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Department of Oncology and Immunology, John Hunter Hospital, Newcastle, Australia
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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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, TNF-related apoptosis-inducing ligand
(TRAIL)3/apo-2 ligand (4, 5), and apo-3 ligand (4, 5, 6). TRAIL appears of
particular importance in that it is able to induce apoptosis in a wide
range of transformed cell lines but not normal cells (4, 5). mRNA for TRAIL can be detected in most normal tissues, and
in view of this it has been postulated that regulation of TRAIL-induced
apoptosis occurs at the level of receptor expression (3).
Apoptosis induced by TRAIL appears to be mediated by two receptors
referred to as DR4 (TRAIL-R1) (7, 8) and
DR5/TRAIL-R2/TRICK 2 (9, 10, 11). These receptors, like TRAIL,
were found to be widely expressed on normal tissues, but the latter are
believed to be protected from apoptosis by two additional receptors,
TRAIL-R3/TRID/DcR1/LIT (12, 13, 14, 15) and TRAIL-R4/DcR2/TRUNDD
(16, 17, 18). The mechanisms of protection by these receptors
was postulated to involve competition for TRAIL (i.e., act as decoys)
or activation of antiapoptotic signals perhaps via activation of
NF-
B (3, 18). A fifth receptor, osteoprotegerin exists
in a secreted form and appears to inhibit TRAIL-induced apoptosis by
competitive inhibition of binding of TRAIL to the death receptors
TRAIL-R1 and -R2 (19). We have shown previously that TRAIL but not other members of the TNF family was able to induce varying degrees of apoptosis in approximately two-thirds of the melanoma lines tested (20, 21, 22, 23). Resistance of some lines to TRAIL was due to absence of all receptors for TRAIL, whereas other cell lines had mRNA for TRAIL-R but lacked cell surface expression of the receptors. The latter applied particularly to TRAIL-R3 and -R4 decoy receptors, which appeared located predominantly within the cell (23). Their location within the cell suggested that expression of the receptors may involve regulated movement from intracellular compartments to the membrane.
In the present studies we have used confocal microscopy and flow cytometry to identify the cellular location of the receptors and their movement in response to TRAIL. The results indicate that TRAIL-R3 and -R4 are located predominantly in the nucleus, whereas TRAIL-R1 and -R2 are located in the Golgi apparatus. TRAIL-R1 and -R2 appear to orchestrate relocation of the decoy receptors from the nucleus to the cytoplasm and cell membrane. The site and location of decoy receptors after exposure to TRAIL may have an important bearing on resistance of melanoma cells to TRAIL-induced apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melanoma cell lines with the prefix Mel were isolated from fresh surgical biopsies from patients attending the Sydney and Newcastle Melanoma Units and established in the laboratory. FH, CV, LT, AT, and RMu were from lymph nodes. MC and MM were from skin. RM and JG were from bowel. The cell lines had been in culture for 2–6 mo at the time of these studies. MM200, Me1007, Me10538, and Me4405 were from primary melanoma. The derivation of MM200, Me1007, Me10538, and Me4405 are described elsewhere (23). All melanoma cell lines were positive for tyrosinase and MART-1 (melanoma Ag recognized by T cells) mRNA by RT-PCR tests described elsewhere (24), except for Mel-SP, which was positive for tyrosinase but not MART-1. All cell lines were cultured in DMEM containing 5% FCS (Commonwealth Serum Laboratories, Melbourne, Victoria, Australia).
mAbs and recombinant proteins
Recombinant human TRAIL (lot 6321-19) prepared as described elsewhere (4) was supplied by Immunex (Seattle, WA). The preparation was supplied as a leucine zipper fusion protein, which required no further cross-linking for maximal activity. The mAbs against TRAIL-R1 (IgG2a huTRAIL-M271, Lot 7136-07), TRAIL-R2 (IgG1 huTRAIL-R2-M413, Lot 5274-96), TRAIL-R3 (IgG1 huTRAIL-R3-M430, Lot 7313-17), TRAIL-R4 (IgG1 huTRAIL-R4-M444, Lot 7136-15), and the mAb against the leucine zipper on TRAIL (M15) were also supplied by Immunex. The specificity of the Abs are described elsewhere (25). Isotype control mAbs used were the ID4.5 (IgG2a) mAb against Salmonellae typhi supplied by Dr. L. Ashman (IMVS, Adelaide, Australia) and the 107.3 IgG1 mAb purchased from PharMingen (San Diego, CA). The Golgi was identified with rhodamine-labeled wheat germ lectin (Sigma, Castle Hill, NSW Australia; catalogue no. L-5266) and the trans-Golgi network (TGN) with rabbit Ab to p230 kindly supplied by Dr. P. A. Gleeson (Monash University, Melbourne, Australia) and described elsewhere (26). Rabbit IgG purchased from Sigma (catalogue no. I5006) was used as control. Abs (goat IgG) against the transferrin receptor (CD71) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA; catalogue no. SC7088). Control Abs were goat IgG purchased from Sigma (catalogue no. I5256). Second Abs were TRITC-conjugated rabbit anti-goat IgG (Sigma; catalogue no. T6028) and Alexa 594 goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Brefeldin A (BFA) was supplied by Sigma (catalogue no. B-7651). It was dissolved in ethanol and used at 5 µg/ml. The calpain proteosome inhibitor, N-acetyl-leucinyl, leucinyl, nor leucinyl (LLnL) was supplied by Sigma (catalogue no. A6185). It was dissolved in DMSO and made up in a stock solution of 25 mM. Cycloheximide (CHX) and actinomycin D (Act-D) were supplied by Sigma (catalogue no. 7698 and A1410, respectively). The pan caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone), was purchased from Calbiochem (La Jolla, CA; catalogue no. 627610).
Immunofluorescence and confocal microscopy
Melanoma cells were seeded onto sterile glass coverslips in 24-well plates (Falcon 3047; Becton Dickinson, Lane Cove, NSW, Australia) 16–24 h before fixation. In studies on relocalization of TRAIL receptors, cells were pretreated with recombinant TRAIL (200 ng/ml) at 37°C for 30 min. Cells were washed in PBS, fixed in 2% paraformaldehyde for 5 min, and permeabilized with 0.1% saponin in PBS containing 10% human AB serum for 10 min. Cells were then incubated with primary Abs diluted in PBS containing 1% human AB serum at 4°C for 45 min. After washing with PBS, cells were incubated with Alexa 488 goat anti-mouse IgG conjugate (Molecular Probes) or FITC-conjugated sheep anti-mouse (Silenus, Amrad Biotech, Boronia, Victoria, Australia) secondary Abs at 4°C for 45 min. For double labeling, cells were washed and incubated with cellular organelle-specific fluorescent reagents at 4°C for 30 min. Coverslips were mounted in Gel-Mount (Biomeda, Foster City, CA) and examined using a Zeiss Axiophot microscope (Oberkochem, Germany) coupled to a Bio-Rad (MRC 600; Hemel Hempstead, Herts, U.K.) confocal attachment and COMOS software (Bio-Rad). In some cases, cells were pretreated with BFA (5 µg/ml) at 37°C for 1 h before fixation.
Flow cytometry
Analysis was conducted using a Becton Dickinson (Mountain View, CA) FACScan flow cytometer. In studies on relocalization of TRAIL receptors, TRAIL (100 ng/ml) was added to melanoma cells grown overnight in 24-well plates. Adherent cells were removed by trypsinization in 0.25% trypsin at 37°C for 5 min, washed twice in cold DMEM and once in PBS at 4°C, and fixed in 4% paraformaldehyde. Appropriate concentrations of mAbs were added to the cells in 100 µl PBS containing 20% human AB serum and incubated for 7 min at room temperature. Cells were either washed twice with PBS and analyzed if directly labeled, or (if indirectly labeled) cells were then incubated with F(ab')2 fragment affinity isolated FITC-conjugated sheep anti-mouse Ig (Silenus, catalogue no. 985051020) plus 20 µl 100% human serum to block Fc receptors for 7 min at room temperature. A minimum of 5000 cells was analyzed. Studies on permeabilized cells were similar to the methods of Jung et al. (27). The cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% saponin in permeabilization buffer, and the Ab added for 30 min at 4°C. The cells were washed and then stained by FITC labeled F(ab')2 fraction of affinity-isolated sheep anti-mouse Ig (Silenus; catalogue no. 3155) at 1/100 dilution for 30 min at 4°C. After washing the cells were analyzed by flow cytometry. The TRAIL-R negative Me10538 cell line (23) was included as a negative control in studies on permeabilized cells. The percentage of cells expressing the receptors was calculated as the difference in positive area between the positive and negative control histograms. The positive area was that to the right of the intersection of the two curves (28).
Apoptosis
Apoptotic cells were determined by the propidium iodide method
(29). In brief, melanoma cells were adhered overnight in a
24-well plate (Falcon 3047; Becton Dickinson) at a concentration of
1 x 105/well in 10% FCS. Cells in
suspension were added on the day of the assay. Medium was removed, and
cells were treated for 4 h with different concentrations of
chemotherapeutic agents in 500 µl of fresh medium plus 10% FCS
before the further treatment for 20 h with the drugs in the
presence of the reagents TRAIL, human Fas ligand, TNF-, or human
CD40 ligand. Cells were incubated for a further 24 h at 37°C,
the medium removed, and adherent and suspended cells washed once with
PBS. The medium and PBS were placed in 12 x 75 mm Falcon
polystyrene tube and centrifuged at 200 x g. A
hypotonic buffer 1 ml (50 µg/ml propidium iodide in 0.1% sodium
citrate plus 0.1% Triton X-100; Sigma) was added directly to the cell
pellet of cells grown in suspension or to adhered cells in the 24-well
plate and gently pipetted off, then added to the appropriate cell
pellet in the Falcon tube. The tubes were placed at 4°C in the dark
overnight before flow cytometric analyses. The propidium iodide
fluorescence of individual nuclei was measured in the red fluorescence
using a FACScan flow cytometer (Becton Dickinson) and the data
registered in a logarithmic scale. At least 104
cells of each sample were analyzed. Apoptotic nuclei appeared as a
broad hypodiploid DNA peak, which was easily distinguished from the
narrow hyperdiploid peak of nuclei in the melanoma cells.
Transfection of melanoma cells with cDNA for TRAIL-R1, -R3, and -R4
Mel-MC, which lacks mRNA expression for TRAIL-R3 and -R4 receptors, Mel-SP, which expressed only TRAIL-R3 (23), MM200, and Me4405 were transfected by electroporation (650 V/cm, 960 µF) with 40 µg p-TARGET (Promega) encoding full-length cDNA of TRAIL-R3 receptor. The pDC409 vector carrying TRAIL-R4 cDNA was a gift from Immunex. Forty micrograms of plasmid pDC409 encoding TRAIL-R4 (18) was cotransfected with 4 µg of p-TARGET. Mel-SP, which lacks mRNA for TRAIL-R1, -R2, and -R4, was transfected at 900 V/cm and 960 µF with 40 µg of p-TARGET encoding the full-length cDNA for TRAIL-R1. Stable transfectants were grown in DMEM containing 5% FBS and G418 (Sigma-Aldrich, Sydney, NSW, Australia) at a concentration of 400 µg/ml for 14 days. Expression of TRAIL-R1, -R3, and -R4 was confirmed by reactivity with mAbs M271, M430, and M444, respectively, by flow cytometry and immunofluorescence microscopy of cells grown on coverslips.
Western blot analysis
Methods used for identification of TRAIL-R3 and -R4 in melanoma cells were as described previously (23). Nuclear and cytoplasmic extracts of the cells were prepared as described by Dignam et al. (30).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In view of the predominant intracellular location of some of the
TRAIL receptors shown in our previous studies, we investigated whether
binding of TRAIL may regulate surface expression of its receptors in
melanoma cells. Melanoma cells were pretreated with TRAIL (200
ng/ml) in DMEM at 37°C for 30 min, and then both surface and total
expression of receptors were examined. As shown in Fig. 1
for studies on Me4405, TRAIL induced
marked down-regulation of TRAIL-R2 expression, whereas there was an
increase in the surface expression of TRAIL-R3 and -R4. Exposure to
TRAIL did not appear to have significant effects on TRAIL-R1 expression
in cells from the Mel-FH line.
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. The pattern of
changes in TRAIL-R expression illustrated in Fig. 1
were seen to
varying degrees in most of the lines. The main exceptions were small or
no reduction in TRAIL-R2 surface expression in Me1007 and Mel-LT, which
were resistant to TRAIL-induced apoptosis. TRAIL-R3 and -R4 surface
expression increased in all the lines except in the TRAIL-resistant
cell line Mel-LT and the sensitive line Mel-JG. TRAIL-R4 was also not
up-regulated in MM200 cells, and TRAIL-R3 was not up-regulated in
Mel-SP cells, which lacked TRAIL-R1 and -R2 (23). TRAIL-R1
showed an increase in the TRAIL-resistant lines Me1007 and Mel-LT but
was otherwise unchanged (Mel-FH, Mel-AT, and Mel-JG) or
showed a decrease similar to TRAIL-R2 (Me4405, SK-Mel28, Mel-CV, and
Mel-RMu).
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). A small
reduction in TRAIL-R2 expression was still evident after exposure to
TRAIL (e.g., Mel-FH, MM200), but this was not as marked as the
down-regulation noted in studies on intact cells. Similarly, changes in
TRAIL-R1, -R3, and -R4 expression induced by TRAIL were small compared
with changes observed in studies on intact cells. The results shown are
representative of at least two studies on the cell lines. Subcellular localization of TRAIL receptors in melanoma cells
The subcellular localization of TRAIL receptors was studied on
melanoma cells from the Mel-FH and Me4405 cell lines by confocal
microscopy. The photomicrographs in Fig. 2 show that TRAIL-R2 was located on the
membrane of the cells and within intracellular organelles. The latter
was identified as the Golgi network by colocalization studies with
wheat germ lectin, which is known to stain the Golgi network
(31). In contrast, TRAIL-R3 and -R4 showed virtually no
membrane expression on Mel-FH cells and appeared to be entirely located
in the nucleus, as shown by colocalization studies with propidium
iodide (Fig. 2). The expression was diffuse throughout the nucleoplasm
but not in nucleoli. TRAIL-R1 in Me4405 cells displayed the same
staining pattern as for TRAIL-R2 with expression in the cell membrane
and the Golgi apparatus (Fig. 2). This pattern of expression was found
in studies on Me1007, Mel-JG, and Mel-RMu. In contrast, TRAIL-R1 was
predominantly within the nucleus of Mel-FH. The reason for
this different distribution in Mel-FH is not known. The nuclear
location of TRAIL-R3 and -R4 was confirmed by Western blot studies on
extracts from isolated nuclei from Mel-FH and Me4405, which showed
characteristic bands at 27 and 42 kDa, respectively (Fig. 3).
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, A—C, shows the
results for TRAIL-R2. BFA is a fungal metabolite which is known to
inhibit the export of proteins from the Golgi network and causes the
different Golgi compartments to redistribute in a characteristic manner
(32). Culture of Me4405 cells in BFA (5 µg/ml) resulted
in disruption of the Golgi network and appearance of a perinuclear
staining pattern characteristic of the TGN (32, 33) (see
Fig. 4D). Colocalization studies with mAbs against TRAIL-R1
and -R2 in Me4405 treated with BFA showed an identical localization
with the TGN (data not shown). No staining was evident with the isotype
controls.
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Mel-FH and Me4405 cells, grown on coverslips, were pretreated with
TRAIL (200 ng/ml) for 30 min, and the localization of TRAIL-R examined
by confocal microscopy. As shown in Fig. 2, exposure to TRAIL resulted
in reduced surface expression of TRAIL-R2 and appearance of punctate
staining throughout the cytoplasm. The Golgi-associated staining
pattern of TRAIL-R2 appeared to be retained. TRAIL-R1 in Me4405
displayed the same changes in expression as noted for TRAIL-R2
following exposure to TRAIL. Marked changes were seen in the
distribution of TRAIL-R3 and -R4 in that the receptors in the nucleus
became dispersed throughout the cytoplasm and cell membrane in all
lines except Mel-AT, Mel-JG, and, in the case of TRAIL-R4, MM200. The
subcellular location of TRAIL-R3 and -R4 in the cytoplasm after
exposure to TRAIL remains under study. The pattern of distribution of
TRAIL-R3 was punctate whereas that of TRAIL-R4 was more granular, but
the organelles involved have not been identified.
Studies on cell lines transfected with cDNA for TRAIL-R
To confirm the localization of TRAIL-R3 and -R4 in the nucleus,
Mel-MC that lacks mRNA expression for TRAIL-R3 and -R4
(23) was transfected with cDNA for TRAIL-R3 and -R4 or
vector alone. The levels of expression of the receptors to TRAIL,
assessed by flow cytometry are shown in Table II. TRAIL-R4 but not TRAIL-R3 was
detected on the cell surface. This correlated with the level of
apoptosis induced by TRAIL in that there was a marked reduction in the
cells transfected with TRAIL-R4 but only a slight reduction in the
TRAIL-R3 transfected cells. As shown in Fig. 5, TRAIL-R3 and -R4 were identified in
the nuclei of the transfected but not the nontransfected Mel-MC cells.
Some perinuclear staining with mAbs to TRAIL-R4 was also evident.
Mel-SP cells, which expressed only TRAIL-R3 (23), were
transfected with cDNA for TRAIL-R1. Despite good levels of surface
expression (Table II), the Mel-SP cells remained insensitive to
TRAIL-induced apoptosis. Fig. 5 shows that the transfected TRAIL-R1
were located in the Golgi network, as expected from studies on the
nontransfected cell lines. Control studies showed no reactivity of mAbs
against TRAIL-R1 in the parental Mel-SP cells or mAbs against TRAIL-R3
and R4 in the parental Mel-MC cells. The isotype control mAbs 104.5
(IgG2a) and 107.3 (IgG1) did not react with the transfected Mel-SP or
Mel-MC cells (data not shown).
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). Transfection of cDNA for TRAIL-R3 in this line
also resulted in increased TRAIL-R3 surface expression, but this
resulted in only a small decrease in apoptosis (Table II). Transfection
of cDNA for TRAIL-R3 and -R4 into Me4405 cells resulted in increased
levels of expression within the cell, but there were only small
increases in surface expression of TRAIL-R3 and -R4. Similarly, there
were only small reductions in apoptosis induced by TRAIL in these cells
(Table II). Confocal microscopy indicated that the location of the
receptors were as shown in Fig. 2. The results shown are representative
of at least two experiments on the transfected and parental cell,
vector alone, combinations. TRAIL-R1 and -R2 undergo internalization after exposure to TRAIL
As indicated in Table I, the total expression of each receptor
assessed in permeabilized melanoma cells remained relatively constant
after treatment with TRAIL, which suggested the changes in surface
expression were the result of relocation of the receptors into or out
of the interior of the cell. The down-regulation of TRAIL-R1 and -R2
from the cell surface in Mel-RMu is shown in Fig. 6A. This indicates the
kinetics of the down-regulation of TRAIL-R1 and -R2 was similar with a
rapid initial decrease occurring as soon as 2 min after exposure and
nearing completion within 10 min. Thirty minutes after exposure to
TRAIL the level of expression was relatively stable. Fig. 6B
indicates that TRAIL identified by mAb against the leucine zipper could
be detected in low amounts on the cell surface but in greater amounts
within the cell, indicating internalization of the ligand. TRAIL within
the cell appeared to be within endosomes, as shown by colocalization of
mAbs to TRAIL and the Abs against the transferrin receptor (CD71) which
identified endosomes (Fig. 7)
(34).
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B, there was a gradual reduction in the amount of
TRAIL on the cell surface so that by 30 min the levels were barely
detectable. This suggests that the continued detection of TRAIL on the
cell surface was due to continuing expression of TRAIL-R2, even though
the TRAIL/TRAIL-R2 complex was being endocytosed. The results shown are
representative of three similar experiments.
Exposure of cells to hypertonic medium was reported to inhibit receptor
induced endocytosis (35). This was confirmed in the
present studies by confocal microscopy studies showing that TRAIL
induced uptake of the transferrin receptor was inhibited in the
presence of the hypertonic medium (data not shown). We therefore used
this approach to study internalization of TRAIL-R1 and -R2. Melanoma
cells were pulsed for 2 min with TRAIL at 4°C, then at 37°C for 30
min in DMEM alone or in DMEM containing 0.45 M sucrose that rendered
the medium hypertonic. The cells were fixed and the surface expression
of the receptors studied by flow cytometry. Table III shows that the down-regulation of
TRAIL-R1 and -R2 was inhibited in the presence of the hypertonic
medium. These results suggest that internalization is at least one of
the mechanisms that lead to down-regulation of TRAIL-R1 and -R2
following exposure to TRAIL. Similar results were found in a repeat of
the experiment.
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As shown above in Table I, there was no up-regulation of TRAIL-R3
in cells which expressed this receptor alone (Mel-SP). To further
delineate the signals involved in up-regulation of TRAIL-R3 and -R4 by
TRAIL, we conducted studies on the cell line Mel-RM that expresses
TRAIL-R2 on the surface and TRAIL-R3 within the nucleus
(23). However, it lacks mRNA for TRAIL-R1 and -R4.
Previous studies have shown that preincubation of cells with the mAb
M413 for 1 h at 37°C blocks the binding of TRAIL to TRAIL-R2
(25). TRAIL-induced apoptosis of Mel-RM cells was
completely abrogated by the mAb M413. Under the same experimental
conditions the surface expression of TRAIL-R3 induced by exposure to
TRAIL was also completely inhibited (Table IV). This indicated that binding of TRAIL
to TRAIL-R2 is required for TRAIL to induce expression of TRAIL-R3 on
the cell surface.
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, pretreatment with
either mAb attenuated TRAIL-induced apoptosis, and TRAIL-induced
up-regulation of TRAIL-R3 and -R4. When the two mAbs were used in
combination, TRAIL failed to induce apoptosis and completely inhibited
relocalization of TRAIL-R3 and -R4. It was of note that TRAIL-R1 mAb
M271 appeared to have greater effect on TRAIL-induced apoptosis and
receptor relocalization than the mAb M413 against TRAIL-R2. Whether
this reflects differing affinities of the mAbs or activity of the
receptors is not known. Relocalization of TRAIL-R3 and -R4 from the nucleus is blocked by the proteasome inhibitor LLnL and by BFA but not by the pan caspase inhibitor zVAD-fmk or protein synthesis inhibitors
TRAIL-R1 and -R2 are known to be complex receptors that can
activate several different signal pathways. One of these leads to
activation of caspases and apoptosis, whereas others lead to activation
of NF-B (8). To assess the role of caspases in the
relocation of TRAIL-R3 and -R4 we used the Pan caspase inhibitor
zVAD-fmk (at 20 nM) to block the caspase pathway (25). We
found that although this completely inhibited apoptosis, it had no
effect on TRAIL induced relocalization of TRAIL-R3 and -R4 (data not
shown). To assess the role of NF-B, the proteasome inhibitor LLnL
was used to prevent degradation of IB and hence activation of
NF-B (36). It was found that treatment of cells with
LLnL before exposure of cells to TRAIL blocked the up-regulation of
TRAIL-R4 and to a lesser extent, TRAIL-R3 (Table V). However, there was no effect on
TRAIL-induced down-regulation of TRAIL-R2. This experiment was repeated
with the Mel-CV line with similar results, i.e., there was no effect on
the down-regulation of TRAIL-R1 and -R2 but up-regulation of TRAIL-R3
and -R4 was inhibited by LLnL. (Percent expression of TRAIL-R3 and R4
induced by TRAIL in absence of LLnL was 13.1 and 13.4, respectively,
and in presence of LLnL, 5.4 and 2.5, respectively.) Similarly BFA,
which blocks export of proteins from the Golgi apparatus, was found to
have no effect on the expression of TRAIL receptors on the cell surface
in cells not exposed to TRAIL but partially inhibited the up-regulation
of TRAIL-R3 and -R4 after exposure to TRAIL (Table V). This may be
secondary to effects of BFA on TRAIL-R2 in that pretreatment with BFA
was found to further decrease the expression of TRAIL-R2 induced by
TRAIL. This may indicate that TRAIL-R2 levels on the cell are
contributed to by export from the Golgi and when the latter is blocked
by BFA the TRAIL-R2 receptor levels are reduced (see also Fig. 6B). To determine whether TRAIL induced relocalization of
TRAIL receptors involved protein synthesis, we cultured Mel-FH cells or
Me4405 cells in the presence of CHX (100 µg/ml) to block protein
synthesis or Act-D (3 ng/ml) to block transcription, before adding
TRAIL. It was found that neither CHX nor Act-D treatment affected
TRAIL-induced relocalization of the receptors, which suggested the
relocation of TRAIL-R3 and -R4 is a posttranslational process.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The appearance of TRAIL-R1 in most of the melanoma cells was identical to TRAIL-R2 except in cells from the Mel-FH line, where TRAIL-R1 was located in the cell membrane and nucleus rather than the Golgi network. The distribution of TRAIL-R1 in melanoma cells that had been transfected with cDNA for TRAIL-R1 was typically that associated with the TGN and suggests that this is the normal location of both death receptors. The reason for the different distribution of TRAIL-R1 in the Mel-FH cells is unknown.
These results are similar to studies suggesting that TNF-R1 in human endothelial and monocyte lines was predominantly located in the TGN (33). Similarly, p53 activation was reported to result in increased expression of Fas on vascular smooth muscle cells due to transport from the TGN (37). These and our own findings suggest that localization in the TGN may be a common feature of TNF family receptors inducing apoptosis. Another similarity between TRAIL-R1 and -R2 and TNF-R1 appears to be internalization of the receptor/ligand complex. This was shown previously for the TNF-R1/TNF complex (33, 38) and was suggested in the present studies by internalization of TRAIL and a decrease in TRAIL-R1 and -R2 expression on the membrane, shown by confocal microscopy and flow cytometry. Confocal microscopy indicated colocalization of TRAIL and markers of endosomes, suggesting that the TRAIL/TRAIL-R2 complex was endocytosed. Flow cytometry revealed a rapid increase of TRAIL within the cell in permeabilized cells. It was also noted that treatment of cells exposed to TRAIL with BFA resulted in inhibition of the surface expression of TRAIL-R1 and -R2 on Me4405 cells, which may indicate that TRAIL-R1 and -R2 undergo continual export from the Golgi after interaction with TRAIL.
In contrast to the localization of the death receptors for TRAIL in the Golgi network, the TRAIL decoy receptors were localized in the nucleus. After exposure to TRAIL there was rapid relocation of TRAIL-R3 and -R4 to the cytoplasm and cell membrane in all but two of the lines. The cytoplasmic staining was punctate or granular in pattern but apart from some colocalization in the Golgi network the organelles involved have not been identified. This pattern of expression was also shown in cell lines transfected with cDNA for TRAIL-R3 and -R4. We do not believe the nuclear location of TRAIL-R3 and -R4 was merely due to overexpression of the proteins as similar transfection of TRAIL-R1 resulted only in localization to the TGN and the transfected cells had identical staining patterns to normally expressed TRAIL-R3 and -R4. The transfected receptors were also functional, and in the case of the TRAIL-R4 transfectants were more resistant to TRAIL-induced apoptosis than the untransfected cells. The TRAIL-R3 transfectant had only slightly reduced sensitivity to TRAIL-induced apoptosis. These results, showing increased resistance to apoptosis in cells transfected with TRAIL-R3 and -R4, are consistent with previous studies (13, 18) but at variance with studies on nontransfected melanoma cells where there was no correlation between expression of TRAIL-R3 and -R4 and sensitivity to TRAIL-induced apoptosis (23).
Several inferences can be made from the studies on transfected cells. In studies on the MM200 line the endogenous TRAIL-R4 was not relocated from the nucleus after exposure to TRAIL, whereas the transfected receptors underwent the expected relocation. Such a result would be consistent with aberrant structure of the endogenous receptor and account in part for the susceptibility of this line to TRAIL-induced apoptosis. In the studies on the Me4405 line there was very little increase in the surface expression of receptors in the transfected cells and a small decrease in apoptosis, even though there was a marked increase in receptor content within the cell. It is not clear why the transfected TRAIL-R3 and -R4 were not expressed at higher levels in these cells, but cell surface rather than nuclear expression of the receptors may be needed for inhibition of apoptosis. The ability of the cell to up-regulate TRAIL-R3 and -R4 may therefore be as important as the total amount of receptors in the cell.
Signals from TRAIL-R1 and -R2 appear responsible for relocation of TRAIL-R3 and -R4. This was suggested by the observation that melanoma cells that expressed only TRAIL-R3 receptors and not TRAIL-R1 or -R2 did not undergo relocation from the nucleus and was confirmed by use of mAbs which block interaction of TRAIL with TRAIL-R1 and -R2 (25). In one line (Mel-RM) which expressed only TRAIL-R2 and -R3 the relocation of R3 was completely inhibited by mAb against TRAIL-R2. Similarly, in a line (Me4405) which expressed all TRAIL-R, inhibition of TRAIL-R2 alone was insufficient to stop relocation of TRAIL-R3 and -R4, and it was also necessary to inhibit TRAIL-R1. These results indicate that the signals for relocation of the decoy receptors are dependent on signals from TRAIL-R1 or -R2. A precedent for these findings is the shedding of the TNF-R2 receptor from the cell surface, which was shown to be dependent on signals from TNF-R1 (38).
The nature of the signals from TRAIL-R1 and -R2 responsible for the
relocation of TRAIL-R3 and -R4 were investigated on the basis of the
signal pathways known to be activated by TRAIL receptors, i.e., the
caspases and NF-B (8). Inhibitors against activation of
NF-B but not those against activation of the caspase pathway
suggested that the relocation of the decoy receptors may be dependent
on activation of the transcription factor NF-B by TRAIL-R1 and/or
TRAIL-R2. This was more evident for up-regulation of TRAIL-R4 than
TRAIL-R3. The proteosome inhibitor LLnL used in these studies is not
specific for NF-B, and more specific inhibitors of NF-B, such as
mutated forms of IB which are resistant to degradation
(39), are needed to confirm these results. However,
involvement of NF-B would be consistent with its role in inhibition
of apoptosis (40). TRAIL-R4 was reported to activate
NF-B (3, 18) so that it is possible the relocation to
the cell membrane may serve to amplify signals from TRAIL-R3 and -R4
which activate NF-B. The latter is believed to inhibit apoptosis by
mechanisms such as induction of inhibitors of apoptosis 1 and 2
(40).
In summary, the present studies show that the death and decoy receptors for TRAIL have different locations and undergo different movement within the cells on contact of the death receptors with TRAIL. They also raise the possibility that abnormalities in the localization and movement of the receptors may underlie the failure of the decoy receptors to protect against TRAIL-induced apoptosis. The basis for the nuclear localization and export from the nucleus remains to be studied, but it is of interest that TRAIL-R3 and -R4 have sequences compatible with those needed to bind to exportin 1, which is a nuclear export factor (41, 42).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter Hersey, Department of Oncology and Immunology, John Hunter Hospital, Room 443, David Maddison Clinical Sciences Building, Corner of King & Watt Streets, Newcastle, NSW 2300, Australia.
3 Abbreviations used in this paper: TRAIL, TNF-related apoptosis inducing ligand; TRAIL-R, receptors for TRAIL; CHX, cycloheximide; Act-D, actinomycin-D; LLnL, acetyl leucinyl, leucinyl, nor leucinyl; BFA, brefeldin A; zVAD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone.
Received for publication September 2, 1999. Accepted for publication January 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B. Immunity 7:831.[Medline]
B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity 7:813.[Medline]
-helical domains and a granin motif. J. Biol. Chem. 271:8328.B activation. J. Biol. Chem. 41:24518.
B activation attenuates apoptosis resistance in lymphoid cells. Blood 91:4624.B. Science
274:784.
B antiapoptosis: induction of
TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation.
Science 281:1680.
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