The Journal of Immunology
Removal of Syndecan-1 Promotes TRAIL-Induced Apoptosis
in Myeloma Cells
Yung-Hsuan Wu,*,†Chen-Ying Yang,*,†Wen-Li Chien,*,†Kuo-I Lin,‡and
Syndecan is the major transmembrane proteoglycan in cells. Of the four syndecans, syndecan-1 is the dominant form expressed in
multiple myeloma and is an indicator of poor prognosis. In the current study, we observed that early TRAIL-induced apoptotic
processes were accompanied by cleavage of syndecan-1 intracellular region, and explored the possibility whether removal of
syndecan-1 promotes apoptotic processes. We found that syndecan-1 knockdown by specific small interfering RNA in multiple
myeloma enhanced TRAIL-induced apoptosis, even though the expression of TRAIL receptors and several apoptosis-
associated molecules was unaffected. The enhanced TRAIL-mediated apoptosis in syndecan-1–deficient cells was not due to
a decrease in surface heparan sulfate or a reduction in TRAIL receptor endocytosis. The increase in TRAIL-induced cell death
was accompanied by an elevated caspase-8 activation and an enhanced formation of death-inducing signaling complexes, which
could be attributed to an increased expression of TRAIL receptor O-glycosylation enzyme in syndecan-1–deficient cells. We also
found that in H9 lymphoma and Jurkat cells, knockdown of the predominant syndecan member also led to an increase in Fas
ligand-induced apoptosis. Our results demonstrate that syndecan plays a negative role in death receptor-mediated cell death,
suggesting potential application of syndecan downregulation in the treatment of myeloma in combination with TRAIL.
Journal of Immunology, 2012, 188: 2914–2921.
brane domain for dimerization, an intracellular domain for linkage
to the actin cytoskeleton, and a PDZ domain. Syndecans play
important roles in different physiological functions, including
embryo development, vasculation, neural migration, signaling,
inflammation, angiogenesis, and focal adhesion (3–7). There are
four members of the syndecan family in vertebrates, as follows:
syndecan-1, -2, -3, and -4. Syndecan-1 is the dominant form of
syndecans in plasma cells and multiple myeloma, and is an indi-
cator of poor prognosis in multiple myeloma (8). Syndecan-1 is
a target gene of Blimp-1 (9), which regulates the differentiation of
B cells into plasma cells (9, 10). Syndecan-1 is involved in the
growth and metastasis of multiple myeloma in vivo (11, 12).
Removal of the heparan sulfate chain by bacterial heparinase
III profoundly suppresses the growth of myeloma in vivo (11).
in the cell (1, 2), and are composed of an extracellular
domain that contains heparan sulfate chains, a transmem-
Syndecan-1 also regulates the activation of avb3and avb5integ-
rins in angiogenesis (13).
Engagement of Fas and TRAIL receptors (death receptor [DR] 4
and DR5) by the Fas ligand (FasL) and TRAIL, respectively, leads
to formation of death-inducing signaling complexes (DISC) con-
taining Fas-associated protein with death domain (FADD) and
procaspase-8 at the cell membrane (14–16). For the TNF-a re-
ceptor, the formation of DISC does not occur at the cell mem-
brane: TNFR type 1-associated death domain protein (TRADD)
has to be dissociated from membrane-bound TNFRI complex, and
FADD and procaspase-8 become then associated with TRADD
in the cytoplasm (16–19). DISC is serving as a platform for
procaspase-8 to undergo autoproteolytic cleavage generating ac-
tive caspase-8. Caspase-8 in turn activates downstream caspases
and leads to irreversible cell damage. DRs like Fas are associated
with actin filaments through binding to ezrin/radxin/moesin (20).
Syndecan binds directly to the actin cytoskeleton (21) and is in-
directly linked to Fas.
Since the identification of Fas and TRAIL receptors, the pos-
sibility of DR agonists to induce apoptosis in tumor cells has been
intensively explored. TRAIL and TRAIL receptor agonists trigger
apoptosis in tumor cells while leaving normal cells unaffected, and
are promising biologic drugs for anticancer therapy, shown by
results from many clinical trials (22–26). A few cancer cells are
relatively resistant to TRAIL, and TRAIL therapeutics often in-
volve sensitization of the transformed cells to TRAIL-induced
apoptosis. Myeloma cells are moderately responsive to TRAIL
(27–29). In the current study, we observed that TRAIL-induced
apoptosis is preceded by removal of the intracellular domain of
syndecan-1 in myeloma cells, and investigated in this study how
TRAIL-induced apoptosis is modulated by syndecan-1 removal.
We found that syndecan-1 knockdown leads to a significant in-
crease in the formation of DISC and in TRAIL-induced cell death
in myeloma cells. In addition, downregulation of the major form
of syndecan in T lymphoma cells enhanced FasL-induced apo-
*Institute of Microbiology and Immunology, National Yang-Ming University, Taipei
Taiwan;‡Genomic Research Center, Academia Sinica, Taipei 11529, Taiwan;
andxInstitute of Immunology, National Taiwan University, Taipei 10051, Taiwan
†Institute of Molecular Biology, Academia Sinica, Taipei 11529,
Received for publication July 15, 2011. Accepted for publication January 3, 2012.
This work was supported by National Science Council Grant NSC98-2321-B001-015
and an Academia Sinica Investigator Award.
Address correspondence and reprint requests to Prof. Ming-Zong Lai, Institute of
Molecular Biology, Academia Sinica, Taipei 11529, Taiwan. E-mail address: mblai@
The online version of this article contains supplemental material.
Abbreviations used in this article: DISC, death-inducing signaling complex; DR,
death receptor; FADD, Fas-associated protein with death domain; FAP-1, Fas-
associated phosphatase-1; FasL, Fas ligand; GALNT, N-acetyl galactosamine trans-
ferase; PI, propidium iodide; shRNA, short hairpin RNA; shSDC1, short hairpin
syndecan-1; siRNA, small interfering RNA; siSDC1, syndecan-1–specific siRNA;
TRADD, TNFR type 1-associated death domain protein.
ptosis. Our results illustrate a negative role of syndecan in TRAIL-
and FasL-induced apoptosis, and suggest that knockdown of syn-
decan may be used to increase the efficacy of TRAIL in targeting
Materials and Methods
Recombinant human TRAIL and anti-His were obtained from R&D Sys-
tems (Minneapolis, MN). FLAG-TRAIL, His-TRAIL, FLAG-FasL, and
anti–c-FLIP (NF6) were purchased from ENZO Life Sciences (Plymouth
Meeting, PA). Anti–syndecan-1 (DL-101), anti–syndecan-3 (M-300), anti–
syndecan-4 (5G9), anti-DR4 (H-130), anti-Fas (C-20), anti–Mcl-1 (S-19),
anti–Bcl-2 (N-19), anti-FADD (H-181), and anti–caspase-3 (H-277) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–
syndecan-1 (BV-600) and annexin V-Cy5 were obtained from Biovision
(Mountain View, CA). Anti-human syndecan-1 (B-B4) was purchased
from Serotec. Z-IETD and Z-VAD were obtained from Calbiochem-Merck
(San Diego, CA). Heparinase III from Flavobacterium heparinum was
obtained from Sigma-Aldrich (St. Louis, MO). Anti–caspase-8 (1C12),
anti-active caspase-8 (18C8), and anti-active caspase-3 were purchased
from Cell Signaling (Beverly, MA). Allophycocyanin-conjugated anti-
human DR4 (DJR1), allophycocyanin anti-DR5 (DJR2-4), DyLight649
anti-rabbit IgG, and allophycocyanin anti-mouse IgG were obtained from
BioLegend (San Diego, CA). Anti-Bax (6A7) was purchased from eBio-
science (San Diego, CA). Anti–b-tubulin (clone AA2) was purchased from
Upstate (Lake Placid, NY). The SSTN92–119peptide (80–90% purity) was
purchased from GenScript (Piscataway, NJ). Goat anti-rabbit Ig and rabbit
anti-mouse Ig conjugated with HRP were obtained from Amersham Bio-
sciences (Buckinghamshire, U.K.). Rabbit anti–syndecan-2 was a gift of
Y.-P. Hsueh (Institute of Molecular Biology, Academia Sinica).
Cell culture and apoptosis
Invitrogen), 10 mM glutamine, 100 U/ml penicillin, 100 mg/ml strepto-
mycin, and 20 mM 2-ME. Two different methods were used for apoptosis
measurements after DR ligand treatments. Cells were stained with allo-
phycocyanin annexin V, and annexin V+populations were quantified by
flow cytometry. Cells were also stained with propidium iodide (PI) in
hypotonic solution (50 mg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100)
overnight at 4˚C. Fractions of cells with sub-G1DNA content were de-
termined using the CELLFIT program on a FACSCalibur flow cytometer
(BD Biosciences, Mountain View, CA). All apoptosis events were con-
firmed by both measurements.
Syndecan-1 was downregulated by two different methods. Multiple
myeoloma cells (5 3 106) were transfected with syndecan-1–specific small
interfering RNA (siRNA) by electroporation on an MP-100 Microporator
(Digital Bio) at 1600 mV using two pulses of 15-ms duration, and cellular
syndecan-1 was analyzed 48 h later. The sequence of siRNA for syndecan-
1 was 59-CAC CUG GCA UCG CAC CAU U-39. Alternatively, syndecan-
1, syndecan-2, or syndecan-4 knockdown lentiviral constructs were pro-
duced by incorporating syndecan-1–, syndecan-2–, or syndecan-4–specific
short hairpin RNA (shRNA) sequence into the pLentiLox vector (pLL3.7;
obtained from I-C. Ho, Harvard Medical School, Boston, MA). The fol-
lowing shRNA sequences were employed: short hairpin syndecan-1
(shSDC1), 59-CAG GTG CTT TGC AAG ATA-39; shSDC2, 59-GCT
TCA GGA GTG TAT CCT A-39; and shSDC4, 59-GGT GAG GTC AAC
CTA ATA-39. Lentiviruses were harvested from culture supernatants of
293FT cells transfected with pLL3.7 or pLL3.7-shSDC (20 mg), psPAX2
(15 mg), and pMD2.G (6 mg). H929 cells, RPMI 8226, U266, H9, or Jurkat
cells were infected with recombinant lentivirus, and GFP-expressing cells
were isolated by sorting on a FACSVantage SE system (BD Biosciences)
48 h postinfection.
Overexpression of syndecan-1
Human syndecan-1 cDNA was subcloned into pTRIP-IRES-GFP to gen-
erate pTRIP-SDC1-IRES-GFP. The 293T cells were transfected with 20 mg
pTRIP-IRES-GPF or pTRIP-SDC1-IRES-GFP, 15 mg psPAX2, and 6 mg
pMD2G, and lentivirus-containing culture supernatants were harvested.
Cells were infected with recombinant lentivirus with pTRIP-IRES-GPF or
pTRIP-SDC1-IRES-GFP, and GFP-expressing cells were isolated 48 h
postinfection by sorting on a FACSVantage SE.
For staining of surface syndecans, cells were incubated with anti–
syndecan-1, anti–syndecan-2, anti–syndecan-3, or anti–syndecan-4 in PBS
containing 1% FBS. After washing with PBS, cells were stained by
allophycocyanin-conjugated anti-mouse IgG or DyLight649 anti-rabbit
IgG, and analyzed on a FACSCalibur flow cytometry system. For TRAIL
receptor internalization, H929 and RPMI 8226 cells were incubated with
FLAG-tagged TRAIL (2 mg/ml) on ice for 30 min, followed by incubation
at 37˚C for 5, 10, or 30 min. Receptor internalization was stopped by
adding ice-cold PBS containing 0.5% sodium azide. Cells were then
washed with ice-cold PBS and stained with anti-FLAG, followed by
allophycocyanin-labeled anti-mouse IgG for FACS analysis.
Caspase-8 activity determination
Caspase-8 activity was quantitated by the Caspase-Glo 8 assay (Promega,
Madison, WI). Myeloma cells were treated with TRAIL for 1.5 h, and an
equal volume of Caspase-Glo 8 reagent was then added and incubated for
additional 30 min. The aminoluciferin released by caspase-8–mediated
cleavage of precursor was determined using a luminescence reader Victor3
1420 Multilabel Counter (PerkinElmer, Shelton, CT).
Aliquots of 2 3 107H929 cells were incubated with His-TRAIL (5 mg/
ml) on ice for 30 min and then at 37˚C for 30 or 60 min. Cells were lysed
in DISC immunoprecipitation buffer (30 mM Tris-HCl [pH 8.0], 150 mM
NaCl, 1% Triton X-100, 10% glycerol, and protease inhibitors [Roche,
Mannheim, Germany]). Cell lysates were incubated with Ni-NTA aga-
rose (Qiagen, Hilden, Germany) for 2 h. DISC complexes were eluted
from Ni-NTA agarose by 200 mM imidazole. The eluent was resolved by
SDS-PAGE and probed with Abs for DR4, DR5, caspase-8 (Cell Sig-
naling), FADD, and b-tubulin. DISC precipitation in H9 cells using
FLAG-FasL and anti-FLAG beads was performed, as previously de-
For detection of TRAIL receptor clustering, 2 3 107cells were incu-
bated with human rTRAIL (200 ng/ml; R&D Systems) for the indicated
times. Cells were lysed in DISC immunoprecipitation buffer. A total of 2
mg cell lysates was incubated overnight with 2 mg anti–caspase-8 (C-20;
Santa Cruz Biotechnology) preloaded on 20 ml protein G-Sepharose at
4˚C. The precipitants were washed and resolved in 4–20% nonreducing
SDS-PAGE and transferred to polyvinylidene difluoride membranes (Mil-
lipore). The membrane was probed with anti–caspase-8 (1C12; Cell Sig-
naling) and anti-DR4 (Santa Cruz Biotechnology).
Total RNA from control and syndecan-1 knockdown H929 cells was iso-
lated using TRIzol (Invitrogen). cDNAs were prepared and analyzed for the
expression of GALNT3 and GALNT14 on a LightCycler 480 Real-Time
PCR System (Roche). The PCR protocol is 95˚C for 10 min, followed by
45 cycles of 95˚C for 10 s, 60˚C annealing for 10 s, and 72˚C extension for
8 s. The PCR primers are as follows: GALNT3, forward, 59-AAA GCG
TTG GTC AGC CTC TA-39 and reverse, 59-AAC GAG ACC TTG AGC
AGC AT-39; GALNT14, forward, 59-CTG AGATGC ACA CTG CTG GT-
39 and reverse, 59-CAT TTC ACC TTG GGC AAC TT-39.
Processing of syndecan-1 occurs early during TRAIL-induced
apoptosis in myeloma cells
Syndecan-1 was the most abundant syndecan on the surface of
multiple myeloma H929, RPMI 8228, and U266 cells (Supple-
mental Fig. 1A, not shown for U266). We found that treatment
with TRAIL was accompanied with the appearance of a low
molecular mass (,15 kDa) fragment of syndecan-1, as detected
by immunoblot analysis of H929 cells (Fig. 1A). The genera-
tion of the syndecan-1 fragment coincided with the activation of
caspase-8 (Fig. 1A). Further support that the cleavage of syn-
decan-1 was mediated by caspase was obtained by the complete
blockage of the processing by the addition of Z-VAD, a cell-
permeable caspase inhibitor (Fig. 1C). In addition, the cleavage
of syndecan-1 was partially prevented by the caspase-8 inhibitor
Z-IETD, indicating an involvement of caspase-8. Notably, there
was no apparent reduction in the surface level of syndecan-1
The Journal of Immunology 2915
(Fig. 1B), in contrast to the disappearance of syndecan-1 in cell
lysates 90 min after TRAIL treatment (Fig. 1C, DMSO). Because
different syndecan-1 Abs were used for Western blotting (BV-
600) and FACS staining (clone DL-101), we investigated whether
the Ab used for immunoblotting detected the C-terminal portion
of syndecan-1 and whether the low molecular mass fragment
represented the intracellular part of syndecan-1. To this end,
syndecan-1 with a Myc tag attached to the C-terminal end of
syndecan-1 was expressed in H929 cells. Treatment of cells ex-
pressing syndecan-1-Myc with TRAIL led to the generation of
the low molecular mass fragment detected by anti-Myc (Fig.
1D). These results suggest that immediately following TRAIL
receptor ligation, the intracellular region of the major trans-
membrane glycosaminoglycan was cleaved by early-activated
Downregulation of syndecan-1 increases the sensitivity to
TRAIL-induced apoptosis in myeloma cells
To explore the possibility that removal of syndecan-1 intracel-
lular domain enhances DR-mediated cell death, syndecan-1 was
knocked down in these myeloma cell lines by transfection with
syndecan-1–specific siRNA (siSDC1). Fig. 2A illustrates that
syndecan-1 was nearly eliminated in H929 cells transfected with
siSDC1. This was accompanied by diminished expression of sur-
face syndecan-1, with a reduction by .90% (Fig. 2B). Syndecan-1
knockdown did not affect the growth and viability of myeloma
cells in vitro (data not shown), similar to a previous report (12).
Syndecan-1 was similarly downregulated in RPMI 8226 myeloma
following TRAIL stimulation. (A and B) TRAIL treatment generated a low
molecular mass fragment of syndecan-1. H-929 cells were incubated with
TRAIL and harvested at the indicated time points. Cell lysates (A) were
prepared in the presence of Z-VAD to prevent any further caspase cleav-
age, resolved by SDS-PAGE, and analyzed by immunoblotting with anti–
syndecan-1 (BV-600) and anti–caspase-8. TRAIL-stimulated H-929 cells
were also stained with anti–syndecan-1 (DL-101) at the indicated time
points, and surface contents of syndecan-1 were analyzed by FACS (B). (C)
Inhibition of syndecan-1 cleavage by z-VAD and Z-IETD. H-929 cells
were treated with TRAIL (200 ng/ml) in the presence of DMSO, Z-VAD
(80 mM), or Z-IETD (40 mM). Cell lysates were prepared at the indicated
time points, and the levels of syndecan-1 and caspase-8 were determined.
(D) Cleavage of syndecan-1 at C-terminal region during TRAIL-induced
apoptosis. Syndecan-1 fused to a Myc tag at the C terminus was expressed
in H929 cells. Cells expressing YFP (control) or syndecan-1-Myc were
treated with TRAIL (200 ng/ml). Cell lysates were prepared, and the
contents of syndecan-1-Myc were determined by anti-Myc.
Processing of syndecan-1 in H929 cells occurs immediately
TRAIL in multiple myeloma cells. (A, B, and E) Knockdown of syndecan-
1 by siRNA. H929 cells and RPMI 8226 were mock transfected or
transfected with siSDC1. The expression of total syndecan-1 (SDC1)
protein in H929 (A) and RPMI 8226 (E) lysates was then determined by
immunoblotting. Cell surface-expressed syndecan-1 in H929 cells was
quantitated by staining with anti–syndecan-1 and subsequent FACS anal-
ysis (B). Solid line, mock; thin line, syndecan-1 knockdown (siSDC1);
shadowed curve, secondary Ab-only control (Ctl). (C, D, and F) Increased
TRAIL-induced apoptosis in syndecan-1–deficient myeloma cells. Mock
control and syndecan-1–downregulated H929 (C, D) or RPMI 8226 (F)
cells were treated with TRAIL at the doses indicated for 6 h, and annexin
V-positive cells (C, F) were determined. In separate experiments, hypotonic
PI solution was added 23 h after TRAIL stimulation, and the percentage of
cells with sub-G1DNA content was quantitated (D). (G and H) Syndecan-1
knockdown does not affect TNF-a–induced apoptosis in RPMI 8226
cells. RPMI 8226 cells were transduced with pLL3.7 or pLL3.7shSDC1,
and GFP+cells were isolated by sorting. Knockdown of syndecan-1 was
confirmed by immunoblots (G). Control (pLL) and shSDC1-expressing
RPMI 8226 cells were treated with TNF-a at the indicated doses, and cell
death was determined 48 h later (H). Each data point represents triplicate
determinations in a single experiment, and each experiment has been re-
peated at least three times (C, D, F, H). ***p , 0.001 for paired t test.
Syndecan-1 knockdown enhances apoptosis induced by
2916SYNDECAN KNOCKDOWN ENHANCES TRAIL-INDUCED CELL DEATH
cells. The effectiveness of syndecan-1 knockdown was demon-
strated by immunoblotting of cell lysates from RPMI 8226 cells
The multiple myeloma cell lines varied with regard to their
susceptibility to DR ligands. In agreement with previous obser-
vations that myeloma cells are modestly sensitive to TRAIL (27–
29), TRAIL triggered a moderate extent of apoptosis in control
H929 and RPMI 8226 cells (Fig. 2C, 2D, 2F). By contrast, these
myeloma cell lines, which express very low levels of Fas, were
refractory to FasL engagement (data not shown). Downregulation
of syndecan-1 significantly increased the extent of apoptosis in-
duced by TRAIL in H929 cells, as assessed by both annexin V
staining and measurement of sub-G1DNA content (Fig. 2C, 2D).
TRAIL-induced apoptosis was also enhanced in syndecan-1–
deficient RPMI 8226 cells relative to their control (Fig. 2F). In
separate experiments, syndecan-1 was also effectively downreg-
ulated by transduction of lentivirus-containing syndecan-1–spe-
cific shRNA in H929, RMPI 8226, and U266 cells (Fig. 2G, data
not shown). Treatment with TRAIL promoted the apoptosis in
these myeloma cell lines relative to the vector control (data not
shown). In control experiments, shSDC1-mediated downregula-
tion of syndecan-1 in H9 T lymphoma cells, which minimally
expresses this protein (Supplemental Fig. 1B), did not affect
TRAIL-induced cell death (data not shown).
TNF-a stimulation triggered a moderate cell death in RPMI
8226 cells (Fig. 2H), similar to a previous report (27), but did not
affect the viability of H929 and U266 cells (data not shown).
Knockdown of syndecan-1 did not affect TNF-a–induced apo-
ptosis in RPMI 8226 cells (Fig. 2H). Taken together, our results
indicate that decreased syndecan-1 expression leads to an increase
in TRAIL- but not TNF-a–triggered death in multiple myeloma
Syndecan-1 overexpression inhibits TRAIL-induced apoptosis
We also examined whether syndecan-1 overexpression in H929
cells also affected TRAIL-mediated cell death. H929 cells were
infected with recombinant lentivirus for syndecan-1 expression.
The elevated syndecna-1 expression was confirmed by flow
cytometry (Fig. 3A). Increased expression of syndecan-1 in H929
cells suppressed TRAIL-induced apoptosis (Fig. 3B), consistent
with a negative role of syndecan-1 in TRAIL-initiated cell death in
Normal expression of DR4/DR5 and apoptosis-associated
molecules in syndecan-1 knockdown myeloma cells
Enhanced TRAIL sensitivity in some cancer cells has been linked
to an increase in surface TRAIL receptors (22–26). The expression
of total TRAIL receptors, as determined by labeling with FLAG-
TRAIL, was not altered in syndecan-1 knockdown H929 cells
(Fig. 4A). Neither was any increase in the surface level of DR4 or
DR5 detectable in syndecan-1 knockdown H929 cells (Fig. 4B).
The total cellular levels of DR4 or DR5, as measured by immu-
noblotting, were comparable in control and syndecan-1–down-
regulated H929 cells (Fig. 4C). Downregulation of syndecan-1
also did not affect the levels of cell surface and total DR4 and DR5
in RPMI 8226 cells (Fig. 4D, 4E). The observed enhanced TRAIL
sensitivity in syndecan-1 knockdown myeloma cells is therefore
not associated with any change in the expression of DR4 and DR5.
As the major transmembrane proteoglycan, syndecan-1 trans-
duces signals (5–7) and modulates the expression of apoptosis-
associated molecules. c-FLIPLis an anti-apoptotic protein linked
to resistance of cancer cells to TRAIL (22–26). Increased Bim
expression and reduced Mcl-1 expression have been observed in
myeloma cells deficient in Blimp-1 (31), the molecule that reg-
ulates syndecan-1 expression. We determined whether syndecan-1
downregulation affected the levels of these apoptosis-related
proteins. The expression of FADD, caspase-8, c-FLIPL, Bcl-2,
Bim, Bax, and Mcl-1 was not altered in syndecan-1 knockdown
H929 cells (Fig. 4C) and syndecan-1–deficient RPMI 8226 cells
(Fig. 4E). Therefore, the increase in TRAIL-triggered apoptosis in
syndecan-1–deficient myeloma cells cannot be attributed to a
change in the expression of these proteins.
Effect of syndecan-1 knockdown in TRAIL-mediated apoptosis
is independent of heparan sulfate or integrin association
proteoglycan decreases the viability of myeloma in vivo (11, 32).
Thus, the increased sensitivity to TRAIL-mediated apoptosis
could be due to a diminished quantity of cell surface heparan
sulfate in syndecan-1 knockdown cells. To examine this possi-
bility, H929 cells were treated with heparinase III. The removal of
heparan sulfate was confirmed by the reduction of the apparent
molecular mass of syndecan-1 from 230 kDa to that of the 75-kDa
core protein (Supplemental Fig. 2A, 2C). In contrast to syndecan-
1 knockdown H929 cells, TRAIL-induced cell death was de-
creased in heparinase III-treated H929 cells (Supplemental Fig.
2B). Similarly, TRAIL-triggered apoptosis was reduced in
heparinase-treated RPMI 8226 cells (Supplemental Fig. 2D). Re-
moval of heparan sulfate proteoglycan therefore inhibits the ca-
pacity of TRAIL to induce apoptosis in myeloma cells. These
results suggest that enhanced apoptosis observed in syndecan-1–
deficient myeloma cells was not due to a reduction in cell surface
We also explored the possibility that the increased ability of
TRAIL to induce apoptosis is associated with a reduced binding of
syndecan-1 to integrin (13). To this end, we used the peptide in-
hibitor synstatin that has been previously shown to disrupt the
interaction between syndecan-1 and integrin. We found that syn-
statin did not have any effect on TRAIL-triggered apoptosis in
H929 and RPMI 8226 cells (Supplemental Fig. 2E, 2F). There-
fore, TRAIL-induced cell death in myeloma cells is independent
of integrin association, and increased TRAIL-mediated apoptosis
ptosis in H929 cells. (A) Syndecan-1 overexpression in H929 cells. H929
cells were infected with vector alone or pTRIP-SDC1. Cells were sorted
based on GFP expression, and cell surface syndecan-1 levels were deter-
mined by flow cytometry. Solid line, mock (pTRIP alone); thin line,
syndecan-1 transduced (SDC1); shadowed curve, secondary Ab-only con-
trol (Ctl). (B) Overexpression of syndecan-1 attenuated TRAIL-triggered
cell death. Control and syndecan-1–transduced H929 cells were stimulated
with TRAIL at the indicated doses for 6 h, and cell death was determined.
**p , 0.01 for paired t test, ***p , 0.001.
Syndecan-1 overexpression inhibits TRAIL-induced apo-
The Journal of Immunology 2917
cannot be attributed to a diminished interaction with integrin in
Normal TRAIL receptor endocytosis in syndecan-1–deficient
Inhibition of endocytosis of TRAIL receptors has been shown to
enhance TRAIL-induced cell death (33, 34). Because syndecan-1
is anchored to cortical actin, we examined whether TRAIL re-
ceptor internalization was altered by downregulation of syndecan-
1 in H929 cells. FLAG-TRAIL was used to measure the surface
levels of TRAIL receptors in H929 cells. Fig. 5A illustrates that
TRAIL stimulation led to the disappearance of TRAIL receptors
from the cell surface in H929 cells. Knockdown of syndecan-1 did
not affect both the degree and the speed of DR4/DR5 endocytosis
in H929 cells (Fig. 5A, 5B). Similar kinetics in the endocytosis of
TRAIL receptors was also found in RPMI 8226 cells with or
without syndecan-1 (Fig. 5C). Therefore, syndecan-1 is not in-
volved in DR4/DR5 internalization, and the increased apoptosis
in syndecan-1 knockdown myeloma cells is unrelated to TRAIL
Accelerated caspase-8 activation and enhanced DISC
formation in syndecan-1–deficient cells
We measured the biochemical processes following TRAIL stim-
ulation in H929 cells. In syndecan-1 knockdown H929 cells, the
cleavage of procaspase-8, the generation of the p43 caspase-8
intermediate, and the emergence of active p18 caspase-8 pro-
ceeded earlier than in wild-type H929 cells (Fig. 6A). This was
associated with an increased processing of procaspase-3 in syn-
decan-1–deficient H929 cells (Fig. 6A). We further quantitated
caspase-8 activity in these cells. Consistent with an accelerated
caspase-8 processing, caspase-8 activity was much higher in
syndecan-1 knockdown cells than the control H929 cells stimu-
lated by different doses of TRAIL (Fig. 6B).
Because activation of caspase-8 is among the earliest biochem-
ical events following DR ligation, we examined whether syndecan-
1 deficiencyaffected DISC formation. His-tagged TRAIL was used
to immunoprecipitate DISC from H929 cell lysates. Before TRAIL
stimulation, His-tagged TRAIL did not pull down FADD or
caspase-8 in H929 cells (Fig. 6C). TRAIL treatment led to the
assembly of a complex containing caspase-8 and FADD in H929
cells. Syndecan-1 knockdown significantly increased the amounts
of FADD and caspase-8 in the DISC (Fig. 6C). Therefore,
syndecan-1 knockdown increased TRAIL-induced recruitment of
FADD and caspase-8 in H929 cells, leading to accelerated acti-
vation of caspase-8 and caspase-3.
Increased N-acetyl galactosamine transferase 3 expression and
enhanced receptor clustering in syndecan-1 knockdown H929
Of the several processes that may affect TRAIL receptor DISC
formation, we found that the expression of peptidyl N-acetyl ga-
lactosamine transferase (GALNT) 3 was increased in syndecan-1
knockdown H929 cells (Fig. 7A). GALNT3 and GALNT14
mediate the O-glycosylation of TRAIL receptors and promote
of TRAIL receptors and apoptosis-associated molecules. (A) TRAIL recep-
tors were not affected by syndecan-1 downregulation. Vector control (pLL)
and syndecan-1 knockdown (shSDC1) H929 cells were stained by FLAG-
TRAIL and anti-FLAG Abs at 4˚C. (B and D) Normal surface DR4 and DR5
expression in syndecan-1 knockdown myeloma cells. The levels of cell sur-
face DR4 and DR5 on mock and syndecan-1 knockdown H929 (B) or RPMI
8226 cells (D) were determined by FACS. (C and E) Normal expression of
proapoptotic and anti-apoptotic proteins in syndecan-1 knockdown myeloma
cells. Total cell lysates from mock and syndecan-1 knockdown H929 (C) or
RPMI 8226 cells (E) were analyzed for the expression of syndcan-1, DR4,
DR5, FADD, c-FLIPL, procaspase-8, Bcl-2, Bim, Bax, and Mcl-1 proteins.
Downregulation of syndecan-1 does not affect the expression
TRAIL receptor. H929 (A, B) and RPMI 8226 (C) cells were treated with
FLAG-TRAIL (2 mg/ml) for 30 min at 4˚C, followed by incubation at 37˚C
in a water bath for the indicated time. Endocytosis was stopped by adding
ice-cold medium containing 0.5% sodium azide. Washed cells were stained
with anti-FLAG and allophycocyanin anti-mouse IgG on ice, and the
quantity of surface TRAIL receptors was determined by FACS (A). The
kinetics of the reduction of cell surface TRAIL receptors of H929 (B) and
RPMI 8226 cells (C) are shown in a bar graph after TRAIL stimulation.
The mean fluorescence intensity (MFI) of TRAIL receptors before TRAIL
addition was set as 100%. Each data point represents triplicate determi-
nations in a single experiment, and each experiment was repeated three
Syndecan-1 knockdown does not affect endocytosis of
2918SYNDECAN KNOCKDOWN ENHANCES TRAIL-INDUCED CELL DEATH
TRAIL-induced DR4/DR5 clustering in cancer cell lines, without
affecting the surface levels of TRAIL receptors (35). Levels of
GALNT3 and GALNT14 mRNA correlate with sensitivity to
TRAIL-triggered apoptosis in colorectal cancer and pancreatic
cancer cell lines, respectively (35). Consistent with the specific
expression of distinct GALNT isoforms in different cancer cells,
GALNT14 mRNA was not altered in syndecan-1–deficient H929
cells (Fig. 7A). To evaluate the consequence in TRAIL receptor
O-glycosylation, TRAIL-induced clustering of TRAIL receptors
was determined in syndecan-1–deficient cells. Immunoprecipita-
tion with caspase-8 revealed enhanced translocation of high mo-
lecular mass DR4 into DISC after TRAIL treatment in syndecan-
1–downregulated cells (Fig. 7B), in accordance with increase in
GALNT3-mediated O-glycosylation of TRAIL receptors.
Increased FasL-induced apoptosis in syndecan-4 knockdown
H9 cells and syndecan-2–deficient Jurkat cells
We found that for human T lymphoma H9 cells, syndecan-4 was
the major form of syndecans on the cell surface (Supplemental
Fig. 1B). Syndecan-4 was downregulated by specific shRNA, and
the effectiveness of knockdown was confirmed by FACS (Sup-
plemental Fig. 3A). Knockdown of syndecan-4 prominently in-
creased FasL-triggered apoptosis (Supplemental Fig. 3B). By con-
trast, TRAIL-induced cell death in H929 and U266 cells was not
affected by downregulation of syndecan-4 (Supplemental Fig. 4A,
4B). Knockdown of syndecan-4 did not alter the expression of Fas
and c-FLIP in H9 cells (Supplemental Fig. 4C, 4D). The enhanced
FasL-induced cell death was associated with an accelerated
capase-8 cleavage and elevated level of active p18 caspase-8
(Supplemental Fig. 3C). This was accompanied by a prominent
increase in the emergence of active caspase-3. Syndecan-2 is the
dominant form of syndecan in the acute T leukemia cell line
Jurkat. Syndecan-2 was also knocked down by specific shRNA,
as shown by diminished syndecan-2 cell surface levels (Supple-
mental Fig. 3D). FasL-triggered apoptosis was significantly in-
creased in syndecan-2 knockdown Jurkat cells (Supplemental Fig.
3E). Because Fas does not contain the O-glycosylation site on
TRAIL receptors (35), the increased Fas-initiated cell death in
syndecan-4 knockdown H9 and syndecan-2–deficient Jurkat cells
is apparently independent of GALNT3 or GALNT14. We exam-
ined molecules that specifically modulate FasL-induced apoptosis.
Fas-associated phosphatase-1 (FAP-1) is a negative regulator of
FasL-triggered cell death, and high levels of FAP-1 are correlated
with resistance to Fas-mediated apoptosis in selective types of
cancer cell (36, 37). The FAP-1 protein levels, however, were
comparable between control and syndecan-4 knockdown H9 cells
before and after FasL stimulation (Supplemental Fig. 4E), sug-
gesting that enhanced sensitivity to FasL-induced apoptosis in
syndecan-4–deficient H9 cells is not due to change in FAP-1
levels. These results demonstrate that syndecan also negatively
regulates the cell death initiated by Fas, with mechanisms dif-
ferent from how syndecan-1 modulates TRAIL sensitivity.
In this study, we examined the roles of syndecan in DR-mediated
cell death. Because syndecan-1, syndecan-4, and syndecan-2 are
predominantly expressed in multiple myeloma, H9, and Jurkat
cells, respectively, we could delineate the contribution of syndecan
to cell death by downregulating a single syndecan in each cell line.
We found that syndecan-1 knockdown in multiple myeloma cells
greatly enhanced the sensitivity of cells to TRAIL-induced apo-
ptosis (Fig. 2). We also observed that knockdown of syndecan-4 in
H9 cells and downregulation of syndecan-2 in Jurkat cells led to
an increased sensitivity to FasL (Supplemental Fig. 3). Therefore,
irrespective of the type of syndecan, removal of syndecan en-
hances TRAIL- and FasL-stimulated apoptosis. Our results illus-
trate that syndecan plays a negative role in the apoptosis initiated
by TRAIL and FasL. We further found that increased TRAIL-
mediated apoptosis in syndecan-1 knockdown cells was not due
to changes in the expression of TRAIL receptors, c-FLIP, Mcl-1,
Bcl-2, Bim, or Bax (Fig. 4). Neither did a decrease in syndecan-1–
associated heparan sulfate proteoglycan or integrin binding play
a role in the observed increase in TRAIL-induced apoptosis
(Supplemental Fig. 2). Furthermore, syndecan-1 knockdown did
not affect the endocytosis of TRAIL receptors (Fig. 5). Instead, the
enhanced TRAIL- and FasL-induced apoptosis in syndecan-1–
deficient myeloma and lymphoma cells was associated with an
increase in DISC formation and accelerated caspase-8 activation
(Fig. 6, Supplemental Fig. 3). In addition, for TRAIL-induced ap-
optosis, the increased cell death in syndecan-1–deficient cells is
partly attributed to an increase in O-glycosylation of TRAIL re-
ceptors, as a consequence of elevated GALNT3 expression.
syndecan-1 knockdown H929 cells. (A) Enhanced cleavage of procaspase-
8 and procaspase-3 in syndecan-1–deficient cells. Control (mock) and
syndecan-1 knockdown (siSDC1) H929 cells were stimulated with TRAIL
(200 ng/ml), and cell lysates were prepared at the time points indicated.
The quantities of procaspase-8 (p55/53), active caspase-8 (p18), caspase-3,
and b-tubulin in cell lysates were determined by immunoblotting. (B)
Enhanced caspase-8 activity in syndecan-1 knockdown cells. Control and
syndecan-1 knockdown H929 cells were treated with TRAIL for 90 min at
the indicated doses. The activity of caspase-8 was determined by the
cleavage of Z-LETD aminoluciferin. ***p , 0.001 for paired t test. (C)
Increased DISC formation in syndecan-1–downregulated H929 cells. Cell
lysates of control and syndecan-1 knockdown H929 cells (2 3 107) were
prepared before (0 min) and 30 or 60 min after His-TRAIL (2 mg) treat-
ment. DISC in the lysates was pulled down by Ni-NTA agarose. DISC
components were then released from the beads by incubation with 200 mM
imidazole and analyzed for the expression of caspase-8, FADD, and His-
Accelerated caspase-8 cleavage and DISC formation in
The Journal of Immunology2919
We observed that cell death triggered by TNF-a, another DR
ligand, was not affected by syndecan-1 knockdown in H929 cells
(Fig. 2H). TRAIL and FasL are different from TNF-a in the
process of DISC formation: whereas FADD is recruited by TRAIL
and Fas to form DISC at the cell surface, FADD becomes asso-
ciated with TRADD for the assembly of DISC in the cytosol after
TRADD is dissociated from the membrane-bound TNFRI com-
plex (17–19). The insensitivity of TNF-a–induced apoptosis to
syndecan-1 knockdown suggests the possibility that syndecan
affects the membrane-situated DR complex, but not with the cy-
toplasmic FADD-containing complex.
Syndecan-1 is a target gene of Blimp-1. Blimp-1 knockdown has
been shown to increase spontaneous cell death in H929 and U266
cells (31). The fact that we observed elevated apoptosis in syn-
decan-1–deficient myeloma cells upon long-term culture (.2 mo)
is consistent with an impaired growth of syndecan-1 knockdown
myeloma in vivo (11). However, we did not find any difference in
cell viability within the few weeks during which these experi-
ments were performed with each cell line. Thus, the increased
DR-induced apoptosis in syndecan-1 knockdown cells observed in
the current study was not due to elevated spontaneous cell death.
The difference between Blimp-1 knockdown and syndecan-1 knock-
down myeloma cells suggests that, in addition to syndecan-1,
other Blimp-1 downstream genes also contribute to the survival
of plasma cells and multiple myelomas. This is illustrated by
the fact that Blimp-1 deficiency led to increased Bim and reduced
Mcl-1 levels (31), whereas the levels of these two proteins were not
altered by knockdown of syndecan-1 in myeloma cells (Fig. 4).
Syndecan is linked to the actin cytoskeleton through the C1
region of the intracellular domain (21). Fas is also associated to the
actin filament through binding to ezrin/radxin/moesin (20, 38). We
have recently demonstrated that knockdown of ezrin enhances the
formation of DISC (25), suggesting that the linkage of Fas to
ezrin-actin imposes a restriction that hampers the association of
FADD and procaspase-8 with DRs to form DISC. The presence
of syndecan at the cell membrane, through the association with
cortical actin, may also exert constraints inhibiting the effective
recruitment of FADD and procaspase-8. The removal of major
syndecan most likely relieves these constraints and facilitates ef-
ficient assembly of DISC. In the current study, we also observed
that TRAIL binding was accompanied with an immediate cleav-
age of the intracellular region of syndecan-1 (Fig. 1A, 1C, 1D),
indicating that removal of the intracellular portion of syndecan-1
could be part of the processes for full assembly of DISC.
We have identified an additional mechanism that is TRAIL
specific on the enhanced DISC formation in syndecan-1 knock-
down cells. The expression of GALNT3, the enzyme that mediates
the O-glycosylation of TRAIL receptor, was elevated in syndecan-
1–downregulated myeloma cells (Fig. 7A). The sensitivity of
cancer cells to TRAIL-triggered cell death has been found asso-
ciated with the expression of GALNT isoforms (35). Levels of
GALNT14 are correlated with sensitivity to TRAIL in pancreatic
carcinoma, non–small-cell lung carcinoma, and malignant mela-
noma cell lines, whereas the expression of GALNT3 correlates
with the susceptibility to TRAIL-induced apoptosis in colorectal
cancer lines (35). Downregulation of O-glycosylation enzymes by
siRNA confers resistance to TRAIL-triggered apoptosis in can-
cer cell lines. Overexpression of GALNT14 in TRAIL-resistant
pancreatic cancer cell line leads to increased TRAIL-triggered
cell death, accompanied with DISC formation and activation of
caspase-8 and caspase-3 (35). As a consequence of increased O-
glycosylation of TRAIL receptors, we observed increased DR4
clustering in syndecan-1 knockdown cells (Fig. 7B), similar to the
enhanced translocation of DR4/DR4 oligomers into DISC com-
plex previously reported for GALNT14 (35). It may be noted that
we do not know how syndecan-1 downregulation promotes the
expression of GALNT3. Syndecan-1 has been shown to transmit
or modulate various signal pathways (5–7), and whether any of the
signaling cascades is linked to GALNT3 expression is being de-
TRAIL triggers apoptosis in multiple myeloma cell lines (27–
29). TRAIL kills myeloma cells regardless of their resistance to
chemotherapeutic drugs, and TRAIL is a potential therapeutic
reagent for incurable multiple myeloma. However, many myeloma
cells are only modestly sensitive to TRAIL, as also shown in the
current study. Syndecan-1 is overexpressed in multiple myeloma
cells, and contributes to the growth and metastasis of myeloma
tumor (8, 11, 12). Syndecan-1 has thus become a target for treat-
ing myeloma, with approaches including specific knockdown of
syndecan-1 (11) or anti–syndecan-1 immunoconjugate (39). Our
results illustrate that, by downregulation of syndecan-1, myeloma
cells become highly susceptible to TRAIL killing. Because de-
TRAIL-induced DR4 clustering in syndecan-1
knockdown H929 cells. (A) Enhanced expression of
GALNT3 mRNA in syndecan-1–deficient H929
cells. The expression of GALNT3, GALNT14, and
SDC1 in control (mock) and syndecan-1 knock-
down H929 cells was determined by quantitative
PCR. (B) Increased TRAIL-induced DR4 clustering
in syndecan-1 knockdown H929 cells. Control and
syndecan-1–deficient H929 cells were treated with
TRAIL, and cell lysates were isolated at the indi-
cated time points. Cell lysates were incubated over-
night at 4˚C with anti–caspase-8–loaded protein
G agarose, and precipitates were resolved by gra-
dient SDS-PAGE. Arrows indicate DR4 and oligo-
mers of DR4.
Increased GALNT3 expression and
2920SYNDECAN KNOCKDOWN ENHANCES TRAIL-INDUCED CELL DEATH
livery of syndecan-1–specific siRNA has been shown to knock Download full-text
down syndecan in vivo (11), the combinatory therapy using
syndecan-1 siRNA and TRAIL is now possible. Whether such
combinatory treatment will be effective against multiple myeloma
deserves further investigation.
Among different DR agonists, TRAIL and TRAIL receptor
agonists have displayed promising therapeutic effect toward solid
tumors and lymphomas in series of Phase I and Phase II clinical
trials (22–26). Notably, a few tumor cells are resistant to treatment
with TRAIL alone. The resistance is often circumvented by
combination of TRAIL with reagents such as cytotoxic drugs,
proteasome inhibitors, or histone deacetylase inhibitors. Most of
these reagents promote TRAIL-induced apoptosis by downreg-
ulating anti-apoptotic molecules (e.g., c-FLIP, Bcl-2), by upregu-
lating proapoptotic proteins (e.g., Bax, Bak), or by increasing the
expression of TRAIL-R1 and TRAIL-R2 (22–26). In the current
study, we demonstrate that knockdown of syndecan-1 provides
a distinct mechanism to sensitize tumor cell to TRAIL killing.
Downregulation of syndecan-1 did not affect the expression of
the aforementioned apoptosis-associated molecules and the surface
levels of TRAIL receptors (Fig. 4). Instead, syndecan-1 knock-
down promotes TRAIL receptor clustering and increased DISC
formation. This suggests that syndecan downregulation could be
used not only in promoting TRAIL-mediated apoptosis, but also in
enhancing the efficacy of the current TRAIL combinatory therapy.
For cancer cells that predominantly display a specific type of syn-
decan, the feasibility of such an approach is being explored.
We thank Drs. I-Chen Ho, Yi-Ping Hsueh, and Garry Nolan for providing
reagents; Yamin Lin and the FACS Core of the Institute of Molecular Bi-
The authors have no financial conflicts of interest.
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