14 acts as a TWEAK receptor.
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A) and
induce proliferation of HUVEC in a low-serum condition (Fig. 1B). Thus, we first examined whether CD8-TWEAK could induce
the proliferation of hFn14/L5178Y and hFn14/P815 cells in a low (1%)
or high (10%) FCS condition. As shown in Fig. 1B, both
hFn14/L5178Y and hFn14/P815 cells did not proliferate, but rather
progressively lost viability in response to CD8-TWEAK in a
dose-dependent manner in the 1% FCS condition, which was completely
reversed by the neutralizing anti-TWEAK mAb. This suggested that
TWEAK could induce cell death in these hFn14 transfectants. Because
hFn14/L5178Y cells exhibited a higher sensitivity to CD8-TWEAK than
hFn14/P815 cells (Fig. 1B), we used hFn14/L5178Y cells to
further characterize the TWEAK-induced cell death in Fn14
transfectants. As represented in Figs. 1C and 2A,
hFn14/L5178Y cells, but not parental L5178Y cells, started to die at
24 h in the 10% FCS condition. Neither a transcription inhibitor
actinomycin D nor a translation inhibitor cycloheximide affected the
level or kinetics of cell death (data not shown), suggesting that TWEAK
could induce cell death in hFn14/L5178Y cells independently of de novo
protein synthesis.
To explore whether Fn14-mediated cell death is dependent on caspase
activation, we next examined the effect of a pan-caspase inhibitor
z-VAD-fmk on TWEAK-induced cell death in hFn14/L5178Y cells. As shown
in Fig. 1C, z-VAD-fmk, which alone was not toxic for the
cells, markedly accelerated and enhanced the TWEAK-induced cell death,
which was completely inhibited by anti-TWEAK mAb CARL-1. Similar
results were obtained with another pan-caspase inhibitor, Boc-D-fmk
(data not shown), but not vehicle (DMSO) alone. To date, we and others
have observed a similar enhancing effect of z-VAD-fmk on TWEAK-,
TNF--, or anti-Fas mAb-induced cell death in certain tumor cell
lines, which was mediated by ROI or lysosomal cathepsin B, and thus
abrogated by antioxidants such as BHA or a cathepsin B inhibitor CA074
Me (7, 20, 21, 22). As shown in Fig. 2A, the TWEAK-induced cell
death in hFn14/L5178Y cells either in the presence or absence of
z-VAD-fmk was also significantly inhibited by both BHA and CA074 Me.
These results suggested the involvement of ROI and cathepsin B in the
Fn14-mediated cell death, which was negatively regulated by
z-VAD-fmk-sensitive caspase(s). To investigate whether caspases were
activated in hFn14/L5178Y cells by stimulation with CD8-TWEAK, we
measured caspase activities in the cell lysate by using fluorogenic
peptide substrates, Ac-DEVD-MCA, Ac-IETD-MCA, Ac-LEHD-MCA, and
Ac-YVAD-MCA for caspase-3-, -8-, -9-, and -1-like activities,
respectively. As a control, we also measured caspase activities in
IFN--treated HT-29 cells after TWEAK stimulation. As we previously
reported (7), substantial levels of caspase-3- and -8-like
activities were induced in HT-29 cells upon CD8-TWEAK stimulation (Fig. 2B). In hFn14/L5178Y cells, only a marginal level of
caspase-3-like activity was detected (Fig. 2B). The levels
of caspase activation in these cells were correlated with their
sensitivity to TWEAK-induced cell death (Fig. 2B).
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C). These results
indicated that TWEAK could primarily induce apoptotic death in
hFn14/L5178Y cells, and that z-VAD-fmk could sensitize hFn14/L5178Y
cells to TWEAK-induced cell death by necrosis via ROI- and cathepsin
B-dependent pathway.
We next measured the intracellular ROI level in TWEAK-stimulated
hFn14/L5178Y cells by flow cytometry using a fluorogenic substrate
DHR123. As shown in Fig. 2D, the ROI level was markedly
increased by CD8-TWEAK in the presence of z-VAD-fmk, with a peak at
24 h. The ROI increase was blocked by the pretreatment with BHA or
CA074 Me, suggesting that cathepsin B activity was required for the
maximal increase of intracellular ROI level in response to
TWEAK.
We next examined whether TWEAK could induce the release of cathepsin B
from lysosome to cytosol in the Fn14 transfectants. We previously
demonstrated the cathepsin B release from lysosome in TWEAK-stimulated
HT-29 cells undergoing necrosis (7). As shown in Fig. 2E, hFn14/L5178Y cells contained two active forms of
cathepsin B, single-chain form and two-chain form, in the whole cell
lysate, which were not increased by CD8-TWEAK stimulation. Subcellular
fractionation showed that a substantial amount of the two-chain form of
cathepsin B was present in the cytosol of TWEAK-stimulated hFn14/L5178Y
cells. Pretreatment with CA074 Me, which specifically inhibits
cathepsin B activity by masking active site cysteine residue
(23), resulted in accumulation of the single-chain form in
the cytosol (Fig. 2F), possibly due to blocking of
autocatalytic processing to the two-chain form. This indicated that the
cathepsin B activity per se was not required for the cathepsin B
release. Notably, the TWEAK-induced cathepsin B release was totally
abrogated by BHA (Fig. 2F). This suggested that ROI played a
crucial role to induce the cathepsin B release from lysosome.
Recently, both lysosomal damage and oxidative stress have been
implicated in some models of cell death (24, 25, 26, 27, 28). For
example, cathepsin B was responsible for oxidative stress-induced cell
death in neuronal cells (27). In contrast, p53-induced
cell death involved early lysosomal damage, followed by mitochondria
damage, which was blocked by BHA (25). Cathepsin D release
preceded ROI production in the course of retinoid-induced HL60 cell
death (28). In TWEAK-stimulated hFn14/L5178Y cells,
cathepsin B release was abrogated by antioxidant BHA (Fig. 2F), and ROI production was inhibited by cathepsin B
inhibitor CA074 Me (Fig. 2D), while both BHA and CA074 Me
effectively inhibited the TWEAK-induced cell death (Fig. 2, A and C). These results suggested that ROI and
cathepsin B constituted a positive feedback loop in mediating the
TWEAK-induced cell death in hFn14/L5178Y cells.
Because ROI have been linked with NF-B activation (29),
we also investigated whether TWEAK could activate NF-B and whether
z-VAD-fmk might affect the NF-B activation in hFn14/L5178Y cells by
Western blotting analysis for IB degradation, which reflects
NF-B activation (17). As shown in Fig. 2G,
IB was degraded at 10 min, and then gradually resynthesized from
30 to 60 min after CD8-TWEAK stimulation. However, z-VAD-fmk did not
affect the level or kinetics of the IB degradation. These results
suggested that TWEAK-induced cell death in hFn14/L5178Y cells was not
critically regulated by NF-B activity.
Characterization of anti-hFn14 mAbs
To further characterize the expression and function of Fn14, we
generated four mAbs that specifically bind to hFn14 and exhibit
cytotoxic activity against hFn14/L5178Y cells. Hybridomas were prepared
from splenocytes from mice immunized with the hFn14/P815 cells. Four
hybridomas producing ITEM-1 (IgG1/), ITEM-2 (IgA/), ITEM-3
(IgG2b/), and ITEM-4 (IgG2b/) mAbs were selected by their
cytotoxicity against hFn14/L5178Y cells in the presence of z-VAD-fmk.
As represented in Fig. 3A, all
mAbs reacted with hFn14/L5178Y cells, but not with parental L5178Y
cells, as estimated by cell surface staining. These mAbs exhibited
cytotoxic activity against hFn14/L5178Y, but not L5178Y, cells in a
dose-dependent manner in the presence of z-VAD-fmk (Fig. 3B). ITEM-1 exhibited the strongest cytotoxic effect even in
the absence of z-VAD-fmk. The ITEM-1-induced cell death in hFn14/L5178Y
cells showed apoptotic or necrotic morphology in the absence or
presence of z-VAD-fmk, respectively, as estimated by electron
microscopy (data not shown), as was the TWEAK-induced cell death (Fig. 2C). These results further substantiated that hFn14
expressed on L5178Y cells could induce both apoptotic and necrotic cell
death.
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We next examined the expression and function of Fn14 on human
tumor cell lines that undergo different modes of TWEAK-induced cell
death. We previously demonstrated that TWEAK induced caspase-dependent
apoptosis and cathepsin B-dependent necrosis in HT-29 cells, only
caspase-dependent apoptosis in HSC3 cells, and endogenous
TNF--mediated apoptosis in Kym-1 cells (7). All these
TWEAK-sensitive tumor cell lines expressed Fn14, as estimated by cell
surface staining with ITEM-1 mAb (Fig. 4A). We also examined the
expression of Fn14 on several TWEAK-resistant tumor cell lines.
Although lymphoma cell lines such as Jurkat (Fig. 4A) did
not express Fn14, some nonhemopoietic tumor cell lines such as SK-Hep-1
(Fig. 4A) and Hep3B (not shown) hepatomas and G361 and A375
melanomas (not shown) expressed substantial levels of Fn14. This
suggested that the TWEAK sensitivity of tumor cells is not solely
determined by the cell surface expression of Fn14.
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, B and C). Both soluble and immobilized ITEM-1
efficiently induced cell death in HT-29 cells, which was further
enhanced by z-VAD-fmk. The dead HT-29 cells in the absence or presence
of z-VAD-fmk exhibited apoptotic or necrotic morphology, respectively
(data not shown). The necrotic death in the presence of z-VAD-fmk was
inhibited by CA074 Me (Fig. 4, B and C), but not
BHA (data not shown). Consistently, intracellular ROI level was not
increased in HT-29 cells (data not shown). This suggested that the
contribution of ROI to Fn14-mediated necrosis was cell type dependent.
In HSC3 cells, especially immobilized ITEM-1 strongly induced
caspase-dependent cell death, which was abrogated by z-VAD-fmk. In
Kym-1 cells, especially immobilized ITEM-1 strongly induced cell death,
which was mostly abrogated by anti-TNF- mAb. All modes of the
ITEM-1-induced cell death in these cell lines were just reminiscent of
the TWEAK-induced cell death we observed in the previous study
(7). In contrast, TWEAK-resistant Jurkat and SK-Hep-1
cells were also resistant to ITEM-1. These results indicated that
ligation of Fn14 could induce caspase-dependent apoptosis, cathepsin
B-dependent necrosis, or endogenous TNF--mediated cell death in
particular cellular contexts, just like TWEAK stimulation.
We further examined whether the TWEAK-induced cell death in these tumor
cell lines was solely mediated by Fn14. For this purpose, we used
ITEM-2, which acted agonistic against hFn14/L5178Y cells (Fig. 3B), but could inhibit the binding of CD8-TWEAK to
hFn14/L5178Y cells most efficiently (data not shown). Similar blocking
effects of agonistic anti-death receptor mAbs on ligand binding
have been observed for Fas-Fas ligand or DR4-TRAIL interaction
(30, 31). As shown in Fig. 5A, the binding of CD8-TWEAK
to HT-29, HSC3, and Kym-1 cells was almost completely blocked by the
preincubation with ITEM-2. Moreover, soluble ITEM-2 alone exhibited
little cytotoxicity against these cell lines and rather completely
blocked the CD8-TWEAK-induced cell death in these cell lines (Fig. 5B), which seemed to result from a weaker agonistic effect
of ITEM-2 than CD8-TWEAK against these cell lines. These results
suggested that Fn14 was solely responsible for the TWEAK-induced cell
death in these cell lines.
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B and
unpublished results). However, the signaling pathways leading to the
multiple modes of cell death and proliferation remain unknown. Because
Fn14 has been reported to lack a DD, but contain a TRAF binding domain,
which interacts with TRAF1, 2, and 3 (13), it is possible
that TRAFs and TRAF-binding proteins such as receptor-interacting
protein (RIP) and TNFR-associated DD (32) may regulate the
multiple pathways. It is well known that DD-containing TNFR1 and
DD-lacking TNFR2 can also mediate multiple pathways leading to cell
activation or death in a cell-dependent manner. Recently, it has been
reported that TNFR2 signaling leading to cell death or proliferation of
T cells was dramatically affected by intracellular RIP levels
(33). In this respect, Fn14-mediated proliferation or
death may be also determined by RIP levels in individual cell lines.
Namely, like TNFR2 signaling in resting T cells that express RIP at a
low level (33), Fn14-mediated cell proliferation may be
caused by NF-B and c-Jun N-terminal kinase activation via TRAF in
endothelial cells. In contrast, like TNFR2 signaling in activated T
cells that express RIP at a high level (33), DD-containing
proteins such as RIP and TNFR-associated DD may be sufficiently
recruited to the death-inducing signaling complex upon ligation of Fn14
in hFn14/L5178Y, HT-29, and HSC3 cells.
In this study, we also revealed that the inhibition of caspases by
z-VAD-fmk sensitized hFn14/L5178Y and HT-29 cells to Fn14-mediated
necrotic death. However, the mechanisms for the negative regulation of
necrotic death by caspases remain unknown. Recently, Holler et al.
(34) have reported that Fas ligand, TRAIL, or TNF-
could induce caspase-independent necrosis via RIP. Lin et al.
(35) have reported that RIP was cleaved by caspases during
TNF-induced cell death. In these respects, Fn14 may primarily activate
caspases, which cleave and inactivate RIP, resulting in apoptosis in
hFn14/L5178Y and HT-29 cells. When caspases are inactivated by
z-VAD-fmk, Fn14 may predominantly mediate RIP-dependent and
caspase-independent pathway leading to lysosomal or oxidative stress,
resulting in necrosis.
In Kym-1 cells, Fn14 mediates endogenous TNF--dependent indirect
cell death, like other DD-lacking TNFR family members such as CD30,
CD40, and TNFR2 (8, 9). However, it remains unknown why
Fn14 could not mediate a direct death signal in Kym-1 cells. Further
studies are now underway to characterize the Fn14-associated
death-inducing signaling complex in TWEAK-sensitive tumor cells by
using the anti-Fn14 mAbs established.
Fn14 was originally identified as a growth factor-inducible gene in fibroblast (15). It has also been reported that Fn14 mRNA expression was induced during liver regeneration and highly expressed in hepatocellular carcinomas (14). In our preliminary experiments with anti-Fn14 mAbs, Fn14 is highly expressed on HUVEC and in some cancer tissues, but not on freshly isolated PBMCs (our unpublished data). The anti-hFn14 mAbs we established in the present study will be useful for further exploring the expression and function of Fn14 in physiological and pathological conditions.
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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: DD, death domain; 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; BHA, butylated hydroxyanisole; CA074 Me, [L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester; DHR, dihydrorhodamine; Fn14, fibroblast growth factor-inducible 14; hFn14, human Fn14; RIP, receptor-interacting protein; ROI, reactive oxygen intermediate; TRAF, TNFR-associated factor; TRAIL, TNF-related apoptosis-inducing ligand; WST, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)[2H]tetrazolium monosodium salt; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.
Received for publication July 1, 2002. Accepted for publication November 1, 2002.
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