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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2679-2688
Syndecan-1 Is a Multifunctional Regulator of Myeloma Pathobiology:
Control of Tumor Cell Survival, Growth, and Bone Cell Differentiation
By
Madhav V. Dhodapkar,
Etsuko Abe,
Allison Theus,
Marie Lacy,
J.
Kevin Langford,
Bart Barlogie, and
Ralph D. Sanderson
From the Divisions of Hematology-Oncology and Endocrinology and the
Departments of Pathology and Anatomy, University of Arkansas for
Medical Sciences and VA Medical Center, Little Rock, AR.
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ABSTRACT |
Multiple myeloma is characterized by an accumulation of malignant
plasma cells in the bone marrow coupled with an altered balance of
osteoclasts and osteoblasts, leading to lytic bone disease. Although
some of the cytokines driving this process have been characterized,
little is known about the negative regulators. We show that syndecan-1
(CD 138), a heparan sulfate proteoglycan, expressed on and actively
shed from the surface of most myeloma cells, induces apoptosis and
inhibits the growth of myeloma tumor cells and also mediates decreased
osteoclast and increased osteoblast differentiation. The addition of
intact purified syndecan-1 ectodomain (1 to 6 nmol/L) to myeloma cell
lines in culture leads to induction of apoptosis and dose-dependent
growth inhibition, with concurrent downregulation of cyclin D1. The
addition of purified syndecan-1 in picomolar concentrations to bone
marrow cells in culture leads to a dose-dependent decrease in
osteoclastogenesis and a smaller increase in osteoblastogenesis. In
contrast to the effect on myeloma cells, the effect of syndecan-1 on
osteoclastogenesis only requires the syndecan-1 heparan sulfate chains
and not the intact ectodomain, suggesting that syndecan's effect on
myeloma and bone cells occurs through different mechanisms. When
injected in severe combined immune deficient (scid) mice,
control-transfected myeloma cells (ARH-77 cells) expressing little
syndecan-1 readily form tumors, leading to hind limb paralysis and
lytic bone disease. However, after the injection of
syndecan-1-transfected ARH-77 cells, the development of
disease-related morbidity and lytic bone disease is significantly
inhibited. Taken together, our data demonstrate, both in vitro and in
vivo, that syndecan-1 has a significant beneficial effect on the
behavior of both myeloma and bone cells and therefore may represent one
of the central molecules in the regulation of myeloma pathobiology.
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INTRODUCTION |
GROWTH OF TUMOR and normal cells in the
tumor microenvironment is regulated by a balance between positive and
negative regulators. Multiple myeloma (MM) is a B-cell malignancy
characterized by the accumulation of malignant plasma cells in the bone
marrow.1 A number of cytokines, including interleukin-6
(IL-6), the related gp 130 family, CD40 ligand, and insulin-like growth
factor, have been shown to promote myeloma cell growth in
vitro.2 However, little is known about the naturally
occurring negative regulators of myeloma cell growth and survival in
vivo. IL-4 and interferon  inhibit the growth of myeloma cells in
vitro, but these effects are indirect and are overcome by the addition
of exogenous IL-6.3,4
Lytic bone destruction and osteoporosis is a characteristic feature of
myeloma. Histo-morphometric studies suggest that abnormal bone
remodeling is one of the earliest features of myeloma, and the
development of lytic bone disease is due to an imbalance of bone cells
with increased osteoclasts and decreased osteoblasts.5,6 The interaction between tumor cells and the bone marrow
microenvironment, including bone cells, although thought to be critical
for myeloma pathogenesis, is poorly understood.7 Since the
original description of osteoclast-activating factor (OAF) by Mundy et
al,8 a number of cytokines, including IL-6, IL-1 , and
tumor necrosis factor  (TNF), have been postulated to mediate the
increased osteoclastogenic activity in myeloma.5 Recently,
it was shown that myeloma cells upregulate IL-6 but downregulate
osteocalcin production by an osteoblast cell line via cell-cell
contact.9 However, it is not known if counter-regulatory
bone-preserving factors exist in myeloma.
Syndecan-1 is a member of a family of cell surface transmembrane
heparan sulfate (HS) proteoglycans.10 During murine B-cell development, syndecan-1 is expressed at the pre-B-cell stage, is lost
in mature B cells, and is re-expressed strongly in the mature plasma
cells.11 The expression of syndecan-1 by most primary
myeloma cells and myeloma cell lines12 has been used for
purification of myeloma cells from clinical samples.13,14 Previous studies in our laboratory have shown that syndecan-1 mediates
specific adhesion of myeloma cells to type I collagen, inhibits their
invasion into type I collagen gels, and mediates cell-cell adhesion
between myeloma cells.12,15-17 We now show that this
proteoglycan is shed from the surface of myeloma cells in culture and
that this shed syndecan-1 inhibits myeloma cell growth in vitro. In
addition, syndecan-1 also affects bone cell differentiation; it
increases osteoblast development and inhibits osteoclast formation in
murine bone marrow cell cultures. These in vitro results are supported
by our in vivo studies using scid mice injected with myeloma
cells transfected with either vector only or syndecan-1. In these
studies, the expression of syndecan-1 on myeloma cells is associated
with an inhibition and delay in the development of myeloma-induced
morbidity and decreased lytic bone disease. These data suggest that
syndecan-1 forms part of a potentially beneficial regulatory loop that
inhibits myeloma cell growth and bone loss in vivo.
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MATERIALS AND METHODS |
Cell Lines and Cell Culture
Myeloma cell lines (RPMI 8226 and ARH-77) were obtained from American
Tissue Type Culture (ATCC; Rockville, MD) or derived at our institution
from patients with myeloma (arp, ark, and mer; kindly provided by Dr J. Epstein, University of Arkansas for Medical Sciences).
Cells were maintained in RPMI 1640 supplemented with 10% fetal calf
serum and 2 mmol L-glutamine. None of the cell lines used require the
addition of IL-6 for growth. For scid mice experiments,
syndecan-1-negative ARH-77 cells transfected with either a control
vector carrying the neomycin resistance gene (ARH-77neo) or
a full length syndecan-1 construct (ARH-77syn-1), as
previously described,16 were used.
Detection of Syndecan-1 Shed Into the Conditioned Media
Immuno-dot blotting.
Shed syndecan-1 was partially purified using diethylaminoethyl
(DEAE) chromatography followed by detection using
immuno-dot blotting as previously described.12 Briefly,
media conditioned by MM cells was brought to 2 mol/L urea and 50 mmol/L
sodium acetate (pH 4.5). The medium was clarified by centrifugation at
15,000g for 10 minutes at 10°C. DEAE sepharose beads were
added to the media and the mixture placed on a rocker for 1 hour at
room temperature. DEAE beads were pelleted by gentle centrifugation
(1,200 rpm for 7 minutes), placed in a clean 0.5-mL microcentrifuge
tube, washed 4 times with phosphate-buffered saline (PBS) by
centrifugation, and eluted with PBS containing 1 mol/L NaCl. The DEAE
eluates were adjusted to a final NaCl concentration of 0.15 mol/L by
dilution with 10 mmol/L Tris, pH 7.4. Samples were loaded on to
Genetrans (Plasco Inc, Woburn, MA), a cationic nylon membrane using a
immuno-dot blot apparatus (Miliblot D; Milipore, Bedford, MA).
Membranes were removed from the apparatus and the remaining binding
sites were blocked for 1 hour with a solution containing 3% nonfat dry milk, 0.5% bovine serum albumin, 10 mmol/L Tris, pH 8.0, 0.15 mol/L
NaCl and 0.3% Tween-20. Blots were probed overnight at 4°C with a
1:100 dilution of an anti-syndecan-1 antibody B-B4
(Serotec Inc, Oxford, UK). After washing in PBS, blots
were incubated for 30 minutes at room temperature with biotinylated
goat antimouse secondary antibody, followed by incubation with
Vectastain Elite ABC reagent in accordance with manufacturer's
protocol (Vector Laboratories, Burlingame, CA), and visualized by
diaminobenzidine as a substrate.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blotting.
Samples eluted from DEAE beads were desalted by passage over G-5
excellulose columns equilibrated with 0.1% SDS and run on 4% to 12%
SDS-PAGE gels. For heparitinase digestion, before desalting, some
samples were diluted to a concentration of 0.15 mol/L NaCl and
incubated with 33 µIU/mL of Flavobacterium heparinum heparitin sulfate lyase (heparitinase; Seikagaku, Rockville, MD) for
30 minutes at 42°C, followed by another incubation for 30 minutes with 33 µIU/mL of heparitinase. After transfer of gels to a cationic nylon filter, syndecan-1 was detected, as described above.
Isolation of Syndecan-1 Ectodomain
Syndecan-1 ectodomain was purified from the conditioned media of
ARH-77syn-1 cells. Sepharose beads conjugated to monoclonal
antibody 281.2, specific for the murine syndecan-1 core
protein,18 were blocked by preincubation with complete
media and heparin (10 µg/mL) for 1 hour at room temperature and
washed twice with PBS containing 0.1% Triton X-100. Material eluted
from DEAE as described above was added to 281.2-conjugated beads and
incubated for 4 hours on a rocker at room temperature or 4°C
overnight. Beads were washed twice with PBS with 0.25 mol/L NaCl and
0.1% Triton X-100 and transferred to a clean 0.5-mL microcentrifuge
tube. Bound syndecan-1 was eluted using 50 mmol/L triethylamine (pH
11.5) and 0.1% Triton X-100 and the pH was immediately neutralized by
addition of 1.0 mol/L Tris, pH 7.4. Purified syndecan-1 or, as a
control, complete media (RPMI with 10% fetal calf serum) not
conditioned by cells was passed over G-5 Excellulose columns (Pierce,
Rockford, IL) equilibrated with complete medium.
Syndecan-1 was quantitated by immunoblot analysis using
I125-labeled antibody 281.2 and compared with purified
syndecan-1 of known quantity (kindly provided by Dr M. Bernfield,
Harvard Medical School, Boston, MA). To determine if the
intact syndecan-1 ectodomain was required for the biologic effect, in
some experiments, purified and quantitated syndecan-1 was pretreated
with 100 µIU/mL Flavobacterium heparinum heparitin sulfate lyase
(heparitinase; Seikagaku) for 30 minutes at 42°C, followed by
another 100 µIU/mL of heparitinase for additional 30 minutes.
Glycosaminnoglycans (GAGs) from syndecan-1 ectodomain were generated
using alkaline sodium borohydride, as previously
described.15 In these experiments, intact ectodomain was
used as a control.
Growth Inhibition Experiments
Myeloma cells (2 × 105 cells/mL) were incubated for
72 hours in complete medium containing various concentrations of
syndecan-1 or control media, obtained as described above. Cell growth
was determined by 3H-thymidine labeling and viable cell
counting. Cell growth was expressed as a percentage of that seen in
control media containing no exogenous syndecan-1.
Apoptosis, Cell Cycle Progression, and Cyclin D1 Expression
Induction of apoptosis by syndecan-1 ectodomain was determined with the
TdT-mediated dUTP nick end-labeling (TUNEL) method, using a flow
cytometry-based in situ cell death detection kit (Boehringer Mannheim,
Indianapolis, IN) following the manufacturer's protocol.
To determine the effect of syndecan-1 on cell cycle progression, DNA
content of cells growing in log phase was analyzed using flow cytometry
after propidium iodide (PI) staining. Cells (106) were
incubated with media containing syndecan-1 or control
media for 48 hours before fixation and DNA content analysis. Expression of cyclin D1 was determined by flow cytometry. Cells were fixed with
alcohol and stained with fluorescein isothiocyanate (FITC)-conjugated anticyclin D1 antibody (PharMingen, San Diego, CA), using
the manufacturer's protocol. Cyclin D1 levels were expressed as the ratio of median fluorescent intensities of cells stained with cyclin D1
versus the isotype-matched control provided in the kit.
Osteoclast Formation in the Coculture System
The osteoblastic cell line (2107 cells), established from neonatal
murine calvaria, was used in coculture assays for the evaluation of
osteoclastogenesis as previously described.19 Briefly,
murine marrow cells (2 × 105 cells per 500 µL) were
cocultured with 40,000 calvaria cells for 8 days in the presence of 10 nmol/L 1,25(OH)2D3 with or without 1% (% volume) conditioned media from vector-transfected or
syndecan-1-transfected myeloma cells. The number of osteoclasts was
determined by counting tartrate-resistant acid phosphatase
(TRAP)-positive cells containing greater than 3 nuclei per cell.
Bone Marrow Cell Culture for Osteoclast and Osteoblast
Differentiation
Bone marrow cell culture assays for the evaluation of
osteoclastogenesis and osteoblastogenesis were performed as previously described.19,20 Briefly, bone marrow cells (106
cells) from 8-week-old Swiss Webster male mice were cultured in 0.5 mL
of -Minimum Essential Medium (-MEM) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma, St Louis,
MO) in 24 multiwell dishes. After 8 days in culture,
osteoclast- and osteoblast-like cell formation was examined, as
previously described.19 1,25(OH)2D3
and/or purified syndecan-1 were added for the last 4 days of
the total 8-day culture period. For osteoclast-like cell formation, the
bone marrow cells (106) were cultured with
10 8 mol/L 1, 25(OH)2D3 alone or together
with 0.65 pmol/L to 2 nmol/L syndecan-1 and stained for TRAP. For
osteoblast-like cell formation, cells were cultured with 0.65 pmol/L to
2 nmol/L syndecan-1 and stained for alkaline phosphatase (ALP). Cells
staining positive for TRAP (only those with >3 nuclei, representing
osteoclasts) or colonies containing ALP-positive cells (representing
osteoblasts) were counted under a light microscope using 200×
magnification.
SCID Mice Experiments
Female CB.17/Icr-severe combined immune deficient (scid) mice, 6 to 8 weeks old, were obtained from Harlan Bioproducts for Science
(Indianapolis, IN). They were housed and monitored as required in our animal facility, and the experiments were performed according to the protocols approved by the Institutional Animal Care
and Use Committee. Irradiated mice (150 cGy) were injected with 3 × 106 ARH-77syn-1 or
ARH-77neo cells via the lateral tail vein. Successful
transplantation was determined and monitored by measuring the human  chain content of sera beginning weekly after day 7. The animals were
euthanised when hind limb paralysis occurred, when large
visible/palpable tumors were observed, or when animals appeared morbid,
as determined by extreme emaciation and lethargy. Development of lytic
bone lesions was determined by evaluation of whole body skeletal
radiographs in a blinded fashion after the mice were sacrificed.
Statistical Analysis
The Student's t-test was used for comparison of various
experimental groups, and significance was set at P < .05.
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RESULTS |
Syndecan-1 Is Expressed and Shed by Myeloma Cell Lines
Expression of cell surface syndecan-1 on myeloma cell lines was
examined by flow cytometry using anti-syndecan-1 antibody B-B4. As
shown in Fig 1, arp, ark, and RPMI 8226 cells strongly express cell surface syndecan-1. Syndecan-1 expression
in mer cells is significantly lower, whereas ARH-77 cells have only
very faint expression of syndecan-1 (Fig 1). The sensitivity of flow cytometric detection is thus higher than the previously reported immuno-dot blot analysis of cell lysates in which syndecan-1 could not
be detected in ARH-77 and mer cells and was weakly expressed in RPMI
8226 cells.12 To determine if syndecan-1 is shed from the
tumor cell surface, media conditioned by these cells were analyzed by
an immuno-dot blot assay. Shed syndecan-1 is detected in the
conditioned media of all three cell lines that highly express cell
surface syndecan-1 (arp, ark, and RPMI 8226), but not in conditioned
media from ARH-77 or mer cells (Fig 2A).
Syndecan-1 ectodomain shed by myeloma cells migrates on Western blots
as a broad smear that is reduced to a narrow smear just above 60 kD
after treatment with heparitinase (Fig 2B), indicating that the
syndecan-1 ectodomain is shed as an intact molecule bearing predominantly heparan sulfate glycosaminoglycan (GAG) chains. Intact
syndecan-1 ectodomain is also shed by ARH-77 cells engineered to
express syndcecan-1 (ARH-77syn-1; Liu et al, unpublished
data). The level of shed syndecan-1 ectodomain in the
myeloma cell conditioned media is dependent on a number of variables,
including cell density, length of culture, and cell surface expression,
but can reach 1 to 2 nmol/L concentration.
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| Fig 1.
Expression of syndecan-1 by myeloma cell lines. Myeloma
cell lines (ark, arp, RPMI-8226, mer, and ARH-77) were stained with FITC-labeled anti-syndecan-1 antibody (B-B4) (thick line) or isotype control (thin line), and fluorescence intensity was analyzed using flow
cytometry.
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| Fig 2.
Syndecan-1 ectodomain is shed from the surface of myeloma
cell lines. (A) Presence of shed syndecan-1 ectodomain in the media conditioned by myeloma cell lines. Syndecan-1 was detected using an
immuno-dot blot analysis with anti-syndecan-1 antibody (B-B4). (B)
Western blot of partially purified syndecan-1 from media conditioned by
ark cells probed with B-B4, showing the presence of intact syndecan-1
ectodomain and ectodomain after digestion with heparitinase.
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Shed Syndecan-1 Inhibits the Growth of Myeloma Cell Lines In
Vitro
ARH-77 and mer cells that express and shed little or no syndecan-1 were
incubated with various concentrations of purified syndecan-1 for 72 hours. Cell growth was determined using 3H-thymidine
proliferation assays, and similar data were obtained using cell
counting (not shown). The addition of syndecan-1 at nanomolar
concentrations inhibits the growth of both cell lines in a
dose-dependent fashion (Fig 3A and B). This
growth-inhibitory effect is not limited to syndecan-1-negative cells,
because a similar growth-inhibition is seen in ark and arp cells that
express and shed syndecan-1 (Fig 3C and D). Thus, shed syndecan-1 is
able to inhibit the growth of all myeloma cell lines tested, regardless of whether they express cell surface syndecan-1.
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| Fig 3.
Dose-dependent inhibition of myeloma cell growth by
syndecan-1 ectodomain. (A) ARH-77 cells; (B) mer cells; (C) ark cells; (D) arp cells. Effect of addition of purified syndecan-1 ectodomain on
the growth of myeloma cell lines (ARH-77, mer, arp, and ark) in
culture. Cells (3 × 104) were incubated with various
concentrations of syndecan-1 or in media alone for 72 hours, and cell
growth was analyzed by 3H-thymidine proliferation assays.
Cell growth is expressed as a percentage of control (media only).
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Pretreatment of purified syndecan-1 with heparitinase (to remove the HS
chains), largely abolishes the growth-inhibitory activity, suggesting
that the presence of HS chains is essential for this effect
(Fig 4). This finding also suggests that
the growth inhibition is due to the syndecan-1 ectodomain and not due
to an impurity copurifying in association with GAG chains. Growth of
syndecan-1-negative ARH-77 cells was not altered in media pretreated
with heparitinase, indicating that heparitinase was not mediating its
effect through modification of other HS containing proteins in the
system (not shown). The addition of either heparin alone (up to 10 µg/mL) or GAG chains isolated from purified syndecan-1 fails to
significantly inhibit myeloma cell growth. Thus, the inhibition of
myeloma cell growth by syndecan-1 requires an intact ectodomain.
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| Fig 4.
Syndecan-1-mediated growth inhibition requires an intact
proteoglycan. ARH-77 cells were incubated with intact purified
syndecan-1 ectodomain (4 nmol/L), syndecan-1 (4 nmol/L) pretreated with
heparitinase (Heparitinase), glycosaminoglycan (GAGs) chains purified
from syndecan-1, or heparin (1 and 10 µg/mL) or in media alone (as control) for 72 hours. Cell growth is expressed as a percentage of
control.
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To evaluate the possibility that the growth inhibition is due to
depletion of growth factors that bind to syndecan-1 HS chains, myeloma
cells were grown in culture media preincubated with syndecan-1 immobilized on sepharose beads via the core protein, leaving the HS
chains free to interact with potential ligands.21 Normal growth of cells in this pretreated media suggests that syndecan-1 does
not exert its effects by depleting the media of growth factors (Fig 5A). Likewise, upon physical
separation of myeloma cells in transwell inserts from immobilized
syndecan-1, the cell growth is not inhibited (Fig 5B). Taken together,
these data suggest that the inhibitory effect requires close proximity
of syndecan-1 to the cell surface. Addition of IL-6 (10 ng/mL) at a
concentration known to inhibit dexamethasone-mediated growth-inhibition
and apoptosis22 fails to reverse the growth-inhibitory
effect of syndecan-1 (data not shown).
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| Fig 5.
Inhibition of growth requires close proximity of
syndecan-1 to the cell surface. (A) Effect of media preincubated with
immobilized syndecan-1 on myeloma cell growth. ARH-77 cells were grown
in media preincubated with syndecan-1 immobilized on sepharose beads or
beads only (as control). Cell growth is expressed as a percentage of
control. (B) Transwell assay. ARH-77 cells were grown in transwell inserts, physically separated from syndecan-1 (4 nmol/L) immobilized on
sepharose beads, or beads only (as control) in the lower wells. Cells
grown in the absence of a transwell but with exogenous, soluble
syndecan-1 (4 nmol/L) serve as a positive control (Syn-1 no transwell).
Cell growth is expressed as a percentage of control.
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Syndecan-1 Mediates Apoptosis and Affects G1-S Progression in Myeloma
Cells
The addition of purified syndecan-1 ectodomain at concentrations  2
nmol/L leads to an induction of apoptosis of myeloma cells as
determined by TdT mediated dUTP end labeling (TUNEL) assay (Fig 6A through D). This induction of
apoptosis is seen both with syndecan-1-nonexpressing (ARH-77) and
syndecan-1-expressing (arp) cells. The degree of apoptosis was
somewhat higher in arp cells (which are also more sensitive to
dexamethasone22 and melphalan) than in ARH-77 cells.
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| Fig 6.
Induction of apoptosis of myeloma cells by syndecan-1.
Myeloma cell lines ([A] and [B] ARH-77 cells; [C] and [D], arp
cells) were incubated with media alone (A and C) or purified syndecan-1 (4 nmol/L) for 48 hours (B and D). Apoptosis was examined using flow
cytometry-based TUNEL (TdT-mediated dUTP nick end labeling) assay
(Boehringer Mannheim) using the manufacturer's protocol. For negative
control (thin line), cells were stained with label solution in the
absence of TdT. For test sample (thick line), cells were stained with
TUNEL reaction mixture according to manufacturer's protocol. The
apoptotic population is marked by an arrow. Figures represent one of
three representative experiments.
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Cell cycle analysis of exponentially growing ARH-77 cells transfected
with the cDNA for syndecan-1 (ARH-77syn-1) or the neo gene
only (ARH-77neo) demonstrates that syndecan-1 expression
inhibits G1-S transition (Fig 7A). A
similar effect was seen upon the addition of purified syndecan-1
ectodomain to ARH-77 cells (Fig 7B). The delay at the G1-S progression
by syndecan-1 is further supported by the downregulation of cyclin D1
upon the addition of purified syndecan-1 to ARH-77 cells (Fig 7C).
Taken together, these data indicate that syndecan-1 may affect the
growth and survival of myeloma cells by inducing apoptosis and
downregulating cyclin D1 expression.
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| Fig 7.
Effect of syndecan-1 on cell cycle progression and cyclin
D1 expression. (A) Expression of syndecan-1 inhibits G1-S progression: DNA content of exponentially growing ARH-77 cells, transfected with
vector only (ARH-77neo) or syndecan-1
(ARH-77syn-1), was determined by flow cytometry after PI
staining. (B) Shed syndecan-1 inhibits G1-S progression: ARH-77 cells
were incubated with syndecan-1 (2 nmol/L) or media alone for 48 hours
and stained with PI. DNA content analysis was performed using flow
cytometry. (C) Shed syndecan-1 inhibits cyclin D1 expression. ARH-77
cells were incubated with syndecan-1 (2 nmol/L) or media alone for 48 hours and stained for cyclin D1 using FITC-conjugated anti-cyclin D1
antibody using the manufacturer's protocol (PharMingen). Expression of
cyclin D1 is expressed as the ratio of median fluorescent intensity of
test sample to that of isotype control (*P < .05).
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Syndecan-1 Inhibits Osteoclastogenesis and Promotes
Osteoblastogenesis In Vitro
Osteoclast activation and recruitment are a characteristic feature of
myeloma, and supernatants from myeloma cell lines have been shown to
promote osteoclast differentiation in vitro.8 In initial
screening experiments using a previously described bone
marrow-calvarial coculture assay,19 media from
control-transfected myeloma cells promote a marked increase in the
number of osteoclasts as expected, whereas the addition of media from
syndecan-1-transfected cells leads to significantly less stimulation
of osteoclasts (Fig 8). Larger amounts of
media led to further decrease in osteoclast formation (data not shown).
These data suggest the presence of an inhibitor of osteoclastogenesis
in the media conditioned by syndecan-1-transfected cells (versus
neo-transfected cells).
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| Fig 8.
Media from syndecan-1-expressing cells inhibit
osteoclast formation in coculture assay. Marrow cells were cocultured
with calvaria cells for 8 days in the absence (control) or presence of
10 nmol/L 1,25(OH)2D3, or 1 µL of conditioned
media from vector-transfected (neo) or syndecan-1-transfected (syn-1)
myeloma cells. The number of osteoclasts was determined by counting
TRAP-positive cells with greater than 3 nuclei per cell (*P < .05).
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To further characterize this effect and to determine the effect on
osteoblasts, we utilized a murine bone marrow cell culture assay and
examined the effect of purified syndecan-1 on both osteoclastogenesis and osteoblastogenesis. The addition of syndecan-1 ectodomain leads to
a significant dose-dependent decrease in multinucleate TRAP-positive
cells (representing osteoclasts) and a modest increase in ALP-positive
cells (representing osteoblasts; Fig 9A and
B, respectively). The effect of syndecan-1 ectodomain on bone cell development was observed even at picomolar concentrations that have no
effect on myeloma cell growth. Interestingly, in direct contrast to the
effect on myeloma cells, the inhibition of osteoclastogenesis and
stimulation of osteoblastogenesis by syndecan-1 does not require an
intact ectodomain, because a similar effect was seen with syndecan-1 pretreated with heparitinase and with GAGs purified from syndecan-1 ectodomain (Fig 9C and D). However, this effect was specific for syndecan-1, because the addition of heparin (1 µg/mL) leads to an
increase in osteoclastogenesis and inhibition of osteoblastogenesis in
this assay.
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| Fig 9.
Effect of syndecan-1 on multinucleate (> 3 nuclei)
TRAP-positive and ALP-positive cell formation in murine bone marrow
cultures. Bone marrow cells (106 cells/well) were cultured
in -MEM containing media in multiwell dishes for a total of 8 days.
(A) Syndecan-1 inhibits osteoclastogenesis: Either 108
mol/L 1,25(OH)2D3 alone or 108
mol/L 1,25(OH)2D3 with 0.65 to 65 pmol/L
syndecan-1 purified by immunoaffinity chromatography was added during
the last 4 days in the culture period and cells were stained for TRAP.
One of three representative experiments is shown. (B) Syndecan-1
promotes osteoblastogenesis: 0.65 to 65 pmol/L syndecan-1 was added
during the last 4 days in the culture period and cells were stained for ALP. One of three representative experiments is shown. (C) Inhibition of osteoclast development by syndecan-1 does not require the intact syndecan ectodomain. Purified intact syndecan-1 ectodomain (45 pmol/L),
syndecan-1 pretreated with heparitinase (Hase), purified syndecan-1
GAGs, or heparin (1 µg/mL) was added for the last 4 days in the
culture period and cells were stained for TRAP. (D) Promotion of
osteoblast development by syndecan-1 does not require the intact
syndecan ectodomain. Purified intact syndecan-1 ectodomain (45 pmol/L),
syndecan-1 pretreated with heparitinase (Hase), GAGs purified from
syndecan-1, or heparin (1 µg/mL) was added for the last 4 days in the
culture period and cells were stained for ALP (*P < .05;
**P < .01).
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Syndecan-1 Inhibits Myeloma-related Morbidity in scid Mice
A scid mouse model, previously well characterized for myeloma
cell growth and bone disease,23,24 was used to examine
whether syndecan-1 affected myeloma pathobiology in vivo. In this
model, injection of ARH-77 cells into scid mice results in the
development of myeloma-related morbidity in most mice, defined as hind
limb paralysis due to spinal cord compression, visible soft tissue tumors, or extreme emaciation and lethargy. Irradiated C.B-17 scid mice were injected with either ARH-77syn-1 or
ARH-77neo cells via the lateral tail vein. The majority of
the mice injected with the syndecan-1-negative ARH-77neo
cells developed overt disease and lytic bone lesions
(Fig 10), thereby replicating the results
obtained by other investigators using this cell line. However, after
transfection of ARH-77 cells with syndecan-1, the development of
myeloma-related morbidity as defined earlier was significantly
inhibited (35% v 85%, P < .01) and delayed (mean
time to morbidity 84 days v 58 days, P < .01)
relative to control mice injected with the neo-transfected cells
(Table 1). Furthermore, only 30% of mice
injected with ARH-77syn-1 cells developed lytic bone
disease, compared with 80% in controls (P < .05; Table 1).
View larger version (107K):
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| Fig 10.
Radiologic examination of mice injected with ARH-77
cells. Note the presence of lytic bone lesions at the proximal tibia
and ilium (arrows) in a representative mice.
|
|
| |
DISCUSSION |
Results from the present work support the conclusion that syndecan-1
plays a major role in regulating the pathobiology of myeloma. First, we
provide evidence that syndecan-1 is shed from the surface of myeloma
cells as an intact ectodomain, and this shed syndecan-1 leads to a
striking induction of apoptosis and inhibition of cell growth in vitro.
Second, we show that syndecan-1 ectodomain may also affect the cells in
the tumor microenvironment by inhibiting osteoclast and promoting
osteoblast differentiation. Third, we demonstrate that, when myeloma
cells are engineered to express syndecan-1 on their surface and then
injected into scid mice, tumor development and disease-related
morbidity is retarded relative to their syndecan-1-negative
counterparts. Taken together with our previous studies showing that
syndecan-1 is highly expressed by most malignant plasma
cells12,13 and that cell surface syndecan-1 mediates both
cell-cell and cell-extracellular matrix adhesion and inhibits the
invasion of cells through type I collagen,12,13,16,17 we
conclude that syndecan-1, in both the cell surface and shed form, may
play an important role in regulating the progression of this disease.
To our knowledge, this is the first report that an HS proteoglycan can
induce cellular apoptosis. The mechanism of this striking induction of
apoptosis of myeloma cells mediated by syndecan-1 is not known and may
involve interaction of heparan sulfate chains with a cellular
receptor17 or growth factor, with resultant modulation of
growth factor signaling.25 In addition, the observation that different myeloma cell lines show differing levels of apoptosis in
response to syndecan-1 (Fig 6) suggests that the extent of syndecan-1-induced apoptosis may be modulated by other factors (eg,
endogenous levels of bcl-2). Induction of apoptosis by
syndecan-1 may not be restricted to malignant cells, because it was
recently shown that the presence of in situ apoptosis in the
extrafollicular foci of antibody forming cells in the spleen correlated
with the expression of high levels of syndecan-1.26
In addition to inducing apoptosis, purified syndecan-1 also induces an
inhibition of G1-S progression and downregulation of cyclin D1. The
fact that this growth-inhibitory effect requires the intact ectodomain,
is not induced by heparin and requires close contact of syndecan-1 with
the cells suggests that a specific interaction between syndecan-1 and
the cell surface may be occurring. In sharp contrast to some other
myeloma growth inhibitors (eg, dexamethasone, IL-4, and
-interferon), exogenous IL-6 failed to reverse the
syndecan-1-mediated effect,4,22 suggesting that the growth
inhibition by syndecan-1 differs from these inhibitors in its mechanism
of action. Similar to what we observe in myeloma, a growth-inhibitory
effect of the intact syndecan-1 ectodomain has also been noted in
carcinoma cells but not in normal epithelial cells.27
However, in contrast to our observations, these investigators indicate
that suppression of carcinoma growth was not associated with induction
of apoptosis, suggesting that myeloma and carcinoma cells may differ in
their response to syndecan-1. Interestingly, elevated levels of
syndecan-1 promote the growth of the transformed renal epithelial cell
line (293 T cells).28 Thus, the effect of syndecan-1 may
vary between different cell types. The growth-regulatory effect of
proteoglycans is not limited to syndecan-1. Recently, it was shown that
the expression of perlecan, a basement membrane HS proteoglycan, was
associated with a decrease in growth and invasiveness of fibrosarcoma
cells.29 Negative growth regulation by the secreted
chondroitin sulfate proteoglycan, decorin, has been attributed to its
ability to bind transforming growth factor- and upregulate
cyclin-dependent kinase inhibitor p21.30 Although the
molecular mechanism of syndecan-1-mediated growth suppression is not
known, it may similarly upregulate p21, leading to downregulation of
cyclin D1 expression and to apoptosis, as we have observed.
Because syndecan-1 is shed from the surface of myeloma cells, it is
potentially capable of exerting both a paracrine and systemic effect in
myeloma patients. Clearly, the concentration of syndecan-1 ectodomain
in vivo in the bone marrow microenvironment is important to
syndecan-1-mediated paracrine effects in patients. In this regard, we
have recently discovered that markedly elevated levels of intact
syndecan-1 ectodomain are present in the serum of some myeloma
patients.31 Moreover, shed syndecan-1 could be the source of the heparin-like anticoagulant occasionally responsible for coagulopathy in myeloma.32 Recently, it was shown that the
shedding of syndecan-1 by cultured endothelial cells is highly
regulated by the activation of at least two distinct receptor classes,
G protein-coupled and protein tyrosine kinase,33 suggesting
that shed syndecan-1 may have important regulatory roles in vivo. The regulation of shedding of syndecan-1 by myeloma cells and the clinical
importance of serum syndecan-1 in myeloma patients remains to be
examined.
In addition to its effects on myeloma cell growth, syndecan-1 also
exerts significant effects on both osteoclast and osteoblast development in vitro and therefore may form part of a bone-preserving regulatory loop in myeloma. To our knowledge, these data represent the
first evidence that syndecans may affect the development of bone cells.
In sharp contrast to the effects on myeloma cells, the effect of
syndecan-1 on bone cell development occurs at much lower (picomolar)
concentrations and does not require an intact ectodomain, suggesting a
different mechanism of action. The finding that heparitinase treatment
does not abolish syndecan-1 effect suggests that fragments of
syndecan-1 GAG may exert biological activity on bone cell precursors.
However, we cannot exclude the possibility that this effect is due to
growth-regulatory factors that are bound to the purified syndecan-1 HS
chains and remain active after heparitinase digestion. The in vitro
effects on bone cell development are further supported by the in vivo
data showing diminished lytic bone disease in mice injected with
syndecan-1-expressing cells. However, whether the observed reduction
in lytic bone disease is due to a direct effect of syndecan-1 on bone
or via tumor bulk reduction is not addressed in this study.
The finding that syndecan-1 may have a beneficial effect on bone is
surprising, because bone loss is a well-recognized complication of
heparin therapy.34 Our finding that heparin leads to an
increase in osteoclastogenesis and inhibition of osteoblastogenesis in vitro is consistent with prior in vivo observations that heparin promotes osteoclast-mediated bone resorption35 and inhibits osteoblast formation36 and may be the mechanism of
heparin's effect on bone. Recent studies have shown that the
osteopenic effect is dependent on both size and sulfation of the
heparin fragments and is less with low molecular weight heparin than
with unfractionated heparin.35,36 Thus, the effect of
various heparan sulfate proteoglycans (HSPG) on the bone may differ,
depending on the nature and fine structure of GAGs. Indeed, HSPGs have
been shown to bind a number of cytokines and soluble factors with
ability to both stimulate or inhibit osteoclasts and osteoblasts in
vitro.37,38 Syndecan-1 is expressed transiently during
mammalian tooth development and may play an important role in this
process.39 Expression of syndecan-1 and bone morphogenetic
protein-4 is specifically reduced in the dental mesenchyme with mutant
Msx1, a member of Msx homeobox family, critical for tooth morphogenesis
and craniofacial development.40 Taken together with the
data presented in this report, these observations suggest that HSPGs,
including syndecan-1, may play an important role in the regulation of
bone formation in vivo and that the nature of this regulation may
depend on the specific GAG structure and nature of the proteoglycan.
In summary, we have shown that syndecan-1 has a significant impact on
the behavior of cells in the tumor microenvironment in myeloma, both in
vitro and in vivo. It may therefore constitute part of a potentially
beneficial regulatory loop to counteract the net effect of other
molecules (eg, IL-6) that promote myeloma cell proliferation/survival
and bone loss. Further understanding of the mechanism and regulation of
these biologic effects may lead to novel therapies for myeloma. These
data also have obvious implications for improved therapy of other
hematologic (eg, Hodgkin's disease and primary effusion
lymphoma)41,42 or epithelial malignancies (eg, laryngeal
cancer) associated with altered syndecan-1 expression43 and
for metabolic bone disease.
| |
FOOTNOTES |
Submitted October 15, 1997;
accepted January 20, 1998.
Supported in part by Florence Carter Fellowship in Leukemia Research
from AMA-ERF (to M.V.D.) and Grants No. CA 68494 (to R.D.S.), PO-1 AG
139181 (to E.A.), and CA71145 (to J.K.L.) from the National Institutes
of Health. M.V.D. is a recipient of a Clinical Research Career
Development Award from the American Society of Clinical Oncology.
Address reprint requests to Ralph D. Sanderson, PhD, Department of
Pathology, Slot 517, 4301 W Markham, Little Rock, AR 72205; e-mail:
SandersonRalphD{at}exchange.uams.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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C. Y. Pumphrey, A. M. Theus, S. Li, R. S. Parrish, and R. D. Sanderson
Neoglycans, Carbodiimide-modified Glycosaminoglycans: A New Class of Anticancer Agents That Inhibit Cancer Cell Proliferation and Induce Apoptosis
Cancer Res.,
July 1, 2002;
62(13):
3722 - 3728.
[Abstract]
[Full Text]
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Y. Yang, S. Yaccoby, W. Liu, J. K. Langford, C. Y. Pumphrey, A. Theus, J. Epstein, and R. D. Sanderson
Soluble syndecan-1 promotes growth of myeloma tumors in vivo
Blood,
June 28, 2002;
100(2):
610 - 617.
[Abstract]
[Full Text]
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F. Silvestris, P. Cafforio, M. Tucci, and F. Dammacco
Negative regulation of erythroblast maturation by Fas-L+/TRAIL+ highly malignant plasma cells: a major pathogenetic mechanism of anemia in multiple myeloma
Blood,
February 15, 2002;
99(4):
1305 - 1313.
[Abstract]
[Full Text]
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P. W. B. Derksen, R. M. J. Keehnen, L. M. Evers, M. H. J. van Oers, M. Spaargaren, and S. T. Pals
Cell surface proteoglycan syndecan-1 mediates hepatocyte growth factor binding and promotes Met signaling in multiple myeloma
Blood,
February 15, 2002;
99(4):
1405 - 1410.
[Abstract]
[Full Text]
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D. M.-Y. Sze, G. Giesajtis, R. D. Brown, M. Raitakari, J. Gibson, J. Ho, A. G. Baxter, B. Fazekas de St Groth, A. Basten, and D. E. Joshua
Clonal cytotoxic T cells are expanded in myeloma and reside in the CD8+CD57+CD28- compartment
Blood,
November 1, 2001;
98(9):
2817 - 2827.
[Abstract]
[Full Text]
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H. M. Lacy and R. D. Sanderson
Sperm protein 17 is expressed on normal and malignant lymphocytes and promotes heparan sulfate-mediated cell-cell adhesion
Blood,
October 1, 2001;
98(7):
2160 - 2165.
[Abstract]
[Full Text]
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J.F. Manakil, P.B. Sugerman, H. Li, G.J. Seymour, and P.M. Bartold
Cell-surface Proteoglycan Expression by Lymphocytes from Peripheral Blood and Gingiva in Health and Periodontal Disease
Journal of Dental Research,
August 1, 2001;
80(8):
1704 - 1710.
[Abstract]
[PDF]
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C. Seidel, M. Borset, O. Hjertner, D. Cao, N. Abildgaard, H. Hjorth-Hansen, R. D. Sanderson, A. Waage, and A. Sundan
High levels of soluble syndecan-1 in myeloma-derived bone marrow: modulation of hepatocyte growth factor activity
Blood,
November 1, 2000;
96(9):
3139 - 3146.
[Abstract]
[Full Text]
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M. Borset, O. Hjertner, S. Yaccoby, J. Epstein, and R. D. Sanderson
Syndecan-1 is targeted to the uropods of polarized myeloma cells where it promotes adhesion and sequesters heparin-binding proteins
Blood,
October 1, 2000;
96(7):
2528 - 2536.
[Abstract]
[Full Text]
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C. Seidel, A. Sundan, M. Hjorth, I. Turesson, I. M. S. Dahl, N. Abildgaard, A. Waage, and M. Borset
Serum syndecan-1: a new independent prognostic marker in multiple myeloma
Blood,
January 15, 2000;
95(2):
388 - 392.
[Abstract]
[Full Text]
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J. K. Langford, M. J. Stanley, D. Cao, and R. D. Sanderson
Multiple Heparan Sulfate Chains Are Required for Optimal Syndecan-1 Function
J. Biol. Chem.,
November 6, 1998;
273(45):
29965 - 29971.
[Abstract]
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W. Liu, E. D. Litwack, M. J. Stanley, J. K. Langford, A. D. Lander, and R. D. Sanderson
Heparan Sulfate Proteoglycans as Adhesive and Anti-invasive Molecules. SYNDECANS AND GLYPICAN HAVE DISTINCT FUNCTIONS
J. Biol. Chem.,
August 28, 1998;
273(35):
22825 - 22832.
[Abstract]
[Full Text]
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Abstract
Introduction
Abstract